CN101060855A - Gastrointestinal proliferative factor and uses thereof - Google Patents

Abstract

The invention relates to pharmaceutical compositions comprising gastrointestinal proliferative factor (GIPF) polynucleotides and polypeptides. The invention further relates to the therapeutic use of GIPF to prevent or treat conditions or disorders associated with the degeneration of the epithelial mucosa.

Description

Gastrointestinal proliferative factor and uses thereof

1. background

1.1 invention field

The present invention relates generally to contain the compoistion and method of use of gastrointestinal proliferative factor polypeptide and polynucleotide.

1.2 sequence table

This paper provides sequence table.

1.3 background

Ionizing radiation therapy and cytotoxic chemotherapy can cause damage to oral cavity stomach function regulating intestinal mucosa, and for standing the patient of antineoplaston, this is serious problem always.Mucositis is a mucosal inflammation, and with treatment or to alleviate be that purpose is used in chemotherapy and the radiotherapeutic patient colony, this is a general especially problem.The gastrointestinal tract mucous damage that (gastrointestinal region) radiation and chemotherapy cause comprises that pit cell is destroyed, fine hair shortens and the ulcer and necrosis (the Berthrong M of gastrointestinal epithelial, World J Surg 10:155-170 (1986)), this formation comprises the basis of the disease of gastrointestinal mucositis and enterocolitis.For patients, this may mean stomachache, bloody diarrhea, malabsorption, means bacterial translocation (Guzman et al., J Surg Res 46:104-107 (1989)) sometimes.In addition, chemotherapy and ionizing radiation also can influence other mucosa, comprise oropharynx and lip mucosa and esophageal mucosa membrane injury.As everyone knows, use radiation and chemotherapeutic combination type therapy can in the head and neck cancer patient, produce the stomatitis of high sign simultaneously, in the small cell lung cancer patient, produce esophagitis.Chemotherapy and radiation causes damage by direct and indirect toxicity to oral cavity stomach function regulating intestinal mucosa.Direct catarrhal mechanism is to carry out non-specific cell and kill and wound being in substrate epithelial cell in the quick division, causes epithelium attenuation, inflammation, cell to upgrade and reduces and the final ulcer that forms.These poignant damages also can make the risk of local and systemic infection increase to some extent.Mucosa toxicity is chemotherapy-inductive myelosuppressive side-product indirectly, and it makes direct mucosa injury position that antibacterial and viral infection take place.The seriousness of these influences can hinder the progressively increase of dosage, delay treatment, and assurance can make dosage reduce, and therefore limited the effectiveness of cancer therapy.

The prevention of chemotherapy and radiation-inductive (mucosa) gastrointestinal damage (mucositis) and treatment must be accepted the chemotherapy or the radiotherapy prescription of suboptimal dose usually, change dosage in the course of treatment after toxicity subsequently produces downwards, or after the methotrexate of medium-dosage or high dose, use specific antidote, as formyl tetrahydrofolic acid (leucovorin) (Allegra CJ.Antifolates.In:Chabner and Collins, eds.Cancer Chemotherapy:Principles and Practice.Philadelphia, Pa.JP LippincottCo; 1990:110-153.).

Damage to gastrointestinal mucosa is also relevant with the gastrointestinal tract chronic inflammatory diseases that is generically and collectively referred to as inflammatory bowel disease.Therapy based on cytokine can be used for treating inflammatory bowel disease (Bouma and StroberNature Rev 3:521-533 (2003)).Yet, suffer from inflammatory bowel disease, often need excise small intestinal as the patient of segmental enteritis Crohn disease (Crohn ' s disease).Also essential by the surgical resection small intestinal after wound, vascular accident and cancer.The surgical discectomy that only stays the survival small intestinal that is shorter than 200cm places the patient and develops into short bowel syndrome (short-bowel syndrome) risk (SBS).The definition clinically of this disease of SBS is: malabsorption, diarrhoea, body fluid and electrolyte disturbance and malnutrition.SBS patient's treatment often needs to use for a long time or all the life parenteral nutrient substance (DiBaise etal., Am JGastroenterol 99:1823-1832 (2004)).

Therefore, be necessary to seek a kind of medicament, strengthen toleration, advance the up-to-date therapy of treatment inflammatory bowel disease, and recover digestion and absorption process impaired behind the small intestine meridian surgical resection antineoplaston in order to preventative or therapeutic ground.

2. summary of the invention

The present invention's part is based on following discovery, and promptly gastrointestinal proliferative factor (GIPF) can be induced gastrointestinal tract epithelial cell propagation.Therefore, contain GIPF, the compositions of its fragment or its analog can be used for treating the disease that needs form epithelium, as be used for the treatment of disorder of gastrointestinal tract, comprise the mucositis that chemotherapy and radiation causes, the mucositis of oropharynx, lip and esophagus, inflammatory bowel disease, other comprises that the disease of wound, burn, oculopathy and any needs stimulate epithelial cell proliferation or regenerated disease.

Therefore, in one embodiment, the present invention relates to contain the GIPF polypeptide for the treatment of effective dose and the compositions of drug acceptable carrier.

Compositions of the present invention comprises the polynucleotide of separated coding GIPF polypeptide, and it comprises recombinant DNA molecules and cloned genes or its degeneracy variant, and particularly natural variant is as allele variant.Particularly, polynucleotide of the present invention are based on separating from the GIPF in cDNA library polynucleotide (SEQIDNO:2), and described library is prepared by people's periderm skin mRNA.

Compositions of the present invention also comprises carrier, as contains the expression vector of polynucleotide of the present invention, contains the cell of described polynucleotide after genetic engineering modified and can express the cell of described polynucleotide after genetic engineering modified.

Compositions of the present invention contains separative polynucleotide, and described polynucleotide include but not limited to the GIPF polynucleotide, its fragment or its mutant; Polynucleotide (the SEQID NO:4 for example that contains the full length protein coded sequence of SEQID NO:2 or 3; GIPFwt); (the SEQID NO:6 for example of the polynucleotide through the protein coding sequence of V5-His-labelling that contains SEQID NO:5; GIPFt); The polynucleotide (for example SEQID NO:10) of nucleotide sequence that contain the dominance mature protein coded sequence of SEQID NO:9; The polynucleotide (for example SEQID NO:12) of nucleotide sequence that contain the mature protein coded sequence of SEQID NO:11; The polynucleotide (for example SEQID NO:14) of nucleotide sequence that contain thrombospondin (thrombospondin) domain of SEQID NO:13; The polynucleotide (for example SEQID NO:16) of the SEQID NO:15 of the nucleotide sequence of the dominance mature protein sequence that containing encodes lacks furin (furin) cleavage site; The polynucleotide (SEQID NO:18) of SEQ ID NO:17 of nucleotide sequence that contain the coding GIPF polypeptide of sudden change furin cracking site; And coding contains the polynucleotide (SEQ ID NOs:84,86,88,90,92,94,96,98,100,102,104 and 177) of different length GIPF polypeptide.Polynucleotide compositions of the present invention also includes, but not limited under stringent hybridization condition polynucleotide (a) the SEQID NO:2 with following nucleotide hybridization, 3,5,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or the complementary series of 177 described arbitrary nucleotide sequences; (b) coding SEQ IDNO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 and 178 nucleotide sequence; () polynucleotide for example, allele variant have 70% sequence homogeneity (for example 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% and 99%) at least with above-mentioned arbitrary polynucleotide to above-mentioned arbitrary polynucleotide variant; Encode the kind homologue of above-mentioned arbitrary polypeptide (for example directly to the polynucleotide of congener (ortholog); Or coding comprises the polynucleotide of the polypeptide of SEQ ID NO:4 or 6 polypeptide ad hoc structure territories or truncate.

The present invention further provides the cloning vehicle or the expression vector that comprise above-mentioned polynucleotide at least a portion, and with these expression vector transformed host cells or organism.Useful carrier comprises plasmid well known in the art, cosmid, lambda phage derivant, phasmid etc.Accordingly, the present invention also provides carrier that comprises polynucleotide of the present invention and the host cell that comprises described polynucleotide.Generally speaking, carrier is included in the replication origin that works at least a organism, but the suitable restriction restriction enzyme site and the selected marker of host cell.Carrier of the present invention comprises that expression vector, replicating vector, probe produce carrier and sequencing vector.Host cell of the present invention can be prokaryotic cell or eukaryotic cell, can be the part of unicellular organism body or multicellular organisms.

Pharmaceutical composition of the present invention includes, but not limited to contain SEQ ID NO:4,6,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103, the polypeptide of the isolated polypeptide of 105 or 178 aminoacid sequence.Polypeptide of the present invention also comprises by the biologically active polypeptide of following polynucleotide encoding (a) and comprises SEQ ID NO:2,3,5,9,11,13,15,17,84,86,88,90,92,94,96,98,100, and arbitrary polynucleotide of 102,104 or 177 described nucleotide sequences; Or (b) under stringent hybridization condition with (a) in the polynucleotide of complementary sequence hybridization of polynucleotide.Also comprise SEQ ID NO:4,6,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103, the biological activity of 105 or 178 described arbitrary protein sequences or immunocompetence analog, and the basic equivalent of retains biological activity.Polypeptide of the present invention chemosynthesis wholly or in part obtains, but preferably utilizes genetically modified cell of the present invention (as host cell) to generate by recombination form.The present invention includes the NO:4 with SEQ ID, arbitrary sequence has at least 85%, 90% in 6,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 and 178,92%, 94%, 96%, 98% or 99% identical polypeptide.The present invention also comprises 20,15,10,9 on the sequence form, 8,7,6,5,4,3,2 or 1 amino acid residues and SEQ ID NO:4,6,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103, the different polypeptide of 105 or 178 arbitrary sequences.It can be that guard or nonconservative that aminoacid changes.

The invention still further relates to the method that produces the GIPF polypeptide, be included under the condition that is suitable for the destination protein expression, in proper culture medium, cultivate the host cell that contains expression vector, this expression vector comprises the GIPF polynucleotide passage of at least one code book invention GIPF polypeptide, and protein purification or polypeptide from culture medium or in the host cell.Embodiment preferred comprises the method that produces maturation protein or dominance maturation protein formal protein.

Polypeptide of the present invention can be used for multiple being applied at present in other proteic conventional process and the method.For example, polypeptide of the present invention can be used for producing the antibody of specificity in conjunction with this polypeptide.This antibody, particularly monoclonal antibody, detect or quantitative tissue in during this polypeptide of great use.

In another embodiment, the present invention relates to stimulate the method for epithelial cell proliferation.This method comprises epithelial cell is contacted with compositions that said composition contains GIPF polypeptide, its fragment or its analog for the treatment of effective dose, and pharmaceutically acceptable carrier.Particularly, to the individuality of needs stimulation epithelial cells (comprising cytoprotective, propagation and/or differentiation), treat GIPF albumen, its fragment or its analog effective dose or the prevention effective dose.

In the method that all are mentioned, epithelial cell can be in contact with one another in external or body with the GIPF polypeptide.

Prevention, treatment also are provided or have improved the method for disease, comprised the compositions that gives mammalian subject treatment effective dose, said composition comprises peptide of the present invention and pharmaceutical acceptable carrier.

Particularly, GIPF polypeptide of the present invention can be used for inducing gastrointestinal pit cell (crypt cell) propagation and/or differentiation, thereby makes gastral epithelial layer regeneration.Therefore, GIPF polypeptide of the present invention and polynucleotide can be used for mucositis, enterocolitis and the inflammatory bowel disease (inflammatory bowel disease) that therapeutical chemistry or radiotherapy cause.Also can be used for treatment and comprise that wound, burn, oculopathy and needs stimulate epithelial cell proliferation or regenerated any other disease.

Polynucleotide of the present invention and polypeptide also can be used as the label of gastrointestinal epithelial histo-differentiation and growth.

Method of the present invention also provides method for the disease that treatment is mentioned herein, comprise the symptom that shows disease association described herein or the mammalian subject of tendency, treat the compositions of effective dose, said composition comprises polynucleotide of the present invention and polypeptide, and pharmaceutical acceptable carrier.In addition, the present invention includes the method for the disease that treatment mentioned herein, comprise and contain chemical compound and other material that can regulate target gene product overall activity, and the compositions of pharmaceutical acceptable carrier.Chemical compound and other material can influence target gene/protein expression level, or the active adjusting of target protein.Especially, also provide prevention, treatment or improvement to comprise the method for diseases such as mucositis, inflammatory bowel disease, wound, comprise and give mammalian subject, include but not limited to the people, the compositions that comprises polypeptide of the present invention of treatment effective dose or comprise the compositions of the binding partners of GIPF polypeptide of the present invention.Whether the individuality that particular disorder or pathology mechanism will indicate polypeptide of the present invention or its binding partners that needs are treated is useful.

The present invention further provides the method for preparing the medicine that is used for said method.

The invention still further relates to the method for the existence that detects middle polynucleotide of the present invention of sample (as tissue or sample) or polypeptide.This method for example can be used for prognosis and diagnosis disease described herein, and differentiates vulnerable crowd.

The invention provides the method for polypeptide of the present invention in the test sample, comprise under certain condition, with sample with described polypeptide in conjunction with and the chemical compound that forms complex contact sufficiently long a period of time, thereby formation complex, detect the formation of this complex, if complex forms, just can detect this polypeptide.

The present invention provides simultaneously and has comprised polynucleotide probes and/or monoclonal antibody, and the test kit of quantitative criterion product randomly, to realize the inventive method.And, the invention provides in the clinical experiment of the above-mentioned disease of treatment, estimate the method for curative effect of medication, monitor patients process.

The present invention also provides the discrimination method of adjusting (for example increase or reduce) polynucleotide of the present invention and/or expression of polypeptides or active chemical compound.This method can be used for, and for example differentiates the chemical compound that strengthens GIPF polypeptide therapeutic activity, improves disease symptoms described herein.This method includes, but not limited to identification and analysis and polypeptide of the present invention interact chemical compound and other material of (for example combining).

The invention provides discrimination method with the bonded chemical compound of polypeptide of the present invention, comprise under certain condition, make chemical compound contact the sufficiently long time with described polypeptide, thereby form polypeptide/compound complex, detect this complex, if detected this polypeptide/compound complex, just identify and the bonded chemical compound of polypeptide.

The present invention also provides the discrimination method with the bonded chemical compound of polypeptide, comprise chemical compound is contacted the sufficiently long time to form polypeptide/compound complex with polypeptide in the cell, wherein this complex promotes the expression of reporter gene sequence in the cell, thereby express the detection complex by detecting reporter gene sequence, if detected described polypeptide/compound complex, just identified and the bonded chemical compound of polypeptide.

Another embodiment of the invention is by transmitting the GIPF polypeptide, providing gene therapy method for treating disease described herein.

In the related embodiment, the present invention relates to the application of carrier in the medicine of preparation treatment disease described herein, this carrier comprises the gene of coding GIPF polypeptide, expresses the GIPF polypeptide thereby this gene and expression regulation sequence can be operatively connected.More specifically, the invention provides the application of adenovirus vector of the present invention (for example, describing in detail hereinafter) in the medicine of preparation treatment mucositis or inflammatory bowel disease.

Except foregoing method and purposes, the invention provides a kind of new viral vector, comprise the gene of the coding GIPF polypeptide that can be operatively connected with expression regulation sequence.In the preferred embodiment, viral vector is an adenovirus vector.Viral vector of the present invention can provide the gene of any GIPF polypeptide of coding, and is as indicated above.

The present invention also provides pharmaceutical composition, comprises arbitrary viral vector of the present invention and pharmaceutical acceptable carrier.

On the other hand, the present invention pays close attention to transgenosis construct, comprises coding natural human GIPF albumen, its analog or its segmental nucleic acid, and under the control of transcriptional regulatory sequences, this nucleic acid is expressed in the B cell.Transgenosis construct preferably includes the B cell specificity promotor, as the immunoglobulin kappa chain promoter.

On the other hand, the present invention pays close attention to transgenic nonhuman mammal, but in its B cell, produce the natural human GIPF albumen of detection level, its analog or its fragment, the wherein said transgene mammal natural human GIPF albumen of will having encoded, its analog or segmental nucleotide sequence with natural human GIPF protein biological activity stably are incorporated in the chromosome, and can be operatively connected with the transcription regulating nucleotide sequence that instructs this nucleotide sequence to express in the B cell.Transcription regulating nucleotide sequence preferably includes the B cell specificity promotor, as the immunoglobulin kappa chain promoter.The non-human transgenic mammal can be, but be not limited to for example mice, rat, rabbit, pig, sheep, goat or cattle.

On the other hand, the present invention pays close attention to the screening technique of the drug candidates of treatment disease described herein, comprise that (a) gives transgenic mice with drug candidates, express the GIPF polypeptide in this mouse B cell, and have the intestinal expansion (intestinal distension) relevant with the epithelial cell hyper-proliferative; (b) the evaluate candidate medicine is to the influence of epithelial cell hyper-proliferative.Described drug candidates can be regulated (as increasing or reducing) polynucleotide of the present invention and/or polypeptide expression or activity.

The present invention also comprises a kind of method for the treatment of or improving disease, and described disease comprises mucositis, inflammatory bowel disease and wound, and this method comprises and gives mammalian subject, includes but not limited to the people, the GIPF polypeptide and the cytokine of treatment effective dose.

On the other hand, the present invention includes pharmaceutical composition, said composition comprises polypeptide of the present invention, second kind of therapeutic agent, for example cytokine, and pharmaceutical acceptable carrier.

According to following enforcement detailed description of the present invention, other aspects of the present invention and advantage are obvious to those skilled in the art.

3. accompanying drawing summary

The DNA sequence (SEQ ID NO:2) that Fig. 1 describes total length GIPF (A) and amino acid sequence corresponding (SEQ ID NO:4) (B).SEQ ID NO:2 comprises 5 ' end and 3 ' end untranslated region and open reading frame.

Fig. 2 describes the expression of GIPF mRNA in people's tissue (A) and rat tissue (B)

Fig. 3 is the sketch map of the GIPF polypeptide of the present composition.The SEQ ID NOs of the digital corresponding polypeptide of underscore, other numerals are SEQID NOs of coded polynucleotide sequence.

Fig. 4 A is the BLASTP aminoacid sequence comparison of GIPF albumen (being SEQ ID NO:4) with the human stem cell growth A1 SEQID NO:23 (the SEQ ID NO:10 of PCT WO 01/77169 A2) of SEQ ID NO:2 or 3 codings, illustrate in these two sequences, the 11-257 amino acids residue of the 10-251 amino acids residue of SEQ ID NO:4 and SEQ ID NO:23 has 63% similarity, and the 11-257 amino acids residue of the 10-251 amino acids residue of SEQ ID NO:4 and SEQ ID NO:23 has 46% homogeneity.

Fig. 4 B is the GIPF albumen (being SEQ ID NO:4) of SEQ ID NO:2 or 3 codings and specific region (the 501-657 amino acids residue of SwissProt registration number P07996 of human blood platelets reactive protein 1; SEQID NO:28) BLASTP aminoacid sequence comparison.This figure shows that in these two sequences, the 501-657 amino acids residue of the 14-166 amino acids residue of SEQ ID NO:4 and SEQ ID NO:28 has 36% similarity and 26% homogeneity, wherein A=alanine, the C=cysteine, D=aspartic acid, E=glutamic acid, the F=phenylalanine, the G=glycine, H=histidine, I=isoleucine, K=lysine, the L=leucine, M=methionine, N=agedoite, the P=proline, the Q=glutamine, R=arginine, S=serine, the T=threonine, the V=valine, W=tryptophan, Y=tyrosine.Breach dots.

Fig. 5 A-5R has described generation GIPF of the present invention and has knocked in (knock-in, GIPF-KI) method step of carrier.Also described and produced the method for optimizing of expressing the transgenic mice of GIPF in its B cell.

Fig. 6 has described the position of the probe that is used for screening the ES clone that homologous recombination produces in the Southern engram analysis, and the size of the EcoRI digestion fragment of the mouse gene group DNA of experience homologous recombination or non-homogeneous reorganization.

Fig. 7 is the macropathology of GIPF-KI mouse intestinal: matched group (A) GIPF-KI (B).

Fig. 8 is respectively the H﹠amp of the small intestinal slices across (transverse section) of GIPF-KI mice (A) and contrast gomphosis mouse (B); E dyeing.

Fig. 9 is the small intestinal section H﹠amp of Fig. 8 of seeing under high-amplification-factor more; E dyeing.The GIPF-KI section of A in A and the C corresponding diagram 8, the intestinal section of the contrast gomphosis mouse of the B in B and the D corresponding diagram 8.

Figure 10 is the Ki67 dyeing of the small intestinal slices across (cross-section) of contrast gomphosis mouse (A and C) and GIPF-KI mice (B and D).

Figure 11 is a control mice (A and C) and with 1 * 10 ¹⁰The small intestinal slices across of the mice (B and D) that virion is handled.Section obtains after 3 days in injection blank respectively and injection GIPF adenovirus.

Figure 12 is a control mice (A and C) and with 1 * 10 ¹⁰The small intestinal slices across of the mice (B of Figure 12 and D) that virion is handled.Section obtains after 5 days in injection blank respectively and injection GIPF adenovirus.

Figure 13 is that BrdU mixes control mice (A and C) and with 1 * 10 ¹⁰The pit cell of the propagation of mice (B and the D) small intestinal that virion (VP) is handled.

Figure 14 is the Ki67 dyeing of the pit cell of control mice (A and C) and the GIPF-adenovirus propagation of handling mice (B and D) small intestinal.

Figure 15 is a control mice (A and C) and with 1 * 10 ⁹The H﹠amp of the small intestinal slices across of the mice (B and D) that virion is handled; E dyeing.

Figure 16 is the H﹠amp that control mice (A and C) and GIPF-adenovirus are handled mice (B and D) colon slices across; E dyeing.

The proteic dissolubility requirement of GIPF of Figure 17 (A and B) natural V5-histidine-labelling that purification obtains from Chinese hamster ovary celI.

The proteic pharmacokinetics of GIPF of the V5-histidine-labelling in Figure 18 (A and B) mice serum.

Figure 19 is the H﹠amp of control mice (A and C) and mice (B and D) the small intestinal slices across handled with the GIPF albumen of purification; E dyeing.

Figure 20 is that BrdU mixes control mice (A and C) and the pit cell of the propagation of mice (B and the D) small intestinal handled with the GIPF albumen of purification.

Figure 21 is the H﹠amp that the GIPF albumen of control mice (A) and purification is handled mice (B) colon slices across; E dyeing.

Figure 22 is that BrdU mixes control mice (A and C) and the pit cell of the propagation of mice (B and the D) colon handled with the GIPF albumen of purification.

Figure 23 is the H﹠amp of the radiation murine small intestinal slices across of not raying irradiation mice (A) and saline (B), KGF (C) or GIPFwt (D) processing; E dyeing.

Figure 24 is that 5-FU is to control mice and the influence of accepting the tumor size of GIPFwt mice.

Figure 25 is GIPF to the influence of the macropathology of the small intestinal of normal mouse (E and F) and tumor-bearing mice (A-D) and colon.

Figure 26 is normal mouse (A) and accepts 5-FU and/or the H﹠amp of the small intestinal of the tumor-bearing mice of GIPF and colon slices across; E dyeing.

Figure 27 is to little morphometry of the intestinal villus length and the crypts degree of depth, shows the influence of GIPF to the small intestine epithelium of accepting the 5-FU mice.

Figure 28 is control mice (A and B) and handles and the epithelial Ki67 dyeing of veutro tongue (ventral tongue) propagation of the mice (C-E) of whole body raying irradiation with KGF or GIPF.

Figure 29 is control mice (A and B) and handles and the epithelial Ki67 dyeing of dorsal part tongue (dorsal tongue) propagation of the mice (C and D) of whole body raying irradiation with KGF or GIPF.

Figure 30 is the epithelial proliferation index of mice veutro tongue with KGF or GIPF processing and whole body raying irradiation.

Figure 31 is the H﹠amp with the mice tongue section that GIPF handles and the whole body raying is shone; E dyeing.

Figure 32 is GIPF induces the activity index (IBDAI) of colitis mice inflammatory bowel disease to DSS influence.

Figure 33 is GIPF induces colitis mice the weight of animals total to DSS influence.

Figure 34 is GIPF induces colitis mice feces viscosity score to DSS influence.

Figure 35 is GIPF induces colitis mice hemorrhage of rectum total to DSS influence.

Figure 36 is that GIPF is to the small intestinal of control mice and DSS inducing mouse and the influence of colon macropathology.

Figure 37 is that DSS and/or GIPF handle the small intestinal of mice and the H﹠amp of colon slices across; E dyeing.

Figure 38 shows that to little morphometry of the intestinal villus length and the crypts degree of depth GIPF induces the effect of the small intestine epithelium of colitis mice to DSS.

Figure 39 is that BrdU mixes the small intestinal of the mice of accepting DSS and/or GIPF and the pit cell of colon propagation.

Figure 40 is GIPF induces the small intestine epithelium propagation of colitis mice to DSS influence.

Figure 41 is the influence of GIPF to beta-catenin in human endocrine and the renal epithelial cell white (catenin) stability.GIPF induces the Stabilization that beta-catenin is white in the HEK293 cell to be dose dependent (A) and time dependence (B).Boiling (C) does not influence the Stabilization of GIPF.

Figure 42 is the effect of GIPF to GSK3 β phosphorylation.

Figure 43 is designed for and measures the example that the GIPF zones of different is stablized the GIPF polypeptide analog of the white ability of beta-catenin.Segment number 1-11 is corresponding SEQ ID Nos:85 respectively, and 87,89,91,93,95,97,99,101,103 and 105 polypeptide.

Figure 44 is the GIPF analog shown in Figure 43 Stabilization white to beta-catenin.

Figure 45 is that people and Mus GIPF are to the active comparison of mouse intestinal macropathology.

Figure 46 is the influence of GIPF to the intestinal crypt degree of depth.

Figure 47 is the influence of GIPF to the Stabilization that beta-catenin is white in the isolating pit cell.

Figure 48 is GIPF to having the influence of body weight that TNBS induces the animal of colitis.

Figure 49 is GIPF to having the influence of colitis score that TNBS induces the animal of colitis.

Figure 50 is that GIPF is to the influence by the inductive chronic colitis of DSS.

Figure 51 is that GIPF induces the villus length of chronic colitis animal and the influence of the crypts degree of depth to having DSS.

To be GIPF induce the influence of the crypts proliferation index of chronic colitis animal to having DSS to Figure 52.

Figure 53 be GIPF to radiation after the influence of crypt survival.

Figure 54 A-N drives the transgenic structure sketch map that GIPF expresses with villin in the transgenic mice epithelium.

The embryo of Figure 55 GIPF in transgenic mice enteric epithelium regulating liver-QI expresses.

Figure 56 expresses white stable of beta-catenin in the transgenic mice of GIPF.

Figure 57 expresses the H﹠amp of small intestinal section of the transgenic mice of GIPF; E dyeing.

Figure 58 A-C drives the transgenic structure sketch map that (villin-driven) expresses GIPF and Wnt3a with villin in transgenic mice.

Figure 59 GIPF and Wnt3a express the embryo of transgenic mice small intestinal and large intestine.

Figure 60 expresses white stable of beta-catenin in the transgenic mice of GIPF and Wnt3a.

Figure 61 expresses the H﹠amp of small intestinal section in the Transgenic Mice Embryo of GIPF and Wnt3a; E dyeing.

Figure 62 A-K is a RS-KO vector construction sketch map.

Figure 63 is natural and reorganization RS-KO cloned genes picture group spectrum.

Figure 64 A-K is the structure sketch map of knocking in carrier pCk m4 KI of expressing GIPF deletion mutant (SEQ ID NO:91) in transgenic mice.

Figure 65 A-C is the structure sketch map of knocking in carrier pPS m4 KI of expressing GIPF deletion mutant (SEQ ID NO:91) in transgenic mice.

Figure 66 is natural and recombinant C k m4 KI cloned genes picture group spectrum.

Figure 67 is natural and reorganization PS m4 KI cloned genes picture group spectrum.

Figure 68 A-C expresses GIPF mutant (SEQ ID NO:177 in transgenic mice; GenBank registration number AK098225) the structure sketch map of knocking in carrier pCk VR KI.

Figure 69 A-C expresses GIPF mutant (SEQ ID NO:177 in transgenic mice; GenBank registration number AK098225) the structure sketch map of knocking in carrier pPS VR KI.

Figure 70 is natural and recombinant C k VR KI cloned genes picture group spectrum.

Figure 71 is natural and reorganization PS VR KI cloned genes collection of illustrative plates.

Figure 72 is the small intestinal of control mice and the transgenic mice of expressing GIPF deletion mutant SEQ ID NO:91 and the comparison of large intestine.

Figure 73 is the H﹠amp that expresses the small intestinal slices across of GIPF deletion mutant SEQ ID NO:91 transgenic mice; E dyeing (low amplification).

Figure 74 is the H﹠amp that expresses the small intestinal slices across of GIPF deletion mutant SEQ ID NO:91 transgenic mice; E dye (high-amplification-factor).

Figure 75 is the stable of axin-2 (Axin-2) in the expression GIPF deletion mutant SEQ ID NO:91 transgenic mice.

Figure 76 expresses GIPF mutant (SEQ ID NO:177; The small intestinal of transgenic mice GenBank registration number AK098225) (PSVR KI) and the comparison of large intestine and control animal.

Figure 77 is control mice and expresses GIPF mutant (SEQ ID NO:177; GenBank registration number AK098225) H﹠amp of transgenic mice (PSVR KI) small intestinal slices across; E dyeing (low amplification).

Figure 78 is control mice and expresses GIPF mutant (SEQ ID NO:177; GenBank registration number AK098225) H﹠amp of transgenic mice (PSVR KI) small intestinal slices across; E dye (high-amplification-factor).

Figure 79 is control mice and expresses GIPF mutant (SEQ ID NO:177; GenBank registration number AK098225) axin-2 stable in the transgenic mice.

Figure 80 is the H﹠amp of the large intestine section of the inductive chronic IBD animal of control animal and T cell transfer (embodiment 37); E dyeing.

4. detailed Description Of The Invention

Polypeptide of the present invention will be described in detail as shown in Figure 3 below.

The GIPF polypeptide of SEQ ID NO:4 is 263 amino acid whose protein, the about 29kDa of estimated molecular weight, not glycosylation. SEQ ID NO:2 is the cDNA of coding GIPF polypeptide. Initial methionine is from 603 of SEQ ID NO:2, and the termination codon of supposition is from 1392 of SEQ ID NO:2. With BLAST algorithm (Altschul S.F.et al., J.Mol.Evol.36:290-300 (1993) and Altschul S.F.et al., J.Mol.Biol.21:403-10 (1990), incorporated herein by reference) the retrieval protein pool, show SEQ ID NO:4 and SEQ ID NO:23 stem cell factor A-1 (the SEQ ID NO:10 of PCT WO01/77169 A2) (Fig. 4 A) and human blood platelets reactive protein 1 (SEQ ID NO:28) (Fig. 4 B) homology.

The 1-20 amino acids residue of SEQ ID NO:4 is the signal peptide (SEQ ID NO:8) of about 20 residues of expection. Extracellular part self is useful. The signal peptide zone is with neutral net signal P VI.I program (Nielsen etal., Int.J.Neural Syst.8:581-599 (1997) is incorporated herein by reference) and/or neutral net signal P V1.I program (Nielsen et al, (1997) Int.J.Neural Syst. 8,581-599) predict. It will be recognized by those skilled in the art that cleavage site may be different from the site of computer program prediction accurately. SEQ ID NO:10 is the GIPF polypeptide that lacks the SEQ ID NO:4 that infers signal peptide (SEQ ID NO:8).

In mammalian cell cultures clone and purifying be derived from the polypeptide of two species of SEQ ID NO:4. SEQ ID NO:10 is the polypeptide that purifying obtains from the cell culture medium of the Chinese hamster ovary cell (CHO) of using the vector construct transfection that comprises SEQ ID NO:3 nucleotide sequence. The polypeptide of SEQ ID NO:10 is the dominant mature form of GIPF. SEQ ID NO:9 is the nucleotide sequence of coding SEQ ID NO:10 polypeptide. The N terminal sequence of this polypeptide is by Edman degraded order-checking (Speicher, D.W.. Methods 6:248-261 (1994); Tempst et al., Methods6:248-261 (1994)) determine. SEQ ID NO:12 separates the mature polypeptide form that obtains from the cell culture of human embryo kidney 293 cells of using the vector construction body transfection that comprises SEQ ID NO:3. SEQ ID NO:11 is the nucleotide sequence of SEQ ID NO:12 polypeptide of encoding accordingly. By Edman degraded order-checking, determined that SEQ ID NO:12 polypeptide lacks front 31 amino acid residues of SEQ ID NO:4. These 31 amino acid whose peptides comprise the conserved site (SEQ ID NO:20) (Zhou et al., J Biol Chem 274:20745-20748 (1999), incorporated herein by reference) of furin cleavage site.

Utilize Pfam software program (Sonnhammer etal., Nucleic Acids Res., Vol.26 (1) pp. 320-322 (1998) is incorporated herein by reference), in check GIPF polypeptide (SEQ ID NO:4) with the domain of known peptide domain homology. The GIPF polypeptide of expection SEQ ID NO:4 has 1 type thrombospondin domain (SEQ ID NO:14, by SEQ ID NO 13 nucleotide sequence coded). The Pfam scoring that is included in 1 type thrombospondin domain among the SEQ ID NO:4 is 0.0034, and expection is from the 151-206 amino acids residue of SEQ ID NO:4. Described thrombospondin domain self is useful.

Utilize eMATRIX software kit (Stanford University, Stanford, CA) (Wu et al., J. Comp.Biol., vol.6, pp.219-235 (1999), incorporated herein by reference), the GIPF polypeptide of expection SEQ ID NO:4 contains the domain that following table is listed, wherein the A=alanine, the C=cysteine, D=aspartic acid, E=glutamic acid, the F=phenylalanine, G=glycine, H=histidine, the I=isoleucine, K=lysine, L=leucine, the M=methionine, N=asparagine, P=proline, the Q=glutamine, R=arginine, S=serine, the T=threonine, the V=valine, the W=tryptophan, Y=tyrosine:

Sequence number	The P value	Identify number	EMATRIX domain title	Amino acid sequence (position)
					24	8.63e-10	IPB001862A	Membrane attack complex composition/perforin/complement C9	PAQCEMSEWSPW   GPCS(145-160)
25	9.03e-10	IPB002174A	Woods albumen sample cysteine enrichment region not	GKRQRRISAEGSQ   ACAKGCELCSEV   NGCLKCS(26-57)
					26	9.80e-08	IPB000433	ZZ zinc digit symbol	IEHCEACFSHNFC   TKCKP(99-115)

Main by Chinese hamster ovary celI and/or dominant maturation (dominant mature) polypeptide of 293 cells generation or the generation of mature polypeptide form (being respectively SEQID NO:10 and SEQ ID NO:12) in order to regulate and control, prepared synthetic construct. SEQ ID NO:16 is the nucleotide sequence that the carrier system of expression polypeptide (SEQ ID NO:16) comprises, wherein the dominant mature form (SEQ ID NO:10) of the contiguous 293 cells generation of the signal peptide (SEQ ID NO:8) of expection. SEQ ID NO:17 is by the constructs of direct mutagenesis (Weiner et al., Gene 126:35-41 (1993)) in the generation thereby generation suddenlys change of total furin cleavage site (SEQ ID NO:22). This sudden change becomes first arginine residues (R) of SEQ ID NO:20 into glutamine (Q). Sudden change from the arginine to the glutamine is so that 293 cells produce the dominant mature form of GIPF (SEQ ID NO:10).

Thrombospondin is gang's extracellular matrix protein, participates in the contact (Lawler et al., Curr.Opin.Cell Bio.12:634-640 (2000)) of cell-cell, cell-matrix. Known have five kinds of different thrombospondins of surpassing, and the Tissue distribution of different mode is arranged. Some tissues, after one's own heart, cartilage and brain express most of thrombospondin gene outcomes. 1 type thrombospondin is hematoblastic main component. 1 type thrombospondin plays a role at cell surface, and memebrane protein is taken to cell factor and other soluble factor. The memebrane protein that combines with 1 type thrombospondin comprises whole catenin, whole catenin GAP-associated protein GAP (CD47), CD36 and proteoglycans. Transforming growth factor β (TGF β) and platelet-derived growth factor also combine with 1 type thrombospondin.

1 type thrombospondin is the large protein with many different structures territory. Its N end and C end contain the spherical structure territory, with the zone of precollagen (procollagen) homology, and three kinds of repetitive sequence motifs, be called respectively thrombospondin (TSP) 1 type, 2 types and 3 types and repeat. TSP1 repeats to exist in multiple different albumen, comprise complement component (C6, C7, C8A etc.), extracellular matrix protein such as ADAMTS, mindin, aixs cylinder instruct molecule (axonal guidance molecule) such as the TRAP albumen of F-f spondin semaphorins (F-spondin semaphorins), SCO-spondin and plasmodium (plasmodium).

Thrombospondin 1 type repeats (TSP1) can activate the TGFp epithelial tissue, participates in regulating Growth of Cells, differentiation, adhesion, migration and death. Thrombospondin 1 type repeats also to participate in protein combination, Heparin-binding, cell adherence, neural process to outgrowth, inhibition propagation, anti-angiogenesis and activation apoptosis. Protoplast ring spore (plasmodium circumsporozoite) (CS) TSP1 domain and the zygoblast (sporozoite) of albumen and TRAP albumen to invade salivary gland relevant.

The feature of TSP1 sequence is the cysteine of guarding, tryptophan and the cluster alkaline residue of tight spacing. The steric configuration of TSP1 sequence show two with heparin-bounding β-lamellar structure territory (Kilpelainen et al (200) J.Biol Chem.275,13564-13570, incorporated herein by reference). The similar space folding of Heparin-binding growth correlation molecule (HB-GAM) has been described. HB-GAM is identical with following molecule: mitogenesis and neural process are outwards grown and are promoted protein pleitrophin; POSTN-1; Heparin-binding neurotrophic factor; Heparin is affine adjusting peptide. The expression of HB-GAM is relevant with the extracellular matrix of aixs cylinder bundle and cynapse, and is also relevant with the basilar memebrane in the cartilage matrix outward with brain. Recently, N-syndecan (N-syndecan) is considered to the acceptor of HB-GAM in the brain, and works in regulating hippocampus long term potentiation (participating in brain plasticity (plasticity) form of memory and study). Therefore, the albumen that contains TSP1 can be used as the growth promoter and shows that GIPF is active.

In addition, synthetic in the marrow and be deposited on thrombospondin in the extracellular matrix and can be used as former generation multipotency CFU-GM and be divided into the adhesion molecule of the HPC of erythron, granulocyte series and megakaryocytic series. Therefore, to grow for haemocyte be important (Long and Dixit (1990) Blood 75,2311-2318, incorporated herein by reference) to thrombospondin.

GIPF polypeptide of the present invention and polynucleotides can be used for inducing propagation or the differentiation of stomach and intestine pit cell. They can be used for the epitheliogenic illness for the treatment of needs, as treat enterogastric diseases, comprise the catarrh that chemotherapy and radiation causes, the catarrh of oropharynx, lip and esophagus, inflammatory bowel disease or comprise the Other diseases of wound, burn, illness in eye, and any disease that needs to stimulate epithelial cell proliferation or regeneration. Thereby polynucleotides of the present invention and polypeptide also can be used for producing the patient that new organization and neologism auxiliary curing need transplanted tissue.

4.1 definition

When description is of the present invention, adopt following noun, it is defined as follows.

It should be noted that unless expressly stated otherwise,, " " of singulative, " one " and " some " comprise plural implication in this paper and additional claim.

Noun " GIPF " refers to that epithelial cell is had active especially " gastrointestinal proliferative factor ".

Among the present invention, noun " GIPF albumen " or " GIPF polypeptide " refer to by 1 determined full-length proteins of methionine to 263 alanine (SEQ ID NO:4), its fragment or its analog.

Noun " total length GIPF ", " elongated GIPF ", " wild type GIPF " or " natural GIPF " refer to comprise the polypeptide (SEQID NO:4) of 263 amino acid residues, as shown in Figure 1B.

Noun " GIPFwt " or " hGIPF " refer to people's wild type, total length GIPF polypeptide (SEQ ID NO:4); Noun " GIPFt " refers to the people GIPF polypeptide (SEQ ID NO:6) of V5His6-mark; " mGIPFt " refers to derive from the GIPF (SEQ ID NO:69) of the V5His6-mark of mouse. GIPF polypeptide mutant is isoleucine rather than valine the 50th of SEQ ID NO:4.

Noun " fragment " refers to be derived from natural GIPF, but does not comprise the polypeptide of GIPF full sequence. This class fragment can be the truncated variant of full-length molecule, such as SEQ ID NO:9 and 12, and the polypeptide of inner disappearance, such as SEQ ID NO:16. By detecting GIPF in vivo or external impact on epithelial cell proliferation, find that the GIPF fragment has the GIPF biologically active, as described herein.

Noun " analog " refers to the derivative of correlation molecule. Analog is retains biological activity as mentioned above. In general, the compound that noun " analog " refers to have natural polypeptides sequence and structure, compare with natural molecule, one or more amino acid have occured inserted, replace (its character is guarded usually) and/or disappearance, as long as this modification does not destroy activity. SEQ ID NO:18 is the example of GIPF analog. Preferred analog has the biologically active identical with parent's molecule at least, even can be higher than the activity of parent molecule. The method for preparing polypeptide analog is known in the art. Particularly preferred analog comprises natural conservative replacement, namely occurs in the replacement between gang's side chain related amino acid. Particularly, amino acid generally is divided into four families: (1) acidic amino acid: aspartic acid and glutamic acid; (2) basic amino acid: lysine, asparagine and histidine; (3) nonpolar amino acid: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; (4) neutral polar amino acid: glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine. Phenylalanine, tryptophan and tyrosine are divided into aromatic amino acid sometimes. For example, can replace leucine with isoleucine or valine by reasonable prediction, replace aspartic acid with glutamic acid, replace threonine with serine, perhaps carry out similar conservative replacement with structurally associated amino acid, can keep the biologically active of GIPF.

By contrasting the sequence of specific polypeptide and homeopeptide, make the amino acid sequence variable number in height homology zone (conservative region) minimum, or use the consensus sequence substituted amino acid, can determine which amino acid residue can replace, adds or lack, and not destroy targeted activity.

In addition, the encode restructuring analog of these same or similar polypeptide can be that " redundancy " that synthesizes or utilize genetic codon selected to obtain. Can in plasmid or viral vectors, introduce various codons and replace, as produce the silence change of various restriction sites, to optimize clone or the expression in specific protokaryon or the eucaryon system. The sudden change of polynucleotide sequence can reflect in other peptide domain of polypeptide or adding polypeptide, has modified the character of the arbitrary part of polypeptide, has changed such as character such as ligand binding compatibility, interchain affinity or degraded/cycling rates.

Preferably, amino acid " replacement " is the amino acid whose result of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor who has analog structure and/or chemical property with another, namely " guards " 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. " guard " 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor and be the polarity, electric charge, dissolubility, hydrophobicity, hydrophily and/or the amphipathic similitude that participate in residue and be the basis. For example, nonpolar (hydrophobic) amino acid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; Polar amino acid comprises glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; Positive charge (alkalescence) amino acid comprises arginine, lysine and histidine; Negative electrical charge (acidity) amino acid comprises aspartic acid and glutamic acid. " insertion " or " disappearance " preferably in about 1-20 Amino Acid Range, more preferably 1-10 amino acid. Use recombinant DNA technology, systematically insertion in peptide molecule, disappearance or substituted amino acid, and detect the activity of the restructuring variant that obtains, can determine the variation that allows.

In addition, when needs change function, can insert, disappearance or non-conservative change be with mutagenic polypeptide. This change is passable, for example, changes one or more biological functions or the biochemical property of polypeptide of the present invention. For example, this change can change the character of polypeptide, such as ligand binding affinity, interchain compatibility or degraded/cycling rate. In addition, can select this change to be more suitable in the polypeptide of expressing and expand (scale up) etc. at the host cell that is used for expressing with generation. For example, can lack cysteine residues or replace to eliminate disulfide bond with another amino acid residue.

Noun " is derived " and is referred to the polypeptide modified with following technicalization: ubiquitination, mark (as with radionuclide or various enzyme), covalent polymer combination, such as PEGization (deriving with polyethylene glycol), amino sour with the chemical synthesis that non-natural is present in the human protein, such as ornithine, insert or replace.

Noun " polypeptide " and " protein " refer to the polymer of amino acid residue, and do not limit the minimum length of product. Except as otherwise noted, this noun also comprises and does not change the peptide modified of amino acid sequence, for example glycosylation, acetylation and phosphorylation form. Based on purpose of the present invention, polypeptide or protein can be synthesize or restructuring produce, or separate from natural origin and to obtain.

For polypeptide or polynucleotides, the molecule of " purifying " and " separation " feeling the pulse with the finger-tip exists and other similar large biological molecule does not exist substantially. Noun " purifying " refers to that the similar large biological molecule that exists in the sample preferably accounts for 75% (weight) at least herein, more preferably at least 85% (weight), more preferably at least 95 % (weight), more preferably at least 98% (weight). In one embodiment, polynucleotides or peptide purification extremely except water, buffer solution and other little molecule, especially molecular weight are lower than outside the molecule of 1000Da, contain 95% purpose large biological molecule at least.

" polynucleotides of the separation of the specific polypeptide of encoding " refer to substantially not contain the nucleic acid molecules of other nucleic acid molecules of the desired polypeptides of not encoding; Yet this molecule can comprise that some do not damage other base or the part of said composition fundamental property.

Noun " natural polypeptides " refers to the polypeptide without genetic engineering modified cell generation, particularly each peptide species of polypeptide posttranslational modification generation, modifies and includes but not limited to acetylation, carboxylation, glycosylation, phosphorylation, esterification and acidylate.

Noun " translation encoding histone part " refer to the encode sequence or the job sequence of the full-length proteins that comprises any leader.

Peptide or proteinic sequence that noun " dominance mature protein coding sequence " refers to encode and do not contain any guide/signal sequence." dominance maturation protein part " refers to not contain the protein portion of guide/signal sequence.The GIPF polypeptide of " maturation " form hypodactylia guide/signal sequence and furin cleavage site.Can in the course of processing of cell, remove the guide/signal sequence and/or the furin cleavage site of peptide, or prepare albumen by synthetic method or with the polynucleotide of encoding mature albumen coded sequence only.Maturation protein or dominance maturation protein part can comprise or not comprise initial methionine residues.Initial methionine is removed in the peptide course of processing usually.

Noun herein " isolating " refers to that the another kind of at least composition (for example, nucleic acid or polypeptide) in nucleic acid or polypeptide and its natural origin is separated.In one embodiment, nucleic acid or polypeptide are present in only solvent, in buffer system, ion or this solution in the solution of naturally occurring other composition.Noun " isolating " and " purification " do not comprise nucleic acid or the polypeptide that exists in its natural origin.

For polypeptide or protein, noun herein " reorganization " refers to from the polypeptide or the protein in recombinant expression system (as microorganism, insecticide or mammal) source." microorganism " refers to the recombinant polypeptide or the protein that produce in antibacterial or fungus (as yeast) expression system.As product, " recombinant microorganism " refers to not contain natural endogenous substance substantially and without Natively glycosylated polypeptide or protein.In most of antibacterial culturing, as escherichia coli, polypeptide expressed or protein lack glycosylation modified; In yeast polypeptide expressed or protein have usually with in mammalian cell, express different glycosylation form.

" recombinant polypeptide " refers to the polypeptide with recombinant DNA technology preparation described herein.In short, the gene of clones coding desired polypeptides, and in the organism that transforms, express, state as follows.Host organisms expression alien gene under expression condition produces polypeptide.In addition, can change the promoter of the endogenous expression of polypeptides of regulation and control to produce recombinant polypeptide.

Noun " activity " refers to keep the biological activity and/or the immunocompetent polypeptide form of any natural polypeptides.According to the present invention, noun " biologic activity " or " biological activity " refer to have the protein or the peptide of structure, adjusting or the biochemical function of natural molecule." biologic activity " or " biological activity " refers to natural, reorganization or synthetic GIPF peptide or its fragments of peptides arbitrarily equally, in suitable animal or cell, brings out the particular biological reaction and in conjunction with the ability of specific antibodies.

Noun " excretory " comprises wears film or transmembrane protein, is included in when expressing in the suitable host cell aminoacid sequence transhipment under the signal sequence effect." excretory " albumen includes but not limited to secrete fully the protein of (as soluble protein) or merocrine secretion (as receptor) from express cell." excretory " albumen also includes but not limited to stride the albumen of endoplasmic reticulum transhipment." excretory " albumen comprises that also the albumen that has the atypia signal sequence is (as il-1 β, see Krasney, P.A. and Young, P.R. (1992) Cytokine 4 (2): 134-143) and the albumen of the factor that discharges from damaging cells (as interleukin-1 receptor antagonist, see Arend, W.P.et.al. (1998) Annu.Rev.Immunol.16:27-55).

Noun herein " polynucleotide " or " nucleic acid molecules " refer to the nucleotide polymer of the random length of ribonucleotide or deoxyribonucleotide.This noun only refers to the primary structure of molecule, therefore comprises double-stranded and single stranded DNA and RNA.Also comprise known modified types, labelling as is known, methylate, add medicated cap, replace one or more natural nucleotides with analog, modify between nucleotide, as not charged connection (as the methyl acid phosphate salt compound, phosphotriester, phosphamide, amino methyl class or the like) with charged the connection (as the phosphorus thioesters, phosphorodithioate etc.), contain suspended portion, for example protein (comprises for example nuclease, toxin, antibody, signal peptide, poly-l-lysine etc.), have intercalating agent and (sting (acridine) as bifurcation, psoralen etc.), contain chelate (metal for example, radioactive metal, boron, metal-oxide etc.), contain alkylating agent, have and modify to connect the non-modified forms of (as α anomer (anomeric) nucleic acid etc.) and polynucleotide.In general, nucleic acid fragment provided by the invention is assembled by genomic fragment and short oligonucleotide joint or is assembled by a series of oligonucleotide or one nucleotide, produce synthetic nucleic acid, can in the reorganization transcriptional units that contains the regulating element that is derived from microorganism or viral operon or eukaryotic gene, express.

Noun " oligonucleotide fragment " or " polynucleotide passage ", " part " or " fragment " or " probe " or " primer " are used interchangeably, refer at least about 5 nucleotide, more preferably at least about 7 nucleotide, more preferably at least about 9 nucleotide, more preferably at least about 11 nucleotide, most preferably at least about the nucleotide residue sequence of 17 nucleotide.Described fragment preferably is less than about 500 nucleotide, preferably is less than about 200 nucleotide, more preferably less than about 100 nucleotide, more preferably less than about 50 nucleotide, most preferably is less than about 30 nucleotide.Preferably about 6-200 the nucleotide of probe, preferably about 15-50 nucleotide, more preferably from about 17-30 nucleotide, most preferably from about 20-25 nucleotide.Preferred described fragment can be used for polymerase chain reaction (PCR), various hybridization program or the microarray assay identical or relevant portion with evaluation or amplification mRNA or dna molecular.Fragment can be differentiated each polynucleotide sequence of the present invention specifically.Preferred described fragment comprises the sequence similar substantially to the part of following sequence: SEQ IDNO:1,2,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177.

Probe for example can be used for determining the similar nucleotide sequence that whether has the specific mrna molecule in the cell or tissue or be used to separate chromosomal DNA, as (Walsh, P.S.et al., 1992, PCR Methods Appl 1:241-250) as described in the Walsh et al..Available nick translation, Klenow fill reaction, PCR or other known method and carry out labelling.Probe of the present invention, its preparation and/or labelling be referring to Sambrook, J.et al., 1989, Molecular Cloning:A Laboratory Manual, Cold Spring HarborLaboratory, NY; Or Ausubel, F.M.et al., 1989, Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, New York NY introduces herein as a reference.

Nucleotide sequence of the present invention also comprises the NO:1 from SEQ ID, 2,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98, the sequence information of arbitrary sequence of 100,102,104 or 177.Sequence information can be specific recognition or represent SEQ ID NO:1,2,5,7,9,11,13,15,17,84,86,88,90,92, the SEQ ID NO:1 of 94,96,98,100,102,104 or 177 sequence informations, 2,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100, the fragment of 102,104 or 177 sequences.One of this fragment can be the nucleotide sequence of 20mer, because the probability of the 20mer that mates fully with the people's gene group is 1/300.In the people's gene group, 3,000,000,000 base pair is arranged on every group chromosome.Because may have 4 ²⁰Individual 20mer nucleotide sequence has the base pair of 300 multiple amounts of 20mer nucleotide sequence on lineup's chromosome.Utilize same analysis, the probability of the 17mer that mates fully with the people's gene group is about 1/5.When these fragments are used for expression analysis, can use the 15mer fragment.In the expressed sequence fully the segmental probability of 15mer of coupling be about 1/5 equally be less than about 5% whole genome sequence because the sequence of being expressed comprises.

Same, when utilizing sequence information to detect single mispairing, but fragment 25mer.With the probability (1/4 that mates fully ²⁵) multiply by the mispairing probability (3 * 25) that each nucleotide site raises, can calculate the probability of the 25mer that has single mispairing that occurs in the people's gene group.The detected probability that contains the 18mer sequence of single mispairing is about 1/5 in the expression study test.The segmental probability of 20mer that has single mispairing in the people's gene group is about 1/5.

Noun " open reading frame (ORF) " is meant a series of nucleotide triplets that do not contain the coded amino acid of termination codon, and can translate into protein.

Noun " can be operatively connected " or " operable association " refers to the nucleotide sequence that function is relevant.For example, if the promoter regulation coded sequence transcribe, then promoter and coded sequence be can be operatively connected or operable association.When the nucleotide sequence that can be operatively connected is successive and in same reading frame, some genetic elements such as suppressor gene are not continuous the connections with coded sequence, but still can control transcribing/translating of coded sequence.

Noun herein " recombinant DNA molecules " or " recombination of polynucleotide " are meant genome polynucleotide, cDNA, semi-synthetic or synthetic, can be divided into (1) according to its source or processing method does not link to each other with all or part of of its natural polynucleotide of following, (2) do not link to each other with its natural polynucleotide that are connected, (3) are non-natural.Therefore, this noun comprises " synthetic deutero-" nucleic acid molecules.

Noun " complementary " or " complementarity " refer to the natural combination of polynucleotide that base pairing produces.For example, sequence 5 '-AGT-3 ' and complementary series 3 '-TCA-5 ' combines.The complementarity of two single chain molecules can be a part, and promptly only some nucleic acid combine, or complete, and is complementary fully between the promptly whole single chain molecule.The interchain complementary degree of nucleic acid has material impact for the effectiveness and the intensity of nucleic acid chains intermolecular hybrid.

Noun " rigorous " refers to the rigorous condition of this area common sense.Rigorous condition comprises that (65 ℃ combine DNA with film and hybridize highly rigorous condition, and reaction system is 0.5M NaHPO ₄, 7% sodium lauryl sulphate (SDS), 1mM EDTA, 68 ℃ with 0.1 * SSC/0.1%SDS washing) and the rigorous condition of moderate (promptly 42 ℃ wash with 0.2 * SSC/0.1%SDS).The example of other hybridization conditions illustrates in an embodiment.

When deoxy-oligonucleotide is hybridized, other exemplary rigorous hybridization conditions is included in 6 * SSC/0.05% tetrasodium pyrophosphate at 37 ℃ (oligonucleotide of 14 bases), 48 ℃ (oligonucleotide of 17 bases), 55 ℃ (oligonucleotide of 20 bases) wash under 60 ℃ of (oligonucleotide of 23 bases) conditions.

" basic identical " refers to nucleotide sequence and aminoacid sequence herein, the mutant nucleotide sequence that for example is different from canonical sequence owing to one or more replacements, disappearance or interpolation, the net effect of these changes do not cause the alienation (adverse functional dissimilarity) of the reverse functions of canonical sequence and target sequence.Typically, this basic identical sequence and sequence as herein described different are equal to or less than about 35% (promptly compare with corresponding canonical sequence, the residue quantity that replaces, adds and/or lack in the basic identical sequence equals about 0.35 or littler divided by the total residue number in the basic identical sequence).This sequence and listed sequence have 65% sequence homogeneity.In one embodiment, basic identical sequence of the present invention is as mutant, with different 30% (the 70% sequence homogeneity) that are equal to or less than of described sequence; In the modification of this embodiment, difference is no more than 25% (75% sequence homogeneity); In another modification, difference is no more than 20% (80% sequence homogeneity); In another modification, difference is no more than 10% (90% sequence homogeneity); In another modification, difference is no more than 5% (95% sequence homogeneity).Basic identical aminoacid sequence of the present invention as mutant, preferably has 80% sequence homogeneity at least with listed aminoacid sequence, more preferably 90% sequence homogeneity.Consider for example repeatability or the degeneracy of genetic code, the basic identical nucleotide sequence of the present invention can have lower sequence homogeneity.The preferred nucleic acid sequence is identical at least about 65%, and is more preferably identical at least about 75%, more preferably identical at least about 95%.For purpose of the present invention, the sequence with basic identical biological activity and basic identical expression character is considered to essentially identical.In order to determine similarity, ignore the truncate (as by producing the sudden change of pseudotermination codon) of mature sequence.(Hein, J. (1990) MethodsEnzymol.183:626-645) can determine sequence homogeneity with Jotun Hein method.Also available other known method as by changing hybridization conditions, is determined the homogeneity between the sequence.

Noun " carrier " refer to transport connect the nucleic acid molecules of another nucleic acid.Noun " expression vector " comprises the proteic plasmid of GIPF, cosmid or the phage that can synthesize each recombination coding that is carried by carrier.Preferred plasmid can self-replicating and is expressed the nucleic acid that is connected.

Noun " conversion " refers to DNA is incorporated in the proper host cell, and DNA is duplicated as extra-chromosomal element or by chromosomal integration.

Noun " transfection " refers to that appropriate host cell absorbs expression vector, and no matter arbitrarily coded sequence whether express by reality.Noun " infection " refers to virus or viral vector nucleic acid be introduced in the proper host cell.

Noun " transcription regulatory element " and " transcriptional regulatory sequences " can be exchanged use, refer to the necessary DNA sequence of the expression that can be operatively connected with coded sequence in specific organism.Be fit to procaryotic regulating and controlling sequence and comprise, for example promoter, operon sequence, and ribosome binding site arbitrarily.Eukaryotic cell utilizes promoter, enhancer, shear signal and polyA signal.These nouns comprise enhancing or regulate all elements of transcribing, the necessary core parts of basic interaction, upstream element, enhancer and the response element (Lewin that comprise promoter, RNA polymerase and transcription factor, " Genes V " (OxfordUniversity Press, Oxford) pages 847-873).

When RNA polymerase was transcribed into mRNA with coded sequence, under coded sequence " was in the adjusting of transcribing in the cell with the translational control sequence ", the mRNA of generation randomly carried out trans RNA and shears and be translated as the coded sequence encoded protein.

Noun " tissue-specific promoter " refers to the nucleotide sequence as promoter, promptly regulates the expression of the selected DNA sequence that can be operatively connected with promoter, influences selected DNA sequence at specific cells, as the expression in the B cell.In an illustrative embodiment, utilize the gene construct of B cell specificity promotor to be preferred for instructing the expression of GIPF albumen in the B cell or protein fragments.

Noun " express regulate fragment (EMF) " refers to the nucleotide that ORF that a series of adjustings can be operatively connected or another EMF express.

When EMF exists the change sequence to express, claim sequence " adjusting can be operatively connected the expression of sequence " herein.EMFs includes but not limited to that promoter and promoter are regulated sequence (inducing element).One class EMFs is the nucleic acid fragment of inducing the ORF response particular adjustments factor that is operatively connected or physiological event to express.

Noun " recombinant expression carrier " refers to plasmid, phage or virus or the carrier of expressible dna (RNA) polypeptide of sequence.Expression vector comprises transcriptional units, comprise the element that plays regulating action in (1) genetic elements or the gene expression, for example promoter or enhancer, (2) can be transcribed into mRNA and translate into proteinic structure or coded sequence, transcription initiation and the terminator sequence suitable as (3).The used construction unit of yeast or eukaryotic expression system preferably includes and makes the host cell exocytosis translate proteic targeting sequencing.In addition, when not containing the expression of recombinant proteins of leading or transit sequence, amino terminal may contain methionine residues.This residue may excise from the recombiant protein of expressing subsequently or be not cut, produces end-product.

Noun " recombinant expression system " refers to recombinate the transcriptional units stable integration to the host cell of chromosomal DNA, or carries the host cell of dyeing vitro recombination transcriptional units.In case introduce regulating element and is connected with dna fragmentation to be expressed or synthetic gene, the recombinant expression system that this paper defines is with regard to expressing heterologous polypeptide or protein.The host cell of the element (as promoter or enhancer) that this noun has also referred to stable integration genetic recombination element or regulator gene are expressed.In case introduce regulating element and is connected with gene that the endogenous dna fragment maybe will be expressed, the recombinant expression system that this paper defines is with regard to express cell endogenous polypeptide or protein.Cell can be prokaryotic cell or eukaryotic cell.

Noun " transgenic " refers to that for the transgenic animal of introducing this nucleic acid or cell be part allos or whole allos, it is external nucleotide sequence, be homologous perhaps for the transgenic animal of introducing this nucleic acid or the endogenous gene of cell, but need to insert the genome that is inserted into cell in the animal gene group with change, for example insert the position different with natural gene.Transgenic is operably connected to one or more transcriptional regulatory sequences and any other nucleic acid, as optimizes the necessary intron of selected expression of nucleic acid.

Accordingly, " transgenic constructs " refers to comprise transgenic, and optional comprising as transcriptional regulatory sequences, and the nucleic acid of other nucleic acid such as polyA site, replication origin, marker gene is generally used for transgenic is inserted the genome of host organisms.

Noun " transgenic " is used for describing the character that for example comprises genetically modified animal or construct.For example, " transgenic organism " can be any animal herein, preferred non-human mammal, and wherein one or more zooblasts contain by manual intervention, as use known transgenic technology, the heterologous nucleic acids of introducing.By careful genetic manipulation, as microinjection or use recombinant virus infection, introduce cell thereby nucleic acid directly or indirectly can be introduced cell precursors.Nucleic acid can be integrated in the chromosome, perhaps as the DNA of extrachromosomal replication.In transgenic animal described herein, transgenic makes cellular expression or overexpression GIPF albumen.

Noun " versatility " phalangeal cell is divided into the ability of the cell type of the multiple different differentiation that exist in the adult organism body.Multipotential cell is compared with totipotent cell, and differentiation capability is limited.

Noun " embryonic stem cell (ES) " comprises sexual cell, refers to produce in embryo or adult the cell of the cell type of many differentiation.Noun " plant lineage stem cells (GSCs) " refers to come from primordial stem cell (primordial germ cell), can stablize and provides stem cell to produce the stem cell of gamete continuously.Noun " primordial stem cell (PGCs) " refers to come from other cell line in embryo's forming process, particularly comes from a small set of cell of yolk sac, barrier film (mesentery) or gonad ridge, has the potential of sexual cell of being divided into (germcell) or other cell.PGCs is the source of GSCs and ES cell.PGCs, GSCs and ES cell all can self renewals.Therefore, these cells not only can component species system and produce the multiple terminally differentiated cells of forming the special organ of adult, can also regenerate oneself.Noun " totipotency " phalangeal cell is divided into the ability of ripe organism all cells kind.Noun " versatility " phalangeal cell is divided into the ability of the multiple differentiated cell types that exists in the adult organism body.Multipotential cell is compared with totipotent cell, and differentiation capability is limited.

Noun " original cell line (founder line) " and " original animal (founder animal) " refer to the sophisticated animal of implanted genetically modified fetal development, promptly by inserted DNA and implant among the one or more hosts of agency fetal development and must animal.

Noun " offspring " and " transgenic animal offspring " refer to every monobasic any and all offsprings after the initial mammal that transforms.

Noun " non-human mammal " refers to mammiferous all members except that human." mammal " refers to any mammiferous animal that is listed in, comprise the mankind, poultry and support in the farm, the animal or the house pet of zoo, sports ground, as mice, rat, rabbit, pig, sheep, goat, cattle and more high primate.

Noun " treatment " or " disposal " refer to therapeutic and preventative or preventing property measure, with prevention or alleviate undesirable physiological change or situation, and the mucositis that causes as chemotherapy or radiotherapy.Whether for purpose of the present invention, clinical effectiveness useful or expectation includes but not limited to, relief of symptoms, the order of severity that palliates a disease, stable disease state, no matter can perceive.

" disease " refers to the free position of the molecular therapy that available transgenic animal model of the present invention is identified.Comprise the chronic and acute disorder or the disease that cause the disorder of mammal pathology state.The non-limitative example of the disorder that this paper treated comprises mucositis, inflammatory bowel disease and skin injury.Preferably mucositis of the disease that this paper treated.

" inflammatory bowel disease (IBD) " refers to the spontaneity or the chronic inflammatory disease of small intestinal and/or large intestine herein, comprises the IBD IBD relevant with antibiotic that Crohn disease (Croh ' s disease), ulcerative colitis, the source of infection cause.

" mucositis " herein refers to comprise the inflammation of the gastrointestinal mucosal of oropharynx, lip, esophagus, large intestine and small intestinal etc.

" short bowel syndrome " or " SBS (Short Bowel Syndrome) " refer to quite to grow the anatomical of one section small intestinal or alimentation that loss of functionality causes bad.

Noun " effective dose " or " pharmacy effective dose " refer to reach the effective but nontoxic dosage of expection biological effect.Described effect can be the change that alleviates and/or improve the living things system of sign, symptom, the disease cause of disease or other any expection.For example, the segmental effective dose of GIPF that is used for the inventive method is to be enough to stimulate the amount that epithelial cell is excited or breed, the amount that the regeneration of individual gastrointestinal epithelial is increased, described individuality stand mucositis, the inflammatory bowel disease that chemotherapy or radiotherapy causes or other disease that needs epithelial cell proliferation.Described dosage is described hereinafter.Those skilled in the art can determine suitable " effective dose " of each case according to routine test.

" medicine can be accepted " or " pharmacy can be accepted " refer to the material do not expected on abiotic or other undesirable material, can give individuality and not cause the biological agent of any non-expectation or not with the material of the arbitrary component interaction in harmful mode and the compositions that comprises it.

" physiological pH " or " pH in the physiological range " refers to pH value about 7.0～8.0.Between the preferred physiology pH value about 7.2～7.6.

Noun " individuality " comprises mammal and nonmammalian herein.Mammiferous example includes but not limited to any member of mammal kind: people, non-human primate such as orangutan, ape, monkey etc.; Agricultural animal such as cattle, horse, sheep, goat and pig; Domestic animal such as rabbit, Canis familiaris L. and cat; Laboratory animal comprises rodent, as rat, mice, Cavia porcellus or the like.The example of nonmammalian includes but not limited to bird, fish or the like.Do not specify specific age or sex.

4.2 pharmaceutical composition of the present invention

4.2.1 nucleic acid compositions

The present invention is based on following discovery: contain the pharmaceutical composition of the polynucleotide of epithelial cell growth factor polypeptide, GIPF and coding GIPF polypeptide, stimulate intestinal epithelial cell, comprise pit cell growth and propagation.Therefore, these pharmaceutical compositions can be used for diagnosis and treat needing to stimulate epithelial cell proliferation or regenerated disease.

Isolating polynucleotide of the present invention include but not limited to, comprise SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or the polynucleotide of 177 arbitrary nucleotide sequences; Comprise SEQ ID NO:3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100, (polynucleotide of 102,104 or 177 full length protein coding sequence are for example encoded SEQID NO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 or 178 sequence); The polynucleotide that comprise the coding nucleotide sequence of the maturation protein of SEQ ID NO:4 polypeptide and dominance maturation protein.Polynucleotide of the present invention also include but not limited to, under rigorous condition with polynucleotide (a) the SEQID NO:2 of following sequence hybridization, 3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or the complementary series of 177 any nucleotide sequences; (b) coding SEQID NO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101, the polynucleotide of 103,105 or 178 arbitrary polypeptide; (c) polynucleotide of the allelic variant of above-mentioned any polynucleotide; (d) polynucleotide of the above-mentioned arbitrary protein kind of coding homologue; Or (e) coding comprise SEQID NO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101, the ad hoc structure territory of 103,105 or 178 polypeptide or the polynucleotide of truncate.The purpose domain comprises extracellular region, strides film district or cytoplasmic domain or its combination; And catalysis and substrate binding structural domain.

Polynucleotide of the present invention comprise natural or all or part of synthetic DNA, for example cDNA, genomic DNA and RNA, for example mRNA.Polynucleotide can comprise the part of all coding regions or the cDNA coding region of cDNA.

The present invention also provides the compositions of the corresponding gene that comprises cDNA sequence described herein.Corresponding gene can be utilized sequence information as herein described known method purification.These class methods comprise with known array information and prepare probe or primer identify and/or the increase gene of suitable genomic library or other genome material source.5 ' can obtain by means known in the art with 3 ' terminal sequence.For example, can be corresponding to the full-length cDNA or the genomic DNA of any polynucleotide of SEQID NO:2 by under suitable hybridization conditions, with SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177 any polynucleotide or its part be as probe, screens suitable cDNA or genome dna library and obtain.In addition, SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177 polynucleotide can be used as in genomic DNA or cDNA library to be identified and/or the basis of the suitable primer of amplification gene.

Nucleotide sequence of the present invention can be by ESTs with from one or more public databases, and the sequence (comprising cDNA and genome sequence) that obtains as dbEST, gbpri and UniGene assembles.Est sequence can provide the new segment information of identifying sequence information, typical segments information or full-length gene.

Polynucleotide of the present invention also provide the polynucleotide that comprise with the essentially identical nucleotide sequence of above-mentioned polynucleotide.For example, polynucleotide of the present invention and above-mentioned polynucleotide sequence have an appointment 65% at least, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least about 90%, 91%, 92%, 93% or 94% even more typically at least about 95%, 96%, 97%, 98% or 99% sequence homogeneity.

Nucleotide sequence of the present invention is included under the rigorous condition and SEQ ID NO:1,6,8,10,12,14,17,84,86,88,90,92,94,96,98,100, the any nucleotide sequence in 102,104 or 177 or the nucleic acid sequence fragments of its complementary sequence hybridization, this fragment is greater than about 5 nucleotide, preferred 7 nucleotide are more preferably greater than 9 nucleotide, most preferably greater than 17 nucleotide.For example can select the fragment (promptly with the arbitrary polynucleotide specific hybrid of the present invention) of 15,17 or 20 or more a plurality of nucleotide.Other polynucleotide sequence of polynucleotide sequence of the present invention and homologous genes family can be differentiated with the probe of polynucleotide specific hybrid, or people's gene and other species gene are differentiated, and be preferably based on unique nucleotide sequence.

Sequence in the scope of the invention is not limited only to these particular sequences, also comprises its allele variant and kind variant.With SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177 sequence, its representative segment or with SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177 at least 90% is identical, and preferred 95% identical nucleotide sequence compares with sequence in another separator of same species, can determine allele variant and kind variant routinely.And, in order to adapt to the codon transmutability, the present invention includes the nucleic acid molecules that coding and specific ORFs described herein have same acid sequence.That is to say, in the ORF coding region, comprise the codon that replaces another coding same amino acid with a codon.

The nearest neighborhood of nucleic acid of the present invention can utilize algorithm or program search data base to obtain.Preferably use BLAST, promptly basic local sequence alignment gopher is retrieved local sequence alignment (Altshul, S.F.JMol.Evol.36 290-300 (1993) and Altschul S.F.et al.J.Mol.Biol.21:403-410 (1990)).

The present invention provides described polynucleotide and proteinic kind homologue (or directly to congener) equally.Prepare suitable probe or primer by sequence provided herein, screening is derived from the suitable nucleic acid of required species, can separate and differentiate kind homologue.

The present invention also comprises described polynucleotide or proteinic equipotential mutant; Be the natural multi-form of isolating polynucleotide, the albumen of coding and described polynucleotide encoding is identical, homology or relevant protein.

Nucleotide sequence of the present invention also comprises the sequence of the described nucleic acid analog of encoding.Use means known in the art, with suitable nucleotide change introduce natural or the variation polynucleotide in, can prepare these aminoacid sequence analog.Two kinds of variable factors are arranged: mutational site and emergent properties in making up the aminoacid sequence mutant.The nucleic acid of encoding amino acid sequence analog preferably makes up with coding alpha-non-natural amino acid sequence by the sudden change polynucleotide.These nucleic acid change can occur in the site (mutational site) that makes nucleic acid be different from other kind nucleic acid or in high conservative zone (constant region).Typically, this class site can be modified by serial of methods, for example guard replacement (as replacing another hydrophobic amino acid) earlier with a different hydrophobic amino acid, carry out other replacement (as replacing hydrophobic amino acid) again, on the purpose site, delete or insert then with charge residue.The aminoacid sequence deletion is about 1～30 residue usually, preferred about 1～10 residue, and normally successive.Aminoacid insertion comprises that aminoterminal and/or c-terminus merge length 1～100 or more a plurality of residue, and inserts single or multiple amino acid residues in the sequence, inserts about 1～10 amino acid residue usually in the sequence, preferred 1～5 residue.The terminal example of inserting comprises and is used for secretion or locatees the sequence that necessary allos signal sequence and for example poly-His etc. are beneficial to the purification expressing protein in different host cells carry out born of the same parents.

In a kind of method for optimizing, the polynucleotide of coding amino acid sequence change by rite-directed mutagenesis.This method utilizes oligonucleotide sequence to change the polynucleotide of coding expection amino acid mutation body, and makes two ends, amino acid mutation site have enough contiguous nucleotides to stablize diploid all to produce at any end that experiences the site that changes.General, site-directed mutagenesis technique is known to those skilled in the art, at Edelman et al., DNA 2:183 has explanation in (1983).Zoller and Smith, Nucleic Acids Res.10:6487-6500 (1982) disclose a kind of general and effective method that produces rite-directed mutagenesis in polynucleotide sequence.PCR also can be used for producing the aminoacid sequence mutant of novel nucleic acids.When with a small amount of template DNA during as parent material, the primers slight different with the sequence of template DNA respective regions can produce the amino acid mutation body of expection.Pcr amplification obtains one group of product D NA fragment, and is different in the primer specificity site with the polynucleotide template of coded polypeptide.Product D NA fragment has substituted the respective regions in the plasmid, and produces the polynucleotide of coding expection amino acid mutation body.

Another technology of preparation amino acid mutation body is the cassette mutagenesis technology, and as Wells et al., Gene 34:315 (1985) is described; And other mutating technology known in the art, as Sambrook et al., ibid and Current Protocolsira Molecular Biology, Ausubel et al.Because the degeneracy of genetic code self, the DNA sequence of encode basic identical or functional equivalent aminoacid sequence also can be used for the present invention clone and express these novel nucleic acids.This class DNA sequence is included under the rigorous condition and the sequence of suitable novel nucleic acids sequence hybridization.

The polynucleotide of code book invention preferred polypeptide truncate can be used for preparing polynucleotide, and coding contains the chimeric or fusion rotein of one or more domains of the present invention and heterologous protein sequence.

Polynucleotide of the present invention also comprise the complement of above-mentioned any polynucleotide.Described polynucleotide can be DNA (genome, cDNA, amplification or synthetic DNA) or RNA.The method and the algorithm that obtain these class polynucleotide are known those skilled in the art, can comprise, for example determine hybridization conditions so that conventional method of separating the polynucleotide with required sequence homogeneity.

Polynucleotide sequence of the present invention comprises dominance maturation protein or mature protein coding sequence, SEQID NO:6 or 8 any coded sequence or its functional equivalent body can be used for preparing the recombinant DNA molecules that in suitable host cell guiding nucleic acid or its functional equivalent body surface reach.The cDNA insert that also comprises any clone that this paper identifies.

Polynucleotide of the present invention can utilize existing recombinant DNA technology (to see SambrookJ et al. (1989) Molecular Cloning:A Laboratory Manual, Cold Spring HarborLaboratory NY) links to each other with any other nucleotide sequence.The nucleotide sequence that is used to connect polynucleotide comprises a class carrier well known in the art, as plasmid, cosmid, bacteriophage lambda derivant, phasmid etc.Accordingly, the invention provides carrier that comprises polynucleotide of the present invention and the host cell that contains polynucleotide.General, carrier contains replication origin, restriction endonuclease sites and the host cell selected marker that works at least a organism.Carrier of the present invention comprises that expression vector, replicating vector, probe produce carrier and sequencing vector.Host cell of the present invention can be prokaryotic cell or eukaryotic cell, can be the part of unicellular organism body or multicellular organisms.

The present invention also provides and has contained SEQ ID NO:2, and 3,5,7,9,11,13,15,17,84,86,88,90,92,94,96,98, the recombinant precursor of the nucleic acid of 100,102,104 or 177 any nucleotide sequences or its fragment or any other GIPF polynucleotide.In one embodiment, recombinant precursor of the present invention comprises carrier, as plasmid or viral vector, has wherein inserted and has SEQ ID NO:2,3,5,7,9; 11,13,15,17,84,86,88,90; 92,94,96,98,100,102,104 or 177 arbitrary nucleotide sequences or its segmental nucleic acid.Under the situation of the carrier that comprises one of ORF of the present invention, described carrier also comprises the adjusting sequence, for example comprises the promoter that can be operatively connected with ORF.A large amount of suitable carrier and promoteres are known for those skilled in the art, and can obtain to produce recombinant precursor of the present invention by commercial sources.Provide following carrier with way of example.Bacteria carrier: pBs, phagescript, PsiX74, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); PTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).Eukaryotic vector: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).In one embodiment, the nucleic acid of SEQ ID NO:3 is inserted into the described C of the embodiment of the invention _KIn the P2KI carrier.

Separation polynucleotide of the present invention can be operatively connected with expression regulation sequence, as Kaufman etal., and Nucleic Acids Res.19, described pMT2 of 4485-4490 (1991) or pED expression vector are to produce recombiant protein.Many suitable expression regulation sequences are known in the art.The conventional method of express recombinant protein also is known, at R.Kaufman, and Methods in Enzyniology185,537-566 has statement in (1990).Ding Yi " can be operatively connected " refers to separation polynucleotide of the present invention and the location of expression regulation sequence in carrier or cell herein, makes to use the polynucleotide/expression regulation sequence that connects to transform the described protein of host cell expression of (transfection).

Carrier with CAT carrier or other selective labelling can be selected promoter region from any required gene.Two suitable carriers are pKK232-8 and pCM7.The antibacterial promoter of special name comprises lacI, lacZ, T3, T7, gpt, λ PR and trc.Eukaryotic promoter comprises immediately early stage CMV, the HSV thymidine kinase, early stage or late period SV40, retroviral LTRs and mice metallothionein-1.Select suitable carriers and promoter to belong in those skilled in the art's technical scope.General, recombinant expression carrier comprises replication origin and allows the selected marker of transformed host cell, for example escherichia coli penicillin resistance gene and beer yeast TRP1 gene, and the promoter that instructs the downstream configurations sequence to transcribe from the gene of highly expressing.This class promoter comes from the coding glycolytic ferment, as 3-phosphoglycerate kinases (PGk), and operation of alpha factor, acid p'tase or heat shock protein.The allos structure sequence is by translation initiation sequence and terminator sequence, and the preferred leader that instructs the translation protein excretion to periplasmic space or extracellular matrix, assembles in the suitable time.Choose wantonly, heterologous sequence can be encoded and be comprised the fusion rotein of amino terminal identification polypeptide, and this identification polypeptide is given special nature, as the recombinant products of stably express or simplify its purification.By inserting the proteic structural DNA sequence of coding expection, inserting suitable transcription initiation signal and termination signal and functional promoter, can make up the expression vector that is used for antibacterial in the open reading phase.Carrier comprise one or more Phenotypic Selection labellings and replication origin keep in the host to guarantee carrier, if desired, also in the host, increase.The suitable prokaryotic hosts that is used to transform comprises escherichia coli, bacillus subtilis, Salmonella typhimurium and Rhodopseudomonas, streptomyces, the various strains of staphylococcus, although other only is as selecting to use the host.

A representativeness but non-circumscribed example be, antibacterial can comprise selected marker and antibacterial replication origin with expression vector, and this starting point is derived from the commercialization plasmid that comprises known cloning vehicle pBR322 (ATCC37017) genetic elements.This class commercialization carrier comprises, for example pKK223-3 (PharmaciaFine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotech, Madison, WI, USA).PBR322 " skeleton " part is made up of suitable promoter and the structure sequence that will express.After transforming suitable hosts bacterial strain and host strain and growing into suitable cell density, induce the promoter of selection or suppress with proper method (as transformation temperature or chemical induction), cell is cultivated a period of time again.Typically, by centrifugal cell harvesting, with physics or chemical method smudge cells, the crude extract that obtains carries out next step purification.

Except realizing the present invention with expression vector, the present invention also comprises the new expression vector that contains the promoter element that the polynucleotide sequence with the coding destination protein can be operatively connected.An example of this class carrier is the pcDNA/ carrier, describes in embodiment 8.

4.2.2 host

The present invention also provides the host cell that carries out genetic modification with carrier of the present invention, and carrier can be, for example cloning vehicle or contain the expression vector of polynucleotide of the present invention.For example, this class host cell contains the nucleic acid of the present invention that useful known conversion, transfection or infection method are introduced host cell.Carrier can be, for example forms such as plasmid, virion or phage.The through engineering approaches host cell can be cultivated at conventional Nutrient medium, and culture medium can improve to be suitable for activating promoter, to select transformant or amplification GIPF gene.Condition of culture as temperature, pH etc., is the previous condition of culture of cultivating the host cell that is used to express, knows for those skilled in the art.The present invention also provides the host cell of expressing polynucleotide of the present invention through genetic modification, and wherein said polynucleotide and adjusting sequence allogenic with host cell, that the driving polynucleotide are expressed in host cell can be operatively connected.

Host cell can be more high-grade eukaryotic host cell, as mammalian cell, and the low eukaryotic host cell that waits, as yeast cells, host cell also can be a prokaryotic cell, as bacterial cell.The transfection of calcium phosphate transfection, DEAE, glucosan mediation or electroporation are realized recombinant precursor introducing host cell (Davis, L.et al., Basic Methods in Molecular Biology (1986)).The host cell that contains a kind of polynucleotide of the present invention can conventional method uses with the gene outcome that produces isolated fragment (under the ORF situation) coding or is used for producing heterologous protein under the EMF regulation and control.

Any host/vector system can be used for expressing one or more GIPF polypeptide.These include but not limited to, eucaryon host, and as the HeLa cell, the Cv-1 cell, COS cell and Sf9 cell, and prokaryotic hosts are as escherichia coli and bacillus subtilis.Most preferred cell is not express described polypeptide of specific polypeptide or protein or low expression level or proteinic cell under the normal condition.Under suitable promoter regulation, mammalian cell, yeast, antibacterial or other cell can be expressed maturation protein.With the RNAs that comes from DNA construct of the present invention, utilize cell free translation system can produce this albuminoid.Sambrook, et al. is at Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor, described suitable cloning vehicle and the expression vector that is used for prokaryotic hosts and eucaryon host among the New York (1989), introduced herein as a reference.

Various mammalian cell culture systems can be used for express recombinant protein.The example of mammalian expression systems comprises Gluzman, the described monkey kidney of Cell 23:175 (1981) fibroblast COS-7 system and can express other cell line of compatible carrier, for example C127,3T3, CHO, HeLa and bhk cell system.Mammalian expression vector comprises replication origin, suitable promoter, essential ribosome binding site, polyadenylation site, shears donor and acceptor site, transcription terminator and 5 ' flank non-transcribed sequence.Come from the virus genomic DNA sequence of SV40, the non-transcribed genetic elements of needs is provided providing as SV40 starting point, early promoter, enhancer, shearing point and polyadenylation site.The recombinant polypeptide that antibacterial culturing obtains separates from the initial extract of cell precipitation usually with protein, carries out then that one or many is saltoutd, aquo ion exchange or molecular-exclusion chromatography.If desired, finish the configuration of maturation protein with the refolding proteins step.At last, carry out final purification step with high performance liquid chromatography (HPLC).The microbial cell of expressing protein can destroy with any short-cut method, comprises multigelation, ultrasonication, Mechanical Crushing or uses the lysis agent.

The cell of some types can be used as the suitable host cell of expressing protein.Mammalian host cell comprises, for example the primate cell of monkey COS cell, people's epithelium A431 cell, people Colo205 cell, 3T3 cell, CV-1 cell, other conversion system, normal diploid cell, the outer cultured cells strain of former generation organizer, former generation explant, HeLa cell, mouse Lcell, BHK, HL-60, U937, HaK or Jurkat cell.Preferably use Chinese hamster ovary cell (CHO) and human embryonic kidney 293 cell to express GIPF albumen.

In addition, wait eukaryotic cell such as yeast low, or also may produce protein in prokaryotic cell such as the antibacterial.Potential suitable yeast strains comprises saccharomyces cerevisiae, fission yeast, kluyveromyces, candidiasis, Pichia sp. or can the proteic any yeast strains of expressing heterologous.Potential suitable bacterial strain comprises escherichia coli, bacillus subtilis, Salmonella typhimurium or can the proteic any bacterial strain of expressing heterologous.If in yeast or antibacterial, prepare albumen, need to modify the albumen that is produced, for example carry out phosphorylation or glycosylation, so that obtain functional protein in appropriate site.This covalently bound available known chemistry or enzyme process are finished.

4.2.3 chimeric protein and fusion rotein

The present invention also provides GIPF chimeric protein or fusion rotein.GIPF " chimeric protein " or " fusion rotein " comprise the GIPF polypeptide that effectively is connected with non-GIPF polypeptide herein." GIPF polypeptide " refers to have the polypeptide of the corresponding aminoacid sequence of GIPF albumen, " non-GIPF polypeptide " refers to have and the GIPF albumen polypeptide of homologous proteic aminoacid sequence not substantially, for example is different from the proteic albumen of GIPF and derives from the GIPF albumen of identical or different organism.The corresponding all or part of GIPF albumen of GIPF polypeptide in the GIPF fusion rotein.In one embodiment, the GIPF fusion rotein contains at least a GIPF protein biological activity part.In another embodiment, the GIPF fusion rotein contains at least two kinds of GIPF protein biological activity parts.In another embodiment, the GIPF fusion rotein comprises at least three kinds of GIPF protein biological activity parts.In fusion rotein, " effectively connecting " refers to that GIPF polypeptide and non-GIPF polypeptide are each other at the frame endomixis.Non-GIPF polypeptide can merge with the N end or the C end of GIPF polypeptide.

In one embodiment, fusion rotein is the GST-GIPF fusion rotein, and wherein the C of GIPF sequence and GST (glutathione S-transferase) sequence end merges.This class fusion rotein help recombinating purification of GIPF polypeptide.In another embodiment, fusion rotein is the GIPF albumen that the N end contains the allos signal sequence.In some host cell (as mammalian host cell), expression and/or the secretion of using the allos signal sequence can improve GIPF.Preferred GIPF polypeptide and V5-His label merge, and be so that utilize anti-V5 antibody to detect easily and fast purifying, as be shown in the examples.

GIPF chimeric protein of the present invention or the preparation of fusion rotein available standards recombinant DNA technology.For example, couple together with will the encode dna fragmentation (in-frame) in frame of different peptide sequences of routine techniques, for example utilize to flush end or sticky end connects, Restriction Enzyme digestion with produce suitable end, as need the polishing sticky end, with alkaline phosphatase treatment to avoid unnecessary connection and enzyme catalysis connection.In another embodiment, fusion gene can be with comprising that the synthetic routine techniques of automated DNA is synthetic.Perhaps, with anchor primer genetic fragment is carried out pcr amplification, make between two consecutive gene fragments and to produce complementation and overhang, with after annealing and once more amplification produce chimeric gene sequence and (see Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley ﹠amp; Sons, 1992).In addition, the expression vector of many codings fusion parts (as gst polypeptide) is commercial.The GIPF code nucleic acid can be cloned in this class expression vector, makes that merging part is connected in frame with GIPF albumen.

4.2.4 peptide composition

Pharmaceutical composition of the present invention comprises isolating GIPF polypeptide, includes but not limited to, comprises SEQ IDNO:4, and 6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 or 178 arbitrary amino acid polypeptide of sequence, or comprise SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92, the polypeptide of 94,96,98,100,102,104 or 177 arbitrarily nucleotide sequence coded aminoacid sequences.Polypeptide of the present invention also preferably include have biological or immunocompetent, by the polypeptide of following nucleotide coding: (a) have SEQ ID NO:2,3,5,7,9,11,13,15,17,84,86,88,90,92,94,96, the polynucleotide of 98,100,102 or 104 any nucleotide sequences, or (b) coding SEQ ID NO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101, the polynucleotide of 103,105 or 178 any aminoacid sequences, or (c) under rigorous condition with (a) or (b) polynucleotide of polynucleotide complementary strand hybridization.The present invention also provides SEQ ID NO:4, and 6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101, the biological activity of 103,105 or 178 arbitrary amino acid sequences or immunocompetence mutant; And kept bioactive " basic equivalent " and (as had, at least about 70%, at least about 75%, at least about 80% at least about 65%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typical at least about 90%, 91%, 92%, 93% or 94%, in addition more typical at least about 95%, 96%, 97%, 98% or 99%, the most typical in 99% aminoacid homogeneity).With comprise SEQ ID NO:4,6,8,10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 or 178 polypeptide is compared, the activity that the polypeptide of equipotential mutant code has is similar, improve or reduce.

The present invention also comprises the proteic fragment of the bioactive the present invention of performance.Protein fragments can be linear forms or use known method, H.U.Saragovi for example, et al., Bio/Technology 10,773-778 (1992) and R.S.McDowell, et al., J.Amer.Chem.Soc.114, the described method of 9245-9253 (1992) (introducing herein as a reference) is carried out cyclisation.This class fragment can with carrier molecule, merge mutually as immunoglobulin, its purpose comprises increases tiring of protein binding site.

The present invention also provides described proteic total length and dominance mature form (as not containing signal sequence or precursor sequence) or mature form (as lacking signal sequence and furin cleavage site).Can differentiate albumen coded sequence in the sequence table by translating disclosed nucleotide sequence.By in suitable mammalian cell or other host, expressing the mature form that full-length polypeptide can obtain this albuminoid.The sequence of maturation protein can be determined from the aminoacid sequence of total length form.

Protein composition of the present invention also comprises can accept carrier, as hydrophilic, medicine acceptable carrier.

The present invention also provides the isolated polypeptide by the degeneracy variant coding of nucleic acid fragment of the present invention or nucleic acid fragment of the present invention." degeneracy variant " refers to that nucleotide sequence is different with nucleic acid fragment of the present invention (as ORF), but because the genetic code degeneracy is encoded the nucleotide fragments of identical peptide sequence.The preferred nucleic acid fragment of the present invention is the ORFs of encoding proteins.

Can use various means known in the art to obtain arbitrary isolated polypeptide of the present invention or protein.On the simplest level, with commercialization peptide synthesizer synthetic amino acid array.Because the synthetic protein sequence that makes up has proteic one-level, secondary or tertiary structure and/or conformational characteristic, therefore has the biological property that albumen has usually, comprises protein active.This technology is particularly useful when obtaining the fragment of little peptide or longer polypeptide.For example, fragment can be used for producing the antibody at natural polypeptides.Therefore in the immunologic process of screening treatment chemical compound and exploitation antibody, can be used as the biological activity or the immunocompetence substitute of natural purifying protein.

Polypeptide of the present invention and protein can also obtain by purification from changed into expression desired polypeptides or proteinic cell.Herein, under normal circumstances cell does not produce or with low-level generation polypeptide and protein, and when making cell produce polypeptide or protein by the genetic engineering operation, claims that cell is changed into expression desired polypeptides and albumen.Those skilled in the art are set-up procedure easily, will recombinate or composition sequence is incorporated into eucaryon or prokaryotic cell and expresses, thereby produce the cell of one of synthetic polypeptide of the present invention or albumen.

The invention still further relates to the method for preparing polypeptide, be included in and cultivate host cell of the present invention in the proper culture medium, protein purification from the culture medium of cell or cell growth.For example, the inventive method comprises the process for preparing polypeptide, is included under the effable condition of coded polypeptide, cultivates the host cell of the suitable expression vector that contains polynucleotide of the present invention.Can collect polypeptide from culture, it is more convenient to collect polypeptide from culture medium, perhaps collects from the lysate of host cell and is further purified.Preferred embodiment comprises preparation total length or the proteic method of mature form.

In other method, thereby by purified polypeptide or albumen preparation polypeptide or protein in the bacterial cell that has transformed the GIPF coding DNA.Those skilled in the art can be at an easy rate according to these method isolated polypeptides and protein to obtain a kind of isolated polypeptide of the present invention or protein.These technology include but not limited to, immune chromatograph, HPLC, molecular size exclusion chromatography, ion exchange chromatography and immune affinity chromatographic.Referring to Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning:A LaboratoryManual; Ausubel et al., Current Protocols in Molecular Biology.Kept biological activity/immunocompetent polypeptide fragment and comprised and containing greater than about 100 aminoacid or greater than about 200 amino acid whose fragments, and the fragment of encode specific protein domain.

Purified polypeptide can be used for external in conjunction with test, and this is the test of discriminating known in the art and the bonded molecule of polypeptide.These molecules include but not limited to, for example the molecule of micromolecule, combinatorial library, antibody or other protein.The molecule that identifies in conjunction with test cultivates with known in-vivo tissue or animal model is checked its antagonism or agonist activity.In simple terms, molecule is splashed in cell culture or the animal, detect the prolongation survival ability of cell/animal dead rate or animal/cell then.

Albumen of the present invention can also be as the formal representation of transgenic animal product, and as the milk composition as transgenic milch cow, goat, pig or sheep, the feature of these transgenic animal is the nucleotide sequence that somatic cell or sexual cell contain encoding proteins.

Protein provided by the invention also comprises the albumen that similar aminoacid sequence is arranged to purifying protein, and this albumen may carry out natural modification or manually modified.For example, the modification of peptide or DNA sequence can be finished with known technology by those skilled in the art.The purpose that protein sequence carries out is modified the selected amino acid residue that comprises in change, replacement, replacement, insertion or the disappearance coded sequence.For example, thus can lack or replace one or more cysteine residues and change molecular conformations with other aminoacid.These change, replace, replace, insert or the technology of disappearance is (referring to US4518584) well known to those skilled in the art.Preferred this change, replacement, replacement, insertion or disappearance have kept proteic required activity.Can determine to comprise the alanine scan method with various known method, promptly systematically replace one or a string aminoacid, detect the biological activity of the mutant that contains alanine that obtains then with alanine for the crucial zone of protein function.This analytical method can determine to be substituted aminoacid to the bioactive importance of protein.Also can determine the crucial albumen of protein function zone with the eMATRIX program.

Expection keeps other fragments of protein sequence of protein active and derivant for screening or other immunization methods of great use in whole or in part, and those skilled in the art can easily prepare according to the content of this paper.The present invention includes this modification.

By in one or more insecticide expression vectors, separation polynucleotide of the present invention effectively are connected with suitable regulating and controlling sequence, and utilize insect expression system, can prepare protein.The material of baculovirus/insect cell expression system and method all provide with the commercial kit form, Invitrogen for example, San Diego, Calif., U.S.A. (MaxBat ^TMTest kit), these methods are well known in the art, and as Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987) is described, introduce herein as a reference.Herein, the insect cell that can express polynucleotide of the present invention is " conversion ".

Under the condition of culture that is fit to express recombinant protein, cultivate transformed host cells, can prepare protein of the present invention.Then with the known purification methods marking protein that (being culture medium or cell extract) purification obtains from this culture, as gel filtration and ion exchange chromatography.Protein purification also comprises the affinity chromatography that contains with described protein bound reagent; With affine resin such as concanavalin A-agarose, heparin-toyopearl ^TMOr Cibacrom blue 3GA agarose ^TMCarry out the one or many chromatographic step; Comprise one or more steps of carrying out hydrophobic interaction chromatograph with resin such as phenyl ether, butyl ether or propyl ether, or immune affinity chromatographic.

In addition, protein of the present invention can help the formal representation of purification.For example, with fusion protein form expression, as maltose-binding protein (MBP), glutathione-S-transferase (GST) or sulfur hydrogen reduction albumen (thioredoxin) are (TRX) or with the formal representation of histidine-tagged fusion rotein.(Beverly, Mass.), (Piscataway N.J.) provides purification and the commercial kit of expressing this class fusion rotein respectively with Invitrogen to Pharmacia to NewEngland BioLab.Can also use the epitope labelled protein, reuse is at the specific antibody purification of this epitope.(New Haven Conn.) provides wherein a kind of epitope (FLAG_) by Kodak.

At last,, for example contain the silica gel of dangle methyl or other aliphatic group, carry out one or more reversed phase high efficiency liquid phases (RP-HPLC) step, can be further purified protein with hydrophobicity RP-HPLC substrate.Above-mentioned all or part of purification step can make up in every way, can be used for providing the isolating recombiant protein of basic homogeneity.So the albumen of purification does not contain other mammalian proteins substantially, is defined as " isolating albumen " in the present invention.

Polypeptide of the present invention comprises the GIPF analog.It comprises the GIPF polypeptide fragment, and the GIPF polypeptide that comprises one or more aminoacid deletion, insertion or replacement.Equally, the analog of GIPF polypeptide of the present invention comprises GIPF polypeptide amalgamation protein or the peptide modified thing of GIPF, wherein GIPF polypeptide or its analog and another part, for example target part or the fusion of other therapeutic agent.This analog shows the character of improvement, and is active and/or stable as what improve.The example of the part that merges with GIPF polypeptide or its analog comprises, polypeptide is passed to the targeting moiety of small intestinal, for example small intestinal antibody or the gastrointestinal cell receptor of expressing and the antibody of part.Can comprise with the other parts that the GIPF polypeptide merges and be used for the treatment of gastroenteropathy and other treatment of diseases agent described herein, for example cytokine or other medicines.

4.2.5 gene therapy

The invention provides the gene therapy of treatment disease described herein.The functional gene of code book invention polypeptide is delivered in the suitable cell in ex vivo (exvivo), original position or body with carrier, especially utilize viral vector (as adenovirus, adeno associated virus or retrovirus), or transmit gene at ex vivo with artificial DNA method for transformation (as liposome or chemical treatment).Referring to, Anderson for example, Nature, supplement to vol.392, no.6679, pp.25-20 (1998).More the summary of polygenes treatment technology aspect is referring to Friedmann, Science, 244:1275-1281 (1989); Verma, ScientificAmerican:68-84 (1990); And Miller, Nature, 357:455-460 (1992).

As previously mentioned, " carrier " refers to nucleic acid of the present invention is delivered to arbitrary mode of host cell.Preferred vector is a viral vector, as retrovirus, and herpesvirus, adenovirus and adeno associated virus.Therefore, utilize viral vector in vivo, ex vivo or external introducing coding GIPF albumen or its segmental gene in polypeptide structure territory or nucleotide sequence or directly introduce DNA.By transgene carrier is directed to specific cells, as utilize viral vector or receptor-ligand, or utilize all usefulness of tissue-specific promoter or the two, can be implemented in the expression in the destination organization.

Be generally used for carrier and reverse transcription carrier that body viral vector interior or ex vivo location and treatment is based on DNA.Making up and using the method for viral vector is [referring to Miller and Rosman, BioTeclmiques 7:980-990 (1992)] known in the art.The preferred virus carrier is a replication defect type, that is to say, they can not be in the purpose cell self-replicating.Usually, the genome of replication-defective virus carrier of the present invention lacks viral necessary at least one zone of duplicating in infection cell.These zones can be eliminated (whole or part), perhaps with any technology that those skilled in the art will know that it are become non-functional.These technology comprise that integral body removes, replaces (using other sequence, especially with the nucleic acid that inserts), part deletion or to the one or more bases of essential regional (being used to duplicate) interpolation.These technology can be in external (on separated DNA) or former bit manipulation, utilizes gene manipulation techniques or handles with mutagens.Preferred replication defective virus has kept the necessary genome sequence of packaging virus granule.

Dna viral vector comprises DNA viruses that weaken or defective, such as but not limited to, herpes simplex virus (HSV), human papillomavirus, Epstein-Barr virus (EBV), adenovirus, adeno associated virus (AAV) etc.The preferred defective virus that lacks viral gene all or almost all.Defective virus is noninfectious after introducing cell.Utilize the defective virus carrier can make it arrive specific, regional area in the cell, need not consider that carrier can infect other cell.Therefore, can specific localization in particular organization.The example of specific support includes but not limited to, defective herpesvirus 1 carrier [Kaplitt et al., Molec.Cell.Neurosci.2:320-330 (1991)], the defective herpesvirus carrier [RD 371005A] that lacks glycoprotein L gene, or other defective herpesvirus carrier [WO 94/21807, and 1994-9-29 is open; WO 92/05263,2, and 1994-4-2 is open]; The attenuation adenovirus vector is as the described carrier of Stratford-Perricaudet al [J.Clin.Invest.90:626-630 (1992); Also can be] referring to La Salle et al., Science 259:988-990 (1993); Deficiency adeno-associated virus vector [Samulski et al., J.Virol.61:3096-3101 (1987); Samulski et al., J.Virol.63:3822-3828 (1989); Lebkowski et al., Mol.Cell.Biol.8:3988-3996 (1988)].

When giving in the body, preferably unite and use suitable immunosuppressant therapy and viral vector, adenovirus vector for example is to avoid the immune activation of viral vector and transformant.For example, can give the immunosuppressant cell factor, as il-1 2 (IL-12), interferon gamma (IFN γ) thereby or the anti-CD 4 antibodies blocking-up to the humoral immunoresponse(HI) or the cellullar immunologic response [referring to Wilson, NatureMedicine (1995)] of viral vector.In addition, utilization has superiority by transforming the minimum antigenic viral vector of expression.

In a preferred embodiment, carrier is an adenovirus vector.As shown in the Examples, it is especially effective that adenovirus vector transmits the GIPF polypeptide, showing stimulates enterocyte propagation aspect to have beyond thought effectiveness, causes the crypts epithelial hyperplasia to cause that mucosa is significant, diffusibility thickens and phenomenal growth of crypts length and branch complexity.Adenovirus is an eukaryotic DNA virus, can be modified into nucleic acid efficient transfer of the present invention to various cell types.The adenovirus that has various serotypes.In these serotypes, the present invention preferably utilizes 2 types or 5 type adenovirus hominiss (Ad 2 or Ad 5) or zoogenous adenovirus (referring to WO94/26914).Can be used in the adenovirus that animal sources adenovirus of the present invention comprises dog, cattle, Mus (Virology 75 (1990) 81 for Mavl for example, Beard et al.), sheep, pig, bird and ape (as SAV) source.

Preferred replication-defective adenoviral vector of the present invention comprises ITRs, packaging sequence and purpose nucleic acid.More preferably, the E1 of adenovirus vector zone right and wrong are functional at least.Other zone can be modified, especially E3 district (WO95/02697), E2 district (WO94/28938), any among E4 district (WO94/28152, WO94/12649 and WO95/02697) or the late gene L1-L5.

In a preferred embodiment, the E1 district of adenovirus vector and E3 district disappearance.The example of the adenovirus in disappearance E1 district is seen EP 185,573, introduces herein as a reference.

Replication defect type recombinant type adenovirus of the present invention can be by arbitrary technology preparation (Levrero et al., Gene 101 (1991) 195, EP 185 573 well known by persons skilled in the art; Graham, EMBO J.3 (1984) 2917).Especially carrying out homologous recombination by adenovirus with the plasmid that carries target DNA prepares.Produce homologous recombination after the cell line that described adenovirus and plasmid co-transfection is suitable.Employed cell strain preferred (i) transforms with described element and (ii) comprises the complementary sequence of portion gene group with replication-defective adenoviral, preferably recombinates avoiding with integration form.The example of the cell line of using comprises that the human embryonic kidney cell is 293 (Graham et al., J.Gen.Virol.36 (1977) 59), its genome conformity the left-hand part (12%) of Ad5 adenoviral gene group, and can complementary E1 and the cell line of E4 function, as describing among WO94/26914 and the WO95/02697.Collect and the purification of Recombinant adenovirus with standard molecular biological technique, these technology are known for the professional and technical personnel.

Be used for promoter of the present invention and comprise constitutive promoter and adjustment type (but induction type) promoter.Promoter is used for expression of nucleic acid inherently.Also can be allogenic.Particularly, can be the promoter sequence of eucaryon or viral gene.For example, can be to be derived from the genomic promoter sequence of wanting transfectional cell.Equally, can be to be derived from employed virus genomic promoter sequence, comprise employed adenovirus.In this respect, the promoter that can mention comprises for example ElA, MLP, the promoter of CMV and RSV gene etc.

In addition, by increasing the sequence that activates or regulate sequence or allow tissue specificity or predominant expression (enolase and GFAP promoter etc.), modify promoter.In addition, when described nucleic acid does not comprise promoter sequence, can insert promoter, as sequence being inserted the viral genome downstream.

Being used for promoteres more of the present invention is: omnipresence promoter (HPRT for example, Vimentin (vimentin), actin, tubulin), intermediate filament's promoter is (as desmin (desmin), neurofilament, keratin, GFAP), the therapeutic gene promoter is (as the MDR type, CFTR, Factor IX), tissue-specific promoter's (as actin promoter of smooth muscle cell), the promoter of the splitted cell of priority activation, reply the promoter (as steroid hormone receptor, retinoic acid receptors) of stimulation, tetracycline is regulated and is transcribed adjustment gene, cytomegalovirus immediate early promoter, retrovirus LTR, metallothionein, SV-40, Ela and MLP promoter.The adjustment gene is transcribed in the tetracycline adjusting and the CMV promoter has description in WO 96/01313, United States Patent (USP) 5,168,062 and US 5,385,839, introduces herein as a reference.

Therefore, being used for the regulator gene expression promoter includes but not limited to, cytomegalovirus (CMV) promoter, SV40 early promoter zone (Benoist and Chambon, 1981, Nature 290:304-310), be included in promoter (Yamamoto, et al. during rous sarcoma virus 3 ' length is terminal repetition, 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1441-1445), the adjusting sequence of metallothionein gene (Brinster et al., 1982, Nature 296:39-42); Prokaryotic expression carrier is as beta-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.Natl.Acad.Sci.U.S.A.75:3727-3731) or tac promoter (DeBoer, et al., 1983, Proc.Natl.Acad.Sci.U.S.A.80:21-25); Also can be referring to " Useful proteins from recombinant bacteria " in Scientific American, 1980,242:74-94; From the promoter element of yeast or other fungus, as the Gal4 promoter, ADC (alcoholdehydrogenase) promoter, PGK (glycerophosphokinase) promoter, alkaline phosphatase promoter; Show tissue specificity, be used for the animal transcripting controling area territories of transgenic animal: at pancreatic acinar cell active elastoser I Gene Handling zone (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.Quant.Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); The insulin gene control area (Hanahan, 1985, Nature 315:115-122) of in pancreatic beta cell, enlivening, the immunoglobulin gene control area (Grosschedl et al., 1984, the Cell38:647-658 that in lymphocyte, enliven; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol.Cell.Biol.7:1436-1444), mouse mammary adenoma virus control area (the Leder et al. that in testis, breast, lymph and mastocyte, enlivens, 1986, Cell 45:485-495), at active albumin gene control area (the Pinkert et al. of liver, 1987, Genes and Devel.1:268-276), at active α-fetoprotein Gene Handling zone (Krumlaufet al., 1985, the Mol.Cell.Biol.5:1639-1648 of liver; Hammer et al., 1987, Science 235:53-58), at active alpha1-antitrypsin Gene Handling zone (the Kelsey et al. of liver, 1987, Genes and Devel.1:161-171), betaglobulin Gene Handling zone (the Mogram et al. that in myelocyte, enlivens, 1985, Nature 315:338-340; Kolliaset al., 1986, Cell 46:89-94), MBP gene control area (the Readhead et al. that in the oligodendrocyte of brain, enlivens, 1987, Cell 48:703-712), myosin light chain 2 Gene Handling zone (Sani, 1985 of in skeletal muscle, enlivening, Nature 314:283-286), in the active gonadotropin releasing hormone Gene Handling zone of hypothalamus (Mason et al., 1986, Science234:1372-1378).

Can introduce the gene of arbitrary nucleotide of the present invention or code book invention polypeptide by outer material (transient expression) of chromosome or artificial chromosome (stably express).Under the situation that protein of the present invention exists, can cultured cell in vitro so that cell proliferation or in cell, produce the effect or the activity of expection.The cell of handling is introduced in the body subsequently and is used for the treatment of.Except realizing the present invention with viral vector, the present invention also comprises new support, comprises the polynucleotide sequence operon and the promoter element that can be operatively connected with the coding destination protein.Described new adenovirus vector is the pAdenoVator-CMV5-Intron carrier, has a detailed description in an embodiment.

4.2.6 transgenic animal

Polynucleotide of the present invention also can be used for producing the chimaeric animals of specific expressed GIPF polypeptide in its B cell.This animal can be used as the model of studying polypeptide activity in vivo of the present invention and the model of studying the polypeptides for modulating thing.The preferred embodiment of the invention relates to transgenic and knocks in (KI) mouse model, is designed for the biological function of fast measuring GIPF.Disclosed among transgenic KI animal model such as PCT/JP02/1123 and the WO2003/041495.Transgenic models relates to the GIPF transgenic of coding B cell specific expression GIPF under immunoglobulin kappa light chain promoter regulation.Comprise in the TT2F ES cell of complete heavy chain immunoglobulin and light chain gene seat and introduce transgenic, will contain the genetically modified ES cell of GIPF and implant and lack heavy chain of antibody allele (IgH-KO Δ H ^-/-) the mice body in (Kitamura etal., Nature 350:423-426 (1991) introduce herein as a reference).Therefore, only be derived from the functional B cell of the ES cell of expressing the IG chain, (WO 00/10383 could to express the immunoglobulin kappa light chain; EP1106061A1).Same, B cellular expression GIPF only occurs in the GIPF-KI gomphosis mouse.With contain by kind being that the genetically modified heterozygosis of transmission or the animal of isozygotying are compared, transgenic animal model of the present invention can rapid analysis chimaeric animals phenotype.In addition, genetically modified expression is confined to the B cell, and the GIPF protein excretion to the blood circulation of animal, is therefore organized each and all is exposed to GIPF, can estimate the GIPF or the biological activity of other encoding proteins arbitrarily fast.Transgenic of the present invention system is expected to be used to express and estimate the biological function of any polypeptide.Another advantage of transgenic models of the present invention is genetically modified transient expression.The activity of κ light chain promoter originates in about 14 days of gestation back, observes circulation immunoglobulin concentration after the wean and significantly improves, and has therefore avoided any potential illeffects of GIPF to the mice early development.The example of transgenic animal of the present invention is seen embodiment.

4.2.6.1 the conventional method of preparation transgenic nonhuman mammal

All contain transgenic of the present invention in the various kinds of cell of all transgenic animal of the present invention, this transgenic has changed the phenotype of " host cell ", and with respect to the B cell of specific expressed GIPF, it is secreted into the GIPF polypeptide in the blood circulation of transgenic animal.The each side technology of transgenic animal is known in the art, has a detailed description in following document, as Hogan et al., Manipulating the MouseEmbryo (Cold Spring Harbor Laboratorr, Cold Spring Harbor, N.Y., [1986]).Although the preparation transgenic animal are example with the transgenic mice at this, this only is in order to illustrate, is not limitation scope of the present invention.Those skilled in the art can adjust particular content disclosed herein at an easy rate, utilize aforementioned method and material that the GIPF transgenic sequence is introduced non-human mammal.The animal that is suitable for transgenic experiments can be originated by normal business, and (Germantown N.Y.) obtains as Taconic.

A. transgenic constructs

Use the technique for gene engineering of any appropriate known in the art can make up transgenic, these technology including but not limited to, standard techniques such as digestion with restriction enzyme, connection, conversion, plasmid purification, dna sequencing, as Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., (1989)) describe.

General and the transcriptional regulatory sequences of transgenic of the present invention can be operatively connected as promoter and/or enhancer, thereby regulate genetically modified expression with ad hoc fashion.In some embodiments, useful transcriptional regulatory sequences is to have the active sequence of altitude mixture control from time and aspect, space.Therefore, the promoter of selection is only in particular organization or cell type promoters active.Promoter/the enhancer that is used for regulating in vivo transgene expression includes but not limited to, human cytomegalic inclusion disease virus (CMV) promoter/enhancer (Karasuyama et al., J.Exp.Med.169:13[1989]), people's beta-actin promoter (Gunning et al., Proc.Natl.Acad.Sci.USA 84:4831-4835[1987]), be present in long glucocorticoid inducible type promoter (MMTV LTR) (the Kiessig et al. in terminal repetition of mouse mammary adenoma virus, Mol.Cell Biol.4:1354-1362[1984]), the long terminal repeat of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.[1985] RNA Tumor Viruses, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.), early stage or late period of SV40 regional promoter (Benoist et al.Nature 290:304-310[1981]; Templeton et al.Mol.CellBiol., 4:817[1984]; And Sprague et al., J.Virol., 45:773[1983]), comprise promoter (the Yamamoto etal. during rous sarcoma virus (RSV) 3 ' length is terminal repetition, Cell 22:787-797[1980]), herpes simplex virus thymidine kinase promoter/enhancer (Wagner et al.Proc.Natl.Acad.Sci.USA 82:3567-71[1981]), metallothionein (MT) promoter (Palmiter etal., Nature 300:611-615[1982]) and herpes simplex virus LAT promoter (Wolfe et al.NatureGenetics1:379-384[1992]).Preferred promoter is the P2 promoter of immunoglobulin kappa light chain (REF).

Except transgenic and transcription regulating nucleotide sequence, be used for preparing the genetically modified carrier of the present invention and generally contain one or more and be used to optimize other element that transgenic is expressed at host animal.Therefore transgenic makes up and can comprise the tanscription termination element, as instructs mRNA transcript polyadenylation, and intron sequences.For example, genetically modified 3 ' end can side joint SV40 sequence (the SV40 intron/PA), thus on the transgenic transcript, increase transcription stop signals and polyadenylation signal.In another embodiment, transgenic can comprise intron sequences.In many cases, when having one or more introns in the coded sequence, genetically modified expression improves.

In other embodiments, transgenic constructs can comprise other element, makes to help operation (as in bacterial cell) in cell before inserting the intended recipient cell.For example, carrier can be included in the replication initiation element that increases in the prokaryotic cell.In addition, transgenic constructs can comprise selected marker, is used for from receptor or from the intermediate cell that preparation transgenic animal process produces (bacterial cell of the construct that promptly is used to increase or be used to introduce genetically modified ES cell) isolated cell.The selected marker can be coded in the transfectional cell existence and/or the necessary protein of growing under the selectivity condition of culture.The albumen that typical selected marker's coding has following character, for example (i) can give opposing antibiotic or other toxin, as for prokaryotic host cell, the resistance of ampicillin, tetracycline or kanamycin, for mammalian cell, give the resistance of anti-neomycin, homomycin or methotrexate, or (ii) compensate the auxotrophy of cell.

B. be used to introduce genetically modified cell

In a specific embodiments, prepare transgenic nonhuman mammal of the present invention by the germ cell line of the GIPF transgenic being introduced non-human mammal.The embryo target cell that is in each stage of development can be used for introducing the GIPF transgenic.According to embryo target cell stage of development employing diverse ways.Select healthy, embryo production is good, the specific cells system of embryo's protokaryon visibility height, animal that reproductive fitness (reproductive fitness) is good implements the present invention.

In one embodiment, transgenic constructs is introduced among the single phase embryo (single stage embryo).On the whole, increase ovulation, copulation, collection germ cell with the HORMONE TREATMENT jenny.For example to female mice injection in six ages in week 5IU priatin (PMSG; Sigma), inject (0.1ml i.p.) 5IU human chorionic gonadotropin (hCG after 48 hours again; Sigma) induce the increase ovulation.Adopting FVB in this example is mice.Female Mus immediately with the copulation of spending the night of male kind of Mus.Detect the vagina plug of these female Mus then.Mice through hypergamasis passes through CO ₂Suffocate or cervical dislocation execution, from the fallopian tube that exsomatizes, obtain the embryo and place to contain 0.5% calf serum (BSA; Sigma) in Dulbecco ' the s phosphate buffer.Remove mound cell (cumulus cell) on every side with hyaluronidase (1mg/ml).Washing protokaryon embryo also places Earle ' the s balanced salt solution (EBSS) that contains 0.5%BSA, in 37.5 ℃, and 5%CO ₂, 95% air wet environment in cultivate until injection.

Under the normal condition, in proper culture medium, cultivate the fertilization embryo and occur until pronucleus (pronuclei).At this moment, introduce transgenic female as previously mentioned or male pronucleus in.Some kind, as mice, preferred male pronucleus.For example, in case form male pronucleus, promptly male-pronucleus well separates with female pronucleus and during the two all close cell membrane, just exogenous hereditary material is joined in the early stage male pronucleus.In addition, exogenous genetic material can join in the nucleus of sperm after condensing through inducing.The sperm that contains exogenous genetic material is added in the ovum subsequently or transgenosis construct adds as early as possible after joining in the ovum and removes condensing sperm.

Except the consideration of similar biological aspect, also to consider the factor of physics aspect, as join the amount (as volume) of the exogenous genetic material of fertilized egg cell's nuclear, or form the hereditary material that the fertilized egg cell examines a part.In general, the volume of the exogenous genetic material of insertion is no more than about 10pl.The physical influence that adds material can not arrive the viablity of destroying germ cell greatly.DNA sequence quantity and function different change of multifarious restriction biology according to specific germ cell and exogenous genetic material, this is conspicuous to those skilled in the art, because the hereditary material of the germ cell that obtains, comprising exogenous genetic material, must be biologically can excite and keep the germ cell differentiation and develop into an organism that function is arranged.

The copy number that joins the transgenic constructs in the germ cell depends on the sum of the exogenous genetic material of adding, and is the amount that can carry out genetic transformation.Theoretically, only need a copy, but use many copies usually, for example 1000-2000 transgenic constructs copies, to guarantee that a copy is functional.

C. introduce transgene method

Each transgenic constructs that inserts cell at first must be linear, because the recombination frequency of linear DNA molecule is than ring molecule height.Therefore,, make its linearisation with suitable digestion with restriction enzyme DNA if construct has been inserted in the carrier, this enzyme selectivity cut vector sequence, and do not cut transgenic sequence.

Can transgenic be introduced among the embryo with any means known in the art, as long as this method is not destroyed the cell or the hereditary constitution of cell, nuclear membrane or other existence.Extensively the method that adopts comprises microinjection, electroporation or lipofection.After introducing transgenic, the embryo at the incubated in vitro different time or heavily implant act on behalf of the host or both.A kind of method commonly used be according to its kind with the embryo at the about 1-7 of In vitro culture days, heavily the host is acted on behalf of in implantation then.

Introduce the most handy germ cell of transgenic constructs by the microinjection method and make the purpose receptor.In the mice body, the male-pronucleus diameter reaches about 20 μ m, can duplicate injection 1-2pl dna solution.Use germ cell to have great advantage as the target of gene transformation, because in most cases, the DNA of injection is being integrated into (Brinster et al.Proc.Natl.Acad.Sci.USA 82:4438-4442 (1985)) in the host gene before the cutting for the first time.Therefore, whole cells of transgenic animal will carry the transgenic of integration.This also is reflected in transgenic usually and is delivered to effectively in founder's the filial generation, because 50% sexual cell comprises transgenic

The retrovirus transfection can be used for transgenic is introduced non-human mammal.The non-human embryo of growing can be at In vitro culture to blastocyst stage.In this stage, blastomere can be used as the purpose cell (Jaenich, R.Proc.Natl.Acad.Sci.USA 73:1260-1264 (1976)) of retrovirus transfection.Remove zona pellucida with the enzyme processing and can effectively infect described blastomere (Manipulating the MouseEmbryo, Hogan eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, (1986)).Being used to introduce genetically modified virus carrier system generally is to carry genetically modified replication defect type retrovirus.(Jahner et al.Proc.Natl.Acad.Sci.USA 82：6927-6931(1985)).Van derPutten et al.Proc.Natl.Acad.Sci.USA 82：6148-6152(1985))。On monolayer virus generation cell, cultivate the cleavage-cell and can simple and effectively carry out transfection (Van der Putten, supra; Stewart et al.EMBO is (1987) J.6:383-388).In addition, transfection can be carried out in the stage a little later.Virus or virus generation injection cell are gone into (Jahner et al.Nature298:623-628 (1982)) in the segmentation cavity.Most of founders are chimeric for transgenic, occur over just in the cell subsets that forms transgenic animal because integrate.In addition, the genomic different loci of founder contains various transgenic retrovirus inserts, will separate in the offspring usually.In addition, by moderate gestation embryo is carried out the transfection of intrauterine retrovirus, also transgenic may be introduced system genitale (ibid for Jahner et al. (1982)).

Use the whole bag of tricks known in the art, comprise that for example electroporation, microinjection and calcium phosphate are handled, transgenic constructs can be inserted in the ES cell.Preferred insertion method is an electroporation, wherein with electroporation apparatus ES cell and transgenic constructs DNA is placed under the electric pulse, and uses according to manufacturer's guidance.Behind the electroporation, general suitable condition of culture is collected the ES cell down.There is genetically modified cell in screening then.

D. the embryo implants

To introduce genetically modified embryo in order implanting, to have prepared pseudo-fetus person, nurturer and succedaneum.General nurturer is by preparing with vasectomized buck copulation of the same race.Nurturer's the pseudo-fetus stage is important for moving into that merit plants, and is that kind is dependent.For mice, about 2-3 of pseudo-fetus stage days.The female receptor copulation is simultaneously as female donor.Although following description is relevant with mice, can be used for other any non-human mammal by those skilled in the art's adjustment.When the embryo shifts, lumbar injection 0.015ml 2.5% tribromoethanol (avertin)/g body weight, anesthesia recipient female animal.Expose fallopian tube at otch of dorsal part medisection.Directly over fallopian tube, cut whole body wall.Use watchmaker ' s pliers strip off ovarian bursa.The embryo who shifts is placed among the DPBS (Dulbecco ' s phosphate buffer), pipettes (about 10-12 embryo) with pipet.The pipet point inserts in the funnel, shifts the embryo.Shift the back and sew up two pin closure of incisions.The embryo's quantity that is implanted into specific host is different according to kind, but is suitable with the progeny size of being born naturally usually.

When the ES cell was used to introduce transgenic, the ES cellular integration of conversion was in foregoing embryo, and embryo transfer advances in pseudo-fetus nurturer's the uterus to cultivate.

E. the screening of transgenic existence or expression

Act on behalf of genetically modified existence and/or expression in host's transgenic progeny with arbitrary suitable method screening.When adopting epidermis dithering strategy (as previously mentioned), the offspring that can hatch with chimeric epidermis dithering succedaneum at first.In addition, use usually and the complementary probe of at least a portion transgenic, the DNA from the tail tissue preparation is carried out the Southern trace or pcr amplification also can screen.Utilization carries out the Western engram analysis at the proteic antibody of transgenes encoding or SABC is the other method whether the screening transgene product exists.Perhaps, analyze or RT-PCR detects tissue or cell with the top level express transgenic, detect wherein genetically modified rna expression with Northern.

Other method of estimating the transgenic existence includes but not limited to, suitable biochemistry detection, and as enzyme and/or immune detection, the histological stain of particular marker or enzymatic activity, flow cytometry or the like.Hemanalysis also can be used for detecting the existence of transgene product in the blood, and is used to estimate the influence level of transgenic for all kinds blood cell and other blood constituent.

F. the breeding of transgenic animal

By transgenic animal and suitable gametophyte copulation, or the fertilization of the ovum of render transgenic animal and/or sperm in vitro can obtain the offspring of transgenic animal.When with the gametophyte copulation, gametophyte can be genetically modified, also can be not genetically modified; Transgenic gametophyte in this way, it can comprise identical or different transgenic, or the two includes.In addition, gametophyte can be a parental line.When using external fertilization, the embryo of after fertilization can implant and act on behalf of host or incubated in vitro or common the utilization.No matter use which kind of method, all need to mention with the front whether transgenic exists among method or other proper method evaluation offspring.Usually, according to the purpose of each particular step in the reproductive process, compatriot or parent system is hybridized with offspring's copulation and backcross.

The preferred embodiment of the invention relates to and lacks heavy chain of antibody allele (IgH-KO Δ H ^-/-), and the very low mice (Kitamura et al., Nature 350:423-426 (1991)) of circulating antibody level.On the one hand, the present invention focuses on and produces GIPF albumen or its segmental transgenic nonhuman mammal in the B cell.In the transgenic animal genome stable integration coding GIPF or have the bioactive segmental nucleotide sequence of native protein, this sequence and transcriptional regulatory sequences can be operatively connected, and guide it to express in the B cell.Preferred transcriptional regulatory sequences comprises the B cell specificity promotor, as the immunoglobulin kappa chain promoter.The non-human transgenic animal includes but not limited to for example mice, rat, rabbit, pig, goat or cattle.

4.2.7 pit cell and tissue growth activity

GIPF polypeptide of the present invention shows growth factor activity, and participates in the propagation and the differentiation of intestinal crypt cell.GIPF also shows other epithelial growth factor activity of gastrointestinal tract.With in the polypeptide body of the present invention or ex vivo gives that pit cell can keep and amplification is in all-round stages of cell colony, help damaging or reparation again, transplanting, manufacturing bio-pharmaceutical and the development biosensor of diseased tissue.It is that produce at present must be from inhuman source or people's albumen of obtaining of donor that the essential industry that produces the ability of a large amount of people's cells is used, and implants cell, as gastrointestinal cell, to carry out tissue transplantation's treatment.

A plurality of different exogenous growth factors and/or cytokine can be united use to accomplish the end in view with polypeptide of the present invention, comprise any somatomedin of listing herein, other stem cell is kept the factor, particularly including stem cell factor (SCF), leukaemia inhibitory factor (LIF), Flt-3 part (Flt-3L), arbitrary interleukin, be blended in the recombinant soluble IL-6 receptor of IL-6, macrophage inflammatory protein 1-α (MIP-1-α), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet derived growth factor (PDGF), nerve growth factor, basic fibroblast growth factor (bFGF), body keratinized cell growth factor-2 (KGF2), glucagon-like peptide 2 (GLP-2).

Can use polynucleotide transfection enterocyte of the present invention, comprise pit cell, induce the autocrine of polypeptide of the present invention to express.This can produce useful undifferentiated cell system maybe can be divided into required mature cell type.These stable cell lines can be used as the source that do not break up mRNA to produce cDNA library and PCR experiment pattern.These researchs help to separate and differentiate the gene of differential expression among the pit cell group, and this Gene regulation crypts is bred and/or kept.

The amplification of epithelial stem cell colony and to keep for handling many pathological conditions be useful.The pit cell that polypeptide for example of the present invention can be used for handling cultivation produces the gastrointestinal epithelial cell, is used to increase or replaces because the cell of inflammation, chemotherapy, infection and inflammation damnification that disease, autoimmune disease, unexpected injury or genetic diseases, ionizing radiation cause.

Can also control polypeptide expression of the present invention and, make the controlled cell type that is divided into more differentiation of pit cell the influence of pit cell.A kind of method that obtains the cell type of pure specific differentiation from undifferentiated stem cell colony of extensive use relates to utilizes the cell type specificity promoter that drives selected marker.

The In vitro culture enterocyte comprises pit cell, can be used for determining whether polypeptide of the present invention shows growth factor activity.Separate pit cell from people's depolymerization colon crypts and Mus mucous membrane of colon, with Whitehead et al., the method that Gastroenterology 117:858-865 (1999) describes (introducing herein as a reference) detects clone's activity of GIPF.Polypeptide individualism of the present invention or with the common situation about existing of other somatomedin or cytokine under can detect growth factor activity.

Pharmaceutical composition of the present invention helps enterocyte, comprises the propagation of pit cell, and the regeneration of oral cavity and gastrointestinal tissue, i.e. treatment is because the lasting wound of epithelial layer that the degeneration of epithelium pit cell, death or wound cause.More specifically, this pharmaceutical composition is used for the treatment of the gastroenteropathy of mentioning herein.

Pharmaceutical composition of the present invention also can be used for promoting that healing of wound does not heal faster and better, includes but not limited to ulcer, surgery and traumatic wound etc. that pressure ulcer, vascular insufficiency cause.The active detection of wound healing includes but not limited to method Winter what follows, Epidermal WoundHealing, pp.71-112 (Maibach, Rovee H.1.and, D.T., eds.), Year Book MedicalPublishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.Invest.Dermatol71:382-84 (1978).

4.2.8 immunoregulatory activity

Polypeptide of the present invention shows the activity of regulating the immune system composition, includes but not limited to cytokine generation and/or activity and/or immune system cell.Polynucleotide encoding of the present invention shows the polypeptide of these character.The adjusting of cytokine and/or immune system cell comprises the quantity that improves and/or reduce cytokine levels or immune system specific cells.

Because this immunoregulatory activity, polypeptide of the present invention can be used for treating various immunological diseases.These diseases include but not limited to that inflammatory bowel disease (IBD) comprises ulcerative colitis and/or Crohn ' s disease, and anticancer therapy comprises the mucositis that radiotherapy and/or chemotherapy cause.The inducement of these immunological diseases may be, for example constitutional (unknown cause), heritability, infection reagent cause (as virus, antibacterial, fungus), and/or anticancer therapy (as radiotherapy and/chemotherapy) damage that causes.

Adjusting to immunne response and/or immune system composition can realize by many modes.The form of downward modulation can be immunne response or the prevention induce immune response that inhibition or blocking-up have been carried out.Suppressor T cell is replied or the specific immunologic tolerance of inducing T cell or the two act on the function that can suppress the activated T cell simultaneously.The immunosuppressant of t cell response is a kind of (active), the process of non-antigenic specificity of active normally, and this process need T cell is exposed to inhibitor continuously.Inducing T cell is not replied or unresponsive toleration can be distinguished with immunosuppressant, because tolerate antigenic specificity normally, and still can continue after no longer being exposed to toleragen.From operation, do not exist under the tolerogenic situation, the T cell is exposed to specific antigen again, T cell and no response can be verified tolerance.

Inflammatory bowel disease is nearly all mediated by one of following two approach: excessive t helper cell 1 (Th1)-cell response is followed high-level IL-12, IFN-γ and/or TNF; Or excessive t helper cell 2 (Th2)-cell response is followed high-level IL-4, IL-5 and/or IL-13 (Bouma et al. introduces as a reference) herein.Therefore polypeptide of the present invention mediates the quantity that the mechanism of immunoregulatory activity is downward modulation Th1 and/or Th2 cell colony in disease treatment.Perhaps, also may reduce other activity, as level relevant with inflammatory response and/or the cytokine (as IL-12, IFN-γ, TNF, IL-4, IL-5 and/or IL-13) that transmitting inflammation is replied.

The activity of polypeptide of the present invention can detect by the following method:

T cell or thymocyte proliferation test, include but not limited to Current Protocolsin Immunology as described below, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach, W.Strober, (Chapter 3, Ira Vitro assays for Mouse Lymphocyte Function 3.1-3.19 for Pub.Greene Publishing Associates and Wiley-Interscience; Chapter7, Immunologic studies in Humans); Takai et al., J.Immunol.137:3494-3500,1986; Bertagnolli etal., J.Immunol.145:1706-1712,1990; Bertagnolli etal., Cellular Immunology 133:327-341,1991; Bertagnolli, et al., I.Immunol.149:3778-3783,1992; Bowman et al., 1.Immunol.152:1756-1761,1994..

Cytokine generation and/or splenocyte, lymph-node cell or thymocyte proliferation test, include but not limited to as described below: Polyclonal T cell stimulation, Kruisbeek, A.M.and Shevach, E.M.In Current Protocols in Immunology.J.E.e.a.Coligan eds.Vol 1 pp.3.12.1-3.12.14, John Wiley and Sons, Toronto.1994; And Measurement of mouse andhuman interferon-' y, Schreiber.R.D.In Current Protocols in Immunology.J.E.e.a.Coligan eds.Vol1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto.1994.

Reply test (thereby producing the protein that identification influences APC-T cell interaction and direct T cytological effect) at antigenic T cell clone by detection propagation and cytokine, include but not limited to as described below: Current Protocols in Immunology, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach, W Strober, Pub.GreenePublishing Associates and Wiley-Interscience (Chapter 3,111 Vitro assays forMouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.Natl.Acad.Sci.USA 77:6091-6095,1980; Weinberger et al., Eur.J.Immun.11:405-411,1981; Takai et al., J.Immunol.137:3494-3500,1986; Takai et al., J.Immunol.140:508-512,1988.).

4.2.9 chemotaxis/chemical agonist activity

Polypeptide of the present invention has chemotaxis or chemical agonist activity (chemokineticactivity) to mammalian cell, and mammalian cell comprises for example mononuclear cell, fibroblast, neutrophilic granulocyte, T cell, mastocyte, eosinophilic granulocyte, epithelium and/or endotheliocyte.Polynucleotide encoding of the present invention has the polypeptide of this class character.Chemotaxis and the effect of chemical excitability receptor activation can be used for driving or causing required cell colony arriving required site of action.Chemotaxis or chemical excitability compositions (protein for example of the present invention, antibody, binding partners or regulator) are having unique advantage aspect treatment wound or other tissue injury and the treatment local infection.For example, attraction lymphocyte, mononuclear cell or neutrophilic granulocyte arrival tumor or infection site can improve the immunne response at tumor or infectant.

If protein or polypeptide can directly or indirectly stimulate specific cells group directional or motion, then this protein or polypeptide have chemotactic activity to this cell colony.Preferred protein or polypeptide have the ability of direct irritation cell directed movement.Can determine by any known cell chemotaxis experiment whether there is chemotactic activity in pair cell colony to specific protein with albumen or peptide at an easy rate.

Chemotactic activity detects (can differentiate the protein of inducing or stoping chemotaxis) and comprises test that detects protein induce cell transmembrane transfer ability and the test that detects protein induce cell colony and another cell colony adhesive capacity.Detect to move and include but not limited to Current Protocols in Immunolo gy as described below with adherent suitable test, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Marguiles, E.M.Shevach, W.Strober, (Chapter 6.12, Measurement of alpha and beta Chemokines6.12.1-6.12.28 for Pub.Greene Publishing Associates andWiley-Interscience; Taub et al.J.Clin.Invest.95:1370-1376,1995; Lind etal.APMIS 103:140-146,1995; Muller et al Eur.J.Immunol.25:1744-1748; Gruber et al.J.of knmunol.152:5860-5867,1994; Johnson et al.J.ofImmunol.153:1762-1768,1994.

4.2.10 drug screening

Transgenic nonhuman mammal of the present invention and offspring thereof provide some important use, and are obvious to those skilled in the art.Transgenic animal are being even more important aspect the chemical compound of screening adjusting (improve or reduce) GIPF polypeptide active.The screening active compound comprises and gives transgenic animal with candidate compound with a series of dosage, in the different time points detection compound to the active influence of GIPF.Chemical compound can give in advance or give when abdominal cavity expansion beginning.Form of medication can be oral or by appropriate method injection, depends on the chemical property of detected chemical compound.Utilize suitable biochemistry and/or histology to estimate of the reaction of a period of time inner cell to chemical compound.

Be used for screening and comprise (1) inorganic and organic compound storehouse in conjunction with the source of the test compounds of polypeptide of the present invention or adjusting (promptly improve or reduce) polypeptide active of the present invention, (2) natural product storehouse and (3) comprise at random or the combinatorial library of simulating peptide, oligonucleotide or organic molecule.

Be easy to synthesize or buy compound libraries by many commercial sources, it comprises the analog of known compound or is confirmed to be the chemical compound of guide's thing (hits or leads) by the natural product screening.

The source in natural product storehouse is microorganism (comprising antibacterial and fungus), animal, plant or other trophosome (vegetation) or marine organisms, and the combinatorial library that obtains by the screening of following several method: tunning and extract that (1) obtains from soil, plant or Marine microorganism culture fluid, or the extract of (2) organism self.The natural product storehouse comprises polyketide, non-ribosomal peptides and (non-natural) mutant thereof.Summary is referring to Science282:63-68 (1998).

Combinatorial library comprises a large amount of peptides, oligonucleotide or organic compound, and can be by traditional automatic synthesis method, PCR, clone or the preparation of proprietary synthetic method.People are interested in especially peptide and oligonucleotide combinatorial libraries.Other library comprises peptide, protein, peptide mimics, the synthetic aggregation of multipath, reorganization thing and polypeptide libraries.The summary in combinatorial chemistry and consequent combinatorial chemistry library is referring to Myers, Curr.Opin.Bioteclznol.8:701-707 (1997).The summary in peptide mimics library and example be referring to Al-Obeidi et al., Mol.BotecAlnol, 9 (3): 205-23 (1998); Hruby etal., Curr Opin Chem Biol, 1 (1): 114-19 (1997); Dorner et al., Bioorg Med Cliem, 4 (5): 709-15 (1996).

4.3GIPF medicable disease

On the one hand, the invention provides pharmaceutical agent and the method that is used for the treatment of epitheliogenic disease of needs and disease.The GIPF polypeptide can be used for improving cytoprotective, propagation and/or the differentiation of oral cavity and gastrointestinal tract epithelial cell.Particularly, the GIPF polypeptide can be used for treatment or prevents following disease or disease, include but not limited to gastroenteropathy, gastrointestinal mucositis, pars oralis pharyngis, lip and esophageal mucosa membrane injury inflammation (oral mucositis), inflammatory bowel disease, short bowel syndrome, gastric and duodenal ulcers, gastrointestinal tract erosion, comprise erosive gastritis, esophagitis, esophageal reflux, and other disease comprises that wound, burn, oculopathy and needs stimulate epithelial cell proliferation or regenerated any other disease.Also comprise the disease that treatment causes owing to oral cavity and gastrointestinal tract myxasthenia.

Mucositis comprises oral cavity and gastrointestinal mucositis, is the complication of certain cancers treatment, and wherein the liner tissue (lining) of digestive system is inflamed.GIPF can be used for preventing and/or improves because the gastrointestinal mucosal degeneration that cancer patient chemotherapy and/or radiotherapy cause, or as the complementary therapy of extracing after the tumor.The example of chemotherapeutics includes but not limited to BCNU, busulfan (busulfan), carboplatin, cyclophosphamide, tannorubicin, doxorubicin (doxorubicin), etoposide (etoposide), 5-fluorouracil, gemcytabine, isoendoxan (ifophamide), Irinotecan (irinotecan), melphalan (melphalan), methotrexate, nvelbine (navelbine), totpotecan and paclitaxel.The example of Therapeutic Method includes but not limited to BEAM (busulfan, etoposide, cytosine, cytosine arabinoside and methotrexate); Cyclophosphamide and total body radiation; Cyclophosphamide, total body radiation and etoposide; Cyclophosphamide and busulfan; 5-fluorouracil, formyl tetrahydrofolic acid or levamisole.Before when treatment, treatment or the treatment back use GIPF to help, for example small intestinal and mucous membrane of colon produce cytoprotection promote its regeneration or both, when reducing its possible side effect, can increase therapeutic dose.

Inflammatory bowel disease with the GIPF treatment comprises general inflammatory bowel disease, it is characterized by chronic, recurrent, unknown cause, also comprise Crohn disease, the dysplasia relevant, transitional colitis, ulcerative colitis with inflammatory bowel disease with the inflammatory bowel disease of GIPF treatment; Noninfectious colitis comprises colitis, collagenous colitis, diversion colitis (diversion colitis), acidophilia's colitis, graft versus host disease disease, granulomatous colitis, ischemic colitis, hemorrhagic colitis, malakoplakia (malakoplakia), necrotizing enterocolitis, radiation enteritis, cecitis (typhlitis) that mobile colon inflammation, antibiotic cause; Infectious colitis, comprise colitis that adenovirus and ameba worm colitis, balantidiasis (balantidiasis), HSV/AIDS are relevant and belong to antibacterial, campylobacter jejuni, Clostridium antibacterial, bacillus botulinus belonging to bacterial colitis by trypanosomicide, escherichia coli, Mycobacterium avium, Sotavirus, Salmonella antibacterial, bacillus dysenteriae, and with schistosomicide, spirochetosis, syphilis, trichuriasis, tuberculosis typhoid fever, colitis that vibrio cholera is relevant with yersinia.

Short bowel syndrome is the basket that the people of half or small intestinal over half has been excised in influence.The common reason of cut-out small intestinal is in order to treat Crohn disease (Crohn ' s disease).In addition, may need with surgical resection part small intestinal so that eliminate cancerous growths.Diarrhoea is the cardinal symptom of short bowel syndrome.Other symptom comprises angor, flatulence and heartburn (heartburn).Many patients that suffer from short bowel syndrome are underfed, because the small intestinal that is kept in their body can not absorb enough moisture content, vitamin and other nutrient substance from food.Life-threatening dehydration may take place in patient.The problem relevant with dehydration and malnutrition comprises physical weakness, fatigue, depression, loses weight, bacterial infection and food anaphylaxis etc.By changing diet, intravenous extra-nutrition, vitamin and mineral and using the medicine of relief of symptoms to treat short bowel syndrome.The GIPF polypeptide helps promoting not excise small intestine's propagation, therefore improves the surperficial absorption area of intestinal tissue, improves the symptom of short bowel syndrome.

The cytoprotective of GIPF polypeptide and/or regeneration activity can detect in as drag: radiation causes catarrhal body inner model (Withers and Elkind, Int J Radiat 17:261-267 (1970) introduce as a reference) herein; Chemotherapy causes catarrhal body inner model (Soris etal., Oral Surg Oral MedOral Pathol 69:437-443 (1990); Moore, CancerChemother Pharmacol 15:11-15 (1985); Farell etal., Cell Prolif 35:78-85 (2002) introduces herein as a reference); The dextran sulfate sodium of colitis and small intestinal ulcer or inflammation (DSS) model (Jeffers et al., Gastroenterology 123:1151-1162 (2002), Han etal., Am J Physiol GastrointestLiver Physiol 279:G1011-G1022 (2000); And the surgery model of short bowel syndrome (SBS) (Scott et al.Am J PhysiolG911-G921 (1998); Helmrath et al., J Am Coll Surg183:441-449 (1996)), introduce herein as a reference).

By comparing the GIPF mRNA and the protein expression level of disease cell, tissue and corresponding normal specimens, can determine whether individual the treatment for GIPF responds.The method of detection and quantitative GIPF polypeptide mRNA or protein expression level is standard nucleic acid well known in the art and Protein Detection and quantitative technique, see Sambrook, etal., MolecularCloning:X Laboratory Manual, Cold SpringHarbor Laboratory, NY (1989) or Ausubel, etal., Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, NewYork, NY (1989) introduces herein as a reference.The standard method of detection and quantitative GIPF mRNA comprises that the GIPF ribose probe with labelling carries out in situ hybridization (Gemou-Engesaeth, etal., Pediatrics 109:E24-E32 (2002), introduce herein as a reference), carry out Northern trace and correlation technique (Kunzli, etal., Cancer 94:228 (2002) with the GIPF polynucleotide probes, introduce herein as a reference), carry out RT-PCR with the GIPF Auele Specific Primer and analyze (Angchaiskisiri, etal., Blood 99:130 (2002)), and other amplification detection method, as side chain dna solution cross experiment (Jardi, etal., J.Viral Hepat.8:465-471 (2001), introduce herein as a reference), the amplification of transcriptive intermediate (Kimura, et al., J.Clin.Microbiol.40:439-445 (2002)), the microarray product, as oligomer, cDNA and monoclonal antibody and PCR in real time (Simpson, et al., Molec.Vision, 6:178-183 (2000) introduces herein as a reference).Detect with the quantitative proteic standard method of GIPF and comprise Western engram analysis (Sambrook, et al., Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory, NY (1989), Ausubel, et al., Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, New York, NY (1989)), immunocytochemistry (Racila, et al., Proc.Natl.Acad.Sci.USA 95:4589-4594 (1998) supra) and various immunoassay, comprise enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and enzyme-specific immunoassay (EIA) (Sambrook, etal., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989), Ausubel, etal., Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, New York, NY (1989)).

Disease and disease with the inventive method treatment preferably occur in the mammal.Mammal comprises, for example human and other primate, house pet or companion animals such as Canis familiaris L. and cat, laboratory animal such as rat, mice and rabbit, and domestic animal pack animal, pig, Yang Heniu.

4.3.1 Therapeutic Method

Pharmaceutical composition of the present invention (comprise polypeptide fragment, analog, mutant and antibody, or other binding partner or regulatory factor comprising antisense polynucleotides) can be applicable to various Therapeutic Method.The treatment examples of applications includes but not limited to illustrational example herein.

One embodiment of the invention are to give individuality with the GIPF polypeptide of the present invention of effective dose or other medicines composition compound, and this individuality suffers from the disease of available peptide treatment of the present invention.Though administering mode is not very important, preferred parenteral administration.The example of administering mode is subcutaneous or vein bolus mode (bolus) administration.The dosage of GIPF polypeptide of the present invention or other medicines compositions is usually by prescription doctor decision.Dosage changes according to age of individual patient, body weight, disease condition with to the reaction of medicine.Usually, every dose of polypeptide amount that gives be about 0.01 μ g/kg body weight to the 100mg/kg body weight, preferred about 0.1 μ g/kg body weight is to the 10mg/kg body weight.During parenteral administration, GIPF polypeptide of the present invention and parenteral route can be accepted carrier and make injection preparation jointly.This class carrier is well known in the prior art, and example comprises water, saline solution, Ringer ' s solution, glucose solution and contains a small amount of human serum albumin's solution.Carrier can comprise the additive of keeping polypeptide or other active component isotonicity and stability on a small quantity.The preparation of this class solution is in this area Professional knowledge scope.

4.3.2 pharmaceutical preparation

Albumen of the present invention or other compositions are (regardless of originating, include but not limited to reorganization and non-recombinant sources, comprise the antibody of polypeptide of the present invention and other binding partner) can give patient separately, also can be mixed and made into pharmaceutical preparation with treatment or the dosage that improves various diseases and give patient with suitable carrier or excipient.This based composition randomly comprises (except albumen or other active component and carrier) diluent, excipient, salt, buffer, stabilizing agent, solubilizing agent and other material known in the art.Noun " pharmacy can be accepted " refers to the not innocuous substance of interferon activity composition biological activity effect.The character of carrier depends on route of administration.Pharmaceutical composition of the present invention also can comprise cytokine, lymphokine or other hematopoietic factor and various somatomedin, as FGF, epidermal growth factor (EGF), platelet-derived somatomedin (PDGF), transforming growth factor (TGF-α and TGF-β), insulin like growth factor (IGF), keratinocyte growth factor (KGF) etc., and cytokine as herein described.

Pharmaceutical composition also can comprise active other reagent that improves albumen or other active component, or replenishes its other active or be used for the treatment of reagent.This class interpolation factor and/or the reagent that are included in the pharmaceutical composition can produce synergism or reduce side effect with protein of the present invention or other active component.On the contrary, the protein of the present invention that comprises in the preparation or other active component can be cytokine, lymphokine or other hematopoietic factor, thrombus dissolving or the antithrombotic factor or make the minimized anti-inflammatory substance of coagulation factors side effect (as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroid hormone, immunosuppressant).Protein of the present invention can form active polymer (for example heterodimer or homodimer) or activated complex with self or other albumen.

Second albumen that pharmaceutical composition of the present invention also can comprise first albumen, give simultaneously with first albumen or therapeutic agent (for example give simultaneously,, can different time give) if when perhaps agent combination has reached treatment concentration in the treatment site.The preparation technique of the application's chemical compound and administering mode can be referring to latest edition " Remington ' s Pharmaceutical Sciences, " Mack Publishing Co., Easton, PA.The treatment effective dose refers to that the amount of chemical compound is enough to relief of symptoms, for example cures, heals, prevents or improve the relevant disease situation, perhaps improves the probability of curing, heal, prevent or improve this disease.When active component gave individuality separately, treatment effective dose list referred to the amount of active component.When giving with composition forms, no matter be associating, order or administration simultaneously, the treatment effective dose refers to produce the combination consumption of the active component of therapeutic effect.

When implementing Therapeutic Method of the present invention and using, with the protein of the present invention of treatment effective dose or the mammal that disease takes place other active component.Can give protein of the present invention or other active component separately or unite use according to the inventive method, as using the Therapeutic Method of cytokine, lymphokine or other hematopoietic factor with other therapeutic modality.When giving jointly with one or more cytokines, lymphokine or other hematopoietic factor, protein of the present invention or other active component can with cytokine, lymphokine or other hematopoietic factor, thrombus dissolving or administration simultaneously of the antithrombotic factor or order administration.If the order administration, the attending doctor will determine the suitable order of administration of protein of the present invention or other active component and cytokine, lymphokine or other hematopoietic factor, thrombus dissolving or the antithrombotic factor.

4.3.3 route of administration

Suitable route of administration comprises for example oral administration, rectally, mucosal or enteral administration; Parenteral administration comprises intramuscular injection, subcutaneous injection, intramedullary injection and intrathecal injection, directly ventricle administration, intravenous injection, peritoneal injection, nasal-cavity administration or intraocular injection.Can various conventional methods give protein of the present invention in the pharmaceutical composition or other active component or implement method of the present invention, as oral medication, inhalation, local application or intradermal injection, subcutaneous injection, peritoneal injection (IP), parenteral administration or intravenous injection with various conventional methods.Preferred intravenous administration.

In addition, can the part but not the whole body form administration, for example to accumulate the storehouse or sustained release form is injected directly into chemical compound in the tissue.

In another embodiment, the cell implantation that produces GIPF is needed propagation and/or stimulates (cell therapy) in the epithelial individuality.Polynucleotide with coding GIPF transform the common cell of GIPF or low expression level GIPF of not expressing to produce the GIPF of treatment level.Cell can with described individual cells with kind, perhaps be derived from another kind.Preferred cell is derived from the individuality that needs the GIPF treatment.GIPF albumen is discharged people's cell or inhuman cell implantation individuality with biocompatible, the outer encapsulation object of semipermeability polymer, or without sealing direct implantation.

In another embodiment, consider the vivo gene treatment.The nucleotide sequence of coding GIPF is directly introduced in the individuality and to be prevented or to treat disease as herein described with secretory protein.The nucleotide of coding GIPF can be injected directly in the tissue that needs treatment, or by viral vector, as adenovirus vector or retroviral vector, imports in the ill histiocyte.The suitable carrier that comprises the GIPF code nucleic acid can be finished the physics transmission by many methods, comprises liposome-mediated transmission, direct injection naked DNA, receptor-mediated transmission or microparticle bombardment.

Employing can be applied to polypeptide of the present invention with the effective dose material Transfer to any approach of site of action of expectation.The suitable route of administration of each specific adaptations disease and effective dose fix in this area Professional knowledge scope really.For the treatment wound, preferably will treat chemical compound and be applied directly to wound site.The suitable dose scope of polypeptide of the present invention can be inferred in the similar research according to suitable animal model.The clinicist can adjust dosage as required and reach best therapeutic effect.

4.3.4 compositions/preparation

Pharmaceutical compositions for use of the present invention can conventional method be made preparation, wherein uses to comprise that one or more physiology of excipient and adjuvant can accept carrier.These pharmaceutical compositions can be used the known method manufacturing, as mixing, dissolving, granulation, sugaring garment piece, grinding, emulsifying, parcel or the freeze-drying process of routine.Select correct preparation according to route of administration.When oral protein of the present invention for the treatment of effective dose or other active component, protein of the present invention or other active component can be tablet, capsule, powder, solution or elixir form.When with the tablet form administration, pharmaceutical composition of the present invention comprises in addition as the solid carrier of gelatin or adjuvant.Tablet, capsule and powder comprise about 5～95% protein of the present invention or other active component, preferred about 25～90% protein of the present invention or other active component.When with the liquid form administration, can add liquid-carrier such as water, oil (petroleum), animal oil or vegetable oil, as Oleum Arachidis hypogaeae semen, mineral oil, Oleum Glycines or Oleum Sesami or artificial oil.The pharmaceutical composition of liquid form also can comprise normal saline, glucose or other sugar juice, glycerol such as ethylene glycol, propylene glycol or Polyethylene Glycol.When with the liquid form administration, pharmaceutical composition contains have an appointment 0.5～90% (weight) protein of the present invention or other active component, preferably contains have an appointment 1～50% protein of the present invention or other active component.

When the protein of the present invention for the treatment of effective dose by intravenous injection, epidermis injection or subcutaneous injection or other active component, protein of the present invention or other active component are the acceptable solution forms of no thermal source, parenteral route.It is relevant with aspects such as pH, isotonicity, stability to prepare the acceptable protein of this parenteral route or other active ingredient solution, and in the specialized technical knowledge scope.Preferred intravenous injection, epidermis injection or hypodermic pharmaceutical composition, except comprising protein of the present invention or other active component, also comprise isotonic vehicle such as NaCl injection, Ringer ' s injection, glucose injection, glucose and NaCl injection, newborn acidifying Ringer ' s injection or other excipient known in the art.Pharmaceutical composition of the present invention also comprises stabilizing agent, antiseptic, buffer, antioxidant or additive well known by persons skilled in the art.When with the injection form administration, reagent of the present invention is made aqueous solution preparation, and preferred physiology compatible buffers is as Hank ' s liquid, Ringer ' s liquid or normal saline.When mucosal, need to add the penetrating agent that is suitable for penetration barriers in the preparation.This class penetrating agent is known for the professional.

When oral administration, active substance is mixed and can easily chemical compound be made preparation with drug acceptable carrier well known in the art.This class carrier is made preparation with The compounds of this invention, as the tablet of the oral digestion of patient, pill, coated tablet, capsule, liquid, gel, syrup, unguentum, suspension etc.Oral drugs are following carries out in preparation: solid excipient, randomly pulverize the mixture of acquisition, add suitable adjuvant as required after, process mixture is granulated, and obtains tablet or coated tablet core.Particularly, suitable excipient has filler, as saccharide, comprises lactose, sucrose, mannitol or sorbitol; Cellulosics is as corn starch, wheaten starch, rice starch, potato starch, gelatin, tragakanta, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvidon (PVP).Also can add disintegrating agent as needs, as crospolyvinylpyrrolidone, agar, alginic acid or its salt such as sodium alginate.The coated tablet core is wrapped up with suitable sugar-coat.Use concentrated sugar solution, optional arabic gum, Pulvis Talci, polyvinylpyrrolidone, carbopol glue, Polyethylene Glycol and/or titanium dioxide, lacquer solution (lacquer solution), appropriate organic solvent or the solvent mixture of comprising for this reason.Can in tablet or sugar-coat, add dyestuff or pigment to distinguish or to identify the various combination of reactive compound.

Oral pharmaceutical preparation comprises extruding capsule with gelatin preparation, with gelatin and plasticizer, the soft seal capsule of making as glycerol or sorbitol.Active component mixes with following ingredients in the extruding capsule, filler such as lactose, and binding agent such as starch, and/or lubricant are as Pulvis Talci or magnesium stearate and stabilizing agent randomly.In the soft capsule, active component dissolves or is suspended in suitable liquid, as fatty oil, liquid paraffin or liquid macrogol.Also can add stabilizing agent in addition.All oral formulations should be made the dosage that is fit to this administering mode.During the administration of buccal method, compositions can adopt usual manner to make tablet or lozenge formulations.

During inhalation, The compounds of this invention gives with spray form, the propellant that injection and use are fit to from pressurized bottle or aerosol apparatus, for example dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas.During with the pressurized aerosol form administration, transmit quantitative medicine by valve and determine dosage unit.Be used for for example gelatine capsule of inhaler or insufflator and the mixture of powders that cartridge case can be mixed with inclusion compound and suitable powder substrate such as lactose or starch.Chemical compound can be mixed with by injection with the administration of parenteral administration mode, as bolus injection (bolus injection) or continuous infusion.Ejection preparation can be a unit dosage form, may reside in the ampoule or multi-dose container that has for example added antiseptic.Compositions can be following form: the Emulsion of suspension, solution or oily or watery carrier, can contain reagent preparation, as suspensoid, stabilizing agent and/or dispersant.

The pharmaceutical preparation of parenteral administration comprises the liquid solution of active compounds in water-soluble form.In addition, the suspension of reactive compound can be made by suitable oily injection suspension.Suitable lipophilic solvent or carrier comprise fatty oil, and as Oleum sesami, perhaps Acrawax is as ethyl oleate or triglyceride or liposome.Liquid injection suspension comprises the material that improves suspension viscosity, as sodium carboxymethyl cellulose, sorbitol or dextran.Randomly, suspension also can comprise suitable stabilizers or improve the reagent of compound dissolution, with the preparation highly concentrated solution.In addition, active component can be prepared into powder form, uses preceding and appropriate carrier, mixes as the no heat source water of sterilization.

Chemical compound also can be mixed with the compositions of rectally, for example contains the suppository or the enema,retention of conventional suppository bases such as cocoa butter or other glyceride.Except previously described preparation, chemical compound also can be mixed with long-acting goods.The preparation of this long term can give by implanting (as subcutaneous or intramuscular) or intramuscular injection.For example, chemical compound can be prepared with suitable polymer or hydrophobic material (as accepting the Emulsion that oil is made) or ion exchange resin, or as slightly molten derivant, as dissolved salt slightly.

The pharmaceutical carrier of hydrophobic compound of the present invention is to comprise that benzyl alcohol, non-polar surfactant, water are mixed with the common solution system of organic polymer and water.Solution system can be a VPD molten system altogether altogether.VPD is the mixed solution of 3%w/v benzyl alcohol, 8%w/v non-polar surfactant polyoxyethylene sorbitan monoleate and 65% Liquid Macrogol, is settled to certain volume with pure alcohol.VPD is the (VPD: 5W) contain the VPD aqueous solution that useful 5% glucose dilutes at 1: 1 of solution system altogether.Molten system can be good at the solubilizing hydrophobic chemical compound altogether, and itself is low toxicity when systemic administration.Usually, the ratio of solution system is variable not destroying under its dissolubility and the toxic situation altogether.And melt into divides different altogether: can substitute with other low toxicity non-polar surfactant as polyoxyethylene sorbitan monoleate; The clip size of PEG can be different; Other biocompatibility polymer such as polyvinylpyrrolidone can replace Polyethylene Glycol; The alternative glucose of other saccharide or polysaccharide.In addition, can adopt other dewatering medicament chemical compound to transmit system.Liposome and Emulsion are that known dewatering medicament transmits carrier.Although toxicity is higher usually, some organic solvent can also use, as dimethyl sulfoxine.In addition, with continuing delivery systme, as the semipermeability substrate that contains the solid hydrophobic polymer of therapeutic agent also can be transmitted chemical compound.Existing multiple lasting releasable material, and be well known to a person skilled in the art.Continue release capsule and can continue several thoughtful 100 days release chemical compounds that surpass according to its chemical property.According to medicine chemical property and biological stability, can take other tactful stable protein or other active component.

Pharmaceutical composition also can comprise suitable solid or gel state carrier or excipient.The example of this class carrier or excipient includes but not limited to calcium carbonate, calcium phosphate, various sugar, starch, cellulose derivative, gelatin, polymer such as PEG.Many active component of the present invention and pharmacy compatibility equilibrium ion form salt.This class medicine can be accepted the salt that base addition salts refers to keep free acid biological effectiveness and characteristic, by obtaining described alkali such as sodium hydroxide, magnesium hydroxide, ammonium, trialkylamine, dialkylamine, an alkylamine, binary amino acid, sodium acetate, Potassium Benzoate, triethanolamine or the like with inorganic or organic base reaction.

Pharmaceutical composition of the present invention can be the form of mixtures of albumen of the present invention or other active component and protein or polypeptide antigen.Protein and/or polypeptide antigen can be delivered to the zest signal bone-marrow-derived lymphocyte and T lymphocyte.Bone-marrow-derived lymphocyte resists the former reaction of making by its surface immunoglobulin receptor.Behind the MHC albumen submission antigen, the T lymphocyte resists the former reaction of making by TXi Baoshouti (TCR).MHC and structurally associated albumen comprise host cell I class and II class mhc gene encoded protein, can be with the peptide antigen presentation to the T lymphocyte.Antigenic component also can be simple purification MHC-peptide complexes or with the complex that directly transmits the costimulatory molecules of signal to the T cell.Can with surface immunoglobulin and bonded other antibody of other B cellular elements, and with TCR and the bonded antibody of other T cellular elements, can with pharmaceutical composition of the present invention.

Pharmaceutical composition of the present invention can be the liposome form, wherein can accept carrier except other medicines, albumen of the present invention and amphiphilic substance are as the lipid combination that exists with aggregated forms, lamella in described aggregated forms such as micelle, soluble monomolecular, liquid crystal or the aqueous solution.The suitable lipid of preparation liposome includes but not limited to monoglyceride, DG, lipid, LYSOLECITHIN SUNLECITHIN A, phospholipid, Saponin, cholic acid or the like.Prepare this lipoid plastid preparation and belong in the specialized technical knowledge scope, US 4,235, and 871, description arranged among US 4,501,728, US 4,837,028, the US 4,737,323 (introducing as a reference) herein.

The amount of protein of the present invention or other active component determines according to the character of the treatment order of severity of disease and character, the previous treatment that receives of patient in the pharmaceutical composition of the present invention.Finally, the attending doctor determines to treat each patient's protein of the present invention or other absorption of active ingredient.Originally, the attending doctor can give low dosage protein of the present invention or other active component, and observes patient's reaction.Give more heavy dose of protein of the present invention or other active component then up to reaching optimum therapeuticing effect, dosage will no longer raise afterwards.The various pharmaceutical compositions of implementing the inventive method contain the about 100mg/kg body weight of 0.01 μ g-of having an appointment (the preferred about 10mg of about 0.1 μ g-, the more preferably from about about 1mg of 0.1 μ g-) protein of the present invention or other active component.When the present composition was used for bone, cartilage, tendon or ligament regeneration, Therapeutic Method comprised part, system's medication or local the implantation.During administration, therapeutic combination of the present invention should be that no thermal source physiology can be accepted form.And, thereby pharmaceutical composition can be wrapped or be delivered to bone, cartilage or damaged tissue position with the injection of thickness form.Local application is suitable for wound healing and tissue repair.Except protein of the present invention or other active component, can be included in the treatment reagent in the compositions as previously mentioned arbitrarily, can with the compositions of the inventive method simultaneously or successive administration.Preferentially be used for bone and/pharmaceutical composition of cartilage comprises and can will contain the substrate that protein or other composition of active components are delivered to bone and/or cartilage injury's position, providing framework and best situation for bone and cartilage development is to be absorbed by health.This substrate is made by being used for other material of transplanting purposes at present.

Select host material to be based on biocompatibility, biological degradability, mechanical property, aesthetic property and interfacial property.The special-purpose of compositions determines the dosage form that it is suitable.The potential substrate of forming is biodegradability, chemically is defined as calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and poly-anhydride.Other potential material is biodegradable, biologically early definition is arranged, as ossein or collagen.Other substrate comprises purifying protein or extracellular matrix components.All the other potential substrate right and wrong are biodegradable, chemically are defined as sintering hydroxyapatite, bio-vitric, aluminate or other potter's clay.Substrate can comprise the combination of above-mentioned any type material, as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.In the compositions, biological potter's clay can change over calcium-aluminum-phosphorus form and change aperture, granular size, grain shape and biological degradability through processing.Preferential lactic acid and glycolic are with the copolymer of 50: 50 (molal weight) ratio formation porous particles, and diameter is at 150～800 microns.In some applications, can use chelating agen, separate with substrate to stop protein composition as carboxymethyl cellulose or from the body clot.

One group of preferred sequestering agent is a cellulosic material, as alkylcellulose (comprising hydroxy alkyl cellulose), comprise methylcellulose, ethyl cellulose, hydroxyethyl-cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose and carboxymethyl cellulose carboxymethyl, most preferably the cationic salts of carboxymethyl cellulose (CMC).Other preferred sequestering agent comprises hyaluronidase, alginic acid, PEG, polyoxyethylene, carboxyl ethylene polymer and polyvinyl alcohol.The amount of sequestering agent used herein is the 0.5-20wt% of total formulation weight, preferred 1-10wt%, this amount is to prevent that protein from being resolved by poly substrate and the necessary amounts of easy operating, does not stop CFU-GM to soak into substrate but do not understand arrive more, therefore makes protein can promote the osteogenic activity of CFU-GM.In another pharmaceutical composition, protein of the present invention or other active component can with other agent combination that helps treating skeleton and/or cartilage defects, wound (wound) or problem tissue.These reagent comprise various somatomedin, as epidermal growth factor (EGF), platelet-derived somatomedin (PDGF), transforming growth factor (TGF-α and TGF-β) and the Insulin-Like factor (IGF).

Therapeutic combination can be used for the veterinary.Except the people, specific domestic animal, the horse of fine breed all are the objects with protein of the present invention or the treatment of other active component.The dosage regimen that is used for the protein-contg pharmaceutical composition of tissue regeneration considers that by the attending doctor the various factors that influence protein active determine, type (as bone), patient age, sex, diet, the infection order of severity, administration time and other clinical factor etc. of the situation of the tissue weight that the described factor for example wishes to generate, damage location, damaged tissue, wound size, tissue injury.Dosage is different according to reinventing other albumen of containing in used matrix type and the pharmaceutical composition.For example, added other known somatomedin in the final composition,, may influence dosage as IGF I (insulin like growth factor).For example pass through periodically evaluation of tissue/osteogenesis and/or reparation, come monitoring process with X-ray, histomorphometricall and tetracycline marker.

Polynucleotide of the present invention can be used for gene therapy.These class polynucleotide can be expressed in vivo or in the external introducing mammalian cell.Can use other known method (including but not limited to form) that nucleic acid is introduced cell or organism to give polynucleotide of the present invention with viral vector or naked DNA.Cell also can carry out ex vivo and cultivate and to make cell proliferation or bring about the desired effect or active existing under the proteic situation of the present invention.Cell after the processing is introduced in the body subsequently and is used for the treatment of.

4.3.5 effective dose

Be applicable to that pharmaceutical composition of the present invention comprises the compositions of active substance to accomplish the end in view that comprises effective dose.Concrete, the treatment effective dose refers to effectively to prevent the symptom development of the individuality of receiving treatment or improves the dosage of existing symptom.Determine effective dose in those skilled in the art's limit of power, especially this paper provides detailed description.Be used for chemical compound of the present invention for any, the treatment effective dose begins and can estimate by suitable in vitro tests.For example, calculate the dosage that reaches the circulation composition scope in the animal model, can be used for more accurately determining the human body consumption.For example, calculate the dosage that reaches the circulation composition scope in the animal model, comprise the IC that determines from cell culture ₅₀(being that detected chemical compound reaches the concentration to maximum inhibitory action one half of protein biological activity).This category information can be used for more accurately determining the human body effective dose.

The amount that the treatment effective dose refers to alleviate patient symptom or prolongs the chemical compound of life.The toxicity of this chemical compound and treatment effectiveness can be determined by the conventional medicine testing process in cell culture or the experimental animal, for example measure LD ₅₀(dosage of 50% colony death) and ED ₅₀(the effective dosage of 50% mass treatment).The dosage ratio of toxicity and treatment effectiveness is a therapeutic index, can use LD ₅₀/ ED ₅₀The ratio value representation.Preferably show the chemical compound of high therapeutic index.The dosage that can be used for formulating the people by the data that obtain in these cell culture tests and the zooscopy.The dosage of this compounds is preferably comprising ED ₅₀And in low toxicity or the nontoxic circulation composition scope.Dosage changes according to employed dosage form and route of administration in this scope.Actual dosage form, route of administration and dosage are selected according to patient by the doctor.Referring to for example Fingl et al., 1975, " The Pharmacological Basis of Therapeutics ", Ch.1 p.l.Adjusting the blood drug level that dosage and dosing interval make active component respectively is enough to produce a desired effect or minimum effective drug concentration (MEC).MEC changes with different chemical compounds, but can be estimated by vitro data.Reach the required dosage of MEC according to individual instances with route of administration and different.Yet available HPLC detects or biological detection is determined blood drug level.

The also available MEC value of dosing interval is determined.The scheme that gives chemical compound should make blood level remain on the MEC in 10～90% times, and is preferred 30～90%, and more preferably 50～90%.When topical or selection absorbed, the local valid density of medicine may be irrelevant with blood drug level.

The example of the dosage of polypeptide of the present invention or other compositions is the about 100mg/kg body weight of about 0.01 μ g-every day, preferred about 0.1 μ g/kg-25mg/kg, and it is different with child's dosage to be grown up.Can be administered once every day or giving the equivalent medicine at interval long or short.

Certainly, dosage should be decided according to treatment individuality, patient age and body weight, disease serious property, administering mode and prescription doctor's judgement.

4.3.6 diagnostic detection and test kit

The present invention also provides and has utilized nucleic probe of the present invention or antibody, at random in conjunction with or unite suitable labelling, the method for coming ORFs of the present invention in the characterization test sample or its homologue to exist or express.

Usually, the method that detects polynucleotide of the present invention comprise with sample with polynucleotide of the present invention in conjunction with and the chemical compound that forms complex contact the sufficiently long time to form complex, and detect this complex, if can detect complex, then in sample, detect polynucleotide of the present invention.These class methods also are included under the stringent hybridization condition, with sample with contact with the annealed nucleic acid primer of polynucleotide of the present invention with this understanding, the annealed polynucleotide that increase if therefore polynucleotide are amplified, then detect polynucleotide of the present invention in sample.

On the whole, the method that detects polynucleotide of the present invention comprise with sample with polypeptide of the present invention in conjunction with and the chemical compound that forms complex contact the sufficiently long time to form complex, and detect this complex, if can detect complex, then in sample, detect polypeptide of the present invention.

Particularly, this method comprises specimen with one or more antibody of the present invention or one or more nucleic probe incubations, detects combining of nucleic probe or antibody and specimen composition.

The condition of nucleic probe or antibody and specimen incubation can change.Incubation conditions depends on the type or the character of employed test format, detection method and nucleic probe or antibody.One skilled in the art will appreciate that any one general hybridization, amplification or immunologic detection method can be adjusted at an easy rate is suitable for nucleic probe of the present invention or antibody.The example that this class detects is referring to Chard, T., AnIntroduction to Radioimmunoassay and Related Techniques, Elsevier SciencePublishers, Amsterdam, The Netherlands (1986); Bullock, G.R.et al., Techniques inImmunocytochemistry, Academic Press, Orlando, FL Vol.1 (1982), Vol.2 (1983), Vol.3 (1985); Tijssen, P., Practice and Theory of imrnunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).Specimen of the present invention comprises protein or film extract or biofluid such as expectorant, blood, serum, blood plasma or the urine of cell, cell.Be used for the specimen of said method according to the character of test format, detection method, as the tissue of specimen or extract and different.The protein extract of preparation cell or the method for film extract are well known in the prior art, and are easy to improve to obtain the sample compatible with system for use in carrying.

In another embodiment of the present invention, provide to contain and implemented the test kit that the present invention detects necessary reagent.Concrete, the invention provides the locellus test kit to hold one or more hermetic containers, comprise that (a) contains first container of a kind of probe of the present invention or antibody; (b) comprise one or more other containers of one or more following materials: cleaning mixture, can detect the reagent that bonding probes or antibody exist.

Concrete, the locellus test kit comprises that reagent is stored in any agent box in the container of separation.This class container comprises little glass container, plastic containers, plastic strip or papery band.This class container can allow the reagent in the interval effectively is transferred in another interval, makes sample and reagent be unlikely to cross-contamination, and reagent in each container or solution can quantitative manner join in another interval from an interval.This class container comprises the container that holds specimen, the container that detects with antibody is housed, hold the container of cleaning mixture (as sodium phosphate buffer, Tris buffer etc.), and the container that the reagent that is used to detect binding antibody or probe is housed.If when the kind of detectable comprises two anti-or anti-being labeled of labeling nucleic acid probe, labelling, can with the enzyme or the antibodies reagent of traget antibody reaction.Those skilled in the art will know that probe disclosed by the invention and antibody can merge in the test kit of having determined well known in the prior art easily.

4.3.7 screening experiment

The present invention further provides a kind of method, with isolated protein of the present invention and polynucleotide acquisition and evaluation and SEQ ID NO:2,3,5,9,11,13,15,17,84,86,88,90,92,94,96,98, the open reading frame encoded polypeptide of 100,102,104 or 177 nucleotide sequences or with the method for the bonded adjusting material in ad hoc structure territory of this nucleic acid coding polypeptide.Concrete, said method comprising the steps of:

(a) isolated protein of this material with open reading frame of the present invention or nucleic acid coding of the present invention contacted; With

(b) determine whether this material combines with described protein or described nucleic acid.

Regulatory factor can increase or reduce GIPF to epithelial proliferation activity.

In general, this discriminating and the method for the bonded chemical compound of polynucleotide of the present invention comprise this chemical compound is contacted the long enough time to form polynucleotide/compound complex with polynucleotide of the present invention, detect this complex, if detect the complex of polynucleotide/chemical compound, just identify and the bonded chemical compound of polynucleotide of the present invention.

Equally, this discriminating and the method for the bonded chemical compound of polypeptide of the present invention comprise this chemical compound is contacted the long enough time to form the complex of polypeptide/chemical compound with polypeptide of the present invention, detect this complex, if detect the complex of polypeptide/chemical compound, just identify and the bonded chemical compound of polypeptide of the present invention.

Identification comprises with the method for the bonded chemical compound of polypeptide of the present invention this chemical compound is contacted the long enough time with formation polypeptide/compound complex with polypeptide of the present invention in the cell, this complex drives the expression of target gene sequences in the cell, this complex of detection of expression by the examining report gene order, if detect polypeptide/compound complex, just identify and the bonded chemical compound of polypeptide of the present invention.

Comprise the chemical compound of regulating polypeptide active of the present invention (activity during promptly with respect to this chemical compound not, active improve or reduce) via this method compounds identified.Perhaps, comprise the chemical compound regulating polynucleotide of the present invention and express (expression during promptly with respect to this chemical compound is not expressed and improved or reduce) via this method compounds identified.Chemical compound as by the inventive method compounds identified, can be measured the ability that it regulates activity/expression with standard detecting method well known to those skilled in the art.

The material that screens in the said method includes but not limited to peptide, sugar, vitamin derivative or other pharmaceutical agents.Can be randomly or reasonably screen or design this pharmaceutical agents with the protein model technology.

For random screening, select peptide, sugar, pharmaceutical agents etc. at random, detect itself and the bonded ability of ORF encoded protein of the present invention.In addition, also can reasonably select or design pharmaceutical substances.When selecting pharmaceutical substances, claim " reasonably selecting or the design pharmaceutical substances " herein, according to the configuration of specific protein.For example, those skilled in the art can easily adopt present existent method prepare can with the bonded peptide of particular peptide sequence, pharmaceutical substances etc., thereby produce the peptide of the anti-peptide of appropriate design, or pharmaceutical substances etc., for example referring to Hurby etal., Application of Synthetic Peptides:Antisense Peptides, " In Synthetic Peptides; A User ' s Guide; W.H.Freeman, NY (1992), pp.289-307; and Kaspczak etal., Biochemistry 28:9230-8 (1989).

Except foregoing, the class material that the present invention fully describes can be by combining and regulate gene expression with one of ORFs of the present invention or EMFs.As previously mentioned, this class material can random screening or appropriate design/selection.With ORF or EMF is target, and those skilled in the art can implementation sequence specificity or element specificity substance, regulates single ORF or depends on the expression that same EMF carries out a plurality of ORFs of expression regulation.One class DNA bound substances is to contain the material that triple helix can be hybridized or form to the base residue with DNA or RNA.This class material is based on standard di-phosphate ester, ribonucleic acid skeleton, or various sulfydryl or polymer derivant with base absorbability.

The material that is applicable to these methods comprises 20～40 bases usually, with a certain regional complementarity of transcribing related gene (triple helix-see Lee et al., Nucl.Acids Res.6:3073 (1979); Cooneyet al., Science241:456 (1988); And Dervan et al., Science 251:1360 (1991)), or with self mRNA complementation (antisense-Okano, J.Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).The formation of triple helix easily causes the interruption of DNA to rna transcription, and antisense RNA hybridization blocking-up mRNA molecule is translated as polypeptide.Confirmed that these two kinds of technology are effective in model system.The information that sequence of the present invention comprised is essential for design antisense or triple helix oligonucleotide and other DNA bound substances.

Can be used as diagnostic reagent with the material of the coded protein bound of one of ORFs of the present invention.Can be mixed with pharmaceutical composition with known technology with the bonded material of one of ORFs of the present invention encoded protein matter.

5. embodiment

Embodiment 1 separates SEQ ID NO:1 from the human cDNA library

Obtain the novel nucleic acids of SEQ ID NO:1 from the human cDNA library (Invitrogen) of fetal skin preparation, it uses Standard PC R, checks order with hybridization sequences feature analysis and Sanger sequencing technologies.With to insertion of the sequence-specific primer of the carrier that inserts sub-both sides through the pcr amplification library.These sample spot are detected to obtain sequence signature (sequencesignature) on nylon membrane and with oligonucleotide probe.With clone clustering is the group of similar or identical sequence, and carries out the gel order-checking from representational clone of every group selection.Use classical Sanger sequence measurement then, use 5 ' sequence of insertion of reverse M13 sequencing primer derivation amplification.With the PCR product purification and carry out fluorescent dye terminator cycle sequencing.Carry out the order-checking of one-step method gel with 377 applying biological system (ABI) sequenators.In the world of WO03/ (029405) is open, insertion of SEQ ID NO:1 is described as new sequence.

Embodiment 2

The set of SEQ ID NO:2

Nucleic acid of the present invention (SEQ ID NO:2) is gathered (assemble) from some sequences, wherein said sequence is to obtain from the cDNA library by the method that top embodiment 1 describes, and obtains from one or more public databases under some situation.Gather last sequence with est sequence as seed (seed).The disparate databases (data base of promptly containing the Nuvelo of est sequence by being subordinated to this set then, dbEST version 124, gbpri124 and UniGene version 124) the extra sequence of middle hauling-out, algorithm expands to seed in the bigger set (assemblage) with returning quickly.When not having to enlarge the additional sequences of set from top data base, algorithm stops.Including in based on BLASTN of sequence composition hit expanded set in the set, its BLAST scoring surpass 300 and same percentage surpass 95%.

Use PHRAP (University of Washington) or CAP (Paracel), produce cDNA sequence and its corresponding proteins matter sequence of full-length gene from set.Manual edit is corrected any frameshift and incorrect termination codon.In editing process, use FASTY and BLAST to Genebank (being dbEST version 124, gbpri124 and UniGene version 124, Genpept release 124) checking sequence.Other computer program that may be used for editing process is phredPhrap and Consed (University of Washington) and ed-ready, and ed-ext and cg-zip-2 (Hyseq, Inc.).The nucleic acid of total length and aminoacid sequence are respectively shown in SEQ ID NOS:2 and 4 in the sequence table.

In order to express GIPF (SEQ ID NO:4), with total length GIPF DNA from Marathon-readycDNA library (Clontech) pcr amplification.First PCR product with further amplification of anchor PCR primer (nestedPCR primer), is described as following, and it produces the GIPF polypeptide when expressing in suitable cell line.

Embodiment 3

The expression of GIPF in muroid and human tissue

The tissue distribution of A.GIPF mRNA:

Fig. 2 shows the relative expression of the GIPF mRNA that derives from people (A) and Mus (B) tissue.

(method that CA) provides is from the total mRNA of tissue extraction shown in Figure 2 for Qiagen, Valencia according to the manufacturer.With RNA carry out quantitative PCR in real time (TaqMan) (Simpson etc., Molec Vision6:178-183 (2000)) with judge shown in the relative expression of GIPF in the tissue.The forward and the reverse primer that use in the PCR of human rna reaction are respectively: 5 ' GACCATGCTGCCTGCTCTGACAC3 ' (forward; SEQ ID NO:29) and 5 ' CACCCGCCTCCTTGCTCTCC3 ' (oppositely; SEQ ID NO:30); The forward and the reverse primer that use in the PCR of Mus RNA reaction are respectively: 5 ' GGGGGAGACCACACCACCTGCT3 ' (SEQ ID NO:31) and 5 ' TTGGACCTCGGCTCCTTGCTGTTC3 ' (SEQ IDNO:32).In all samples, the elongation factor 1 of will encoding, the DNA sequence of beta-actin and atp synthase 6 is as positive control and normalization factor.All experiments are carried out three times and are repeated, and gained result's value is average.

Y-axis is represented the copy number of GIPF mRNA in each cell, supposes that each cell has 400,000 of the mRNA transcripts of moderate-length (median length) 1.2kb, and in the cell total RNA 2% be mRNA.Fig. 2 is presented at the middle in a organized way GIPF mRNA of institute of experiment with low expression level.The top level of observed GIPF mRNA is at mouse skin, lung, ovary and brain, and people's small intestinal, skin, skin, ovary, testis and mammary gland.

The proteic distribution of B.GIPF:

With rabbit polyclonal anti--GIPF antibody test human tissue specimen in the expression (table 1) of GIPF.Rabbit polyclonal antibody is predicted that wherein described peptide is immunogenic, and is had aminoacid sequence Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys GlyGln (SEQID NO:67) by producing with the peptide immunize rabbit.From rabbit anteserum, will resist GIPF antibody affinity purification with the GIPF peptide that is coupled to Affi-Gel 10 (Bio-Rad), and be stored in the saline of the phosphate-buffered that contains 0.1% sodium azide.Be immunohistochemical analysis (IHC) (LifeSpan Biosciences, Inc., Seatlle, WA), fixing by being organized in 10% formalin, paraffin embedding and with standard technique section preparation adrenal gland, bladder, mammary gland, colon, kidney, liver, lung, ovary, pancreas, Placenta Hominis, prostate, skin, small intestinal, spleen, stomach, testis, thyroid, tonsil and uterine cancer cell specimen.Section is also developed the color as substrate with the link coupled anti-rabbit two anti-AEC that use of biotin with the special antibody test of GIPF-.

The celluar localization of GIPF is as shown in table 1 in the human tissue.The strongest observed dyeing is in the Cytoplasm of islet cells subgroup and in the Cytoplasm of the last Intradermal neuroendocrine cell of small intestinal stomach function regulating.Significantly dyeing also is present in adrenal cortex, gastric pits epithelium, and renal tubular epithelial.The lymphocyte positive is often arranged and show strong nuclear staining.At skin, the local positive is present in granular layer and the pilosebaceous unit.Some cell types show as low-intensity Cytoplasm and nuclear staining, comprise airway epithelial, II type pneumonocyte, prostatic epithelium, and breast epithelium.Local weak nuclear staining is present in hepatocyte, colonic epithelium, placental trophoblast, breast epithelium, ovary and thyroid follicle epithelium.Ganglionic cell shows as blush dyeing, and the cell type of other experiment is the GIPF feminine gender, comprises neutrophilic granulocyte and fibroblast in glandular epithelium, smooth muscle, endothelium, the blood vessel.

Table 1

Organ-/ tissue	Subcellular Localization	Dyeing
			The adrenal gland, epithelium	Nuclear and Cytoplasm	The part, moderate
Bladder, epithelium	Nuclear and Cytoplasm	Light
			Mammary gland, epithelium	Nuclear and Cytoplasm	Moderate
Colon, epithelium	Nuclear and Cytoplasm	Moderate
			Colon, neuroendocrine cell	Nuclear and Cytoplasm	A little less than

Renal cortex, glomerule	Nuclear	Light
			Renal cortex, epithelium	Cytoplasm	Moderate
Renal medulla, glomerule	Nuclear	By force
			Renal medulla, epithelium	Cytoplasm	Moderate
Liver, hepatocyte	Nuclear	Moderate
			Lung, airway epithelial	Nuclear and Cytoplasm	Moderate
Lung, II type pneumonocyte	Based on nuclear	Moderate
			Lung, pulmonary alveolar macrophage	Based on nuclear	Moderate
Ovary, epithelium	Nuclear	Moderate
			Pancreas, islands of Langerhans	Cytoplasm	By force
Placenta Hominis, trophoderm	Nuclear	Light
			Prostate, epithelium	Nuclear	Moderate
Skin, epithelial layer	Cytoplasm	Local
			Small intestinal, neuroendocrine cell	Cytoplasm, punctured nuclear (punctate nuclear)	By force
Small intestinal, inflammatory cell	Based on nuclear	Moderate
			Spleen, lymphocyte	Based on nuclear	By force
Stomach, neuroendocrine cell	Nuclear and Cytoplasm	The part, moderate is to strong
			Stomach, epithelium	Nuclear and Cytoplasm	Moderate is to strong
Testis, the Leydig cell	Cytoplasm	Light, be dispersed in
			Thymus, lymphocyte	Nuclear and Cytoplasm	Moderate
Thyroid, follicular epithelium	Nuclear	Light
			Tonsil, lymphocyte	Nuclear and Cytoplasm	Moderate
The uterus, theca interna	Nuclear	Moderate is dispersed in

Embodiment 4

The GIPF transgenic animal

The structure of A.GIPF-KI carrier

The structure that transgenic GIPF-gene is knocked in (GIPF-KI) carrier (Fig. 5 A) carries out according to the method that describes below, and is depicted among Fig. 5 B-5R.

Obtain the DNA of encoding murine immunoglobulin kappa constant region (IgC κ) and adjacent domain by two fragments that increase following:

The preparation of Fig. 5 B IgC κ fragment 1

According to from GeneBank (gi:V00777; SEQ ID NO:33) the synthetic forward (igkc1 that is used for PCR of the sequence of mouse immuning ball protein kappa constant region (IgC κ) of Huo Deing and adjacent domain; SEQID NO:34) and oppositely (igkc2; SEQ ID NO:35) primer, and be used for the DNA of amplification coding IgC κ fragment 1.Prepare igkc1:ATCTCGAGGAACCACTTTCCTGAGGACACAGTGATAGG (SEQ ID NO:34) by adding Xho I recognition sequence, and prepare igkc2:ATGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGAC (SEQ ID NO:35) by adding EcoR I recognition sequence in 5 ' end site in 5 ' end site.The clone (WO00/10383) who contains the pBluescript SK II (+) of mice constant region and hinge region with 25ng carries out PCR as template.The PCR product with restriction endonuclease EcoR I and Xho I digestion, and is connected to and uses in restriction endonuclease EcoR I and the predigested pBluescript IIKS of Xho I (-) carrier (Stratagene).Resulting plasmid pIgC κ A contains the cDNA of specific mice IgC κ fragment 1, and the zone does not have nucleotide sequence to replace between Xho I and EcoRI.

The preparation of Fig. 5 C IgC κ fragment 2

According to from GeneBank (gi:V00777; SEQ ID NO:33) the synthetic forward (igkc3 that is used for PCR of the sequence of the downstream area of the mouse immuning ball protein kappa constant region (IgC κ) of Huo Deing; SEQID NO:36) and oppositely (igkc4; SEQ ID NO:37) primer, and be used for the DNA of amplification coding IgC κ fragment 2.Prepare Igkc3:ATGAATTCAGACAAAGGTCCTGAGACGCCACC (SEQ ID NO:36) by adding EcoR I recognition sequence in 5 ' end site, and by adding BamH I in 5 ' end site, Xho I and Sal I recognition sequence prepare igkc4:ATGGATCCTCGAGTCGACTGGATTTCAGGGCAACTAAACATT (SEQ IDNO:37).The clone (WO00/10383) who contains the pBluescript SK II (+) of mice constant region and hinge region with 25ng carries out PCR as template.The PCR product with restriction endonuclease EcoR I and BamH I digestion, and is connected to and uses in restriction endonuclease EcoR I and the predigested pIgC κ of the BamH I A carrier (see above).Resulting plasmid pIgC κ AB contains the specific cDNA fragment 1 and 2 that is derived from the mouse immuning ball protein constant region, and the zone does not have nucleotide sequence to replace between EcoR I and BamH I.

Fig. 5 D puromycin gene inserts pIgC κ AB

Lox-P Puro plasmid (WO00/10383) is handled with restriction endonuclease EcoR I and Xho I digestion and with the T4DNA polymerase.The gained fragment is connected to in restriction endonuclease Sal I predigestion and the pIgC κ AB carrier (see above) with the processing of T4DNA polymerase.After verifying the intersegmental join domain of pIgC κ AB and Lox-P Puro sheet, obtain plasmid pIgC κ ABP.

Fig. 5 E IRES cDNA inserts pIgC κ ABP

According to the synthetic following forward (iresfw that is used for PCR of the IRES region sequence of pIREShyg plasmid (Clontech); SEQ ID NO:38) and oppositely (iresrv; SEQ ID NO:39) primer.Prepare iresfw:ATGAATTCGCCCCTCTCCCTCCCCCCCCCCTA (SEQID NO:38) by adding EcoR I recognition sequence to 5 ' end site, and by preparing iresrv:ATGAATTCGTCGACTTGTGGCAAGCTTATCATCGTGTT (SEQID NO:39) to 5 ' end site interpolation EcoR I and Sal I recognition sequence.Carry out PCR with 150ng pIREShyg plasmid (Clontech) as template.The PCR product is digested with restriction endonuclease EcoR I, and be connected in the pGEM-T carrier (Promega) of using restriction endonuclease EcoR I digestion in advance.Obtained to contain the plasmid IRES-Sal/PGEM of the specific cDNA sequence that no nucleotide sequence replaces.The IRES-Sal/PGEM plasmid is digested with restriction endonuclease EcoR I, and be connected in the pIgC κ ABP plasmid (see above) of using restriction endonuclease EcoR I digestion in advance.After the sequence of join domain, obtain plasmid pIgC κ ABP IRES between checking pIgC κ ABP and IRES-Sal.

The structure of Fig. 5 F P Δ C κ Sal plasmid

IgCk KO carrier (WO 00/10383) with restriction endonuclease SacII digestion, is partly digested with limiting enzyme EcoRI then.In order to replace the LoxP-PGKPuro district with Sal I restriction site, the 14.6Kb band that separates disappearance LoxP-PGK Puro zone, and be connected with SacII/EcoRI compatibility joint, this joint is for producing by following two oligonucleotide (Sal 1 is just negative with Sal 1) annealing.Obtain p Δ C κ Sal plasmid in sequence checking back.

Sal 1 is being just: 5 ' AGTCGACA3 ' (SEQ ID NO:40) and

Sal 1 is negative: 5 ' AATTTGTCGACTGC3 ' (SEQ ID NO:41).

The structure of Fig. 5 G pKI κ plasmid

PIgC κ ABP IRES plasmid with restriction endonuclease XhoI digestion, is connected comprise C district, IRES and the fragment of LoxP-puromycin of gained with the P Δ C κ Sal carrier (see above) that digests with restriction endonuclease SalI in advance.After the sequence checking, obtain pKI κ plasmid.

The segmental preparation of Fig. 5 H pIgC κ Δ IRES

PIgC κ ABPIRES plasmid is partly digested with limiting enzyme EcoRI and BglII, and the pIgC κ Δ IRES fragment of separating obtained disappearance IRES Gene Partial.

Fig. 5 I prepares mice P2 promoter fragment with PCR

According to from GeneBank (gi:aj231225; SEQ ID NO:42) the synthetic following primer that is used for PCR of the mouse immuning ball protein kappa promoter sequence of Huo Deing.

Prepare P2F:CCCAAGCTTTGGTGATTATTCAGAGTAGTTTTAGATGAGTGCAT (SEQ IDNO:43) by adding Hind III recognition sequence, and prepare P2R:ACGCGTCGACTTTGTCTTTGAACTTTGGTCCCTAGCTAATTACTA (SEQID NO:44) by adding Sal I recognition sequence to 5 ' end site to 5 ' end site.Use 25ng mouse gene group DNA (from the genomic DNA of TT2F ES cell) to carry out PCR as template.The PCR product with restriction endonuclease Hind III and Sal I digestion, and is connected in pBluescript II KS (-) carrier that digests in advance with restriction endonuclease Hind III and Sal I (Stratagene).After the sequence checking, the gained plasmid is digested with restriction endonuclease Hind III and Sal I, and separate the Hind III-Sal I fragment that contains mice P2 promoter fragment.

Fig. 5 J prepares portion C κ PolyA fragment by PCR

According to the synthetic following primer that is used for PCR of the mouse immuning ball protein kappa polyA region sequence that obtains from GeneBank (gi:V00777).By adding Sal I, Fse I in 5 ' end site and Nhe I recognition sequence prepares PPF:ACGCGTCGACGCGGCCGGCCGCGCTAGCAGACAAAGGTCCTGAGACGCCACC ACCAGCTCCCC (SEQ ID NO:45), and by preparing PPR:GAAGATCTCAAGTGCAAAGACTCACTTTATTGAATATTTTCTG (SEQIDNO:46) at 5 ' end site interpolation Bgl II recognition sequence.Use 25ng mouse gene group DNA (from the genomic DNA of TT2F ES cell) to carry out PCR as template.The PCR product with restriction endonuclease Sal I and Bgl II digestion, and is connected in the psp72 carrier that digests in advance with restriction endonuclease Sal I and Bgl II (Promega KK).After the sequence checking, use restriction endonuclease Sal I and Bgl II digestion to produce " portion C κ PolyA fragment " plasmid of purification.

Fig. 5 K prepares total length C κ PolyA fragment by PCR

According to the synthetic following primer that is used for PCR of the mouse immuning ball protein kappa polyA region sequence that obtains from GeneBank (gi:V00777).Prepare TPF:GGAATTCAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCC (SEQ ID NO:47) by adding EcoR I recognition sequence, and prepare TPR:CCCAAGCTTGCCTCCTCAAACCTACCATGGCCCAGAGAAATAAG (SEQID NO:48) by adding Hand III recognition sequence in 5 ' end site in 5 ' end site.Use 25ng mouse gene group DNA (from the genomic DNA of TT2F ES cell) to carry out PCR as template.The PCR product with restriction endonuclease EcoR I and Hand III digestion, and is connected to and uses in restriction endonuclease EcoR I and the predigested pBluescript II of the Hand III KS-carrier (Stratagene).After the sequence checking, plasmid is digested to produce " total length C κ PolyA fragment " with restriction endonuclease EcoR I and Hand III.

The structure of the dna fragmentation A that Fig. 5 L is made up of total length C κ PolyA fragment, P2 promoter fragment and portion C κ PolyA fragment

" total length C κ PolyA fragment ", " the P2 promoter fragment " and " portion C κ PolyA fragment " of generation as described above are sequentially connected in advance with in the pBluescript II KS-carrier of restriction endonuclease EcoR I and Bgl II digestion (Stratagene) according to described.After the sequence checking, use restriction endonuclease EcoR I and Bgl II digestion to produce " dna fragmentation A " plasmid of purification.

The structure of Fig. 5 M pIgC κ Δ IRES ProA plasmid

" dna fragmentation A " is connected in isolating as described above " pIgC κ Δ IRES fragment ".Sequence checking back obtains plasmid pIgC κ Δ IRES ProA.

The structure of Fig. 5 N.C κ P2 H plasmid

PIgC κ Δ IRES ProA plasmid is digested with Xho I, and therefore isolating main fragment is connected with the p Δ C κ Sal that digests with restriction endonuclease Sal I in advance, and wherein said main fragment contains upstream gene group district, mice IgC κ, dna fragmentation A and the Lox-P Puro of mice IgC κ.Sequence checking back obtains plasmid C κ P2 H.

The structure of Fig. 5 O.C κ 5 ' geneome plasmid

According to the synthetic following primer that is used for PCR of the sequence dna fragment that contains mouse immuning ball protein kappa J and C district that obtains from GeneBank (gi:V00777).Prepare 5GF:ATAAGAATGCGGCCGCCTCAGAGCAAATGGGTTCTACAGGCCTAACAACCT (SEQID NO:49) by adding Not I recognition sequence in 5 ' end site, and by preparing 5GR:CCGGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGACAATGG, (SEQID NO:50) at 5 ' end site interpolation EcoR I recognition sequence.Use 25ng mouse gene group DNA (from the genomic DNA of TT2F ES cell as template) to carry out PCR.The PCR product with restriction endonuclease Not I and EcoR I digestion, and is connected to and uses in restriction endonuclease Not I and the predigested pBluescript II of the EcoR I KS-carrier (Stratagene).After the sequence checking, obtain C κ 5 ' geneome plasmid.

The structure of Fig. 5 P.C κ P2 KI Δ DT plasmid

C κ P2 H plasmid with restriction endonuclease EcoR I and Xho I digestion, is obtained the 11Kb fragment, and is connected in the C κ 5 ' geneome plasmid of using restriction endonuclease EcoR I and Xho I digestion in advance.Sequence checking back obtains C κ P2 KI Δ DT plasmid.

The structure of Fig. 5 Q.C κ P2 KI carrier

Use restriction endonuclease Xho I the DT-A fragment to be separated from pKI κ plasmid, and be connected in the C κ P2 KI Δ DT plasmid of using restriction endonuclease Xho I and Kpn I digestion in advance with Kpn I.After the sequence checking, obtain C κ P2 KI carrier.

The assembling of Fig. 5 R.GIPF-KI carrier

Use following according to the synthetic PCR primer amplification GIPF cDNA of people GIPF cDNA sequence (SEQ ID NO:2) fragment.By adding Sal I recognition sequence in 5 ' end site and the Kozak sequence prepares SA3F:ACGCGTCGACCCACATGCGGCTTGGGCTGTGTGT (SEQ ID NO:51), and by preparing SA3R:ACGCGTCGACGTCGACCTAGGCAGGCCCTG (SEQID NO:52) at 5 ' end site interpolation Sal I recognition sequence.(fetal skin and tire lung, BD Biosciences CLONTECH) carries out PCR as template with Marathon-Ready cDNA storehouse.The PCR product is digested with restriction endonuclease Sal I, and be connected with using restriction endonuclease Sal I predigested pBluescript II KS-carrier (Stratagene).After the sequence checking, obtained the clone, and confirmation contains correct GIPF cDNA sequence and do not have nucleotide sequence to replace.To clone digestion, with GIPF cDNA fragment purification and be connected in advance in the C κ P2 KI carrier with restriction endonuclease Sal I digestion with restriction endonuclease Sal I.After the sequence checking, obtain GIPF-KI carrier (Fig. 5 A).

The production of B.GIPF-KI transgenic animal

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publish, and 1995 methods of describing are obtained mice embryonic, cultivation, the ES injection cell is gone into the embryo, is transplanted to the general procedure in foster mother uterus.

According to Aizawa Shinichi, " Biomanual Series 8; Gene Targeting ", Yodosha publishes, 1995 methods of describing, the GIPF-KI carrier is transferred in the C57BL/6X CBA F1 source mice TT2F ES cell ((Uchida, 1995), Lifetech oriental) with Not I linearisation and through electroporation.The ES cell of electroporation is suspended in [DMEM (GIBCO) in the 20ml ES culture medium, 18% hyclone (GIBCO), 0.1mM 2 mercapto ethanol (GIBCO), 1000U/ml LIF (leukaemia inhibitory factor, CHEMICON International, and be seeded in planting in advance of two 100mm and have on tissue culturing plastic's plate (Corning) of feeder cells (Invitrogen) Inc.)].After one day, culture medium is replaced by the culture medium that contains 0.75g/ml puromycin (Sigma).After this 7 to 9 days, totally 119 clones that picking forms.Each is cloned in to grow in 12 orifice plates converges, then 4/5ths cultures are suspended in the frozen culture medium of 0.2ml [ES culture medium+10%DMSO (Sigma)] and frozen in-80 ℃./ 5th of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.To carry out 0.8% agarose gel electrophoresis then from the genomic DNA of the TT2F cell separation of puromycin resistance with restriction endonuclease EcoR I (Takara Shuzo) digestion.The separated DNA fragment is transferred to (GeneScreen, NEN on the film ^TMLife Science Products), use from IgJ then _K-C _KThe dna fragmentation of genomic DNA 3 ' district (X720I-EcoR I, 1.3Kb (SEQ ID NO:67): WO 00/10383, and embodiment 48) preparation is hybridized as probe.The clone's of the ES in the hybridization banding pattern does not show as the band of an about 15Kb of molecular weight, and the clone of the ES in the hybridization shows as two bands (Fig. 6) of about 15Kb of molecular weight and 13.4Kb.After analyzing, Southern selects two #10 among the ES clone in 48 hybridization, 12 (the homologous recombination rate is about 4.2%).Also the ES clone who selects is carried out karyotyping, it is according to Aizawa Shinichi, and " Biomanual Series 8, Gene Targeting ", Yodosha publishes, and 1995 methods of describing are carried out.With two ES clone #10 of the caryogram of acting normally, 12 are used for implanting to the embryo.

With the ES cell clone #10 in the frozen hybridization, 12 cell melts, begin to cultivate and be expelled to embryo (the Tomizuka et al.Proc.Natl.Acad.Sci.USA of 8 cell stages that obtain from the male and female mice copulation of heavy chain immunoglobulin knock-out mice kind system, 97:722-727,2000) in; Injection rate is each embryo 10-12 cell.For make the ES cell development become blastocyst with the embryo in culture medium after the overnight incubation, the embryo transfer of about 10 TT2F cell-injections is gone into foster mother ICR mice (CREA JAPAN, INC.) each Aconitum carmichaeli Debx. palace, the pseudo-fetus that wherein this mice has been carried out 2.5 days is handled.As the transplanting result who amounts to 120 embryonal vaccinations, 24 filial generation mice births are arranged.The judgement of mosaic is undertaken by the degree of TT2F cell-source agouti appearance color (dark-brown) in host embryo (ICR)-source albino appearance color in the filial generation.In whole 24 filial generations, think that 11 mices (gene is knocked in Mus) have part agouti appearance color, illustrate the effect of ES cell.To knock in the isolating genomic DNA of Mus tail from gene and be used for pcr analysis.Following 2 primers that are used for PCR are synthetic according to GIPF-KI carrier sequence: SACF:CTGACTAGACTCTATCTTGC (SEQID NO:53), and SACR:CCACGGAGACCACTCGCTCATT (SEQ NID NO:54).DNA carries out PCR as template with 25ng Mus coda gene group.The reaction solution of gained is carried out 0.8% agarose gel electrophoresis, detect the band of 606bp.With of the contrast of normal TT2F cell clone as chimeric Mus generation.

Mus is remained on 12/12-hour dark/illumination circulation (in 8:00 illumination in the morning), and arbitrarily accept the filtering water of 5 μ m and CE-2 food (CLEA JAPAN, INC.).After weaning stage male mouse is raised separately.

Embodiment 5

Estimate the biologic activity of GIPF with transgenic GIPF-KI Mus

The general pathology of above-described transgenic mouse small intestinal of following evaluation and colon changes and Histological change.

Confirm that GIPF transgenic KI Mus begins tangible abdominal distension in small intestinal auxetic growth and the growth when 4 ages in week.Fig. 7 shows when comparing with corresponding contrast KI Mus, the small intestinal quality (mass) that GIPF transgenic KI Mus had tangible little enterectasis (intestinal distension) and increased age 15 weeks.Use h and E (H﹠amp; E) dyeing (Issacson, P.G., and Wright, D.H., 1983) is carried out the histopathology evaluation to the specimens paraffin embedding slices (5 μ m are thick) of various tissues, comprises liver, spleen, lung, kidney, heart, small intestinal and large intestine.The H﹠amp of small intestinal; The E section is shown in Fig. 8 (low power) and Fig. 9 (high power).The IDEXX laboratory provides the histopathology report, and is described below.

Discovery contrast Mus and gene are knocked in unique small intestinal that obviously do not coexist of histology's performance between the mouse tissue.This change comprises tangible mucosa thickening, and it is owing to have crypts length and the obvious crypts epithelial proliferation that increases of branch's complexity.Crypts is arranged with plentiful columnar epithelial cell, the heterogeneous nucleus of its big oval that has basophilous Cytoplasm and the mitosis that is positioned at basilar part easily to see.Many apoptotic bodies are dispersed in whole crypts epithelium, show that the cell multiplication factor raises.The crypts epithelium also is divided into paneth's cell (paneth cell) usually and spreads all over the mucus-secreted cup cell of crypts length.Chorioepithelium does not obviously change.In the relevant mucous membrane of small intestine of aggregate nodules (peyer ' s patch), also observe similar change.Mucous membrane of small intestine arranges and can form adenoid little recessed to aggregate nodules with normal appearance.The GIPF-KI mice, on the surface with also observe hypertrophy in recessed and sexually revise for a short time, this locate its with slight acute inflammation and crypts in the non-viable non-apoptotic cell accumulation, promptly the crypts microabscess is relevant.Therefore described aggregate nodules are difficult to estimate adenoid amount and characteristics by tangential section.But, have minority to mean that the B-lymphocyte transformation becomes antibody-celliferous plasma cell.Organize medium-sized lymphocyte or inflammatory cell group not to have other visible change at small intestinal and other.

For measuring intestinal epithelial cell hypertrophy in the KI mice, according to manufacturer's explanation and the method (Scholzen that described in the past, T.et al.2000), on contrast and the small intestinal specimens paraffin embedding slices of GIPF KI mice with monoclonal rat anti-mouse Ki67 antigen (Dako Ltd., High Wycombe UK) carries out immunohistochemistry.As shown in figure 10, confirm that the positive epithelial cell of Ki67 increases in the GIPF KI mouse small intestine, show by the GIPF protein expression hypertrophy is strengthened.

3 GIPF KI mices of results in the time of 12 months.These 12 the monthly age mice show typical abdominal distension and in younger animal observed small intestinal volume increase.Prepare the various H﹠amp that organize; The E section comprises spleen, liver, adrenal gland, kidney, thymus, heart, lung, small intestinal, large intestine, harmonization of the stomach brain.

The histology of the small intestinals of 12 monthly age GIPF-KI mices section shows that GIPF has induced tangible crypts length to increase, and reaches the observed degree in 15 age in the week GIPF-KI animals that is same as.In addition, from the histologic analysis demonstration of other slices of organs, prolong 12 months later GIPF again and any obvious oncogenic activity do not occur.Sometimes the spontaneous tumor of observed some mice formation is normally with mice or the KI transgenic animal have nothing to do.Observe the adenoma of liver of low incidence rate 12 monthly age control mice.

Embodiment 6

The GIPF adenovirus vector

With Nhe I in the multiple clone site (MCS) and Xba I site, GIPF cDNA (SEQ IDNO:2) is cloned into pAdenoVatorCMV5-Intron to produce the GIPF recombinant adenovirus of V5His6 label.(CA U.S.A) obtains pAdenoVator-CMV5-Intron for Qbiogene, Carlsbad by modifying pAdenoVatorCMV5-IRES-GFP.PAdenoVator-CMV5-IRES-GFP is digested to remove its MCS, IRES and GFP with SpeI, and be connected with Intron-MCS-V5His-BGH polyA from cDNA/Intron carrier pcr amplification, wherein PCR uses primer: 5 '-CACCCCTAGGTCAATATTGGCCATTAGC-3 ' (SEQ ID NO:55) and 5 ' CACCCCT-AGGTAGGCATCCCCAGCATGC-3 ' (SEQ ID NO:56).

According to manufacturer's explanation use electroporation carry out linearizing transfer vector to bacterial cell BJ5183 (Qbiogene, Carlsbad, CA, conversion U.S.A), this cell carries the AdEasy-1 plasmid of encoding adenovirus-5 genome (E1/E3 deletion).(CA produces and amplification in U.S.A), and (Garnier, A., J.Cote et al.1994) the CsCl banding purification by describing in the past for Qbiogene, Carlsbad at the QBI-293A cell with recombinant adenovirus.(Invitrogene Inc., Carlsbad CA) utilize Recombinant Protein Expression in the 293A cell that the Western assay determination infected with recombinant adenovirus to use anti--V5 antibody.According to manufacturer's method, use the quick titration test kit of Adeno-X (BDbiosciences, Palo Alto, U.S.A.) titre of the recombinant virus of mensuration CsCl purification.In brief, by infecting the 293A cell, fixing and with the viral stock of the raji cell assay Raji of the adjacent body of mouse anti-six (hexon) antibody staining transduction back 48 hours of infection then with the recombinant adenovirus stock of serial dilution.With coupling the goat of horseradish peroxidase anti--mouse antibodies detection signal and enhanced 3 with metal, 3 '-diaminobenzidine (DAB) colour developing.

Embodiment 7

Give the biologic activity of GIPF adenovirus as model evaluation GIPF

Give normal mouse with the effect of judgement GIPF with the GIPF recombinant adenovirus, and confirm on the non-transgenic animal, to set up in observed phenotype on the GIPF transgenic mouse to small intestinal and colonic epithelium.The injection adenovirus is preceding with 6-8 BALB/C mice isoflurane anesthesia in age in week.With every mice 1 * 10 ¹⁰Virion inject by vena orbitalis posterior.With the contrast virus of same titre (empty virus) or separately PBS with comparing.Behind virus injection, mice is put to death (for all groups, n=3) when 3 days or 5 days.Put to death preceding 4 hours, peritoneal injection (IP) 1mg bromodeoxyribouridine (BrdU) is to judge epithelial propagation in the body.Collection comprises the various tissues of small intestinal, colon, spleen, liver and bone marrow, and is fixed in the formalin.With paraffin-embedded section h and E (H﹠amp; E) Histological evaluation is carried out in dyeing.As former description (McKinley, J.N.et al.2000) (immunohistochemistry to BrdU is also carried out section in explanation U.S.A.) for Oncogene Researchproduct, Boston according to the manufacturer.As the explanation according to the manufacturer of former description (Scholzen, T.et al.2000), (immunohistochemistry UK) is to estimate the propagation of intestinal epithelial cell for Dako Ltd., High Wycombe also to use monoclonal rat anti-mouse Ki67 antigen.

Adenovirus is injected the H﹠amp that put to death the section of Mus small intestinal in back 3 days; E dyeing (Figure 11) shows that the small intestinal of accepting GIPF adenovirus mice changes obviously, and show with the GIPF transgenic mouse in observed homologue learn feature (Fig. 8 and 9).The Histological change that is caused by GIPF comprises the tangible diffuse thickening of mucosa, and it is owing to have crypts length and the obvious crypts epithelial proliferation that increases of branch's complexity.Crypts is arranged with plentiful columnar epithelium, the heterogeneous nucleus of its big oval that has basophilous Cytoplasm and the mitosis that is positioned at basilar part easily to see.The crypts epithelium also is divided into paneth's cell usually and spreads all over the mucus-secreted cup cell of crypts length.GIPF is to the epitheliogenetic influence of crypts further enhancing in 5 days behind virus injection, as shown in figure 12.For estimating GIPF, in contrast with accept to carry out BrdU in the small intestinal section of GIPF adenovirus mice and mix immunostaining with Ki67 to the outgrowth influence of intestinal epithelial cell.Shown in Figure 13 and 14, the mice of accepting the GIPF adenovirus has the little intestinal crypt of difference BrdU showed increased and Ki67 positive cell.Every Mus 1 * 10 ⁹Also observe the biological action (Figure 15) of GIPF during the low viral dosage of virion to the crypts epithelial cell proliferation.Except the observed effect of small intestinal, GIPF also induces crypts epithelial proliferation in the colon, and it has crypts length obviously to increase and goblet cell number and volume increase (Figure 16).

Embodiment 8

The expression vector of coding GIPF and GIPF analog

Be cloned in the pcDNA/Intron carrier to produce the GIPF (SEQ IDNO:5) of wild type and c-terminus V5His6-label with will the encode cDNA of GIPF (SEQID NO:3) of KpnI and XbaI site.Be derived from pCI mammalian expression vector (Promega by what import design, Madison, chimeric intron WI) is with pcDNA3.1TOPO carrier (Invitrogene Inc., Carlsbad, CA) genetic modification obtains mammalian expression vector pcDNA/Intron.PCI is digested with BG1II and KpnI, and intron sequences is cloned among the pcDNA3.1 that uses BG1II and KpnI digestion.By PCR the GIPF reading frame (SEQ ID NO:3) of SEQ ID NO:2 at first is cloned among the pcDNA3.1V5His-TOPO (Invitrogen), it uses following primer: forward 5 ' CACCATGCGGCTTGGGCTGTCTC3 ' (SEQID NO:57), reverse 5 ' GGCAGGCCCTGCAGATGTGAGTG3 ' (SEQID NO:58), the KpnI-XbaI that will contain the pcDNA 3.1/V5His-TOPO source of whole GIPF reading frames then insert in the pcDNA/Intron carrier that son is connected to modification to produce the pcDNA/Intron construction.

The proteic analog of following production total length GIPF.With primer 5 '-GATCAAGGGGAAA CAGCAGAGGCGGATCAG-3 ' (SEQID NO:59) and 5 '-CTGATCCGCCTCTGC TGTTTCCCCTTGATC-3 ' (SEQID NO:60) carries out the direct mutagenesis sudden change, realizes the sudden change (aminoacid 28R/Q) to the total furin cleavage site of GIPF among the pcDNA/Intron of prediction.Produce GIPF deletion mutant (deletion amino acid residue 21-31) (SEQID NO:16) with the stitching method.Use primer set1:5 '-CACCGCTAGCCTCGAGAATTCACGCGTG-3 ' (SEQ ID NO:61) and phosphoric acid 5 '-GCTGATGGTGAGGTGCGTC-3 ' (SEQID NO:62), set2: phosphoric acid 5 '-ATCAGTGCCGAGGGGAGCCAG-3 ' (SEQID NO:63) and 5 '-GCCCTCTAGAGCGGCAGGCCCTGCAGATG-3 ' (SEQIID NO:64), with two fragments of pcr amplification, subsequently described two fragments are connected.The GIPFcDNA that carries aminoacid 21-31 deletion is used the forward and the reverse primer pcr amplification of SEQ ID NO:61 and 64 respectively, with NheI and XbaI digestion, and with NheI in its multiple clone site and XbaI site sub-clone in pcDNA/Intron.Confirm the sequence of two kinds of mutants.

In order to express mammal, also (nucleotide 451 of GIPF reading frame to nucleotide 618 (SEQ ID NO:13) is cloned in the pcDNA/Intron carrier with thrombospondin (TSP) domain.CDNA:NheI forward primer with following primer PCR amplification coding TSP domain: CCGGCTAGCCACCATGGCGCAATGTGAAATGA (SEQ ID NO:65) and NotI reverse primer: CCATGCGGCCGCCCTCCTCACTGTGCACCT (SEQID NO:66).The PCR product of NheI and the digestion of NotI restriction endonuclease is connected in the pcDNA/Intron carrier of NheI and NotI digestion.For producing recombinant adenovirus, use NheI and NotI the restriction endonuclease next TSP domain of pcr amplification as described above are cloned among the pAdeno Vator-CMV5-Intron.The sequence of the TSP domain of checking PCR-amplification.

The furin sample of describing disappearance GIPF in embodiment 19 is rich in other analog of the different piece in cysteine district.

The method of describing among the embodiment below using is in vivo with the biologic activity of the above-described GIPF analog of in-vitro evaluation.Estimate the biologic activity of GIPF analog with the GIPF transgenic models.

Embodiment 9

The purification of reorganization GIPF

A. the expression of GIPFt and purification in eukaryotic cell:

V5-His label G IPF (GIPFt) (SEQ ID NO:5) is expressed in HEK293 and Chinese hamster ovary celI and following purification:

The stabilized cell culture of HEK293 cell is grown in the serum-free 293free-style culture medium (GIBCO), and wherein said HEK293 cell has been used the GIPF pcDNA/Intron construction transfection of the DNA that comprises coding V5-His-label G IPF polypeptide (SEQID NO:5).Suspension culture is inoculated with cell density 100 ten thousand cells/ml, and after 4-6 days, gathered in the crops.Measure the level that has been secreted into the V5-His label G IPF in the culture medium with ELISA.

The stable culture of Chinese hamster ovary celI is grown in the serum-free EX-CELL302 culture medium (JRH), and wherein said Chinese hamster ovary celI transforms with the pDEF 2S carrier of the nucleotide sequence that comprises coding V5-His-label G IPF (SEQID NO:5).Expression vector contains the DNA sequence of encoding D HFR, and wherein DHFR allows in methotrexate (MTX) positive-selecting and amplification when existing.Measure the V5-His label G IPF level that has been secreted in the culture medium with ELISA.

Results contain the proteic culture medium of excretory GIPF and frozen in-80 ℃.Culture medium 4 ℃ of thawings, and in order to prevent GIPF degraded, is added protease inhibitor with every kind of material final concentration 1mM, and EDTA and Pefabloc (Roche, Basel, Switzerland).Culture medium is filtered by 0.22 μ m filter (Corning), and concentrate 10 times with the TFF system (PallFiltron) that has the 10kDa molecular weight to hold back (cut-off) film.With spissated culture medium buffer and 20mM sodium phosphate, 0.5M NaCl, pH 7 exchanges.In the purge process, it is important keeping the complete dissolubility of V5-His label G IPF when adding 0.5M NaCl to pH 7 in purge process in phosphate buffer.Behind ultrafiltration and diafiltration, 1: 500 (v/v) adds mammalian protease inhibitor mixed thing (Sigma) with final dilution factor.

With HiTrap Ni ²⁺-sequestration affinity column (Pharmacia) is used the 20mM sodium ascorbyl phosphate, and pH 7, the 0.5MNaCl balance.Ni is filtered and be loaded into to the culture medium of buffer-exchanged with 0.22 μ mPES filter ²⁺On-sequestration the affinity column.With Ni ²⁺Post is with 10 column volumes of the 20mM imidazoles of 10 times of column volumes (CV) washing, and with above the imidazoles of the gradient 20mM to 300mM of 35CV with the albumen eluting.With SDS-PAGE and Western engram analysis fraction.To contain the fraction analysis of V5-His label G IPF and mix with the GIPF protein solution of production purity between 75-80%.

To use Ni ²⁺Isolating proteic buffer of GIPF and the 20mM sodium phosphate of containing of post, the 0.3M arginine, pH 7 exchanges are to remove NaCl.In purification step subsequently, in phosphate buffer, replaced NaCl to keep the V5-His proteic complete dissolubility of GIPF that tags with the 0.3M arginine.To use Ni ²⁺The isolating GIPF protein loaded of post is to using the 20mM sodium phosphate, the 0.3M arginine, on the SP agarose gel high-effective cationic exchange column that pH 7 balances are crossed (Pharmacia, Piscataway, NJ).Post with 0.1M NaCl washing 8CV, is used gradient 0.1M to the 1M NaCl eluting that surpasses 30CV.The fraction that will contain V5-His label G IPF mixes with the GIPF protein solution of production purity between 90-95%.

With blended fraction buffer and 20mM sodium phosphate, pH 7, and 0.15M NaCl exchange is concentrated into 1 or 2mg/ml with albumen, and by aseptic 0.22 μ m filter.Pure GIPF prepared product is stored in-80 ℃.

With the protein yield that ELISA, protein B radford measure and HPLC analyzes and quantitative each purification step end obtains.Each step in purge process is judged the proteic response rate of GIPFt, and is shown in the following table 2.

Table 2

Step	The step response rate	Overall recovery
			Culture medium concentrates/diafiltration	100％	100％
The Ni-sequestration is affine	65％	65％
			The SP cation exchange	80％	52％
Last preparation and filtration	95％	49％

Under reduction and non-reduced condition, carry out the proteic SDS-PAGE of the GIPF of purification and analyze, and the demonstration V5-His that is derived from CHO and the 293 cells GIPF albumen that tags all exists with monomer.GIPF albumen be glycosylated and with molecular weight (MW) approximately 42kDa moving on the SDS-PAGE under the non-reduced condition.From the GIPF albumen of Chinese hamster ovary celI purification with at molecular weight minute differences is arranged from the HEK293 cell purification.This difference can be explained by glycosylated degree with GIPF in the different cell types.The analysis of N-terminal sequence shows that the HEK293 cell produces two kinds of form peptides: advantage adult form (dominantmature form) (SEQ ID NO:10), it is equivalent to lack the GIPF albumen of the SEQ ID NO:4 of signal sequence, and adult form (SEQ ID NO:12), it is equivalent to not only lack signal peptide but also lack the GIPF albumen that furin cuts the SEQ ID NO:4 of sequence.On the SP post, two kinds of isolated in form are good, and with adult form about 1: 2 ratio of advantage adult form shown.

NaCl and arginine (Arg) are shown in Figure 17 A to the influence of GIPF protein solubility when judging pH 7.Determine and proteic 50% lose during when not having 0.3M Arg, causing purification.Figure 17 B shows the dissolubility of albumen in PBS (pH 7 for 20mM sodium phosphate, 0.15M NaCl) of purification.Concentration is up to 8mg/ml, and 4 ℃, during pH 7, GIPF albumen kept in solution 7 days.

In brief, carry out V5-His-label G IPF purification by 1 from HEK293 or Chinese hamster ovary celI culture) concentrate and dialysis is present in GIPF albumen the culture medium, 2) carry out Ni ²⁺-sequestration affinity chromatograph and 3) SP cation-exchange chromatography.Purification process is produced the GIPF albumen of＞90% purity.The overall recovery of purification process approximately is 50% at present.It is important adding in buffer in the purge process of culture medium dialysis and Ni-post that 0.5M NaCl GIPF when keeping pH 7 dissolves fully.For GIPF being attached on the SP post, remove NaCl, add 0.3M Arg to keep high dissolubility and to improve protein recovery.Adding 0.5M NaCl and 0.3M Arg show overall recovery and increase to 50% from 25% at least in first and second purification steps.

The condition of describing as embodiment 10 is used for the biologic activity of GIPF in the test body with the tag proteic advantage mature form of GIPF and mature form of V5-His.Induce the obvious hypertrophy of small intestinal crypts epithelial cell consistently by the albumen of present embodiment method purification, this means the little enterectasis of the mice of accepting purification GIPF protein medicine-feeding.

B. the expression of GIPFwt and purification in eukaryotic cell:

With with being used to of describing the tag mode of GIPF protein similar express, wild type GIPF albumen (GIPFwt untagged with purification; SEQ ID NO:4).The stabilized cell culture of HEK293 cell is adapted to suspension growth and is grown in the serum-free 293free-style culture medium (GIBCO) of 25 μ g/ml Geneticins, and wherein said HEK293 cell has been used the pcDNA/Intron carrier transfection of the DNA that contains coding total length GIPF polypeptide (GIPFwt) (SEQ ID NO:4).

Cell culture growth in rotator:, a cell cryopreservation thing is grown in the 293free-style culture medium of having added 0.5% hyclone (FBS) and increase for producing at the rotator middle and small scale.With cell cell density inoculation and amplification with per generation 0.3-0.5 1,000,000/milliliter in rotator.When cell of enough producing when accumulation and cell density reach 100 ten thousand/milliliter, culture medium and the exchange of serum-free 293free-style culture medium with removal 0.5%FBS, and were gathered in the crops after 6 days.The initiator cell vigor is at 80-90%, and it reduces to 30% when results.Measure the level that has been secreted into the GIPFwt in the culture medium with ELISA and Western trace.GIPFwt growth-gen productive rate in rotator is 1.2-1.5mg/ml.

To be used for the large-scale production of bioreactor at the cell culture growth-gen in the Bioreactors-Fed-batch pattern.When passage, with the cell density inoculation of the suspension culture of the adaptation serum-free of HEK293 cell with 0.2-0.4 1,000,000/milliliter.Cell is grown in the serum-free 293free-style culture medium, and is expanded to the 20-50 agitator for inoculation 2001 and 5001 bioreactors shake bottle from 50-500ml.When having accumulated enough cells, the density of cell with 0.2-0.4 1,000,000 cells/ml is inoculated in the bioreactor.When cell density reaches 100 ten thousand cells/ml, add vitamin and MEM aminoacid (GIBCO) to promote and to keep growth.After 6-7 days when cell viability is reduced to 25-30%, from the bioreactor harvesting.Measure the level that has been secreted into the GIPFwt in the culture medium with ELISA and Western trace.The Western of excretory GIPF analyzes and shows do not have protein degradation to take place.Anti--GIPF polyclonal antibody that uses purification that Western analyzes, through the chicken of using purification of the Protein Detection of ELISA anti--the GIPF polyclonal antibody is as trapping antibody, rabbit is anti--the GIPF polyclonal antibody is as detecting antibody.Prepare rabbit and chicken polyclonal antibody at intact proteins.GIPFwt growth-gen output in bioreactor is 2.6-3mg/ml.

Ultrafiltration-diafiltration: centrifugal results contain the proteic culture medium of excretory GIPFwt.Add protease inhibitor, (Roche, Basel is Switzerland) to prevent the GIPF degraded for 1mM EDTA and 0.2mM Pefabloc.Culture medium is filtered by 0.22 μ m PES filter (Corning), and with TFF system (PallFiltron) or concentrated 10 times of the doughnut system (Spectrum) of 10kDa molecular weight mwco membrane is arranged.With spissated culture medium buffer and 20mM sodium phosphate, 0.3M Arg, pH 7 exchanges.In the purge process, it is important adding 0.3M Arg GIPFwt when keeping pH 7 solvable fully in phosphate buffer.After ultrafiltration and diafiltration, 1: 500 (v/v) adds mammalian protease inhibitor mixed thing (Sigma) with dilution factor.

Q anion-exchange chromatography: with 20mM sodium phosphate (NaP) the buffer balance that contain 0.3M Arg of anion exchange Q agarose gel HP post (Amersham) with pH 7.And culture medium that buffer-exchanged cross spissated with 10 times filters and is loaded into 0.22 μ m PES filter on the Q agarose gel post with in conjunction with impurity and nucleic acid.

The SP cation-exchange chromatography: collection contains the Q-agarose gel effluent of GIPFwt and is loaded in conjunction with on the proteic cation exchange SP agarose gel of the GIPF HP (Amersham).With the 20mM NaP of 15 times of column volumes (CV), 0.3M Arg, 0.1M NaCl, pH 7 washing SP agarose gel posts, and with above gradient 0.1M to the 0.7M NaCl of 40 times of column volumes with the GIPF eluting.With SDS-PAGE and Western engram analysis fraction.To contain fraction analysis and the mixing of GIPFwt.With blended fraction buffer and 20mM sodium phosphate, pH 7,0.15M NaCl exchange.When using the Coomassie brilliant blue staining analysis of sds gel, judge that the purity of purifying protein is 92-95%.Albumen is concentrated into 1mg/ml, filters and be stored in-80 ℃ by aseptic 0.22 μ m filter.

Measure the protein yield that each step end of quantitative purge process obtains with ELISA and Bradford, calculate the proteic response rate of GIPF, and be shown in the table 3.

Table 3

Step	The step response rate	Overall recovery
			Culture medium concentrates/diafiltration	100％	100％
Q anion exchange	95％	95％
			The SP cation exchange	75％	71％
Last preparation and filtration	98％	70% 48% (only advantage adult forms)

Measure the level of endotoxin that test kit (Charles River) is analyzed the GIPF protein solution of final preparation with color development LAL (Limulus Amebocyte Lysate), and be judged to be every milligram of GIPF of 0.24EU.

C. the expression of GIPFt and purification in yeast:

From yeast culture, express and purification GIPFt, and contrast the biologic activity of itself and above-described GIPFt from HEK293 cell culture purification.

The nucleotide sequence of GIPFt (SEQ ID NO:5) of will encoding is cloned among the Pichia expression vector pPICZ α A that contains yeast α-factor secretory signal sequence.Pichia Pastoris wild type X-33 strain is used for expressing GIPFt.Use for the Pichia carrier, the method of Recombinant Protein Expression and purification can be from Invitrogen Life Technologies (Carlsbad, CA, USA) obtain, also be described in " PichiaProtocols:Methods in Molecular Biology " (D.R.Higgins and J.Creggeds., TheHumana Press, Totowa, NJ1998)).

In brief, the purification of GIPFt uses the SP cation-exchange chromatography, subsequently at IMAC Ni ²⁺Affinity chromatograph on the post.With Ni ²⁺Post is with the washing of 20mM imidazoles, and with the gradient 20-300mM imidazoles eluting GIPF above 30 times of column volumes.The SDS-PAGE of purified product represents the wide of about 50kDa and the protein band of disperse, shows that GIPFt is by glycosylation in various degree.The interior biologic activity analysis of external and body is described as embodiment 17 and 20 respectively.

The protein induced mouse small intestine epithelial proliferation of the GIPFt that expresses in Pichia Pastoris, it is white to stablize beta-catenin, though the difference a little (result does not show) that its degree obtains than the GIPF albumen with the HEK293 cell purification.

The mice of D.GIPF is directly to the purification of congener-mGIPFt:

The mice of the human GIPF of coding directly is cloned into the pcDNA/Intron carrier to express the albumen mGIPFt of V5-His label to the SEQ of congener ID NO:68.With the murine protein of label at the HEK293 cellular expression, and according to the proteic method purification of above-described purifying human GIPFt.Protein purification is become 80% purity, and in PBS, prepare preparation.Analyze in the mGIPFt body and the extracorporeal biology activity, show to have the hypertrophy characteristic same (embodiment 20) with human GIPF.

E. the feature of the reorganization GIPF of purification

The SDS-PAGE that carries out the GIPF albumen (GIPFt and GIPFwt) of purification under reduction and non-reduced condition analyzes, and shows that the GIPF albumen of the V5-His label that is derived from 293 cells exists with monomer.GIPFt and GIPFwt albumen are glycosylated, and the molecular weight (MW) with about 42kDa and 38kDa moves on SDS-PAGE respectively under non-reduced condition.Substance assistant laser desorpted/MALDI-MS (MALDI) shows that it is 37.8kDa and 32.9kDa that GIPFt and GIPFwt divide other molecular weight, and is respectively 30.2kDa and 26.8kDa for GIPFt that lacks signal peptide and GIPFwt molecular weight.The difference of molecular weight shows may be because proteinic glycosylation.Subsequently, according to manufacturer's explanation, (CA USA) carries out N-and connects the complete deglycosylation that is connected oligosaccharide with O-for Prozyme, San Leandro to use N-and O-dextranase.The proteic SDS-PAGE of deglycosylation analyzes and causes molecular weight obviously to reduce 4-5kDa.When the mensuration in vitro and in vivo described according to embodiment 17 and 20 respectively, deglycosylation does not influence the biologic activity of GIPF.

Protein stability-, GIPF was boiled 5 minutes for the activity of GIPF after the mensuration degeneration, and in cooling rapidly on ice.(seeing embodiment 21) judges that GIPF has kept whole activity in external (seeing embodiment 17) and the body.These find that explanation GIPF is a kind of stable albumen.

The reduction of the capping of cysteine residues-carry out cysteine residues and alkylation are with the activity of cancellation GIPF.At 30mM DTT, among the pH 8 37 ℃ of insulations of GIPF 1h is obtained the disulfide bond reduction of GIPF (1mg/ml).Subsequently, in 37 ℃ of dark, keep 30 minutes with free sulfydryl S-carboxymethylation reaction (Crestfield AM with the 20mM iodoacetamide; Moore S; Stein W H.J.Biol.Chem.1963; 238,622).Freezing cessation reaction is removed excessive DTT and iodoacetamide with the PBS dialysis.Both with the external biologic activity that also adds the GIPF behind the medicated cap with determination and analysis in the body.The biologic activity of GIPF is eliminated (seeing embodiment 17 and 20) by capping.

The analysis of N-terminal sequence shows two kinds of form polypeptide of HEK293 cell generation or GIPFt or GIPFwt: advantage adult form (SEQ ID NO:10), it is equivalent to lack the GIPF albumen of the SEQ ID NO:4 of signal sequence, and adult form (SEQ ID NO:12), it is equivalent to not only lack signal peptide but also lack the GIPF albumen that furin cuts the SEQ ID NO:4 of sequence.On the SP post, two kinds of isolated in form are good, and with the adult form ratio expression about 1: 2 to the advantage adult form.Since all induce small intestinal crypts epithelial proliferation in advantage adult form and the adult form GIPFt body, with the advantage adult form for use in testing for embodiment 11,12, the therapeutical effect of GIPF in 13 and 14 disease animal models of describing.Chinese hamster ovary celI is only expressed the advantage adult form of GIPF.

Furin broken site (Arg ²⁸The mutagenesis of-＞Gln)-produce through the furin natural process for showing from the adult form GIPF of HEK293 cells produce, with the conserved sequence sudden change of furin broken site with the Gln that applies ²⁸-QRR replaces Arg ²⁸-QRR.With mutain (SEQ IDNO:18) at the HEK293 cellular expression and according to the method that is used for the GIPFt purification (embodiment 9A) purification.The N-end sequence of purifying protein confirms only to express the advantage adult form in culture.This discovery confirms that the generation of adult form is the result of the active Proteolytic enzyme cutting of furin in cell.In addition, the overall recovery of the advantage adult form of purification increases to 68% from 50%.

In brief, purge process produces＞90% pure GIPFt albumen and the pure GIPFwt of 92-95%.Using the overall recovery of the GIPF advantage adult form of any purge process approximately is 50%.But, have the albumen of the furin broken site of sudden change can increase output by expression.In the purge process of culture medium diafiltration and Ni post, the complete solvable of GIPF is important when buffer adds 0.5M NaCl for maintenance pH 7.For GIPF is incorporated into the SP post, NaCl is removed, add 0.3M Arg to keep highly dissoluble and to increase protein recovery.

The biologic activity that the GIPFt and the GIPFwt of advantage adult form and adult form is used for GIPF in the test body.Induce the epithelial obvious hypertrophy of little intestinal crypt consistently by the albumen of present embodiment method purification, this is the reason of little enterectasis that causes accepting the mice of purification GIPF protein medicine-feeding.

The influence that the biologic activity of GIPF is not subjected to glycosylation or boils, but eliminated by iodoacetamide capping cysteine residues.

Embodiment 10

Test biology in the proteic body of reorganization GIPF of HEK293 and expressing cho cell

Following in the normal mouse inner evaluation is derived from the proteic body of GIPFt of HEK293 and Chinese hamster ovary celI biological action.

Judge the pharmacokinetics (PK) of the albumen (GIPFt) of reorganization GIPF V5His6-label in the mice.With age in 6-8 week BALB/c mouse through the injection of tail vein single dose or 40mg/KG GIPFt albumen or preparation buffer in contrast.0,30 minute, 1 hour, 3 hours, 6 hours and blood-letting in 24 hours after injection, and with anti-V5 antibody (Invitrogene Inc., Carlsbad CA) analyze each time point serum albumin level (Figure 18 A) through Western.Figure 18 A demonstration does not detect the proteic obvious degradation of serum GIPF.The calculating of GIPF albumen half-life is by (InvitrogeneInc., Carlsbad CA) as the semilog plot of protein concentration after the injection of standard V5 label protein, and are estimated as 5.3 hours (Figure 18 B) with Positope in the serum.

Can produce similar in appearance to knocking in mice at the GIPF gene and for whether studying the reorganization GIPFt albumen of purification with observed phenotype on the mice of recombinant adenovirus injection, with age in 6-8 week BALB/c mouse once a day in contrast through tail vein injection or 4mg/KG GIPFt albumen or preparation buffer, totally 7 days.At the 8th day, last was injected and mice was put to death in back 24 hours.Put to death preceding 4 hours, peritoneal injection 1mg bromodeoxyribouridine (BrdU) is to judge the proliferation activity of GIPF in the body.Collection comprises the various tissues of small intestinal, colon, spleen, liver and bone marrow, and is fixed in the formalin.With paraffin-embedded section h and E (H﹠amp; E) Histological evaluation is carried out in dyeing.(explanation U.S.A.) and in the past described (McKinley, J.N.et al.2000) is also handled section to be used for the immunohistochemistry of BrdU for OncogeneResearch product, Boston according to the manufacturer.In all experiments, analyze 3 animals at least for every group, and experiment repeats at least 2 times.

The H﹠amp of gastrointestinal section; The E demonstration of dyeing is observed small intestinal and the obvious hypertrophy of the medium and small intestinal crypt epithelial cell of colon (seeing Figure 19 and 21 respectively) the mice of accepting GIPF.This result is consistent with the result who knocks in mice at transgenic GIPF gene and accept to obtain in GIPF adenovirus (embodiment above the seeing) Mus.Measure also by both measuring small intestinal (Figure 20) that mixing of BrdU confirms the proteic proliferative effect of GIPF in the colon (Figure 22).

Embodiment 11

GIPF is to the preventive effect of radiation-induced mucositis

Test GIPF is as the effect of prevention and medicine on radiation-induced mucositis animal model.

Use 48 bull BDF1 mices, age 10-12 week.Before supplier's deliver goods and experiment, animal is raised 2 weeks in airy cage separately, and 12 hour daytime: circulate with the stabilate rhythm and pace of moving things night.Allow the water inlet of animal ad libitum access.

Animal is divided into 8 groups, every group of 6 animals, following processing:

1. be exposed to preceding 72,48 and 24 hours of 13 gray(Gy) X-lines (whole body) with 2mg/kg GIPF intravenous injection;

2. be exposed to preceding 72,48 and 24 hours of 13 gray(Gy) X-lines (whole body) with 5mg/kg GIPF intravenous injection;

3. be exposed to preceding 72,48 and 24 hours of 13 gray(Gy) X-lines (whole body) with 125 μ g KGF intravenous injections;

4. be exposed to 13 gray(Gy) X-lines (whole body) and used the saline vehicle intravenous injection in preceding 72,48 and 24 hours;

5. be untreated non-radiative contrast;

6. be exposed to 13 gray(Gy) X-lines (whole body) back 24,48 and 72 hours with 2mg/kg GIPF intravenous injection;

6. be exposed to 13 gray(Gy) X-lines (whole body) back 24,48 and 72 hours with 5mg/kg GIPF intravenous injection;

6. be exposed to 13 gray(Gy) X-lines (whole body) back and used saline IV in 24,48 and 72 hours.

All 15:00 points that are injected at give.Induce the small intestinal infringement in the 15:00 point with single dose 13 gray(Gy) x-ray radiations (with 0.7 gray(Gy)/minute give).

After the radiation 4 days with sacrifice of animal.Take off small intestinal and before carrying out fabric analysis, be fixed in Carnoy ' the s fixative.Be cut into the thick cross-section section of 3 μ m, dye with h and E.Also get duodenum after the execution immediately, middle colon, liver, lung, tongue, spleen, stomach, and pancreas, and in formalin saline, fixedly spent the night before in being stored in 70% ethanol.

Therefore being equivalent to the given length of small intestinal for 10 small intestinal circumference of every animal analysis (circumferences) (60 every group)-circumference, is length baseline unit easily.Count the crypts number of each circumference survival and judge every group meansigma methods (table 4).Only counting contains 10 or how strong H﹠amp; The crypts of E staining cell (except Pan Shi (Paneth) cell) and do not contain the full circumferences of aggregate nodules.

In order to correct the error of calculation, also measure average crypt width (measuring its wideest point) because of the crypts difference in size.The following correction:

All animals are handled the back survival and apparent side effect do not occurred.Figure 23 A-D shows the section of following treated animal small intestinal: untreated fish group 5 (A), saline pretreated group 4 (B), KGF-processed group 3 (C) and GIPF-processed group 2 (D).The kitchen range (the survival crypts that one or more clone's cellulations are arranged) high-visible (Figure 23 B) of regenerating in the tissue slice of saline treatment treated animal (group 4).Except these kitchen ranges, mesenchyme exposes fully, and if existence is more than 4 days after allowing these animal radiation, and they develop into diarrhoea and die from mucositis.GIPF (Figure 23 D) plays protection to the small intestinal structure with the same way as with KGF (Figure 23 C).

Table 4

Experimental group	Crypts number/circumference (mean ± standard deviation)	Crypt width (μ m) (mean ± standard deviation)	Proofread and correct crypts number/circumference (mean ± standard deviation)
				1.	12.96±4.9	51.98±5.4	7.4±3.3
2.	15.4±4.9	45.03±2.8	10.1±3.3
				3.	22.1±3.8	54.46±2.1	12.0±1.2
4.	7.2±2.6	55.27±6.4	3.8±1.2
				5.	109.1±5.3	29.56±3.0
6.	10.7±4.5	57.16±3.9	5.5±2.3
				7.	10.6±4.1	55.50±4.7	5.6±2.1
8.	9.4±1.9	56.85±6.9	5.0±1.4

It is quite tangible increasing the crypts number of surviving after the 13 gray(Gy) radiation greatly with the GIPF pretreatment.GIPF pretreatment (the 1st group) with 2mg/kg dosage increases by 1.95 times (being also referred to as the protection factor) than untreated the 4th group of survival, and further increases by 2.66 times of crypt survival with the GIPF (the 2nd group) of 5mg/kg dosage.This is very good and almost catch up with known optimal dose KGF (the 3rd group) and handle viewed effect, and it increases by 3.16 times of crypt survival.

Therefore, show the influence of not raying of GIPF protection intestinal epithelial cell damage, and can be used as effective preventive drug of arranging to do radiation therapy subject.

Embodiment 12

The inductive mucositis model of chemotherapy

On healthy and tumor-bearing mice, estimate the efficient of the inductive mucositis of recombined human GIPF (GIPFwt) treatment chemotherapy.Experimental technique is previously described based on (Cancer Res 61:687-693 (2001)) such as Boushey.

(VA USA) is subcutaneously injected into the female BALB/c mouse of homology for ATCC, Manassas, allows tumor growth 5 days with 100 ten thousand CT26 mouse colon cancer cells.The animal with the lotus tumor of health is divided into experimental group, 6 every group, and carry out following processing:

1. mice with tumor, the peritoneal injection carrier (50%DMSO) from the 1st day to the 5th day, intravenous injection saline (TVS) from the 0th day to the 7th day;

2. mice with tumor, the peritoneal injection carrier (50%DMSO) from the 1st day to the 5th day, intravenous injection every day was dissolved in the brinish 50 μ g GIPF (TVG) of 100 μ l from the 0th day to the 7th day;

3. mice with tumor, the peritoneal injection 50mg/kg 5-FU from the 1st day to the 5th day, intravenous injection saline (TDS) from the 0th day to the 7th day;

4. mice with tumor, the peritoneal injection 50mg/kg 5-FU from the 1st day to the 5th day, intravenous injection every day was dissolved in the brinish 50 μ g GIPF (TDG) of 100 μ l from the 0th day to the 7th day;

5. healthy Mus, the peritoneal injection 50mg/kg 5-FU from the 1st day to the 5th day, intravenous injection saline (NDS) from the 0th day to the 7th day;

6. healthy Mus, the peritoneal injection 50mg/kg 5-FU from the 1st day to the 5th day, intravenous injection every day was dissolved in the brinish 50 μ g GIPF (NDG) of 100 μ l from the 0th day to the 7th day.

In the weight of animals of record measurement in the 0th, 2,4,6 and 8 day, the size of diarrheal severity and tumor.The reflection of diarrheal 0-3 level is normally to be serious corresponding serious symptom to 3 from 0.The change of body weight is calculated to be the percentage ratio with the untreated fish group body weight.With the length and width and the height of caliper measurement tumor, gross tumor volume is calculated to be (length * wide * height)/2.

With all animals euthanasia the 8th day the time.Downcut big and small intestinal and weighing, measure their length, and the diameter of jejunum in writing down.Cutting-out is downcut apart from one section (1cm) transverse colon of returning the about 4cm of blind connection apart from the about 14-15cm of pylorus one section (1cm) middle jejunum.Intestinal segment is also fixing to carry out histologic analysis with 10% neutral buffered formalin perfusion.With ImagePro software (Imagepro, Ltd., Ashford, Middlesex UK) carries out the histological examination and the morphometry of mucosa on tissue slice.

GIPFwt is summarized as follows the influence of diarrheal seriousness, body weight and tumor size:

Diarrhoea scoring (mean ± standard deviation) body weight (% untreated fish group)

The 3rd group of TDS 2.83 ± the 0.41st 74.7 ± 5.2

The 4th group of TDG 0.33 ± the 0.52nd 85.1 ± 5.7

The 5th group of NDS 3 ± 0 73.2 ± 4.3

The 6th group of NDG 0.5 ± the 0.55th 87.0 ± 6.0

Gross tumor volume (mean ± standard deviation; Mm ³):

The 1st group of TVS 95.8 ± 8.1

The 2nd group of TVG 95.1 ± 4.2

The 3rd group of TDS 21.8 ± 3.0; P＜0.05

The 4th group of TDG 16.7 ± 8.6; P＜0.05

When with the 5th comparing with the scoring of mice with tumor of not accepting GIPF with 3 groups normal, GIPF significantly reduces the 6th and 4 group health and mice with tumor and causes diarrheal seriousness by 5-FU.Similarly, GIPF alleviates the body weight reduction of being handled the animal experience by 5-FU.

The tumor of untreated mice with tumor (the 1st group) is similar with the tumor size of the mice with tumor (the 2nd group) that GIPF handles.Therefore, GIPF does not influence tumor growth.According to expectation, 5-FU reduce the gross tumor volume of the 3rd group of mice, and also reduce the gross tumor volume of the 4th group of mice.Therefore, GIPF does not suppress the activity (Figure 24) that 5-FU reduces gross tumor volume.

The influence that GIPF shows substantially to small intestinal as shown in figure 25, corresponding small intestinal diameter, weight and the length of measuring is listed in table 5.Accept the little intestinal atrophy (Figure 25 E) of the normal and tumor-bearing mice of 5-FU, and observe a large amount of and hemorrhage relevant infringement, and it is obviously normal to have accepted the mouse small intestine outward appearance of GIPF, and with because typical case's expansion (Figure 25 B that GIPF causes the proliferative effect of small intestine epithelium, C, D, and F).

Table 5

The CT26 mice with tumor	The 1st group	The 2nd group	The 3rd group	The 4th group
					Middle jejunum diameter (mm)	2.66±0.15	3.65±0.21 *	2.53±0.15	3.58±0.14#
Weight small intestinal (g) large intestine (g)	1.16±0.09 0.32±0.02	1.47±0.14 * 0.38±0.02 *	0.90±0.01 0.25±0.03	1.51±0.13# 0.39±0.02#
					Length small intestinal (cm) large intestine (cm)	35.2±2.0 8.7±0.3	40.5±1.0 * 9.7±0.3 *	30.2±1.3 6.3±0.3	39.7±2.1# 9.5±0.5#
Normal mouse	The 5th group	The 6th group
					Middle jejunum diameter (mm)	2.38±0.13	3.44±0.13 **
Weight small intestinal (g) large intestine (g)	0.88±0.08 0.25±0.02	1.43±0.13 ** 0.38±0.04 **
					Length small intestinal (cm) large intestine (cm)	30.3±1.9 6.9±0.9	39.3±1.0 ** 9.7±1.0 **

^*P＜0.05 (ANOVA, the 2nd group to the 1st group)

#p＜0.05 (ANOVA, the 4th group to the 3rd group)

^*P＜0.05 (ANOVA, the 6th group to the 4th group)

Demonstration is analysed in the intestinal biopsy tissues credit of all experimental group small intestinals and colon, causes intestinal mucosa fine hair and crypts considerable damage at interval by preventing 5-FU, and GIPF has protected the intestinal structure (Figure 26) of accepting the 5-FU mice.Figure 26 A shows the influence of 5-FU to small intestinal, and Figure 26 B shows the influence of 5-FU to colon.The microscopic morphology of the hollow enteral height of naps and the crypts degree of depth is measured the effect remarkable (Figure 27) of confirming GIPF.

GIPF protection small intestinal and colon are avoided the destruction of 5-FU, and it does not hinder the therapeutical effect of 5-FU.Therefore, GIPF can be used for the uniting of chemotherapeutics to alleviate the destructive side effect of antineoplaston.

Embodiment 13 GIPF are to the preventive effect of chemotherapy and radiation-induced oral mucositis

With the mice study GIPF that accepts the irradiation of X-line or accept 5-FU that describes as embodiment 11 and 12 respectively to back side (cheek side) and the epitheliogenetic influence of veutro.

According to the former method (Scholzen that describes of manufacturer's explanation, T.et al.2000), use monoclonal rat anti-mouse Ki67 antigen (Dako Ltd., High Wycombe, UK) on the tongue specimens paraffin embedding slices of the mice of not shining and shining (among the embodiment 11 the 1st, 2 and 3 group), carry out immunohistochemistry.

When not giving the comparing of GIPF animal, GIPF obviously increases the painted cell nuclei of Ki67 (Figure 28 and 29) in irradiation animal veutro and the dorsal part tongue epithelium.The epithelial proliferation index, it is calculated as Ki67 stained positive veutro epithelial cell percentage ratio, confirms that GIPF significantly reduces the cell loss (Figure 30) by radiation-induced veutro tongue epithelium.The histologic analysis of handling the tongue section of animal (embodiment 12 2-6 group) with 5-FU shows that GIPF has kept the form (Figure 31) of normal and tumor animal dorsal part and the veutro epithelial layer handled with 5-FU.

All are subjected to the infringement of 5-FU obviously to be less than the epithelial layer of not accepting the GIPF laboratory animal with the tongue epithelial layer of GIPF processing animal.

Therefore, can be with GIPF as the medicine that treats and/or prevents chemotherapy and the inductive oral mucositis of radiotherapy.When uniting when giving with other cell toxicant material, can be with the quantitative animal model of oral mucositis (Wardly etal. for example, Arch Oral Biol 43:567-577 (1998); Potten etal., Cell Prolif 35:32-47 (2002)) further studies the treatment characteristic of GIPF, reduce the latent effect that cell is eliminated the seriousness of (deletion) and increased the epithelial layer regeneration rate of oral cavity and enteric epithelium with further evaluation GIPF.

Embodiment 14

GIPF is to the therapeutical effect of the inductive colitis of dextran sodium sulfate

Induce the effect of test recombined human GIPF (GIPFwt) treatment colitis on the mouse model of colitis at dextran sodium sulfate (DSS), and with the effect of GLP-2 (L ' Heureux andBrubaker J Pharmacol Exp Ther 306:347-354 (2003) relatively; Kriegelstein et al., J ClinInvest 110:1773-1782 (2002); Siegmund et al., J Pharmacol Exp Ther296:99-105 (2001)).

With age in 6-8 week female BALB/c mouse (MA USA) raises in airy cage, adapts to 12 hour daytime of 1 week: the circulation at night for Charles River Laboratories, Wilmington.Have only the mice of similar body weight (approximately 20g,＜5% difference) to raise in 4 cages and ad libitum access 4%DSS (v/w) drinkable solutions 7 days with 24.

In the 7th day, write down the body weight of every animal, shown in following table 6, judge the scoring of weight loss, feces denseness and archorrhagia.

Table 6

Scoring	Weight loss (%)	The feces denseness	The visible hemorrhage of rectum of recessiveness/naked eyes
				0	Do not have	Normally	Normally
1	0-5％
				2	5-10％	Loose	Occult blood
3	10-20％
				4	＞20％	Diarrhoea	Naked eyes as seen

Scoring is used to calculate IBD activity index (IBDAI), and this index is used as the indication of colitis seriousness, and is calculated as the scoring average that provides list parameter.Judge weight loss every day, the scoring of feces denseness and hemorrhage of rectum, and write down IBDAI every day at experimental session.

At the 7th day, replace 4% (v/w) DSS drinkable solutions not increase the weight of the effect of DSS to keep the disease activity state with 1% (v/w) DSS solution.Select 16 feed DSS animals of consistent and suitable morbid state, and be divided into 4 one group and carry out following processing:

1. water, every day, (point in the mornings 10) intravenous injection saline was 7 days

2.DSS (1%) 7 day, every day, (point in the mornings 10) intravenous injection saline was 7 days

3.DSS (1%) 7 day, (point in the mornings 10) intravenous injection every day 100 μ g GIPF 7 days

4.DSS (1%) 7 day, (point in the mornings 10) intravenous injection every day 50 μ g GIPF 7 days

5.DSS (1%) 7 day, every day, twice (at 10 in the morning and 6 pm) subcutaneous injection 10 μ g was GLP-27 days.

The GIPF albumen that are used for these experiments are people GIPF albumen (the SEQ ID NO:4 that recombinate; GIPFwt), it is expressed and purification according to the method for describing among the embodiment 9.The analog of GLP-2, h[Gly ²] GLP-2 be synthetic and available from Biosource International (Camarillo, CA, USA).

At the 14th day, from cage, take food away with the emptying of permission intestinal, and all animal necks dislocations are put to death.All animals are at execution injection in preceding 2 hours 4mg/0.1ml BrdU.Downcut big and small intestinal and weighing, measure their length, and the diameter of jejunum in writing down.Cutting-out is downcut apart from one section (1cm) transverse colon of returning the about 4cm of blind connection apart from the about 14-15cm of pylorus one section (1cm) middle jejunum.Intestinal segment is also fixing to carry out histologic analysis with 10% neutral buffered formalin perfusion.With ImagePro software (Imagepro, Ltd., Ashford, Middlesex UK) carries out the histological examination and the morphometry of mucosa on tissue slice.The IBDAIs of experimental group 2-5 mice shown in figure 32, corresponding weight loss, feces denseness and hemorrhage of rectum scoring are respectively shown in Figure 33,34 and 35.These data show that GIPF plays therapeutical effect by the seriousness that alleviated colitis as far back as the 11st day.By the 14th day, the 3rd and 4 group of mice that GIPF handles had obviously low IBDAI (being respectively 1.75 and 1.83) than untreated the 2nd group of mice (3.75).GLP-2 plays medium effect to colitis seriousness, to the 14th day it reduce the IBDAI to 3.25 that DSS handles mice.

1st, the example of 2,3 and 5 groups of mice intestinal and colon general pathology as shown in figure 36.Compare with matched group, accept the brinish animal of DSS and develop into usually and atrophy, hyperemia and the relevant serious colitis of suffering from diarrhoea.Small intestinal and the large intestine of the animal of handling with GIPF demonstrate some expansions, but obviously similar to matched group.These discoveries show can be with the effective medicine of GIPF as the treatment inflammatory bowel disease.As and if GLP-2 plays certain therapeutical effect, and the comparison of the little and large intestine of this group is light slightly according to the infringement of treated animal.Little and large intestine measurement reflects that the significance of change is shown in following table 7.

Table 7

	The 1st group	The 2nd group	The 3rd group	The 4th group	The 5th group
						Middle jejunum diameter (mm)	2.25±0.09	1.72±0.05 *	2.50±0.18#	2.23±0.10#	1.97±0.04 **
Weight small intestinal (g) large intestine (g)	0.94±0.06 0.26±0.02	0.78±0.05 * 0.18±0.01 *	0.91±0.09# 0.23±0.01#	0.89±0.05# 0.22±0.01#	0.88±0.07 0.18±0.01
						Length small intestinal (cm) large intestine (cm)	32.0±1.4 7.3±0.3	26.6±0.9 * 4.9±0.5 *	31.1±0.9# 6.6±0.5#	30.3±0.6# 6.4±0.3#	28.9±0.8 ** 5.4±0.6

^*P＜0.05 (ANOVA, the 2nd group to the 1st group)

#p＜0.05 (ANOVA, the 3rd or 4 group to the 2nd group)

^*P＜0.05 (ANOVA, the 5th group to the 2nd group)

The H﹠amp of specimens paraffin embedding slices; E dyeing shows that DSS causes that the massive inflammatory cells infiltrated of small intestinal and mucous membrane of colon and fine hair and crypts disintegrate (Figure 37) at interval.Consistent with above-described general pathology observation, GIPF reverses the effect that DSS causes, and rebuilds the structure of intestinal crypt and fine hair.With comparing of matched group, the crypts that GIPF handles animal enlarges.Though well below the level of GIPF, GLP-2 plays certain therapeutical effect (result does not show).The curative effect that the micromorphology of fine hair and crypts is measured (Figure 38) confirmation GIPF is reflected in by the obvious reconstruction of the height of naps of colitis heavy damage and the crypts degree of depth.The obvious hypertrophy of pit cell has been emphasized the reparation (Figure 39 and 40) of (underscore) GIPF to the mucosa structure.Crypts hypertrophy index, it is calculated as BrdU stained positive pit cell percentage ratio, handles mice at the DSS that accepts GIPFwt and handles mice (P＜0.5) (Figure 40) apparently higher than the DSS of pump pickle.

Embodiment 15

The therapeutical effect of big section intestinal excision back GIPF

Test GIPF strengthens the effect of the adaptation response of big section intestinal excision on the rat animal model of short bowel syndrome (SBS).Describe this animal model and be used to study intestinal nutrient substance effect (Scottet al.Am J PhysiolG911-G921 (1998); Helmrath et al., J Am Coll Surg 183:441-449 (1996)), here experimental technique is introduced with for referencial use.

Animal is divided into the excision group that 75% hollow enterochirurgia is excised, and intestinal cuts and identical again sham cut is removed operative control group and non-operative control group.Give the GIPF of animal saline or 2mg/kg.Select 75% intestinal excision so that the maximization of any adjustment reaction, and the near-end jejunum and the far-end ileum that keep equivalent are to remove terminal ileum to the specific absorption ability of vitamin B12 and cholic acid and the nutrition complication of ileum lock.Rat, reservation comprises that the animal that 25% small intestinal of ileum distal portions is enough to allow to excise reaches and control animal isometric growth speed.

Experimental session is estimated metabolism, morphology, histology and the functional response of digestive tract to excision and GIPF processing, and also at the 10th day as the end point analysis.Estimate food intake and growth as (the Scott et al., ibid) that describes, cardinal principle and the small intestinal morphology of microscopically and the functional evaluation of mucosa absorption characteristic.

GIPF obviously increase food consumption and alleviate small bowel resection often with weight loss.GIPF also increases length, diameter, weight in wet base and the mucosa weight in wet base of residue intestinal, and increases the absorbability of residue small intestinal.The cross section H﹠amp of small intestinal; E dyeing shows that GIPF had both prolonged height of naps and also prolonged the crypts degree of depth, and increases pit cell hypertrophy in the animal alimentary canal that excises small intestinal.Therefore, GIPF is by strengthening the effect that the intestinal adaptability alleviates the intestinal excision.

Embodiment 16

GIPF is to the influence of tumor cell proliferation

GIPF induces the epithelial strong proliferative effect of intestinal crypt in vivo.For studying external proliferative effect, in the influence of testing in vitro reorganization GIPFwt to kinds of tumors and normal cell system propagation.By measuring ³The H-thymus pyrimidine mixes the cell proliferation speed of measuring following cell line (ATCC):

Caco-2	The people ties rectal adenocarcinoma; Epithelium
		COLO205	The ascites of metastatic human knot rectal adenocarcinoma; Epithelium
HCC70	People's mammary gland gland duct carcinoma; Epithelium
		HCT116	People's colorectal cancer; Epithelium
HT-29	The people ties rectal adenocarcinoma; Epithelium
		IEC-18	Rat ileum; Epithelium
IEC-6	Rat small intestine; Epithelium
		LS513	People's caecum; Colorectal cancer; Epithelium
MCF7	The pleura of metastatic human adenocarcinoma of breast is sent out; Epithelium
		NCI-H1373	Human lung adenocarcinoma
PC-3	The bone of human prostate adenocarcinoma shifts; Epithelium
		SCC-25	People's squamous cell carcinoma of tongue
SCC-4	People's squamous cell carcinoma of tongue
		SK-BR-3	The pleura of metastatic human adenocarcinoma of colon is sent out; Epithelium
SK-MES-1	The pleura of metastatic human prognosis of squamous cell lung cancer is sent out; Epithelium
		SW620	The people ties the lymphatic metastasis of rectal adenocarcinoma; Epithelium
T84	The lung of people's colorectal cancer shifts; Epithelium
		293	People's tire kidney; Epithelium

According to cell line with cell with every hole 10,000-50,000 cell inoculation, and handle with the GIPFwt of dosage (1.37-1000ng/ml) in proportion.GIPF is handled the contrast of the rate of increase and the untreated cell of cell, or with the contrast that is grown in the cell in 10% complete medium (growth medium, the hyclone of 2.5% dialysis, and penicillin/streptomycin).With cell in 37 ℃ of insulations 48 hours, and in insulation in last 20-24 hour with 0.5 μ Ci ³The H-thymus pyrimidine impacts.Harvesting, judgement has been mixed ³The amount of H-thymus pyrimidine is from this judged result of parallel double increment of repeated experiments.

GIPF does not influence the rate of increase of most of tumor cells of test.At IEC28, T84, HCT116 and HT29 cell, have only the GIPF of higher dosage to induce the rate of increase to raise.The degree of propagation is lower than 40% of untreated cell speed.

Therefore, these discoveries show the growth rate of the tumor that GIPF exists in can acceleration bodies, and GIPF can be used for the treatment of the cancer patient who suffers from the mucositis that causes because of antineoplaston.

Embodiment 17

The influence of signal in the GIPF pair cell

Wnt/ beta-catenin white signal path plays pivotal role in growth and stable state.At small intestinal, known wnt signal plays a key effect by stablizing beta-catenin in vain as the outgrowth moderator of intestinal crypt, induces trans-activation (Wetering etal., the Cell 111:241-250 (2002) of T-cytokine (TCF) target gene after it; Batle et al., Cell, 111:251-263, (2002); Perreault et al., J Biol Chem 276:43328-43333 (2001); Booth et al., Nat Med8:1360-1361 (2002)).

For estimating the influence of GIPF, in the white stability of multiple cultured cells system's mensuration beta-catenin to wnt/ beta-catenin white signal path.With cell with 100 ten thousand cell inoculations in every hole in 6 orifice plates, with Eagle ' the s culture medium of Dulbecco ' the s improvement of having added 10%FBS.Second day, cell is grown in the serum-free medium, and with the GIPF of 50ng/ml or the LiCl of 10mM ₂(positive control) under low serum condition (0.1%FBS) handles.Preparation Cytoplasm part according to description such as Haertel-Weismann (J Biol Chem175:32046-32051 (2000)).Albumen is differentiated with gradient (4-20%) SDS-PAGE, and the white rabbit antibody (Abcam) of beta-catenin is used in the evaluation of the level that beta-catenin is white, with second antibody (Cell Signaling) demonstration of horseradish peroxidase.

In the cell line of test, GIPF relies on dosage and the time correlation mode is induced the L cell line (NCI-H716, the result does not show) and white stable (Figure 41 A and the B respectively) of the interior beta-catenin of HEK293 cell of human endocrine.Consistent with the discovery that embodiment 18 describes, the GIPF that boils does not influence the white ability of its stable beta-catenin, and still, this effect is handled by E.C. 3.4.21.64 and cancelled (Figure 41 C) by the DTT reduction.

For further research GIPF cause beta-catenin white pile up via signal path, analyze the activity of GSK3 β in the HEK293 cell.In the wnt of classics signal path, to activate beta-catenin white by suppressing GSK3 β for wnt, the other phosphorylation beta-catenin of GSK3 β white and with its labelling to be destroyed by albuminous body.

GIPF increases the phosphorylation (Figure 42) of GSK3 β in the HEK293 cell in time dependent mode.It is white that these data show GIPF can be passed through classical wnt signal path activation beta-catenin by the GSK3 phosphorylation in vain by the inhibition beta-catenin.But GIPF does not induce white the stablizing of beta-catenin in other cell line, comprises showing that wherein wnt3A is to inducing the white mouse epithelial cell line C57MG with useful effect that activates of beta-catenin.And, Dickkopf-1 (Dkk1), effective inhibitor of Wnt signal path (Kuhnert, PNAS 101:266-271,2004) can not suppress GIPF stable (result do not show) white to beta-catenin in 293 cells fully.It is white that these data show that GIPF may stablize beta-catenin by the approach that is different from the white path of known classical wnt/ beta-catenin.

Embodiment 18

GIPF is to the influence of the white expression of target gene of beta-catenin

The accumulation that beta-catenin is white causes its swivel base to nuclear, and it combines with the transcription factor of TCF/LEF family in nuclear.Because its trans-activation ability, beta-catenin is white-and transcription factor-complex is incorporated into DNA and activates the wnt target gene.For further studying the inductive beta-catenin white signal of GIPF-, we judge the activation of HEK293 and NCI-H716 cell middle and lower reaches target gene with quantitative PCR.

With 1 * 10 ⁶HEK-293 cell and 2 * 10 ⁶NCI-H716 cell (ATCC) is inoculated in the 6-orifice plate and allows and attach 6 hours in complete medium.Then cell is changed into 0.1%FBS and measure culture medium and incubated overnight.Measure the same day, measure culture medium with extra 1ml and add processing to cell.With cell 20mMLiCl (Sigma), 100ng/ml Wnt-3A (R﹠amp; D Systems), 250ng/ml GIPFt or 250ng/ml add the GIPF albumen of medicated cap in 37 ℃/5%CO2 insulation 8 hours.Comprise and maintain the cell of measuring the hole of being untreated in the culture medium background as gene expression.With RNeasy Mini test kit and DNaseI test kit (Qiagen) from the total RNA of two class cell separation, character and the concentration of quantitatively total RNA.To each sample, the total RNA of 4 μ g is guided 3 minutes with 3 μ g random hexamers and every kind of dNTP of 2mM at 70 ℃.To be reflected at cooled on ice 1 minute.With 5X M-MLV buffer (Promega), 25mM MgCl ₂, 0.1M DTT and RNaseOut (Invitrogen) add to 22 μ l with reaction volume.In case add 400 M-MLV of unit reverse transcriptases (Promega), will be reflected at 23 ℃ the insulation 10 minutes, 42 ℃ 50 minutes, and 70 ℃ 5 minutes with cessation reaction.Then with cDNA dilution and with RNaseH (Invitrogen) processing of 1 unit to digest remaining RNA.The quantitative cDNA concentration of OD260nm.To the quantitative SYBR Green of per 10 μ l PCR reaction, use forward and the reverse primer of 2 μ l cDNA (440ng) and every kind 1.25 μ M, and 2X SYBR Green mastermix (Eurogentec).Reaction is repeated three times.Design the quantification PCR primer of the white target gene of following human beta-catenin: Axin-2 (SEQ ID NOs:70 and 71), CD44 (SEQ ID NOs 72 and 73), EpherinB2 (SEQ ID NOs:74 and 75), c-myc (SEQ ID NOs:76 and 77), glicentin former (SEQ ID NOs:78 and 79), and Cox-2 (SEQ ID NOs:80 and 81).People EF1 (SEQ ID NOs:82 and 83) is used as house-keeping gene with the normalized expression level.

GIPF increases the expression of Axin-2 in HEK293 and the NCI-H716 cell, and causes and transfer to the level that is higher than with due to wnt3A or the lithium stimulation on CD44 and the EphrinB2 far away.Cox-2, the expression of c-myc and glicentin protogene are not subjected to the influence (result does not show) of GIPF.

These data provide and have participated in the clue that GIPF induces the activated mechanism of target gene.Further study to illustrate the downstream events of GIPF signal.

Embodiment 19

The active external test of GIPF

With deletion mutant (SEQ ID NOs:84,86,88,90,92,94,96,98,100, the 102 and 104) sub-clone of 11 GIPF in the pIntron/IgK carrier, and in the HEK293 cell transient expression.The position of coded each polypeptide fragment in total length GIPF polypeptide (SEQ ID NOs:85,87,89,91,93,95,97,99,101,103,105 and 178) as shown in figure 43.(the chimeric intron of engineering WI) is with pSectag carrier (Invitrogene Inc., Carlsbad, CA) genetic modification acquisition mammalian expression vector pIntron/IgK for Promega, Madison to be derived from the pCI mammalian expression vector by importing.The pcDNA/Intron carrier is digested with BG1II and NheI, and intron sequences is cloned among the pSectag that uses BG1II and NheI digestion.Be used to increase and sub-clone is equivalent to the forward of polynucleotide passage of SEQ ID NOs:106-119 and reverse primer shown in sequence table.The forward primer of SEQ ID NO:106 and the reverse primer of fragment 1-7 are used (primer SEQ IDNOs:107-113) together; The forward primer of SEQ ID NO:114 and the reverse primer of fragment 8-10 (SEQ ID NOs:115-117) are used together.

The polypeptide fragment transient expression is stablized the white activity of beta-catenin in the HEK293 cell and as the mensuration fragment that embodiment 17 describes.Compare with the fragment of other test, the polypeptide fragment of SEQ ID NO:91 has induced the most stable beta-catenin white.This discovery shows that it may be essential to mediation GIPF proliferative activity that the furin sample of GIPF is rich in cysteine structure territory.But the activity of the polypeptide of SEQ ID NO:91 is lower than total length GIPF (Figure 44).Therefore, beta-catenin is at utmost stablized in vain becomes possibility, and the other parts of GIPF are essential.

Embodiment 20

Measure in the active quick body of reorganization GIPF

Develop the biological activity of measuring in the quick body with the GIPF that detects purification.The activity of test person and mice GIPF (GIPFwt and mGIPFt) on the mice of also following processing that divides into groups:

1. saline, intravenous injection

2.GIPFwt, 100 μ g intravenous injections

3.GIPFwt, 50 μ g intravenous injections

4.mGIPFt, 100 μ g intravenous injections

5.mGIPFt, 50 μ g intravenous injections

6.GIPFwt, boil 100 μ g intravenous injections

7.GIPFwt, add medicated cap, 100 μ g intravenous injections

8.GIPFwt, 100 μ g subcutaneous injections

Use 24 female BALB/c mouse in the experiment.1 injection every day GIPF totally 3 days.At the 4th day with sacrifice of animal.Before the execution, collect 0.5ml blood and carry out analysis of Hematology Changes, and in putting to death preceding 2 hours, with the 1mg/ml BrdU solution of all animal peritoneal injection 0.4ml.As described above small intestinal and colon are downcut and measure, and middle jejunum and colonic segment are carried out histologic analysis by what front embodiment described.

The result as shown in figure 45.The little enterectasis of accepting the proteic mice of mice GIPF is similar to acceptor GIPFwt's.In addition, accept the intestinal phenotype of mice of GIPFwt subcutaneous administration to accept GIPFwt intravenous route administration person similar.As mentioned previously, GIPFwt's boils the ability that it causes enterectasis (distension) that do not influence.These find the measurement consistent (table 8) with intestinal length, weight and the diameter of all animal groups.

Table 8

The animal grouping	Jejunum means standard deviation in the diameter (mm)	Weight (g) small intestinal means standard deviation	Weight (g) large intestine means standard deviation	Length (cm) small intestinal means standard deviation	Length (cm) large intestine means standard deviation
						1	2.31±0.15	0.90±0.04	0.24±0.03	29.5±1.5	6.7±0.3
2	3.64±0.14 *	1.34±0.10 *	0.33±0.02 *	36.0±1.3 *	8.5±0.5 *
						3	3.55±0.12 *	1.32±0.07 *	0.32±0.03 *	36.5±0.5 *	8.7±0.3 *
4	3.68±0.06 *	1.39±0.05 *	0.35±0.02 *	37.8±0.8 *	8.8±0.3 *
						5	3.45±0.09 *	1.23±0.04 *	0.33±0.01 *	36.5±0.5 *	8.5±0.5 *

6	3.07±0.12 *	1.25±0.06 *	0.34±0.01 *	35.5±0.5 *	8.3±0.3 *
						7	2.28±0.11	0.88±0.07	0.21±0.03	31.0±1.3	6.3±0.3
8	3.03±0.10 *	1.12±0.04 *	0.28±0.02 *	34.2±0.8 *	7.8±0.3 *

^*P＜0.05 (ANOVA, 1-8 organizes the 1st group)

The whole blood of animal is learned the result in normal range, therefore shows that GIPF does not produce any direct untoward reaction.GIPFwt and mGIPFt are presented at the crypts degree of depth that the back GIPF of treatment on the 3rd obviously increases healthy mice to the influence of the height of naps and the crypts degree of depth.The effect that increases infected animal small intestinal intestinal villus height with GIPF is opposite, and GIPF does not influence normally, the height of naps of healthy animal (Figure 46).

The recombinate proliferative effect of GIPF of these data show people is not the result of dystopy effect.In addition, intravenous still is that subcutaneous administration GIPF brings into play its biologic activity.

Embodiment 21

The activity of external mensuration GIPF on isolating pit cell

According to the method for (Fujimoto et al., Gastroenterology 117:858-865 (2002)) such as Fujimoteo, on isolating mice colon pit cell, measure the influence of GIPF to conduction of crypts epithelial cell signal and propagation.

Colon was sterilized 15 minutes from the mice cutting-out and with 0.04% liquor natrii hypochloritis.After the PBS flushing, colon is incubated 90 minutes in room temperature in DTT/EDTA solution (containing 0.5mM DTT, the PBS of 3mM EDTA).After the insulation, will be organized among the PBS washing once and add 10ml PBS.Pipe is firmly shaken to separate crypts down from mucosa.The PBS that will contain crypts transfers in the centrifuge tube and repeats concussion and reduce up to crypts output.Wash with crypts centrifugal gently (400 rev/mins, 5 minutes) and with fresh PBS.Crypts is resuspended among the PBS of 20ml 0.3% pancreatinum (Sigma), and, shook once in wherein preceding 30 minutes per 10 minutes, shook once in after this per 30 minutes in room temperature insulation 90 minutes.When insulation finishes, add equal-volume PBS and in 1000 rev/mins centrifugal 5 minutes, reuse EDTA/DTT solution washing 1-2 time is removed up to all mucosas.Pit cell is resuspended in (RPMI 1640, added 5%FCS, glutamine, sodium bicarbonate, insulin, transferrins, selenium, penicillin/streptomycin) in 1 * culture medium.With the 23G syringe needle cell mass is broken up then with the 21G syringe needle that is connected with syringe earlier.With cell counting and be used for following mensuration: the stability that beta-catenin is white, by ³The H-thymus pyrimidine mixes the judgement proliferation activity and is used to measure GIPF influences the ability that the pit cell clone forms.

A. on pit cell in the research body GIPF to the white active influence of beta-catenin, wherein from as mentioned above with 6 hours BALB/c mouse separation pit cell after the 100 μ g GIPFwt intravenous injections.

As shown in figure 47, when with from contrast Mus pit cell observed comparing, GIPFwt induces white obviously stable of beta-catenin in the isolating pit cell kytoplasm.Figure 47 A represents unphosphorylated active beta-catenin white level, and Figure 47 B shows the total beta-catenin white level that is present in the Cytoplasm.Use that (the unphosphorylated beta-catenin of the white antibody recognition of beta-catenin USA) is white for Waltham, MA available from Upstate, and use (Cambridge available from Abcam, MA, the level that the total beta-catenin of TPPA USA) is white, this antibody had both been discerned the unphosphorylated albumen of also identification of phosphorylation.This result shows that the inductive mice crypts of GIPF epithelial hyperplasia may be mediated by the beta-catenin white signal.

B. use ³The H-thymus pyrimidine mixes the influence of external test GIPFwt to isolating pit cell propagation.The result shows that GIPFwt albumen strengthens the propagation of isolating pit cell in dose-dependent mode.

Therefore, GIPF induces isolating pit cell propagation in vain by stablizing beta-catenin, and isolating enterocyte can be used to illustrate the signal path of supporting the GIPF proliferation function.

C. the clone who carries out (Whitehead et al.Gastroenterology, 117:858-865 (1999)) descriptions such as Whitehead forms the ability of mensuration with research GIPF control intestinal mucosa propagation and/or differentiation.In brief, 2 * RPMI culture medium mixed in equal amounts that will contain 1% agar and contain 10%FBS adds in the 35mm plate and forms bottom.With every milliliter of 50,000 cells isolating clone's pit cell is added to upper strata culture medium (0.8% agarose of equivalent and be added with the 2X RPMI culture medium of 10%FBS), every hole packing 2ml cell suspension.With plate have 50,100 and 200ng/ml GIPF situation under in 37 ℃ the insulation 3-4 weeks.After the insulation, inspection plate and counting clone number.The definition clone is the group more than 40 cells.

GIPF stimulates the clone to form.The clone is formed the proliferation activity that mensuration is used for testing in vitro GIPF and GIPF analog.

Embodiment 22

GIPF is to the influence of the inductive colitis of TNBS-

Hapten material 2,4,6-trinitro-benzene-sulfonic acid (TNBS) is induced chronic colitis, it is characterized in that the serious saturating wall inflammation relevant with diarrhoea, proctoptosis and weight loss.These clinical and histopathology characteristics show that the inductive colitis of TNBS is similar to the feature of people's Crohn disease (Neurath et al., JExp Med 182:1281-1290 (1995)).

The therapeutic effect of test GIPF on the mice of inducing colitis with TNBS.As (ibid) as described in the Neurath etc., give 1mg TNBS by the single rectum and on 6-8 week female BALB/c mouse in age (the 2nd group), induce intestinal inflammation.Control animals (the 1st group) is accepted the rectally of independent carrier (45% ethanol).By accept the subcutaneous once a day therapeutical effect that gives hGIPF (4mg/kg or 2mg/kg) the test hGIPF of dosage 100 μ g (the 3rd group) or 50 μ g (the 4th group) of 3 days animal of TNBS to.After 7 days mice is put to death, estimate TNBS inducing colitis.HGIPF obviously weakens TNBS inductive weight loss (Figure 48) on the 2nd treated animal.HGIPF also alleviates the seriousness (result does not show) of serious diarrhoea that the 2nd treated animal that TNBS handles suffers from, colonic ulcer, hemorrhage and atrophy.

Organize the H﹠amp of the specimens paraffin embedding slices of colon with matched group and TNBS; E dyeing evaluation of tissue is learned and is changed.HGIPF weakens saturating wall infiltration of the inductive mice mucous membrane of colon of TNBS and mucosa crypts structural deterioration (Figure 49).The effect of the hGIPF of classification judgement is organized in the block diagram representative of Figure 49 with following colitis:

The histology of colitis (micro-) classification
		Scoring	Standard
	0	Normally
1			Low-level (once in a while) leukocyte infiltration, non-structure changes
	2	Medium leukocyte infiltration is arranged, superficial epithelium damage, no ulcer in lamina pripria
3			The height leukocyte infiltration expands under the mucosa with inflammatory cell, and mucosa corrodes, local ulcer, and colon wall is medium to be thickened
	4	Very high leukocyte infiltration is with saturating wall inflammation, extensive mucosa injury, and goblet cell is lost, high vessel density, colon wall thickens, ulcer

In addition, hGIPF weakens in the inductive mice colon of TNBS the increase as the myeloperoxidase (MPO) of neutrophilic granulocyte sign.With respect to the animal of not handling with GIPF, GIPF handles and obviously alleviates the inductive diarrhoea of TNBS, the thickening of inflammation and colon wall, and the losing of goblet cell.Therefore, GIPF can be treated the patient who suffers from Crohn disease as effective therapeutic agent.

Embodiment 23

GIPF is to the therapeutical effect of the chronic inductive mouse colitis of dextran sodium sulfate

Induce the mouse model (L ' Heureux andBrubaker J Pharmacol Exp Ther 306:347-354 (2003) of colitis at chronic dextran sodium sulfate (DSS); Kriegelstein et al., J ClinInvest 110:1773-1782 (2002); Siegmund et al., J Pharmacol Exp Ther296:99-105 (2001)) go up and test the effect that recombined human GIPF (GIPFwt) treats colitis.

With age in 6-8 week female BALB/c mouse (MA USA) raises in airy cage, adapts to 12 hour daytime of 1 week: the circulation at night for Charles River Laboratories, Wilmington.Fed to mice 4%DSS (v/w) to induce colitis drinking water from the 0th day to the 7th day.From the 7th day to the 21st day, give water that mice do not have DSS to induce for first catabasis.From the 21st day to the 28th day, give mice 4%DSS once more to induce for the first recurrence phase.From the 28th day to the 35th day, give water that mice do not have DSS once more to induce for second catabasis.At the 35th day, treat up to the 42nd day with the mice random packet and at the 35th day beginning GIPF.Monitored the active performance of mouse disease every day from the 35th day to the 42nd day.At the 42nd day, finish experiment, mice is put to death and collects intestinal tissue to analyze.

GIPF obviously alleviates the inductive mouse colitis of DSS-in dose-dependent mode, its be reflected in obvious reduction inflammatory bowel disease activity index (inflammatory bowel disease activity index) (IBDAI) (Figure 50: ^*P＜0.05 (ANOVA, DSS/ saline is organized DSS/hGIPF); #P＜0.05 (ANOVA, DSS/ saline DSS/KGF); ^*P＜0.05 (ANOVA, DSS/ saline is to DSS/GLP-2)).In embodiment 14, provided the definition of IBDAI.The H﹠amp of small intestinal and colon; The E stained shows that hGIPF has prevented the inductive mice destruction of mucosal of DSS-, and has reversed inductive height of naps of DSS-and crypts degree of depth shortening (Figure 51).GIPF has also eliminated DSS to small intestinal pit cell inhibition of proliferation effect (Figure 52; ^*P＜0.05 (ANOVA, DSS/ saline is to water/saline); #P＜0.05 (ANOVA, DSS/hGIPF is to DSS/ saline) ^*P＜0.05 (ANOVA, DSS/KGF is to DSS/ saline); ##P＜0.05 (ANOVA, DSS/GLP-2 is to DSS/ saline)).In embodiment 14, defined the crypts proliferation index.

In brief, the therapeutic effect of GIPF obviously reduces the inductive colitis of the long-term DSS-of mice, illustrates that GIPF may be the very useful therapy of treatment people inflammatory bowel disease.

Embodiment 24

HGIPF alleviates the inductive toxicity of 5-fluorouracil

On normal BDF-1 mice, estimate the effect that hGIPF alleviates the gastrointestinal toxicity of 5-fluorouracil.

Mice is divided into following group:

1) the injection carrier adds saline treatment

2) the 5-FU injection adds saline treatment

3) the 5-FU injection adds the GIPF processing

Beginning in the 3rd day, every day, subcutaneous injection gave the female BDF-1 mice in age in 11-13 week or the hGIPF of saline or every dose 100 μ g.From the 0th day to the 4th day, continuous 4 days 5-FU to every Mus peritoneal injection dosage 50mg/kg.Monitor the mice body weight every day, diarrheal takes place, and mortality rate.

GIPF handles and obviously reduces the inductive gastrointestinal toxicity of 5-FU, comprises that reducing weight limit descends, diarrhoea scoring, and mortality rate (table 9), and therefore explanation GIPF on mice effectively reduces the inductive gastrointestinal toxicity of chemotherapy.

Table 9

		Toxicity
						5-FU	Handle	Weight limit decline (%)	The diarrhoea scoring	Mortality rate	Time-to-live (my god)
Be	Not	33.1±3.6	2.8±0.5	92	8.5±1.2
						Be	hGIPF	12.5±6.9 *	0.9±0.6 *	8.3	10.0±0.0 *
Be	KGF	16.8±7.9 *	1.7±0.8 *	25	9.0±1.0
						Be	GLP-2	17.4±8.3 *	1.9±0.8	42	8.4±1.1

^*P＜0.05 (5-FU/KGF or 5-FU/GLP-2 are to 5-FU/ saline for ANOVA, 5-FU/hGIPF)

Embodiment 25

The activity of GIPF in the non-human primates body

Carrying out the active repeated doses research of GIPF on the stump-tailed macaque to judge the activity of GIPF in non-human primates.

(USA) veterinary personnel or veterinary technician are female to 9 for Everett, Washington by SNBL USA

Monkey carries out prescriptive screening, and carries out hematology and serum chemistry screening.Be confirmed to be in healthy 9 animals, selected 8 and give seminar.Before beginning one's study, the animal of quarantine was in the past complied with 14 days in the research department that SNBL USA facility is arranged at least.

Method: according to the planning of following form femalely be divided into 4 processed group and with 8 through intravenous bolus injection GIPF protein medicine-feeding, carried out 3 days for 1 time every day.At the 4th day, before postmortem, gave intravenous push bromodeoxyribouridines of all animals (BrdU) (50mg/kg) in about 4 hours.Collect the tissue of selecting during postmortem.

Research design

Group	Dosage level (mg/kg)	Concentration (mg/mL)	Dose volume (mL/kg)	Size of animal
					1	0mg/kg	0	3.3mL/kg	2

	(contrast)
					2	0.1mg/kg	1.5mg/mL	0.067 mL/kg	2
3	1.0mg/kg	1.5mg/mL	0.67mL/kg	2
					4	5.0mg/kg	1.5mg/mL	3.3mL/kg	2

Observe and detect: during complying with and the omnidistance following clinical observation of carrying out of experiment.Till beginning to finish from the phase of complying with, carry out mortality rate and stool examination once a day in the morning, carry out the clinical observation of general health and appearance once a day in the afternoon to life.If desired, also can carry out extra clinical observation and record.If clinical observation shows the situation (declining condition) of decline, veterinary personnel or veterinary technician are estimated every animal and are reported the study director.

Blood preparation is collected; Collect in the phase of complying with and once and in postmortem before giving BrdU, to collect a blood to carry out hematology and serum chemistry the same day.

General pathology is checked: during postmortem, and the outer surface of having a physical examination, institute is porose, and cranial cavity, thoracic cavity, abdominal cavity and content thereof.Naked eyes carry out organ weight and histopathological examination, collect and fixedly carry out histopathological examination with 10% neutral buffered formalin.Below the tissue of checking is listed in.

Brain-brainstem-cerebellar-brain	Large intestine a-Ceco-colon-rectum	Small intestinal a-Jejuno-Duodeno-ileum
			Spleen	Liver	Tongue

Measure by using the immunohistochemical hamartoplasia of BrdU: measure the paraffin-embedded section of preparation as BrdU.

The result: the general pathology inspection finds that the organ of checking does not obviously change, and shows the acute toxicity that does not have hGIPF in this therapeutic scheme during postmortem.In addition, the not change of serum biochemistry parameter of the blood preparation hematology who before hGIPF is handled, collects and serum chemistry proof blood cell component and test with the back.

The relevant increase of show dose on small intestinal length.The 1st, 2,3 and 4 groups of average intestinal length (cm) are respectively 120.65,122.555,133.350 and 142.875.In addition, as (table 10) of summing up in the following table, histology's microscopically evaluation of various tissues confirms in all GIPF processed group, duodenum, the crypts hypertrophy of jejunum and ileum.At caecum, colon and rectum are also observed the crypts hypertrophy of the relevant mode of dosage.This result shows that hGIPF has proliferation function to the intestinal crypt epithelial cell of monkey, and this is with observed consistent on mice and rat.

Be to confirm the proliferation function of hGIPF, carry out immunohistochemistry and detect to analyze mixing of in small intestinal and large intestine BrdU.All increase the positive hypertrophy index of BrdU at small intestinal and colon hGIPF.

These discoveries are presented in the non-human primates, and hGIPF increases the hypertrophy of crypts epithelium.

Each histopathology is found

Classification

_: no abnormal change

±: very light

+: slight

2+: moderate

3+: obviously

Table 10

Each histopathology on female stump-tailed macaque is found

Tissue	Find	Group (size of animal)
										1		2		3		4
		#1	#2	#1	#2	#1	#2	#1	#2
										Duodenum	The crypts hypertrophy	-	-	-	±	±	±	+	2+
Jejunum	The crypts hypertrophy	-	-	±	±	±	±	±	+
										Ileum	The crypts hypertrophy	-	-	-	±	+	±	+	+
Caecum	The body of gland length that the crypts hypertrophy increases	-	- -	- -	- -	± -	± -	+ ±	+ +
										Colon	The body of gland length that the crypts hypertrophy increases	- -	- -	- -	- -	± -	± -	± -	+ ±
Rectum	The crypts hypertrophy	-	-	-	-	±	+	±	+
										Liver	-	-	-	-	-	-	-	-	-
Spleen	-	-	-	-	-	-	-	-	-
										Brain	-	-	-	-	-	-	-	-	-
Cerebellum	-	-	-	-	-	-	-	-	-
										Brain stem	-	-	-	-	-	-	-	-	-

Embodiment 26

With the radioactivity labelling ¹²⁵(ADME) research is drained in the proteic absorption distribution of I-HGIPF metabolism

Research purpose: in the judgement mice [ ¹²⁵I]-blood plasma pharmacokinetics and the tissue distribution of hGIPF.

Protein labeling: with the IODO-GEN labeling method (Amersham) with hGIPF albumen with [ ¹²⁵I] labelling.Initial specific activity is 35uCi/ug (1020Ci/mmol) during labelling.Albumen with labelling before giving injected in mice is further purified.

Animal: before injection, make male CD-1 ^_[Crl:CD-1 ^_(ICR) the BR mice was accorded with one's environment 7 days, and raised in the clean wire mesh cage that hangs separately.Rise to cage on cage or on other suitable material, change at least weekly 3 times.Give every mice with the dosage of 1.67mg/kg and contain 3 μ Ci ¹²⁵The proteic hGIPF of I-hGIPF.After accepting radiolabeled dosage, the animal of collecting urine and feces is on schedule raised separately in the metabolism unit.

Research design:

Group	The specimen of collecting	Dosage level	Dose volume	The time of euthanasia	The size of animal of each time point	The sum of animal
							1	Blood and tissue	1.67 mg/kg	10 mL/kg	After the administration 5 and 30 minutes and 1,3,6 and 24 hours	3	18
2	Urine, feces, tissue and corpse	1.67 mg/kg	10 mL/kg	After the administration 24 hours	3	3

All animals of accepting research are handled to stop thyroid to being derived from the picked-up that labelling is tried the free iodide of thing with sodium iodide.About 48,24 and 1 hours oral (gavage) gives 0.1ml 1%NaI solution before giving radio-labeled albumen.The single dose that every animals received intravenous injection gives [ ¹²⁵I]-hGIPF.Animal is divided into two groups also as analyzing of listing in the top research design.Shown in the time point of euthanasia, collect blood preparation, and isolated cell fraction and blood plasma are to analyze.Collect liver, kidney, lung, tongue, spleen, brain, esophagus, stomach, small intestinal, large intestine and comprise the tissue specimen of the large intestine of content, judge in described tissue [ ¹²⁵I]-the mixing of hGIPF.

According to the WIL standard operating procedure on DPC GAMMA-C12 polycrystal γ calculating instrument, carry out [ ¹²⁵I]-analysis that hGIPF mixes.The result of γ counting proofreaied and correct with the isotope half-life.According to the WIL standard operating procedure, the calculating of radioactivity amount uses WIL toxicology data management system or table procedure (spreadsheet) to carry out in the various materials that produce in the research.Usually, only use descriptive statistics (for example, amounting to arithmetic mean, standard deviation, standard error, the coefficient of variation, percentage ratio).If possible, with standard pharmacokinetics Equation for Calculating standard drug kinetic parameter (for example, C _Max, t _Max, AUC (area under curve), T _1/2(half-life)).

The result: table 11-14 shown in the following specimen [ ¹²⁵I]-hGIPF concentration and dynamic (dynamical) data: mice plasma, erythrocyte, liver, kidney, lung, the heart, brain, spleen, esophagus, stomach, small intestinal, and large intestine.

Table 11

Vein give behind the 1.67MG/KG in the mice plasma and erythrocyte [ ¹²⁵I]-normal concentration of hGIPF and kinetics

Time (hour)	Blood plasma	Erythrocyte
			(ng/g) (means standard deviation)	(ng/g) (means standard deviation)
	0.083 0.5 1 3 6 24	11145(113) 3894(485) 2506(490) 1347(68) 476(204) 60(7)			2745 212l 1658 971 295 23
C max(ng/g) t max(h) AUC 0-24(ng-h/g)			11145 0.083 16607	2745 0.083 9458

Latter stage kinetics

(Log concentration is to the linear regression of 3-24 hour time)

Slope (b) Y-y-intercept (ng) coefficient of determination (r 2) elimination rate constant (h -1) half-life (h)

-0.05939 1517.75 0.960 0.1367 5

-0.07225 1161.79 0.967 0.1664 4

N=3 was except 24 hours N=6.

Table 12

Vein gives Mouse Liver behind the 1.67MG/KG, kidney, and lung, the heart, in brain and the spleen [ ¹²⁵I]-normal concentration of hGIPF and kinetics

	Liver	Kidney	Lung	Tongue	Brain	Spleen
							(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)
	0.083hr 0.5hr 1hr 3hr 6hr 24hr	9104(959) 4982(1319) 3635(413) 2706(185) 1757(213) 875(75)	24581(5032) 21283(4731) 17039(1543) 13445(1084) 8933(800) 4707(485)	3332(462) 1997(375) 1575(375) 633(450) 337(106) 63(13)	1157(88) 1124(202) 1044(292) 588(32) 227(91) 35(7)	141(25) 105(28) 95(18) 51(7) 17(9) 5(3)							1956(354) 1905(663) 1380(106) 840(74) 457(193) 157(49)
C max(ng/g) t max(hour) AUC 0-24 (ng-h/g)							9104 0.083 42191	24581 0.083 206968	3332 0.083 9404	1157 0.083 6282	141 0.083 554	1955.54 0.083 11400

Latter stage kinetics

(Log concentration is to the linear regression of 3-24 hour time)

Slope (b) Y-y-intercept (ng) coefficient of determination (r 2) elimination rate constant (h -1) half-life (h)

-0.02106 2742.03 0.936 0.0485 14

-0.01953 13559.24 0.932 0.0450 15

-0.04512 746.08 0.982 0.1039 7

-0.05344 649.87 0.958 0.1230 6

-0.04209 47.18 0.883 0.0969 7

-0.03164 873.39 0.947 0.0728 10

N=3 was except 24 hours

N＝6。

Table 13A and B

Vein gives mice esophagus behind the 1.67MG/KG, stomach, in small intestinal and the large intestine [ ¹²⁵I]-normal concentration of hGIPF and kinetics

A

	Esophagus	Stomach	Small intestinal	Large intestine
					(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)	(ng/g) (means standard deviation)
	0.083 hours 0.5 hour 1 hour 3 hours 6 hours 24 hours	1560(321) 1743(178) 1666(743) 1199(330) 350(63) 48(43)	1960(166) 2855(1140) 5545(3546) 3678(2047) 1021(475) 106(40)	1117(135) 1191(423) 1053(369) 664(28) 222(110) 32(6)					1016(150) 910(135) 1006(189) 694(141) 383(175) 74(13)
C max(ng/g) t max(hour) AUC 0-24(ng-h/g)					1743 0.5 10372	5545 1 29604	1191 0.5 6421	1016 0.083 8344

B

Latter stage kinetics

(Log concentration is to the linear regression of 3-24 hour time)

Slope (b) Y-y-intercept (ng) coefficient of determination (r 2) elimination rate constant (h -1) half-life (h)

-0.06022 1247.25 0.936 0.1387 5

-0.06683 3997.67 0.947 0.1539 5

-0.05745 715.76 0.948 0.1323 5

-0.04408 822.73 0.984 0.1015 7

^*N=3 was except 24 hours N=6.

Table 14A and B

Vein gives 1.67MG/KG normal recovery of mice hGIPF after 24 hours

A

Tissue										Amount to
											Liver	Small intestinal	Colon	Brain	Spleen	Lung	Kidney	Stomach	Esophagus	Tongue
2.39 2.13 2.64	0.07 0.10 0.07	0.06 0.06 0.04	0.01 0.00 0.01	0.02 0.02 0.02	0.02 0.02 0.02	4.83 5.17 4.21	0.03 0.10 0.03	0.01 0.00 0.00	0.01 0.01 0.01												7.43 7.61 7.03

B

Number of animals	The %GIPF/ tissue	The %GIPF/ intestinal contents	The %GIPF/ urine	%GIPF/ feces	The %GIPF/ corpse	Overall recovery _
							1	7.43	0.13	85.70	3.48	1.87	98.6
2	7.61	0.13	76.70	3.12	1.82	89.4
							3	7.03	0.32	87.47	3.53	1.76	100.1
Mean:	7.35	0.19	83.29	3.38	1.82	96.0
							Standard deviation:	0.30	0.11	5.77	0.23	0.06	5.81
_ tissue, the GI content comprises the urine that the cage upper punch washes, the summation of feces and corpse

As desired, organize for example liver highly dabbling, kidney, spleen and lung are observed T the earliest _MaxWith maximum C _Max, and observe the longest half-life (table 11) at the kidney regulating liver-QI.

Gastrointestinal tract comprises esophagus, and organizing of harmonization of the stomach small intestinal all shows the Tmax (table 12) that prolongs.Table 13A shows the tissue and the intestinal contents of 24 hours various organs of animal after the radiolabeled hGIPF administration, urine, radiolabeled hGIPF recovery percent in feces and the corpse.List in table 13B from the hGIPF recovery of various organ-tissues.After the administration of data show intravenous route, therefore the distribution of radiolabeled hGIPF illustrates that hGIPF may have high-affinity to gastrointestinal tissue usually at gastrointestinal tract organ height.

Embodiment 27

Radiation-induced mucositis: the evaluation of therapeutic regimen

The purpose of this research is to determine to provide the therapeutic scheme of the maximum preventive effect of the inductive mucositis of GIPF radioprotective.

Bull BDF-1 mice is 10-12 age in week in use.With animal 12 hours the daytime/night circulation raised for 1 week, and allowed the whole process water inlet of taking food at random.Animal is divided into 6 groups at random, 5 every group (totally 30 mices), carry out following processing:

1. be untreated not radiating contrast.

2. be exposed to the total irradiation of 13Gy X-line) preceding 3,2,1 day intravenous injection 4mg/kg hGIPF.

3. be exposed to the total irradiation of 13Gy X-line) preceding 4,3,2 days intravenous injection 4mg/kg hGIPF.

4. be exposed to the total irradiation of 13Gy X-line) preceding 5,4,3 days intravenous injection 4mg/kg hGIPF.

5. be exposed to the total irradiation of 13Gy X-line) preceding 6,5,4 days intravenous injection 4mg/kg hGIPF.

6. be exposed to the total irradiation of 13Gy X-line) preceding 72,48,24 hours intravenous injection saline.

Animal is exposed to the total irradiation of single dose 13Gy X-line with emission in 2.7Gy/ minute.

Shine after 4 days sacrifice of animal.Put to death preceding 2 hours, with the 4mg BrdU of all animal volume injected 0.4ml.When dissected is measured the length and the weight of small intestinal and large intestine, and small intestinal (middle jejunum) that will about 1cm, colon (transverse colon) tissue segments, and tongue is in esophagus stomach function regulating stuck-at-0% formalin.

Analyze the cross section BrdU picked-up of small intestinal.Count the survival crypts number of each section and judge every group average.Only counting contains 10 or more a plurality of strong H﹠amp; The crypts of the painted cell of E (except Paneth cell) and do not contain the complete tangent plane of aggregate nodules.

In order to proofread and correct the error of calculation, also measure average crypt width (measuring its wideest point) because of the crypts difference in size.The following correction:

The result: be exposed to hGIPF after the radiation to the influence of crypt survival shown in Figure 53.Data show, when when total irradiation gave hGIPF in preceding 24 hours or 48 hours, hGIPF obviously alleviates radiation-induced esoenteritis (table 15).

These find to confirm can be with hGIPF as prophylactic agent remedying the ill effect of radiation-induced esoenteritis, and total irradiation administration in preceding 24 hours provides the maximum protection to intestinal crypt.

Table 15

	The hGIPF treatment
				Radiation dose (Gy)	GIPF (mg/kg/ days)	Treatment time is shown	HGIPF protects multiple (means standard deviation)
13	Do not have	Do not have	1.0±0.5
				13	4	The-3 days to the-1 day	15.8±6.2 *
13	4	The-4 days to the-2 days	9.4±6.8 *
				13	4	The-5 days to the-3 days	4.8±3.3
13	4	The-6 days to the-4 days	5.3±4.4

^*P＜0.05 (ANOVA, 13Gy/hGIPF is to 13Gy/ saline)

Embodiment 28

The hypertrophy that cell lineage relies on the mouse small intestine is measured

Research hGIPF to the influence of intestinal crypt to judge whether hGIPF also influences the transitional type proliferative cell by both influencing the crypts stem cell before morphological change occurs, still be by influence they one of work.

Animal is divided into following group at random:

(1.PBS every group of mice): injection back 1,3,6,12,24 or 48 hours

(2.hGIPF every group of 2 mices, 100ug single injection): injection back 1,3,6,12,24 or 48 hours

Every animal was injected BrdU (4mg/kg) in preceding 2 hours in execution.The crypts degree of depth, the hypertrophy analysis of crypts hypertrophy exponential sum cell position are carried out in middle jejunum section to small intestinal.By single injection hGIPF (100 μ g) back shown in time (3,6,12,24 and 48 hours) measure crypts hypertrophy index by the BrdU immunohistochemistry.The BrdU that analyzes 40 crypts of 2 mices mixes, the result be expressed as means standard deviation ( ^*P＜0.01, ANOVA).

The result: shown in table 16, hGIPF increased the hypertrophy of small intestinal pit cell as far back as 3 hours, and hypertrophy is handled at hGIPF and reached the peak in back 24 hours.The inductive crypts hypertrophy of hGIPF is reversed in 48 hours.In addition, the position analysis of BrdU positive cell (Potten et al., Int J Exp Path78:219-243 (1997)) proves that the hypertrophy of the part pit cell on position 3-5 (the crypts bottom that exists from stem cell) and crypts obviously increases.

These data show that hGIPF both can influence stem cell and also influence just splitted transition cell group.

Table 16

Time (hour)	%BrdU positive cell PBS matched group	%BrdU positive cell hGIPF group
			3	38.0±12.47	47.7±8.38
6	36.45±8.33	49.75±11.3
			12	39.16±8.57	51.24±9.86
24	36.55±9.62	74.97±9.0
			48	33.0±5.32	19.5±6.5

Embodiment 29

HGIPF is to the influence of pit cell differentiation and migration

With sum and the distribution of counting goblet cell and paneth's cell behind the hGIPF processing mice to judge whether hGIPF influences these cell types in the small intestinal.

Carry out Ah Xinlan (Alcian) dyeing with goblet cell in the middle jejunum section that shows PBS and hGIPF processing mice (n=3).Animal is injected hGIPF (100 μ g) or PBS 3 or 7 days every day.For showing paneth's cell (Paneth cell), in same animal, carry out immunohistochemistry (IHC) in the jejunum section with anti-lysozyme antibody.

The result: the immunohistochemical analysis of small intestinal and Ah Xinlan dye proves paneth's cell and the not obviously change of goblet cell number on the small intestinal of hGIPF processing mice.HGIPF does not influence cell ripe and migration along crypts/fine hair axle of differentiation.

Embodiment 30

Express the transgenic gomphosis mouse of hGIPF at enterocyte

1. the preparation of mice villin gene promoter long segment (Figure 54 A)

Villin is found in the actin vascular bundle albumen (actinbundling protein) of the top brush border of absorptive tissue, is by first structural gene of transcriptional activation in embryo's gut entoderm.Growing up, the villin wide expression is in each cell of the enteric epithelium of intestinal vertical axis (crypts is to the fine hair point) and trunnion axis (duodenum is to colon).The tend to act high level expression (J.Biol.Chem.277, p33275-33283,2002) of two kinds of different reporter genes (LacZ and Cre recombinase) in the whole enteric epitheliums of transgenic mouse of the 12.4kb zone of proof mice villin genes such as Madison.For being created in the chimeric Mus of transgenic of enteric epithelium expressing human GIPF, our construction of expression vector wherein is connected in GIPF cDNA this transcriptional regulatory sequences that instructs it to express at enteric epithelium.

Obtain the nucleotide sequence information of the upstream region of mice villin gene from public database (ensembl).With mice BAC (RP23-278N11; The GenBank accession number: AC098570) DNA digests with EcoRI and BamHI (Roche), and carries out 0.8% agarose gel electrophoresis to separate the fragment of about 11kb.With pBluescriptIISK (-) (STRATAGENE) with after EcoRI and BamHI (Roche) digestion, with carrier segments with the separation of 0.8% agarose gel electrophoresis and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that its two ends dephosphorylation all.The dna fragmentation of top about 11kb is connected in dephosphorylized carrier segments, and with connecting mixture transfection XL10-Gold Ultracompetenet cell (STRATAGENE).The primer that use describes below (SEQ ID NO:120 and 121) will carry out pcr amplification from the DNA sample of gained conversion product preparation.The sequence analysis of amplified fragments shows the mice villin gene promoter fragment (pPvil 11.2) that comprises about 11.2kb.PPvil 11.2 usefulness restriction endonuclease ClaI and BamHI are digested, and reactant mixture is carried out 0.8% agarose gel electrophoresis to separate the fragment of about 11.2kb.

PvilEIBI-FW1(SEQ ID NO：120)GATCAGCAGCTGGAACAAACACAG

PvilEIBI-RV1(SEQ ID NO：121)TGCACAATCAGTCAATCAACAGAGC

(2) the short segmental preparation (Figure 54 B) of mice villin gene promoter

Nucleotides sequence with the mice villin gene upstream region that obtains from public database (ensembl) is classified the basis as, synthetic two kinds of synthetic DNAs (SEQ ID NO:122 and 123).

PvilBI-FW(SEQ ID NO：122)GGCGGATCCCTGAGTTGGAGGCCAGTTTGG

PvilBI-NcoIXbaIRV(SEQ ID NO：123)GCTCTAGACCATGGTGGACGAGCCTAGAGGAGAAGGCAT

KOD-puls (TOYOBO) is used for the PCR reaction.The PCR reactant mixture contains every kind of primer and the mice BAC (RP23-278N11 of 10pM; The GenBank accession number: AC098570) DNA is as template.Be 94 ℃ with initial degeneration insulation and carried out pcr amplification in 2 minutes.Carry out 30 circulation degeneration by 94 ℃ of 15 seconds and 68 ℃ of insulations of 2 minutes then, annealing and amplification.With PCR product (approximately 1.9kb) with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With isolating PCR product with BamHI and XbaI digestion after, with the fragment of digestion with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With the fragment of purification be connected in XhoI and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pBluescriptIISK in its two ends (-) (STRATAGENE).To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.The clone that will comprise the correct nucleotide sequence fragment digests with NcoI.With the fragment of digestion with Klenow fragment (TAKARA BIO) digestion process with floating its two ends, with it further with XbaI digestion and with 0.8% agarose gel electrophoresis purification.With the fragment of gained with E.Coli C75 alkaline phosphatase treatment with its two ends dephosphorylation.

(3) the segmental preparation of GIPF (Figure 54 C)

Hy01XhISphIFW(SEQ ID NO：124)CCGCTCGAGGCATGCGGCTTGGGCTGTGTGTGGTGGCCCTG

Hy01BgXb-RV(SEQ ID NO：125)GCTCTAGAAGATCTCTAGGCAGGCCCTGCAGATGTGAGTGGCCC

KOD-puls-(TOYOBO) is used for the PCR reaction.The PCR reactant mixture contains every kind of primer (SEQ ID NO:124 and 125) of 10pM and GIPF cDNA as template.Be 94 ℃ with initial degeneration insulation and carried out pcr amplification in 3 minutes.Carry out 30 circulation degeneration by 94 ℃ of 15 seconds and 68 ℃ of insulations of 2 minutes then, annealing and amplification.With PCR product (approximately 800kb) with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With isolating PCR product with XhoI and XbaI digestion after, connect in XhoI and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pBluescriptIISK in its two ends (-).To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.The clone that will comprise the correct nucleotide sequence fragment digests with SphI.With the fragment of digestion with Blunting high (TOYOBO) digestion process with floating its two ends after, with it further with XbaI digestion and with 0.8% agarose gel electrophoresis purification.

(4) structure of pPvil 2-01 (Figure 54 D)

The GIPF fragment of preparation in (3) is connected in the pPvil2 of preparation in (2), and will connects mixture transfection DH5 α.And by the nucleotide sequencing analysis from the DNA sample of gained conversion product preparation to confirm to insert segmental structure.Selection comprises the clone (pPvil 2-01) of correct nucleotide sequence fragment.

(5) preparation of pIRES-GFP (Figure 54 E)

Behind EcoRI and NotI digestion pIRES2-EGFP (BD Bioscience Clontech), by comprise the fragment in IRES-GFP zone with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With the fragment of purification be connected in XhoI and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pcDNA3 in its two ends (Invitrogen).To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pIRES-GFP) of correct nucleotide sequence fragment.

(6) structure of pUC119 IRES-GFP (Figure 54 F)

Behind BamHI and XbaI digestion pIRES-GFP, by comprise the fragment in IRES-GFP zone with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With the fragment (IRES-GFP) of purification be connected in with BamHI and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pUC119 in its two ends.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pUC119 IRES-GFP) of correct nucleotide sequence fragment.

(7) structure of pUC119 IRES-GFP+As (Figure 54 G)

The DNA of synthetic oligonucleotide (SEQ ID NO:126 and 127) annealing preparation that will be by describing below be connected in EcoRI and BamHI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pUC119 IRES-GFP in its two ends.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pUC119 IRES-GFP+As) of correct nucleotide sequence fragment.

EI-BIAscI-(BI)S(SEQ ID NO：126)AATTCGGATCCGGCGCGCC

EI-BIAscI-(BI)AS(SEQ ID NO：127)GATCGGCGCGCCGGATCCG

(8) structure of pUC119 IRES-GFP+loxP (Figure 54 H)

The DNA of synthetic oligonucleotide (SEQ ID NO:128 and 129) annealing preparation that will be by describing below be connected in NotI and XhoI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pUC119 IRES-GFP+As in its two ends.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pUC119 IRES-GFP+loxP) of correct nucleotide sequence fragment.

Nt-PmloxP-XhS(SEQ ID NO：128)GGCCGTTTAAACATAACTTCGTATAATGTATGCTATACGAAGTTATC

Nt-PmloxP-XhAS(SEQ ID NO：129)TCGAGATAACTTCGTATAGCATACATTATACGAAGTTATGTTTAAAC

(9) the segmental preparation of bovine growth hormone (BGH) polyA (Figure 54 I)

BGHpAFW(SEQ ID NO：130)CGGGATCCGTTTAAACCTGTGCCTTCTAGTTGCCAGCCATC

BGHpARV(SEQ ID NO：131)CGGATATCCCATAGAGCCCACCGCATCCCCAGC

KOD-puls-(TOYOBO) is used for the PCR reaction.The PCR reactant mixture contains IRES-GFP of preparation in every kind of primer (SEQ ID NO:130 and 131) of 10pM and (6) as template.Be 94 ℃ with initial degeneration insulation and carried out pcr amplification in 3 minutes.Carry out 30 circulation degeneration by 94 ℃ of 15 seconds and 68 ℃ of insulations of 2 minutes then, annealing and amplification.With PCR product (approximately 0.2kb) with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With isolating PCR product with BamHI and EcoRV digestion after, connect in BamHI and EcoRV digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pBluescriptIISK in its two ends (-).To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.The clone that will comprise the correct nucleotide sequence fragment is with PmeI and EcoRV digestion, and the zone that will comprise bovine growth hormone (BGH) poly A is with 0.8% sepharose electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.

(10) comprise IRES-GFP, the preparation of the dna fragmentation of bovine growth hormone polyA and loxP sequence (Figure 54 J)

With pUC119 IRES-GFP+loxP with PmeI digestion and by 0.8% agarose gel electrophoresis purification.BGH polyA fragment of preparation in (9) is connected in the also usefulness Intestinum Bovis seu Bubali alkaline phosphatase treatment of purification so that its two ends all dephosphorylized pUC119 IRES-GFP+loxP carrier.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises BGHpoly A fragment and the unidirectional clone of GIPF coded sequence (pIRES-GFP+pA).PIRES-GFP+pA with BamHI and XbaI digestion, and will be comprised IRES-GFP, and the fragment of bovine growth hormone polyA and loxP sequence is with 0.8% sepharose electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.

(11) structure of pPvil 2-01GFP (Figure 54 K)

To comprise IRES-GFP, the dna fragmentation of bovine growth hormone polyA and loxP sequence [seeing (10)] be connected in BglII and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pPvil2GIPF in its two ends.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pPvil 2-01GFP) of correct nucleotide sequence fragment.

(12) structure of pPv-total (Figure 54 L)

With the long segment [seeing (1)] of the mice villin gene promoter of about 11.2kb be connected in BglII and ClaI digestion and with E.coli C75 alkaline phosphatase treatment so that all dephosphorylized pPvil2-01GFP in its two ends.To connect mixture transfection XL10-Gold Ultracompetent cell (STRATAGENE), and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pPv-total) of correct nucleotide sequence fragment.

(13) structure of pLoxP-StneoR (Figure 54 M)

The pLoxP-STneo that WO 00/10383 is described handles with floating its two ends with XhoI digestion and with Blunting high (TOYOBO).Comprise loxP-Neo by 0.8% agarose gel electrophoresis purification ^rThe gained dna fragmentation of-loxP unit.The dna fragmentation of synthetic oligonucleotide (SEQ ID NO:132 and 133) annealing preparation that will be by describing below is connected in PacI and FseI digestion and with the pBlueLAB (WO 00/10383) of 0.8% agarose gel electrophoresis purification.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pBlueLAB2) of correct nucleotide sequence fragment.Comprise loxP-Neo above inciting somebody to action ^rThe dna fragmentation of-loxP unit be connected in EcoRV digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pBlueLAB2 in its two ends.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises and the reverse segmental clone (pLoxP-STneoR) of pLoxP-STneo (WO 00/10383).

AsiSI-S(SEQ ID NO：132)：TAACCGCGATCGCGGCCGG

AsiSI-AS(SEQ ID NO：133)：CCGCGATCGCCCTTAAT

(14) structure of pPv01GFP (Figure 54 N)

PPv-total length plasmid DNA is digested with restriction endonuclease ClaI and XhoI, and will comprise that the dna fragmentation of Pv-GIPF unit passes through 0.8% agarose gel electrophoresis purification.With the dna fragmentation of purification be connected in ClaI and XhoI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pLoxP-StneoR in its two ends.To connect mixture transfection XL10-Gold Ultracompetent cell (STRATAGENE), and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.Selection comprises the clone (pPv01GFP) of correct nucleotide sequence fragment.

(15) be used for the preparation of the pPv01GFP plasmid DNA of mouse ES cells electroporation

(pH7.0, the plasmid DNA (60 μ g) with pPv01GFP in reactant mixture Sigma) digested 5 hours in 37 ℃ with ClaI containing the 1mM spermidine.Then reactant mixture is carried out phenol/chloroform extracting and in-20 ℃ of ethanol precipitation (0.3M NaHCO ₃) 16 hours.Linearizing carrier segments is dissolved in (0.5 μ g/ μ l) in the HBS buffer and is used for following electroporation experiment.

(16) production of the chimeric Mus of transgenic of enterocyte expressing human GIPF and GFP

According to the Shinichi Aizawa that nineteen ninety-five Yodosha publishes, the method for describing in " Biomanual Series 8, GeneTargeting " is finished and is obtained mice embryonic, cultivates, and the ES injection cell to the embryo, is transplanted to the general step in foster mother uterus.

Shinichi Aizawa according to nineteen ninety-five Yodosha publication, the method of describing in " Biomanual Series 8; GeneTargeting ", by electroporation the transfection of linearizing pPv01GFP carrier is derived from mice TT2F ES cell ((Uchida, 1995) C57BL/6X CBA F1 strain, Lifetech oriental).To be suspended in the 20ml ES culture medium and be inoculated in two 100mm tissue culturing plastic plates (Corning) of having inoculated feeder cells in advance through the ES of electroporation cell.After 1 day, culture medium is replaced by the culture medium of the G418 (Invitrogen) that contains 200 μ g/ml.After this 7-9 days, select totally 24 clones of every kind of carrier.Make each be cloned in to grow in 12 orifice plates and converge, then 4/5ths culture is suspended in the frozen culture medium of 0.2ml [ES culture medium+10%DMSO (Sigma)] and frozen in-80 ℃./ 5th of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.To carry out 0.8% agarose gel electrophoresis then from the genomic DNA of the TT2F cell separation of G418 resistance with limiting enzyme EcoRI and XhoI digestion.Use EcoRI-XhoI digestion, judge by the band that detects about 16kb in the G418 resistance clone, to have kept expressed intact unit (comprising the villin promoter, people GIPF cDNA, the GFP cDNA of pPv01GFP and BGH polyA sequence).The separated DNA fragment is transferred to (GeneScreen on the film, NEN Life Science Products), hybridize with probe then, its middle probe is to see (13) by PCR from pPV01GFP[with the primer (SEQ ID NO:134 and 135) that describes below] the preparation of IRES zone dna fragmentation (IRESprobeF1, R1).We are chosen in the clone who shows single 16kb band on the Southern trace.Also the ES clone who selects is carried out karyotyping, it is according to Aizawa Shinichi, and " Biomanual Series 8, Gene Targeting ", Yodosha publishes, and 1995 methods of describing are carried out.The caryogram person that will act normally is used for implanting to the embryo.

IRESprobeF1(SEQ ID NO：134)：CTAACGTTACTGGCCGAAGC

IRESprobeR1(SEQ ID NO：135)：ATTATCATCGTGTTTTTCAAAGGAA

The cell of the ES cell clone #2 of frozen transfection is melted, begin to cultivate and be expelled to that (CREA JAPAN is among the embryo of 8 cell stages of male and female mice copulation acquisition INC.) from MCH (ICR) mice kind system; Injection rate is each embryo 10-12 cell.For the ES cell development becomes blastocyst with the embryo in culture medium after the overnight incubation, the embryo transfer of about 10 ES cell-injections is gone into foster mother ICR mice (CREA JAPAN, INC.) each Aconitum carmichaeli Debx. palace, the pseudo-fetus that wherein this mice has been carried out 2.5 days is handled.The pigmentation that effect in the tissue that is organized in host embryo (albefaction) source in TT2F (agouti) ES clone-source can be by eyes among the embryo and the offspring of survival are judged by the hair color.

(17) expression of people GIPF-GFP mRNA in the chimeric Mus of transgenic

Prepare total RNA sample from the chimeric intestinal in (E13.5, E16.5, E19.5, the 3rd day, the 7th day) pPv01GFP/TT2F-#2 of various stages of development source, and carry out sxemiquantitative RT-PCR and analyze to detect the expression of GIPF-GFP mRNA.Use the synthetic first chain cDNA of Superscript III (Invitrogen), wherein use random hexamer and contrast total RNA of the 500ng of intestinal extraction with Isogen (NipponGene) and RNasy Mini (QIAGEN) from pPv01GFP/TT2F-#2 source chimera and TT2F-source chimera.Using cDNA to carry out sxemiquantitative RT-PCR at every kind of primer to special annealing temperature analyzes.PCR product electrophoresis on 2% agarose gel is also dyeed with the pyridine of bromination second.The integrity of the amplification control RNA of the cDNA that produces by Mus GAPDH.Be used for GIPF (Pv01RT F1, R1; SEQ ID NO:136 and 137), Axin 2 (Axin2 F, R; And mGAPDH (mGAP DH5,3 SEQ ID NO:138 and 139)); SEQ ID NO:140 and 141) below the annealing temperature of nucleotide sequence and primer is listed in.

Pv01RT F1(SEQ ID NO：136)GCTCTGACACCAAGGAGACC

Pv01RT R1(60℃)(SEQ ID NO：137)CCCTAGGAATGCTCGTCAAG

Axin2 F(MUS)(SEQ ID NO：138)CAGGAGCCTCACCCTTCG

Axin2 R(MUS)(60℃)(SEQ ID NO：139)ACGCCGAGGTGCTTGCCC

mGAPDH5(SEQ ID NO：140)CACCATGGAGAAGGCCGGGGCCCAC

mGAPDH3(65℃)(SEQ ID NO：141)

ATCATACTTGGCAGGTTTCTCCAGG

Shown in Figure 55, can detect GIPF-GFP at the chimeric intestinal in pPv01GFP/TT2F-#2 source during at E13.5, and in the liver specimens of all detections, do not detect, this and former research (Madison et al., J.Biol.Chem.277, p33275-33283,2002) very consistent, its genetically modified expression of describing by 13kb villin promoters driven detects in 12.5dpc. embryo hindgut and midgut, and expression mainly is that enteric epithelium is special.To raise gradually with the age be tangible to the expression of GIPF-GFP transcript between the period of development.Report endogenous Axin2 mRNA such as Eek-hoon do not express and are induced (Mol.Cell Biol.22,1172-1183,2002) by the activation of Wnt signal path.Also known Wnt signal plays an important role in intestinal growth.Therefore we detect the expression of Axin2 in the chimera intestinal of pPv01GFP/TT2F-#2 source.Result (Figure 55) shows that when comparing with the contrast chimera Axin2 mRNA is expressed in the 3rd day and obviously rising in the 7th day, shows that people GIPF causes the activation of Wnt signal path in the expression of neonate intestinal.

(18) the white stability of beta-catenin in the chimeric Mus intestinal of transgenic

Be to estimate the influence of GIPF, the white stability of beta-catenin in the small intestinal of measuring the chimeric Mus in pPv01GFP/TT2F-#2 source on the 10th day and colon specimen to Wnt/ beta-catenin white signal path.The method of measuring the white stability of beta-catenin has been described among the embodiment 17.Shown in Figure 56, during with contrast chimera (wild type) contrast, what beta-catenin was white in expression induced strong pPv01GFP/TT2F-#2 (Pv01#2) the source chimera small intestinal of GIPF and the colon specimen stablizes.

(19) evaluation of phenotypic alternation in the chimeric Mus intestinal of transgenic

Newborn pPv01GFP/TT2F-#2 source chimera young baby showed tangible abdominal distension at the 3rd day, and the degree of this phenotype was strengthened gradually with the age.Perusal shows that small intestinal total length diameter obviously increases when chimera postmortem in the 3rd day, and wherein diameter is relevant with enhanced vascular surfaceization.With whole embryo, young baby or gastrointestinal tract are fixed in the Bouin liquid.For carrying out Histological evaluation, with paraffin-embedded section h and E (H﹠amp; E) dyeing.Shown in Figure 57, the H﹠amp of pPv01GFP/TT2F-#2 (Pv01#2); The histopathological analysis of E section shows, compared with the control from embryo the 19.5th day (E19.5) to the 14th day (d14), crypts number and branch increase.

Embodiment 31

Intestinal epithelial cell is expressed the chimeric Mus of transgenic of GIPF and Wnt3a

(1) the segmental preparation of Wnt3a (Figure 58 A)

Wnt3aFW(SEQ ID NO：142)：CGGGATCCCCATGGCTCCTCTCGGATACCTCTTAGTGCT

Wnt3aRV(SEQ ID NO：143)：GCTCTAGAGTTTAAACCTACTTGCAGGTGTGCACGTCATAG

KOD-puls (TOYOBO) is used for the PCR reaction.The PCR reactant mixture contains every kind of primer (SEQ ID NO:142 and 143) of 10pM and Wnt3a cDNA as template.Be 94 ℃ with initial degeneration insulation and carried out pcr amplification in 3 minutes.Carry out 30 circulation degeneration by 94 ℃ of 15 seconds and 68 ℃ of insulations of 2 minutes then, annealing and amplification.With PCR product (approximately 1.06kb) with 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.With isolating PCR product with BamHI and EcoRI digestion after, connect in BamHI and XbaI digestion and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that all dephosphorylized pBluescriptIISK in its two ends (-).To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.The clone that will comprise the correct nucleotide sequence fragment digests with NcoI and PmeI.The fragment that comprises Wnt3a cDNA by 0.8% sepharose electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.

(2) the segmental preparation of IRES/Wnt3a+pA (Figure 58 B)

PIRES/GFP+pA plasmid DNA [seeing embodiment 30-(13)] is digested with NcoI and PmeI, and do not comprise the carrier segments of GFP coded sequence with 0.8% agarose gel electrophoresis purification.To comprise that Wnt3a cDNA[sees (1)] fragment be connected in the carrier segments of purification, its with E.coli C75 alkaline phosphatase treatment so that its two ends dephosphorylation all.To connect mixture transfection DH5 α, and insert segmental structure from the DNA sample that the gained conversion product prepares with affirmation by the nucleotide sequencing analysis.The clone that will comprise correct nucleotide sequence fragment (pIRES/Wnt3a+pA) digests with AscI and XhoI, and comprises the dna fragmentation of IRES/Wnt3a+pA unit by 0.8% agarose gel electrophoresis and QIAquick gel extraction kit (QIAGEN) purification.

(3) structure of pPv01Wnt3a (Figure 58 C)

PPv01-GFP plasmid DNA [seeing embodiment 30-(13)] is digested with restriction endonuclease AscI and XhoI, and the reactant mixture of digestion is carried out 0.8% agarose gel electrophoresis.Separate the carrier segments in no IRES/GFP+pA zone and with the Intestinum Bovis seu Bubali alkaline phosphatase treatment so that its two ends dephosphorylation all.IRES/Wnt3a+pA fragment [seeing (2)] and top carrier segments be connected mixture transfection XL10-GoldUltracompetent cell (STRATAGENE).And by the nucleotide sequencing analysis from the DNA sample of gained conversion product preparation to confirm to insert segmental structure.Selection comprises the clone (pPv01 Wnt3a) of correct nucleotide sequence fragment.

(4) be used for the preparation of the pPv01 Wnt3a plasmid DNA of electroporation mouse ES cells

(pH7.0, the plasmid DNA (60 μ g) with pPv01 Wnt3a in reactant mixture Sigma) digested 5 hours in 37 ℃ with ClaI containing the 1mM spermidine.Then reactant mixture is carried out phenol/chloroform extracting and in-20 ℃ of ethanol precipitation (0.3M NaHCO ₃) 16 hours.Linearizing carrier segments is dissolved in (0.5 μ g/ μ l) in the HBS buffer and is used for following electroporation experiment.

(5) production of the chimeric Mus of transgenic of expressing human GIPF and Wnt3a in the enterocyte

According to the Shinichi Aizawa that nineteen ninety-five Yodosha publishes, the method for describing in " Biomanual Series 8, GeneTargeting " is finished and is obtained mice embryonic, cultivates, and the ES injection cell to the embryo, is transplanted to the general step in foster mother uterus.

Shinichi Aizawa according to nineteen ninety-five Yodosha publication, the method of describing in " Biomanual Series 8; GeneTargeting ", by electroporation the transfection of linearizing pPv01 Wnt3a carrier is derived from mice TT2F ES cell ((Uchida, 1995) C57BL/6X CBA F1 strain, Lifetech oriental).To be suspended in the 20ml ES culture medium and be inoculated in two 100mm tissue culturing plastic plates (Corning) of having inoculated feeder cells in advance through the ES of electroporation cell.After 1 day, culture medium is replaced by the culture medium of the G418 (Invitrogen) that contains 200 μ g/ml.After this 7-9 days, select totally 24 clones of every kind of carrier.Make each be cloned in to grow in 12 orifice plates and converge, then 4/5ths culture is suspended in the frozen culture medium of 0.2ml [ES culture medium+10%DMSO (Sigma)] and frozen in-80 ℃./ 5th of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.To carry out 0.8% agarose gel electrophoresis then from the genomic DNA of the TT2F cell separation of G418 resistance with limiting enzyme EcoRI and XhoI digestion.Use EcoRI-XhoI digestion, judge in the G418 clone by the band that detects about 16kb to have kept the expressed intact unit, this expression unit comprises pPv01 Wnt3a villin promoter, people GIPF cDNA, Wnt3a cDNA and BGH poly A sequence.The separated DNA fragment is transferred to (GeneScreen on the film, NEN Life Science Products), hybridize with probe then, its middle probe is to see embodiment 30-(13) by PCR from pPV01GFP[with the primer that embodiment 30-(16) describes] the preparation of IRES zone dna fragmentation (IRESprobeF1, R1).We are chosen in the clone who shows single 16kb band on the Southern trace.Also the ES clone who selects is carried out karyotyping, it is according to Aizawa Shinichi, and " Biomanual Series 8, GeneTargeting ", Yodosha publishes, and 1995 methods of describing are carried out.The #7 and the #13 of the caryogram of acting normally are used for implanting to the embryo.

The ES cell clone #7 of frozen transfection and the cell of #13 are melted, and beginning to cultivate and being expelled to from MCH (ICR) mice kind is that (CREA JAPAN is among the embryo of 8 cell stages that male and female mice copulation INC.) obtains; Injection rate is each embryo 10-12 cell.For the ES cell development becomes blastocyst with the embryo in culture medium after the overnight incubation, the embryo transfer of about 10 ES cell-injections is gone into foster mother ICR mice (CREA JAPAN, INC.) each Aconitum carmichaeli Debx. palace, the pseudo-fetus that wherein this mice has been carried out 2.5 days is handled.The pigmentation that effect in the tissue that is organized in host embryo (albefaction) source in TT2F (agouti) ES clone-source can be by eyes among the embryo and the offspring of survival are judged by the hair color.

(6) expression of people GIPF/Wnt3a mRNA in the chimeric Mus of transgenic

Prepare total RNA sample from the newborn chimeric intestinal of pPv01 Wnt3a/TT2F-#7 and #13 source, and carry out sxemiquantitative RT-PCR and analyze to detect the expression of GIPF-GFP mRNA.Use the synthetic first chain cDNA of Superscript III (Invitrogen), total RNA of the 500ng that wherein uses random hexamer and extract from pPv01 Wnt3a/TT2F-#7 and #13 source chimera and TT2F-source chimera contrast intestinal with Isogen (Nippon Gene) and RNasy Mini (QIAGEN).Using cDNA to carry out sxemiquantitative RT-PCR at every kind of primer to special annealing temperature analyzes.PCR product electrophoresis on 2% agarose gel is also dyeed with the pyridine of bromination second.The integrity of the amplification control RNA of the cDNA that produces by Mus GAPDH.(Pv01RT F1, R1), (Axin2 F is R) and below the annealing temperature of mGAPDH (mGAPDH5,3) nucleotide sequence and primer is listed in Axin 2 to be used for GIPF.

Pv01RT F1(SEQ ID NO：136)GCTCTGACACCAAGGAGACC

Pv01RT R1(60℃)(SEQ ID NO：137)CCCTAGGAATGCTCGTCAAG

Axin2 F(MUS)(SEQ ID NO：138)CAGGAGCCTCACCCTTCG

Axin2 R(MUS)(60℃)(SEQ ID NO：139)ACGCCGAGGTGCTTGCCC

mGAPDH5(SEQ ID NO：140)CACCATGGAGAAGGCCGGGGCCCAC

mGAPDH3(65℃)(SEQ ID NO：141)ATCATACTTGGCAGGTTTCTCCAGG

Shown in Figure 59, pPv01 Wnt3a/TT2F-#7 and-can detect the GIPF-Wnt3a transcript in the intestinal tissue of the newborn chimera (Pv01 Wnt3a:1 to 4) in #13 source.Result (Figure 59) shows also and contrasts chimera (TT2F:5 and 6) when comparing that Axin2 mRNA expresses obviously and raises.

(7) the white stability of beta-catenin in the chimeric Mus intestinal of transgenic

Be to estimate the influence of GIPF to Wnt/ beta-catenin white signal path, measure pPv01 Wnt3a/TT2F-#7 and-the chimeric small intestinal in #13 source and colon specimen in the white stability of beta-catenin.The method of measuring the white stability of beta-catenin has been described among the embodiment 17.Shown in Figure 60, (wild type: when 3 and 4) contrasting, GIPF and Wnt3a coexpression are induced white the stablizing of beta-catenin in pPv01 Wnt3a/TT2F-#7 embryo (E20.5:1 and 2) duodenum and the colon specimen with the contrast chimera.

(8) evaluation of phenotypic alternation in the chimeric Mus intestinal of transgenic

Newborn pPv01 Wnt3a/TT2F-#7 and-#13 source chimera young baby is at the tangible abdominal distension of performance.Perusal shows that small intestinal total length diameter obviously increases during the postmortem of new life's chimera, and wherein diameter is relevant with enhanced vascular surfaceization.With whole embryo, young baby or gastrointestinal tract are fixed in the Bouin liquid.For carrying out Histological evaluation, with paraffin-embedded section h and E (H﹠amp; E) dyeing.Shown in Figure 61, pPv01 Wnt3a/TT2F-#13 embryo the 20.5th day the embryo (E20.5) H﹠amp; The histopathological analysis of E section shows the villus cell number, and the hypertrophy of irregular branch and fine hair increases.The degree of these phenotypes is better than pPv01 GFP/TT2F-#2 source chimera [embodiment 30-(19)] person, shows by Wnt3a to express the effect that strengthens GIPF.

Embodiment 32

The RS-KO mouse ES cells

The structure of A.RS-KO carrier

The structure of RS-KO carrier (Figure 62A) carries out according to the method that describes below, and is depicted in Figure 62 B-1K.

Figure 62B (Stratagene) adds new restriction enzyme site (NruI, SgrAI and AscI) to pBluescript II SK (-).

The synthetic few dna fragmentation (SEQ ID NO:144 and 145) that is used for adding new restriction enzyme site at pBluescript II SK (-).

Joint A1:TCGAGTCGCGACACCGGCGGGCGCGCCC (SEQ ID NO:144)

Joint A2:TCGAGGGCGCGCCCGCCGGTGTCGCGAC (SEQ ID NO:145)

The joint A1 and the joint A2 of preparation are connected to among restriction endonuclease SalI and the XhoI predigested pBluescript II SK (-).Resulting plasmid pBlueLA contains the restriction enzyme site (NruI, SgrAI and AscI) of new interpolation.

Figure 62 C adds new restriction enzyme site (PacI, FseI and SalI) to pBlueLA.

The synthetic few dna fragmentation (SEQID NO:146 and 147) that is used for adding new restriction enzyme site at pBlueLA.

Joint B1:GGCCGCTTAATTAAGGCCGGCCGTCGACG (SEQ ID NO:146)

Joint B2:AATTCGTCGACGGCCGGCCTTAATTAAGC (SEQ ID NO:147)

The joint B1 and the joint B2 of preparation are connected to among restriction endonuclease NotI and the predigested pBlueLA of EcoRI.Resulting plasmid pBlueLAB contains the restriction enzyme site (PacI, FseI and SalI) of new interpolation.

The segmental preparation of Figure 62 D LoxP-Neo-B

The segmental preparation of LoxP-Neo-B is by handling LoxP-Neo with the T4DNA polymerase, and its pLoxP-STneo (WO 00/10383) from XhoI digestion obtains.

The preparation of Figure 62 E pBlueLAB-LoxP-Neo plasmid

The LoxP-Neo-B fragment is connected in the predigested pBlueLAB with restriction endonuclease EcoRV.Gained plasmid pBlueLAB-LoxP-Neo contains the LoxP-Neo-B fragment.

The segmental preparation of Figure 62 F DT-A

PMC1DT-A (GIBCO BRL) with XhoI and SalI digestion, is separated the DT-A fragment of gained and recovery with agarose gel electrophoresis.

The preparation of Figure 62 G pBlueLAB-LoxP-Neo-DT-A plasmid

The DT-A fragment is connected to among the predigested pBlueLAB-LoxP-Neo of restriction endonuclease XhoI.Gained plasmid pBlueLAB-LoxP-Neo-DT-A contains the DT-A fragment.

The preparation in Figure 62 H RS element 3 ' genome district

So that (accession number: AC090291) the mice sequence of Huo Deing is the upstream (RS3 ' FW2 that is used for PCR according to synthetic from GenBank; SEQ ID NO:148) and downstream (RS3 ' RV3; SEQ ID NO:149) primer, and the DNA in RS element 3 ' the genome district that is used to increase.

The preparation of RS3 ' FW2:TTGGCGCGCCCTCCCTAGGACTGCAGTTGAGCTCAGATTTGA (SEQ IDNO:148) is by adding the AscI recognition sequence at 5 ' end site, and the preparation of RS3 ' RV3:CCGCTCGAGTCTTACTGTCTCAGCAACAATAATATAAACAGGGG (SEQID NO:149) is by adding the XhoI recognition sequence at 5 ' end site.Carry out PCR with yeast artificial chromosome (BAC) clone RP23-435I4 (GenBank accession number AC090291) as template.The PCR product is digested with restriction endonuclease AscI and XhoI, and be connected in restriction endonuclease AscI and the predigested pBlueLAB of XhoI.Resulting plasmid contain specific RS element 3 ' genome district DNA sequence and between AscI and XhoI the zone do not have nucleotide sequence to replace, this plasmid with AscI and XhoI digestion, has been obtained 3 ' the genome district (approximately 2Kb) of RS element then.

Figure 62 I inserts 3 ' genome district of RS element in pBlueLAB-LoxP-Neo-DT-A

3 ' genome district of RS element is connected to among AscI and the predigested pBlueLAB-LoxP-Neo-DT-A of XhoI.Behind the bonding pad between 3 ' the genome district of checking pBlueLAB-LoxP-Neo-DT-A and RS element, obtain plasmid pBlueLAB-LoxP-Neo-DT-A-3 ' RS.

The preparation in 5 ' genome district of Figure 62 J RS element

So that (accession number: AC090291) the mice sequence of Huo Deing is the upstream (RS5 ' FW3 that is used for PCR according to synthetic from GenBank; SEQ ID NO:150) and downstream (RS5 ' RV3; SEQ ID NO:151) primer, and the DNA in RS element 5 ' the genome district that is used to increase.

The preparation of RS5 ' FW3:ATAAGAATGCGGCCGCAAAGCTGGTGGGTTAAGACTATCTCGTGAAGTG (SEQ ID NO:150) is by adding the NotI recognition sequence at 5 ' end site, and the preparation of RS5 ' RV3:ACGCGTCGACTCACAGGTTGGTCCCTCTCTGTGTGTGGTTGCTGT (SEQID NO:151) is by adding the SclI recognition sequence at 5 ' end site.Carry out PCR with yeast artificial chromosome (BAC) clone RP23-435I4 (GenBank accession number AC090291) as template.The PCR product is digested with restriction endonuclease NotI and SalI, and be connected in restriction endonuclease NotI and the predigested pBlueLAB of SalI.Resulting plasmid contain specified RS element 5 ' genome district DNA sequence and between NotI and SalI the zone do not have nucleotide sequence to replace, this plasmid with NotI and SalI digestion, has been obtained 5 ' the genome district (approximately 5Kb) of RS element then.

Figure 62 K inserts RS element 5 ' genome district in pBlueLAB-LoxP-Neo-DT-A-3 ' RS

RS 5 ' element genes group district is connected to among NotI and the predigested pBlueLAB-LoxP-Neo-DT-A-3 ' RS of SalI.Behind the bonding pad between checking pBlueLAB-LoxP-Neo-DT-A-3 ' RS and RS element 5 ' the genome district, made up the RS-KO carrier.

The preparation of B.RS-KO mouse ES cells

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publishes, and 1995 methods of describing are obtained the general procedure of mice embryonic and cultivation.According to AizawaShinichi, " Biomanual Series 8; Gene Targeting ", Yodosha publishes, 1995 methods of describing, just the RS-KO carrier is transferred in the C57BL/6X CBA F1 source mice TT2F ES cell ((Uchida, 1995), Lifetech oriental) with the NotI linearisation and through electroporation.The ES cell of electroporation is suspended in [DMEM (GIBCO) in the 20ml ES culture medium, 18% hyclone (GIBCO), 0.1mM2-mercaptoethanol (GIBCO), 1000U/ml LIF (leukaemia inhibitory factor, CHEMICONInternational, and be seeded in planting in advance of two 100mm and have on tissue culturing plastic's plate (Corning) of feeder cells (Invitrogen) Inc.)].After one day, culture medium is replaced by the culture medium that contains 0.75g/ml puromycin (Sigma).After this 7 days, the clone of the puromycin resistance that picking forms.Each is cloned in to grow in 24 orifice plates converges, then 2/3rds cultures are suspended in the frozen culture medium of 0.2ml [FBS+10%DMSO (Sigma)] and frozen in-80 ℃./ 3rd of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.

Be prepared as follows and be used for 3 ' the KO-probe that Southern analyzes.So that (accession number: AC090291) the mice sequence of Huo Deing serves as according to synthetic RS3 ' Southern FW1 (SEQ ID NO:152) and RS3 ' Southern RV2 (SEQ ID NO:153) primer, and the DNA in long RS element 3 ' the genome district of about 600mer that is used to increase from GenBank.

RS3′Southern FW1：TCTTACTAGAGTTCTCACTAGCTCT(SEQ ID NO：152)

RS3′Southern RV2：GGAACCAAAGAATGAGGAAGCTGTT(SEQ IDNO：153)

To carry out 0.8% agarose gel electrophoresis then from the genomic DNA of the TT2F cell separation of puromycin resistance with restriction endonuclease EcoR I (Takara Shuzo) digestion.

The separated DNA fragment is transferred to (GeneScreen, NEN on the film ^TMLife ScienceProducts), use the dna fragmentation (3 ' KO-probe) for preparing from RS element DNA 3 ' genome district to hybridize then as probe.The clone's of the ES in the hybridization banding pattern does not show as the band of an about 5.7Kb of molecular weight, and the clone of the ES in the hybridization shows as two bands (Figure 63) of about 5.7Kb of molecular weight and 7.4Kb.Also the RS-KO mice ES clone who selects is carried out karyotyping, it is according to AizawaShinichi, and " Biomanual Series 8, Gene Targeting ", Yodosha publishes, and 1995 methods of describing are carried out.The RS-KO mice ES clone of the caryogram of acting normally is used for further experiment.

Embodiment 33

The mutant animal of genetically modified GIPF deletion

The structure of A.pCk m4 KI carrier.

The structure that GIPF deletion mutant 4Ck gene is knocked in (pCk m4 KI) carrier (Figure 64 A) carries out according to the method that describes below, and is depicted in Figure 64 B-64K.

The preparation of Figure 64 B Ck P2 KI+AS KI

The synthetic few dna fragmentation (SEQID NO:154 and 155) that is used for adding new restriction enzyme site at Ck P2 KI.

(top) joint: GGCCAGGCGCGCCTTGC above the AscI (SEQ ID NO:154)

(bottom) joint: GGCCGCAAGGCGCGCCT below the AscI (SEQ ID NO:155)

Plane tie under last plane tie of the AscI of preparation and the AscI is connected to among the predigested Ck P2 of the restriction endonuclease NotI KI.Resulting plasmid Ck P2 KI+AS KI contains the restriction enzyme site (AscI) of new interpolation.

The preparation of Figure 64 C pBlueLAB+Nh

The synthetic few dna fragmentation (SEQID NO:156 and 157) that is used for adding new restriction enzyme site at pBlueLA.

Pac-Nhe-Fse S：TAAGGGCTAGCTAGGGCCGG(SEQ ID NO：156)

Pac-Nhe-Fse AS：CCCTAGCTAGCCCTTAAT(SEQ ID NO：157)

The Pac-Nhe-Fse S and the Pac-Nhe-Fse AS of preparation are connected to among restriction endonuclease PacI and the predigested pBlueLAB of FseI.Resulting plasmid pBlueLAB+Nh contains the restriction enzyme site (NheI) of new interpolation.

The preparation of Figure 64 D pBlueLAB+NhHp

The few dna fragmentation (SEQID NO:158 159) that synthesizes the new restriction enzyme site of in pBlueLAB+Nh interpolation.

S/HpaI/Hd-S：TCGAGTTAAC(SEQ ID NO：158)

S/HpaI/Hd-AS：AGCTGTTAAC(SEQ ID NO：159)

The S/HpaI/Hd-S and the S/HpaI/Hd-AS of preparation are connected to among restriction endonuclease SalI and the predigested pBlueLAB+Nh of HindIII.The restriction enzyme site (HpaI) that contains new interpolation among the resulting plasmid pBlueLAB+NhHp.

The preparation of Figure 64 E pCkpAP2

PCkP2+As KI is separated with agarose gel electrophoresis with HpaI and NheI digestion and with the 952bp fragment of gained and reclaim.The 952bp fragment is connected to among restriction endonuclease HpaI and the predigested pBlueLAB+NhHp of NheI.The plasmid pCkpAP2 of gained contains the 952bp fragment.

The preparation of Figure 64 F pCkpAMCS

Synthesize the few dna fragmentation (SEQ ID NO:160 and 161) that in pCkpAP2, adds restriction enzyme site.

SPFN joint-S:AGCTGTCGACTTAATTAAGGCCGGCCG (SEQ ID NO:160)

SPFN joint-AS:CTAGCGGCCGGCCTTAATTAAGTCGAC (SEQ ID NO:161)

The SPFNlinker-S and the SPFNlinker-AS of preparation are connected to among restriction endonuclease HindIII and the predigested CkpAMCS of NheI.The plasmid CkpAMCS of gained contains the restriction enzyme site (PacI) of new interpolation.

The preparation of Figure 64 G pCkP2 Δ P

PCkpAMCS is digested with HpaI and NheI, and the fragment that about 700bp of gained is long is separated and is reclaimed from agarose gel electrophoresis.The 700bp fragment is connected to among restriction endonuclease HpaI and the predigested pCkP2+As KI of NheI.The plasmid pCkP2 Δ P of gained contains this 700bp fragment.

The preparation of Figure 64 H pBS+PFN

The few dna fragmentation (SEQ ID NO:162 and 163) that synthesizes the new restriction enzyme site of in pBluescript II SK (-) interpolation.

S/PFN/Hd-S：TCGACTTAATTAAGGCCGGCCCTAGCTAGCA(SEQ IDNO：162)

S/PFN/Hd-AS：AGCTTGCTAGCTAGGGCCGGCCTTAATTAAG(SEQ IDNO：163)

The S/PFN/Hd-S and the S/PFN/Hd-AS of preparation are connected to among restriction endonuclease SalI and the HindIII predigested pBluescript II SK (-).The plasmid pBS+PFN of gained contains the restriction enzyme site (PacI, Fse, and NheI) of new interpolation.

The preparation of Figure 64 I pPSs3.8

With the mice sequence that obtains from GenBank (accession number K02159) is upstream (the PsecSP FW1 that is used for PCR according to synthetic; SEQ ID NO:164) and downstream (PsecSP RV; SEQ ID NO:165) primer, and be used for the promoter of amplification coding Ig κ and the DNA of targeting sequencing coding region.Intrinsic intron sequences is contained in the targeting sequencing coding region.

Prepare PsecSP FW1:CCTTAATTAAAGTTATGTGTCCTAGAGGGCTGCAAACTCAAGATC (SEQID NO:164) by adding the PacI recognition sequence, prepare PsecSP RV:TTGGCCGGCCTTGGCGCCAGTGGAACCTGGAATGATAAACACAAAGATTATTG (SEQ ID NO:165) by adding the FseI recognition sequence at 5 ' end site at 5 ' end site.((Uchida, 1995), mice genome Lifetechoriental) carries out PCR as template with being derived from TT2F ES cell.The PCR product is digested with restriction endonuclease PacI and FseI, and be connected to among restriction endonuclease PacI and the predigested pBS+PFN of FseI.The plasmid pPSs3.8 of gained contains the promoter of Ig κ and the dna fragmentation of targeting sequencing coding region.

Figure 64 J m4 (+SP) and m4 (preparation SP)

Sequence (Figure 43 with deletion mutant #4; SEQ ID NO:91) is synthetic upstream (the Sal kozak GIPF F that is used for PCR in basis; SEQ ID NO:166) and downstream (GIPF m4 RV Fse; SEQ IDNO:167) primer, and the DNA of the deletion mutant #4 sequence of the targeting sequencing of the GIPF that is used to increase and GIPF.

Prepare Sal kozak GIPF F:AAG CGTCGA CCA CCA TGC GGC TTG GGC TGT GTG (SEQ ID NO:166) by adding the SalI recognition sequence, prepare GIPF m4 RV Fse:ATG GCC GGC CCT ACATGG TGC CAT TGG CAG (SEQ ID NO:167) by adding the FseI recognition sequence at 5 ' end site at 5 ' end site.Carry out PCR with GIPF KI carrier as template.The PCR product with restriction endonuclease SalI and FseI digestion, is obtained m4 (+SP) fragment then.

(Figure 43 with the sequence of deletion mutant #4; SEQ ID NO:91) is the synthetic upstream (Hy01 (SP) FW that is used for PCR in basis; SEQ ID NO:168) and downstream (GIPF m4 RV Fse; SEQ ID NO:169) primer, and the DNA of the deletion mutant #4 sequence of the GIPF that is used to increase.

(SP) FW:AGCCGGGGGATCAAGGGGAAAAGGCAGAGG (SEQ ID NO:168) prepares GIPF m4 RV Fse:ATG GCC GGC CCTACA TGG TGC CAT TGG CAG (SEQ ID NO:169) by adding the FseI recognition sequence at 5 ' end site by prepare Hy01 at 5 ' end site interpolation phosphoric acid.Carry out PCR with GIPF KI carrier as template.The PCR product with restriction endonuclease FseI digestion, is obtained m4 (SP) fragment then.

The structure of Figure 64 K pCk m4 KI carrier

With m4 (+SP) fragment is connected to among restriction endonuclease SalI and the predigested pCkP2+As KI of FseI.Resulting pCk m4 KI carrier contains m4, and (+SP) fragment, it is used to produce GIPF deletion mutant mice.

The structure of B.pPSm4 KI carrier

The structure that GIPF deletion mutant 4PS gene is knocked in (pPS m4 KI) carrier (Figure 65 A) carries out according to the method that describes below, and is depicted in Figure 65 B-65C.

The preparation of Figure 65 B pPSs3.8m4

(SP) fragment is connected to among restriction endonuclease SfoI and the predigested pPSs3.8 of FseI with m4.The pPSs3.8m4 of gained contains m4 (SP) fragment.

The structure of Figure 65 C pPSm4 KI carrier

PPSs3.8m4 is digested with PacI and FseI, and the fragment that about 1.2Kb of gained is long is separated and is reclaimed from agarose gel electrophoresis.The 1.2Kb fragment is connected to among restriction endonuclease PacI and the predigested CkP2 Δ of the FseI P.The pPSm4 KI carrier of gained contains this 1.2Kb fragment, and it is used to produce GIPF deletion mutant mice.

The production of C.GIPF deletion mutant mice

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publish, and 1995 methods of describing are obtained mice embryonic, cultivation, the ES injection cell is gone into the embryo, is transplanted to the general procedure in foster mother uterus.

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publishes, 1995 methods of describing, with pCkm4 KI carrier and the Not I linearisation of pPSm4 KI carrier, and in electroporation is transferred to the RS-KO mouse ES cells.The RS-KO mouse ES cells of electroporation is suspended in [DMEM (GIBCO) in the 20ml ES culture medium, 18% hyclone (GIBCO), 0.1mM2-mercaptoethanol (GIBCO), 1000U/ml LIF (leukaemia inhibitory factor, CHEMICONInternational, and be seeded in planting in advance of two 100mm and have on tissue culturing plastic's plate (Corning) of feeder cells (Invitrogen) Inc.)].After one day, culture medium is replaced by the culture medium that contains 0.75g/ml puromycin (Sigma).After this 7 to 9 days, 24 clones forming of 24 clones forming of picking pCkm4 KI carrier electroporation RS-KO mouse ES cells and pPSm4 KI carrier electroporation RS-KO mouse ES cells respectively.Each is cloned in to grow in 12 orifice plates converges, then 2/3rds cultures are suspended in the frozen culture medium of 0.2ml [FBS+10%DMSO (Sigma)] and frozen in-80 ℃./ 3rd of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.To carry out 0.8% agarose gel electrophoresis then from the isolating genomic DNA of the RS-KO mouse ES cells of puromycin resistance with restriction endonuclease EcoR I (Takara Shuzo) digestion.The separated DNA fragment is transferred to (GeneScreen, NEN on the film ^TMLife Science Products), use the dna fragmentation of (embodiment 48 for XhoI-EcoR I, 1.3Kb (SEQ ID NO:67): WO00/10383) preparation to hybridize then as probe from IgJk-Ck genomic DNA 3 ' district.The clone's of the ES in the hybridization banding pattern does not show as the band of a molecular weight 15.6Kb.Respectively, the RS-KO clone in the pCkm4 KI hybridization shows as two bands of molecular weight 15.6Kb and 12.9Kb, and the RS-KO clone in the pPSm4 KI hybridization shows as two bands (Figure 66 and 67) of molecular weight 15.6Kb and 12.5Kb.Southern analyzes among 10 RS-KO mice ES clone #3 in (the homologous recombination rate is approximately 41.7%) the picking pCkm4 KI hybridization of back one and Southern and analyzes 7 RS-KO mice ES in (the homologous recombination rate is approximately 29.2%) the pPSm4 KI hybridization of back and clone among the #7 one.Also the ES clone who selects is carried out karyotyping, it is according to Aizawa Shinichi, and " Biomanual Series 8, GeneTargeting ", Yodosha publishes, and 1995 methods of describing are carried out.The clone that clone in the pCkm4 KI hybridization of the caryogram of will acting normally respectively among the RS-KO mice ES clone #3 and the RS-KO mice ES in the pPSm4 KI hybridization clone among the #7 is used for implanting to the embryo.

The cell of RS-KO mouse ES cells clone #7 in RS-KO mouse ES cells clone #3 and the pPSm4 KI hybridization in the frozen pCkm4 KI hybridization is melted, begin to cultivate and be expelled to embryo (the Tomizuka et al.Proc.Natl.Acad.Sci.USA of 8 cell stages that obtain from the male and female mice copulation of heavy chain immunoglobulin knock-out mice kind system, 97:722-727,2000) in; Injection rate is each embryo 10-12 cell.For the ES cell development becomes blastocyst with the embryo in culture medium after the overnight incubation, the embryo transfer of about 10 ES injection cells is gone into foster mother ICR mice, and (pseudo-fetus that this mice has been carried out 2.5 days is handled for CREAJAPAN, each Aconitum carmichaeli Debx. palace INC.).As the transplanting result of every kind of 80 embryonal vaccinations, 15 and 20 filial generation mice births are arranged respectively.Mosaic is judged by the degree of TT2F cell-source agouti appearance color (dark-brown) in host embryo (ICR)-source albefaction appearance color in the filial generation.In whole 15 filial generations, think that 9 mices (the pCkm4 gene is knocked in Mus) have in part agouti appearance color and whole 20 filial generations, think that 16 mices (the pPSm4 gene is knocked in Mus) have part agouti appearance color, illustrate the effect of RS-KO ES cell.As described above, other clone of the RS-KO mouse ES cells in RS-KO mouse ES cells and the pPSm4 KI hybridization obtains GIPF and produces mutant from the pCkm4 KI hybridization.

Mus is remained on 12/12-hour dark/illumination circulation (in 8:00 illumination in the morning), and arbitrarily accept the filtering water of 5 μ m and CE-2 food (CLEA JAPAN, INC.).After weaning stage male mouse is raised separately.

Embodiment 34

Genetically modified GIPF mutant animal

The structure of A.pCk VR KI carrier

The structure that GIPF variant Ck gene is knocked in (pCk VR KI) carrier (Figure 68 A) carries out according to the method that describes below, and is depicted in Figure 68 B-68C.

Figure 68 B has the preparation of GIPF variant and the GIPF variant (VR) of kozak (VR+kz)

Sequence with GenBank (accession number AK098225) is synthetic upstream (the VRCk Fw that is used for PCR in basis; SEQ ID NO:170) and downstream (VR KI Rv; SEQ ID NO:171) primer, and the 5 ' end site that is used to increase has the DNA of the GIPF variant of kozak.

Prepare VR Ck Fw:ACG CGT CGACCA CCA TGA TAT TCC GAG TCA GTG C (SEQ ID NO:170) by adding the SalI recognition sequence, prepare VR KI Rv:GGC CGG CCC TAG GCA GGCCCT GCA GAT GTG AGT GG (SEQ ID NO:171) by adding the FseI recognition sequence at 5 ' end site at 5 ' end site.Carry out PCR with GIPF KI carrier as template.The PCR product with restriction endonuclease SalI and FseI digestion, is obtained VR+kz then.

Sequence with GenBank (accession number AK098225) is synthetic upstream (the VRCk Fw that is used for PCR in basis; SEQ ID NO:172) and downstream (VR KI Rv; SEQ ID NO:173) primer, and the DNA of the GIPF variant that is used to increase.

Prepare VR Fw:ATG ATA TTC CGA GTC AGTGCC GAG GGG AGC CAG (SEQ ID NO:172) by adding phosphoric acid, prepare VR KI Rv:GGC CGG CCC TAG GCA GGC CCT GCA GATGTG AGT GG (SEQ ID NO:173) by adding the FseI recognition sequence at 5 ' end site at 5 ' end site.Carry out PCR with GIPF KI carrier as template.The PCR product with restriction endonuclease FseI digestion, is obtained VR then.

The structure of Figure 68 C pCk VR KI carrier

VR+kz is connected to among SalI and the predigested pCkP2+As KI of FseI.The pCkVR KI carrier of gained contains VR+kz, and it is used to produce GIPF variant mice.

The structure of B.pPS VR KI carrier

The structure (Figure 69 A) that GIPF variant PS gene is knocked in (pPS VR KI) carrier carries out according to the method that describes below, and is depicted in Figure 69 B-69C.

The preparation of Figure 69 B pPSs3.8VR

VR is connected in among restriction endonuclease SfoI and the predigested pPSs3.8 of FseI.The plasmid pPSs3.8VR of gained contains VR.

The structure of Figure 69 C pPS VR KI carrier

PPSs3.8VR is digested with SalI and FseI, and the fragment that about 1.5Kb of gained is long is separated and recovery with agarose gel electrophoresis.The 1.5Kb fragment is connected to among restriction endonuclease SalI and the predigested pCkP2 Δ of the FseI P.The pPS VR KI carrier of gained contains the 1.5Kb fragment, and it is used to produce GIPF variant mice.

The production of C.GIPF variant mice

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publish, and 1995 methods of describing are obtained mice embryonic, cultivation, the ES injection cell is gone into the embryo, is transplanted to the general procedure in foster mother uterus.

According to Aizawa Shinichi, " Biomanual Series 8, Gene Targeting ", Yodosha publishes, 1995 methods of describing, with pCkVR KI carrier and the Not I linearisation of pPSVR KI carrier, and in electroporation is transferred to the RS-KO mouse ES cells.The RS-KO mouse ES cells of electroporation is suspended in [DMEM (GIBCO) in the 20ml ES culture medium, 18% hyclone (GIBCO), 0.1mM2-mercaptoethanol (GIBCO), 1000U/ml LIF (leukaemia inhibitory factor, CHEMICONInternational, and be seeded in planting in advance of two 100mm and have on tissue culturing plastic's plate (Corning) of feeder cells (Invitrogen) Inc.)].After one day, culture medium is replaced by the culture medium that contains 0.75g/ml puromycin (Sigma).After this 7 to 9 days, 24 clones forming of 24 clones forming of picking pCkVRKI carrier electroporation RS-KO mouse ES cells and pPSVR KI carrier electroporation RS-KO mouse ES cells respectively.Each is cloned in to grow in 12 orifice plates converges, then 2/3rds cultures are suspended in the frozen culture medium of 0.2ml [FBS+10%DMSO (Sigma)] and frozen in-80 ℃./ 3rd of a remainder is seeded in 12 orifice plates of gelatin bag quilt and cultivated 2 days.Then, with Puregene DNA separating kit (Gentra System) isolation of genomic DNA.To carry out 0.8% agarose gel electrophoresis then from the isolating genomic DNA of the RS-KO mouse ES cells of puromycin resistance with restriction endonuclease EcoR I (Takara Shuzo) digestion.The separated DNA fragment is transferred to (GeneScreen, NEN on the film ^TMLife Science Products), use the dna fragmentation of (embodiment 48 for XhoI-EcoR I, 1.3Kb (SEQ ID NO:67): WO00/10383) preparation to hybridize then as probe from IgJk-Ck genomic DNA 3 ' district.The clone's of the ES in the hybridization banding pattern does not show as the band of a molecular weight 15.6Kb.Respectively, the RS-KO clone in the pCkVR KI hybridization shows as two bands of molecular weight 15.6Kb and 13.1Kb, and the RS-KO clone in the pPSVR KI hybridization shows as two bands (Figure 70 and 71) of molecular weight 15.6Kb and 12.9Kb.Southern analyzes among 12 RS-KO mice ES clone #3 in (the homologous recombination rate is approximately 37.5%) the picking pCkVR KI hybridization of back one and Southern and analyzes 8 RS-KO mice ES in (the homologous recombination rate is approximately 25%) the pPSVRKI hybridization of back and clone among the #14 one.Also the ES clone who selects is carried out karyotyping, it is according to Aizawa Shinichi, and " Biomanual Series 8, Gene Targeting ", Yodosha publishes, and 1995 methods of describing are carried out.One of a RS-KO mice ES clone clone #14 who clones the RS-KO mice ES clone in #3 and the pPSVR KI hybridization is used for implanting to the embryo in the pCkVR KI hybridization of the caryogram of will acting normally respectively.

The cell of RS-KO mice ES clone #14 in RS-KO mice ES clone #3 and the pPSVR KI hybridization in the frozen pCkVR KI hybridization is melted, begin to cultivate and be expelled to embryo (the Tomizuka et al.Proc.Natl.Acad.Sci.USA of 8 cell stages that obtain from the male and female mice copulation of heavy chain immunoglobulin knock-out mice kind system, 97:722-727,2000) in; Injection rate is each embryo 10-12 cell.For the ES cell development becomes blastocyst with the embryo in culture medium after the overnight incubation, the embryo transfer of about 10 ES injection cells is gone into foster mother ICR mice, and (pseudo-fetus that this mice has been carried out 2.5 days is handled for CREAJAPAN, each Aconitum carmichaeli Debx. palace INC.).As the transplanting result of every kind of 220 embryonal vaccinations, 68 and 60 filial generation mice births are arranged respectively.Mosaic is judged by the degree of TT2F cell-source agouti appearance color (dark-brown) in host embryo (ICR)-source albefaction appearance color in the filial generation.In whole 68 filial generations, think that 47 mices (the pCkVR gene is knocked in Mus) have part agouti appearance color, the effect of RS-KO mouse ES cells is described, in whole 60 filial generations, think that 38 mices (the pPSVR gene is knocked in Mus) have part agouti appearance color, illustrate the effect of RS-KO mouse ES cells.As described above, other clone of the RS-KO mouse ES cells in RS-KO mouse ES cells and the pPSVR KI hybridization obtains GIPF variant mice from the pCkVR KI hybridization.

Mus is remained on 12/12-hour dark/illumination circulation (in 8:00 illumination in the morning), and arbitrarily accept the filtering water of 5 μ m and CE-2 food (CLEA JAPAN, INC.).After weaning stage male mouse is raised separately.

Embodiment 35

Estimate the biologic activity of GIPF deletion mutant with transgenic GIPF deletion mutant-KI mice

The general pathology of above-described transgenic mouse small intestinal of following evaluation and colon changes and Histological change.Results GIPF deletion mutant-KI (Ckm4-KI and PSm4-KI) mice when 4 ages in week.GIPF deletion mutant-KI gastrointestinal shows substantially and compares slightly different (Figure 72) with matched group.Be the histopathology evaluation, gastrointestinal tract is downcut and is fixed in the formalin.Be Histological evaluation, with paraffin-embedded section h and E (H﹠amp; E) dyeing.The H﹠amp of small intestinal; The E section is shown in Figure 73 (low power) and Figure 74 (high power).

With comparing of contrast, the H﹠amp of GIPF deletion mutant-KI mouse small intestine; The E section shows that crypts length and number moderate raise.The rising degree of crypts length and number is tended to the height than Ckm4-KI on PSm4-KI.

Delete small intestinal and the colon analyzing specimen GIPF deletion mutant #4 gene expression and the inducing in mutant-KI mice source to beta-catenin white targeting Axin-2 gene expression with GIPF.Downcut the ileum of 50mg, colon and liver specimen are also used the liquid nitrogen quick freezing.With refrigerated section 1ml ISOGEN (NIPPON GENE) homogenate, and under the condition of suggestion, extract total RNA.For removing genomic DNA, with RNA solution DNA enzyme (WAKO; Deoxyribonuclease RT level) handled 15 minutes in 37 ℃.Then total RNA is used RNasy Mini (QIAGEN) purification under the condition of suggestion.For each sample, under the condition of suggestion, use Super Script III (Invitrogen) from the synthetic cDNA of total RNA of 500ng.Then cDNA is handled 20 minutes to digest residual RNA with the RNaseH (Invitrogen) of 1 unit in 37 ℃.Carry out PCR with synthetic cDNA as template.Two following primers are used to detect GIPF deletion mutant: PSm4RT F1:ATCAAGGGGAAAAGGCAGAG (SEQ ID NO:174) and CkpolyA R2:CGCTTGTGGGGAAGCCTCCAAGACC (SEQ ID NO:175).For detecting Axin-2, two primers below using: Axin2 F (MUS): CAGGAGCCTCACCCTTCG (SEQID NO:138) and Axin2 R (MUS): ACGCCGAGGTGCTTGCCC (SEQ ID NO:139).Mice GAPDH primer: mGAPDH5:CACCATGGAGAAGGCCGGGGCCCAC (SEQ ID NO:140), and mGAPDH3:ATCATACTTGGCAGGTTTCTCCAGG (SEQ ID NO:141) is used to detect the gene expression of house-keeping gene.The preparation of PCR reactant mixture is by adding the 10X LAPCR buffer II (Takara Shuzo) of following material: 2.5ul, every kind of dNTP mixture of the 2.5mM of 4ul (TakaraShuzo), 0.5ul every kind of primer of 10mM, 2ul with the 10X cDNA of sterile distilled water dilution and the LA Taq (Takara Shuzo) of 0.5ul, add sterile distilled water to 25 μ l.Be to detect GIPF deletion mutant and Axin-2, with the PCR reactant mixture 94 ℃ of insulations 2 minutes, with 94 ℃ 30 seconds, 60 ℃ of 30 seconds and 72 ℃ carried out 33 circulation in 30 seconds and react.Be to detect GAPDH, with the PCR reactant mixture 94 ℃ of insulations 2 minutes, with 94 ℃ 30 seconds, 65 ℃ of 30 seconds and 72 ℃ carried out 22 circulation in 30 seconds and react.

Detect GIPF deletion mutant gene expression in PSm4-KI mice ileum and the colon.Compared with the control, the expression of Axin-2 rising (Figure 75) in the PSm4-KI mice.This result shows that the deletion saltant of GIPF has the activity of inducing the white target gene of beta-catenin to express, and activates relevant with the white downstream signal path of beta-catenin.

Therefore this shows that GIPF deletion mutant #4 has the activity that stimulates beta-catenin white signal path.

Embodiment 36

Estimate the biologic activity of GIPF variant with transgenic GIPF variant-KI mice

The general pathology of above-described transgenic mouse small intestinal of following evaluation and colon changes and Histological change.Results GIPF variant-KI (CkVR-KI and PSVR-KI) mice when 4 ages in week.When comparing with control mice, the gross examination of skeletal muscle of the PSVR-KI of results shows its small intestinal weight (Figure 76) with tangible enterectasis and increase.Be the histopathology evaluation, gastrointestinal tract is downcut and is fixed in the formalin.Be Histological evaluation, with paraffin-embedded section h and E (H﹠amp; E) dyeing.The H﹠amp of small intestinal; The E section is shown in Figure 77 (low power) and Figure 78 (high power).

The H﹠amp of small intestinal; The E section shows that PSVR-KI is obviously different with contrast.Shown in Figure 76, to compare with control mice, the pit cell hypertrophy of the PSVR-KI of confirmation has increased the number of crypts length and paneth's cell.On the other hand, CkVR-KI does not show tangible enterectasis or crypts length increase (result does not show).The phenotype difference of estimating observed CkVR-KI and PSVR-KI is because its KI-carrier structure different.

With GIPF variant-small intestinal in KI mice source and inducing of expression of colon analyzing specimen GIPF variant gene and beta-catenin white targeting Axin-2 gene expression.Downcut the ileum of 50mg, colon and liver specimen are also used the liquid nitrogen quick freezing.With refrigerated section 1ml ISOGEN (NIPPON GENE) homogenate, and under the condition of suggestion, extract total RNA.For removing genomic DNA, with RNA solution DNA enzyme (WAKO; Deoxyribonuclease RT level) handled 15 minutes in 37 ℃.Then total RNA is used RNasy Mini (QIAGEN) purification under the condition of suggestion.For each sample, under the condition of suggestion, use Super Script III (Invitrogen) from the synthetic cDNA of total RNA of 500ng.Then cDNA is handled 20 minutes to digest residual RNA with the RNaseH (Invitrogen) of 1 unit in 37 ℃.Carry out PCR with synthetic cDNA as template.Two following primers are used to detect the GIPF variant: PSFLJRT F1:ATAACTTCTGCACCAAGTGTAAGGA (SEQ ID NO:176) and CkpolyA R2:CGCTTGTGGGGAAGCCTCCAAGACC (SEQ ID NO:175).For detecting Axin-2, two primers below using: Axin2 F (MUS): CAGGAGCCTCACCCTTCG (SEQ ID NO:138) and Axin2 R (MUS): ACGCCGAGGTGCTTGCCC (SEQ ID NO:139).Mice GAPDH primer: mGAPDH5:CACCATGGAGAAGGCCGGGGCCCAC (SEQ ID NO:140), and mGAPDH3:ATCATACTTGGCAGGTTTCTCCAGG (SEQ ID NO:141) is used to detect the gene expression of house-keeping gene.The preparation of PCR reactant mixture is by adding the 10X LA PCR buffer II (Takara Shuzo) of following material: 2.5ul, every kind of dNTP mixture of the 2.5mM of 4ul (Takara Shuzo), 0.5ul every kind of primer of 10mM, 2ul with the 10XcDNA of sterile distilled water dilution and the LA Taq (Takara Shuzo) of 0.5ul, add sterile distilled water to 25 μ l.Be to detect GIPF variant and Axin-2, with the PCR reactant mixture 94 ℃ of insulations 2 minutes, with 94 ℃ 30 seconds, 60 ℃ of 30 seconds and 72 ℃ carried out 33 circulation in 30 seconds and react.Be to detect GAPDH, with the PCR reactant mixture 94 ℃ of insulations 2 minutes, with 94 ℃ 30 seconds, 65 ℃ of 30 seconds and 72 ℃ carried out 23 circulation in 30 seconds and react.

Detect GIPF variant gene expression in PSVR-KI mice ileum and the colon.Compared with the control, the expression of Axin-2 rising (Figure 79) in the PSVR-KI mice.This result shows that the GIPF variant has the activity of inducing the white target gene of beta-catenin to express, and activates relevant with the white downstream signal path of beta-catenin.

Therefore this shows the activity that the GIPF variant has stimulates beta-catenin white signal path.

Embodiment 37

CD4+CD45RB ^HighT-cell transfer model

Report such as Aranda is by shifting CD4+CD45RB to the SCID of immunodeficiency mice ^HighThe t lymphocyte subset group induces the chronic enteritis similar to inflammatory bowel disease (J Immunol158:3464-3473).Therefore, following at CD4+CD45RB ^HighTest GIPF is to the therapeutic effect of the colitis of immune induction on the T cell transfer model.

Make 7 the week ages adult female C.B-17/Icr Crj-scid/scid mice (CHARLES RIVERJAPAN, INC.).From supplier's deliver goods before experiment, animal is raised separately 2 weeks in airy cage, and 12 hour daytime: circulate with the stabilate rhythm and pace of moving things night.Allow the water inlet of animal ad libitum access.

In the cell transfer same day (the 0th day), collect CD4+CD45RB from 20 BALB/c female mice spleens ^HighThe T-cell.Splenocyte is suspended from 2%FBS, 2mM EDTA, in the PBS sorting buffer, and with FACS (Becton Dickinson FACS Aria) sorting CD4 positive cell.Then with the CD4 positive cell of sorting with 2 * 10 ⁷Cell/ml is resuspended in the sorting buffer and the gate CD4 positive and CD45RB ^HighThe cell fraction.With the CD4+CD45RB that collects ^HighT-cell centrifugation and with 1.1 * 10 ⁶Cell/ml is suspended among the 6ml PBS.By peritoneal injection with CD4+CD45RB ^HighThe T-cell is with 4.5 * 10 ⁵Cell/mice is transferred to 13 female C.B-17/Icr Crj-scid/scid mices.

After the T-cell transfer, the concentration of monitoring serum amyloid A (SAA) is with monitoring inflammation progress.And, by losing weight the progress of feces denseness and occult blood test monitoring colitis.SAA concentration from shift raise in back 7 days and on T-cell transfer mice inflammation obvious.12 days rare feces and occult blood test positive individuals of appearance after the T-cell transfer.After the T-cell transfer 30 days, observe diarrhoea and macroscopic hemorrhage T-cell transfer mice.Further by 40 days, observe T-cell transfer mice and to lose weight.After the T-cell transfer 61 days, to the GIPF of 4 T-cell transfer mouse mainlines, 100 μ g/ doses injection 3 days.Also to 3 days PBS of the transcellular mouse mainline of T-as negative control.After the T-cell transfer 70 days, with these sacrifice of animal.The cutting-out intestinal also is fixed in the formalin.Be Histological evaluation, with paraffin-embedded section h and E (H﹠amp; E) dyeing.

Shown in Figure 80, compare with the mice of injecting with PBS, inject the H﹠amp of the large intestine section of 100 μ g/ dose GIPF Mus; Gland structure, the mitotic cell number of increase and the non-viable non-apoptotic cell fragment of minimizing of E dyeing clear display.The tissue pathologies change of the big intestinal mucosa that the T-cell transfer causes is alleviated by the GIPF injection really.Therefore, GIPF can be used for the treatment of and suffer from the patient that inflammatory bowel disease comprises the mucositis that Crohn disease causes.

Sequence table

＜110〉NUVELO INC (Nuvelo, Inc.and Kirin Beer Kapushiko Kaisha)

＜120〉gastrointestinal proliferative factor and uses thereof

<130>11926-194WO1

<150>US 60/539,605

<151>2004-01-27

<150>US 60/619,241

<151>2004-10-15

<160>178

<170>PatentIn version 3.3

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gggacactgt ccagcctcca ggcccatgca gaaagagttc agtgctactc tgcgtgattc 240

aagctttcct gaactggaac gtcgggggca aagcatacac acacactcca atccatccat 300

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tactcagtat taaggttggt ttttggagta gtccctgctg acgtgacaaa aagatctctc 360

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tacggcccag gcctgggcct cctgctgctg acccagcccc agaggtgtta gcaagagccg 540

tgtgctatcc accctccccg agaccacccc tccgaccagg ggcctggagc tggcgcgtga 600

ctatgcggct tgggctgtgt gtggtggccc tggttctgag ctggacgcac ctcaccatca 660

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gggagaatgc caacaggaac ctggccagga aggagagcaa ggaggcgggt gctggctctc 1320

gaagacgcaa ggggcagcaa cagcagcagc agcaagggac agtggggcca ctcacatctg 1380

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cacacataca cacacacaca cacacgcaca cctgaggcca ccagaagaca cttccatccc 1680

tcgggcccag cagtacacac ttggtttcca gagctcccag tggacatgtc agagacaaca 1740

cttcccagca tctgagacca aactgcagag gggagccttc tggagaagct gctgggatcg 1800

gaccagccac tgtggcagat gggagccaag cttgaggact gctggtggcc tgggaagaaa 1860

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ccaaatagcc tgctcagtag agggtcatgc agacaaggaa gaaaacttag gggctgctct 2160

gacggtggta aacaggctgt ctatatcctt gttactcaga gcatggcccg gcagcagtgt 2220

tgtcacaggg cagcttgtta ggaatgataa tctcaggtct cattccagac ctggagagcc 2280

atgagtctaa attttaagat tcctgatgat tggcatgtta cccaaatttg agaagtgct 2339

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gccaaaggct gtgagctctg ctctgaagtc aacggctgcc tcaagtgctc acccaagctg 180

ttcatcctgc tggagaggaa cgacatccgc caggtgggcg tctgcttgcc gtcctgccca 240

cctggatact tcgacgcccg caaccccgac atgaacaagt gcatcaaatg caagatcgag 300

cactgtgagg cctgcttcag ccataacttc tgcaccaagt gtaaggaggg cttgtacctg 360

cacaagggcc gctgctatcc agcttgtccc gagggctcct cagctgccaa tggcaccatg 420

gagtgcagta gtcctgcgca atgtgaaatg agcgagtggt ctccgtgggg gccctgctcc 480

aagaagcagc agctctgtgg tttccggagg ggctccgagg agcggacacg cagggtgcta 540

catgcccctg tgggggacca tgctgcctgc tctgacacca aggagacccg gaggtgcaca 600

gtgaggagag tgccgtgtcc tgaggggcag aagaggagga agggaggcca gggccggcgg 660

gagaatgcca acaggaacct ggccaggaag gagagcaagg aggcgggtgc tggctctcga 720

agacgcaagg ggcagcaaca gcagcagcag caagggacag tggggccact cacatctgca 780

gggcctgcc 789

<210>4

<211>263

<212>PRT

＜213〉people (Homo sapiens)

<400>4

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile

20 25 30

Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser

35 40 45

Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu

50 55 60

Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

65 70 75 80

Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys

85 90 95

Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr

100 105 110

Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala

115 120 125

Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser

130 135 140

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

145 150 155 160

Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr

165 170 175

Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp

180 185 190

Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu

195 200 205

Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn

210 215 220

Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg

225 230 235 240

Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro

245 250 255

Leu Thr Ser Ala Gly Pro Ala

260

<210>5

<211>927

<212>DNA

＜213〉artificial sequence

<220>

＜223〉GIPF V5His labelling

<400>5

atgcggcttg ggctgtgtgt ggtggccctg gttctgagct ggacgcacct caccatcagc 60

agccggggga tcaaggggaa aaggcagagg cggatcagtg ccgaggggag ccaggcctgt 120

gccaaaggct gtgagctctg ctctgaagtc aacggctgcc tcaagtgctc acccaagctg 180

ttcatcctgc tggagaggaa cgacatccgc caggtgggcg tctgcttgcc gtcctgccca 240

cctggatact tcgacgcccg caaccccgac atgaacaagt gcatcaaatg caagatcgag 300

cactgtgagg cctgcttcag ccataacttc tgcaccaagt gtaaggaggg cttgtacctg 360

cacaagggcc gctgctatcc agcttgtccc gagggctcct cagctgccaa tggcaccatg 420

gagtgcagta gtcctgcgca atgtgaaatg agcgagtggt ctccgtgggg gccctgctcc 480

aagaagcagc agctctgtgg tttccggagg ggctccgagg agcggacacg cagggtgcta 540

catgcccctg tgggggacca tgctgcctgc tctgacacca aggagacccg gaggtgcaca 600

gtgaggagag tgccgtgtcc tgaggggcag aagaggagga agggaggcca gggccggcgg 660

gagaatgcca acaggaacct ggccaggaag gagagcaagg aggcgggtgc tggctctcga 720

agacgcaagg ggcagcaaca gcagcagcag caagggacag tggggccact cacatctgca 780

gggcctgcca agggcaattc tgcagatatc cagcacagtg gcggccgctc gagtctagag 840

ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt 900

accggtcatc atcaccatca ccattga 927

<210>6

<211>308

<212>PRT

＜213〉artificial sequence

<220>

＜223〉GIPF V5His labelling

<400>6

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile

20 25 30

Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser

35 40 45

Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu

50 55 60

Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

65 70 75 80

Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys

85 90 95

Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr

100 105 110

Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala

115 120 125

Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser

130 135 140

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

145 150 155 160

Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr

165 170 175

Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp

180 185 190

Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu

195 200 205

Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn

210 215 220

Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg

225 230 235 240

Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro

245 250 255

Leu Thr Ser Ala Gly Pro Ala Lys Gly Asn Ser Ala Asp Ile Gln His

260 265 270

Ser Gly Gly Arg Ser Ser Leu Glu Gly Pro Arg Phe Glu Gly Lys Pro

275 280 285

Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His

290 295 300

His His His His

305

<210>7

<211>60

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(60)

<400>7

atg cgg ctt ggg ctg tgt gtg gtg gcc ctg gtt ctg agc tgg acg cac 48

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

ctc acc atc agc 60

Leu Thr Ile Ser

20

<210>8

<211>20

<212>PRT

＜213〉people (Homo sapiens)

<400>8

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser

20

<210>9

<211>732

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(729)

<400>9

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag 288

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc 336

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tcc tca gct gcc aat ggc acc atg gag tgc agt agt cct gcg caa tgt 384

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

gaa atg agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag 432

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg aca cgc agg gtg cta 480

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

cat gcc cct gtg ggg gac cat gct gcc tgc tct gac acc aag gag acc 528

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

cgg agg tgc aca gtg agg aga gtg ccg tgt cct gag ggg cag aag agg 576

Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

agg aag gga ggc cag ggc cgg cgg gag aat gcc aac agg aac ctg gcc 624

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala

195 200 205

agg aag gag agc aag gag gcg ggt gct ggc tct cga aga cgc aag ggg 672

Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly

210 215 220

cag caa cag cag cag cag caa ggg aca gtg ggg cca ctc aca tct gca 720

Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro Leu Thr Ser Ala

225 230 235 240

ggg cct gcc tag 732

Gly Pro Ala

<210>10

<211>243

<212>PRT

＜213〉people (Homo sapiens)

<400>10

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala

195 200 205

Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly

210 215 220

Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro Leu Thr Ser Ala

225 230 235 240

Gly Pro Ala

<210>11

<211>696

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(696)

<400>11

atc agt gcc gag ggg agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc 48

Ile Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys

1 5 10 15

tct gaa gtc aac ggc tgc ctc aag tgc tca ccc aag ctg ttc atc ctg 96

Ser Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu

20 25 30

ctg gag agg aac gac atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc 144

Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys

35 40 45

cca cct gga tac ttc gac gcc cgc aac ccc gac atg aac aag tgc atc 192

Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile

50 55 60

aaa tgc aag atc gag cac tgt gag gcc tgc ttc agc cat aac ttc tgc 240

Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys

65 70 75 80

acc aag tgt aag gag ggc ttg tac ctg cac aag ggc cgc tgc tat cca 288

Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro

85 90 95

gct tgt ccc gag ggc tcc tca gct gcc aat ggc acc atg gag tgc agt 336

Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser

100 105 110

agt cct gcg caa tgt gaa atg agc gag tgg tct ccg tgg ggg ccc tgc 384

Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys

115 120 125

tcc aag aag cag cag ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg 432

Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg

130 135 140

aca cgc agg gtg cta cat gcc cct gtg ggg gac cat gct gcc tgc tct 480

Thr Arg Arg yal Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser

145 150 155 160

gac acc aag gag acc cgg agg tgc aca gtg agg aga gtg ccg tgt cct 528

Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro

165 170 175

gag ggg cag aag agg agg aag gga ggc cag ggc cgg cgg gag aat gcc 576

Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala

180 185 190

aac agg aac ctg gcc agg aag gag agc aag gag gcg ggt gct ggc tct 624

Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser

195 200 205

cga aga cgc aag ggg cag caa cag cag cag cag caa ggg aca gtg ggg 672

Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly

210 215 220

cca ctc aca tct gca ggg cct gcc 696

Pro Leu Thr Ser Ala Gly Pro Ala

225 230

<210>12

<211>232

<212>PRT

＜213〉people (Homo sapiens)

<400>12

Ile Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys

1 5 10 15

Ser Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu

20 25 30

Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys

35 40 45

Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile

50 55 60

Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys

65 70 75 80

Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro

85 90 95

Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser

100 105 110

Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys

115 120 125

Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg

130 135 140

Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser

145 150 155 160

Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro

165 170 175

Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala

180 185 190

Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser

195 200 205

Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly

210 215 220

Pro Leu Thr Ser Ala Gly Pro Ala

225 230

<210>13

<211>168

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(168)

<400>13

agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag ctc tgt 48

Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys

1 5 10 15

ggt ttc cgg agg ggc tcc gag gag cgg aca cgc agg gtg cta cat gcc 96

Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala

20 25 30

cct gtg ggg gac cat gct gcc tgc tct gac acc aag gag acc cgg agg 144

Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg

35 40 45

tgc aca gtg agg aga gtg ccg tgt 168

Cys Thr Val Arg Arg Val Pro Cys

50 55

<210>14

<211>56

<212>PRT

＜213〉people (Homo sapiens)

<400>14

Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys

1 5 10 15

Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala

20 25 30

Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg

35 40 45

Cys Thr Val Arg Arg Val Pro Cys

50 55

<210>15

<211>756

<212>DNA

＜213〉artificial sequence

<220>

＜223〉GIPF+sigP-furin

<220>

<221>CDS

<222>(1)..(756)

<400>15

atg cgg ctt ggg ctg tgt gtg gtg gcc ctg gtt ctg agc tgg acg cac 48

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

ctc acc atc agc atc agt gcc gag ggg agc cag gcc tgt gcc aaa ggc 96

Leu Thr Ile Ser Ile Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly

20 25 30

tgt gag ctc tgc tct gaa gtc aac ggc tgc ctc aag tgc tca ccc aag 144

Cys Glu Leu Cys Ser Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys

35 40 45

ctg ttc atc ctg ctg gag agg aac gac atc cgc cag gtg ggc gtc tgc 192

Leu Phe Ile Leu Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys

50 55 60

ttg ccg tcc tgc cca cct gga tac ttc gac gcc cgc aac ccc gac atg 240

Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met

65 70 75 80

aac aag tgc atc aaa tgc aag atc gag cac tgt gag gcc tgc ttc agc 288

Asn Lys Cys Ile Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser

85 90 95

cat aac ttc tgc acc aag tgt aag gag ggc ttg tac ctg cac aag ggc 336

His Asn Phe Cys Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly

100 105 110

cgc tgc tat cca gct tgt ccc gag ggc tcc tca gct gcc aat ggc acc 384

Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr

115 120 125

atg gag tgc agt agt cct gcg caa tgt gaa atg agc gag tgg tct ccg 432

Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro

130 135 140

tgg ggg ccc tgc tcc aag aag cag cag ctc tgt ggt ttc cgg agg ggc 480

Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly

145 150 155 160

tcc gag gag cgg aca cgc agg gtg cta cat gcc cct gtg ggg gac cat 528

Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His

165 170 175

gct gcc tgc tct gac acc aag gag acc cgg agg tgc aca gtg agg aga 576

Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg

180 185 190

gtg ccg tgt cct gag ggg cag aag agg agg aag gga ggc cag ggc cgg 624

Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg

195 200 205

cgg gag aat gcc aac agg aac ctg gcc agg aag gag agc aag gag gcg 672

Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala

210 215 220

ggt gct ggc tct cga aga cgc aag ggg cag caa cag cag cag cag caa 720

Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln

225 230 235 240

ggg aca gtg ggg cca ctc aca tct gca ggg cct gcc 756

Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

245 250

<210>16

<211>252

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic construct

<400>16

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser Ile Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly

20 25 30

Cys Glu Leu Cys Ser Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys

35 40 45

Leu Phe Ile Leu Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys

50 55 60

Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met

65 70 75 80

Asn Lys Cys Ile Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser

85 90 95

His Asn Phe Cys Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly

100 105 110

Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr

115 120 125

Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro

130 135 140

Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly

145 150 155 160

Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His

165 170 175

Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg

180 185 190

Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg

195 200 205

Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala

210 215 220

Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln

225 230 235 240

Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

245 250

<210>17

<211>789

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the GIPF-furin cleavage site of undergoing mutation

<220>

<221>CDS

<222>(1)..(789)

<400>17

atg cgg ctt ggg ctg tgt gtg gtg gcc ctg gtt ctg agc tgg acg cac 48

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

ctc acc atc agc agc cgg ggg atc aag ggg aaa cag cag agg cgg atc 96

Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Gln Gln Arg Arg Ile

20 25 30

agt gcc gag ggg agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct 144

Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser

35 40 45

gaa gtc aac ggc tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg 192

Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu

50 55 60

gag agg aac gac atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca 240

Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

65 70 75 80

cct gga tac ttc gac gcc cgc aac ccc gac atg aac aag tgc atc aaa 288

Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys

85 90 95

tgc aag atc gag cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc 336

Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr

100 105 110

aag tgt aag gag ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct 384

Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala

115 120 125

tgt ccc gag ggc tcc tca gct gcc aat ggc acc atg gag tgc agt agt 432

Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser

130 135 140

cct gcg caa tgt gaa atg agc gag tgg tct ccg tgg ggg ccc tgc tcc 480

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

145 150 155 160

aag aag cag cag ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg aca 528

Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr

165 170 175

cgc agg gtg cta cat gcc cct gtg ggg gac cat gct gcc tgc tct gac 576

Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp

180 185 190

acc aag gag acc cgg agg tgc aca gtg agg aga gtg ccg tgt cct gag 624

Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu

195 200 205

ggg cag aag agg agg aag gga ggc cag ggc cgg cgg gag aat gcc aac 672

Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn

210 215 220

agg aac ctg gcc agg aag gag agc aag gag gcg ggt gct ggc tct cga 720

Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg

225 230 235 240

aga cgc aag ggg cag caa cag cag cag cag caa ggg aca gtg ggg cca 768

Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro

245 250 255

ctc aca tct gca ggg cct gcc 789

Leu Thr Ser Ala Gly Pro Ala

260

<210>18

<211>263

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic construct

<400>18

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Gln Gln Arg Arg Ile

20 25 30

Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser

35 40 45

Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu

50 55 60

Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

65 70 75 80

Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys

85 90 95

Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr

100 105 110

Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala

115 120 125

Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser

130 135 140

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

145 150 155 160

Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr

165 170 175

Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp

180 185 190

Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu

195 200 205

Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn

210 215 220

Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg

225 230 235 240

Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro

245 250 255

Leu Thr Ser Ala Gly Pro Ala

260

<210>19

<211>12

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(12)

<400>19

agg cag agg cgg 12

Arg Gln Arg Arg

1

<210>20

<211>4

<212>PRT

＜213〉people (Homo sapiens)

<400>20

Arg Gln Arg Arg

1

<210>21

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Tu Bian furin cleavage site

<220>

<221>CDS

<222>(1)..(12)

<400>21

cag cag agg cgg 12

Gln Gln Arg Arg

1

<210>22

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic construct

<400>22

Gln Gln Arg Arg

1

<210>23

<211>272

<212>PRT

＜213〉people (Homo sapiens)

<400>23

Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met

1 5 10 15

Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg

20 25 30

Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys

35 40 45

Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala

50 55 60

Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys

65 70 75 80

Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr

85 90 95

Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr

100 105 110

Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn

115 120 125

Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser

130 135 140

Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr

145 150 155 160

Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val

165 170 175

Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro

180 185 190

Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys

195 200 205

Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn

210 215 220

Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser

225 230 235 240

Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys

245 250 255

Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His

260 265 270

<210>24

<211>16

<212>PRT

＜213〉people (Homo sapiens)

<400>24

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

1 5 10 15

<210>25

<211>32

<212>PRT

＜213〉people (Homo sapiens)

<400>25

Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly Ser Gln Ala Cys Ala

1 5 10 15

Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly Cys Leu Lys Cys Ser

20 25 30

<210>26

<211>17

<212>PRT

＜213〉people (Homo sapiens)

<400>26

Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys

1 5 10 15

Lys

<210>27

<211>4

<212>PRT

＜213〉people (Homo sapiens)

<400>27

Asn Gly Thr Met

1

<210>28

<211>1170

<212>PRT

＜213〉people (Homo sapiens)

<400>28

Met Gly Leu Ala Trp Gly Leu Gly Val Leu Phe Leu Met His Val Cys

1 5 10 15

Gly Thr Asn Arg Ile Pro Glu Ser Gly Gly Asp Asn Ser Val Phe Asp

20 25 30

Ile Phe Glu Leu Thr Gly Ala Ala Arg Lys Gly Ser Gly Arg Arg Leu

35 40 45

Val Lys Gly Pro Asp Pro Ser Ser Pro Ala Phe Arg Ile Glu Asp Ala

50 55 60

Asn Leu Ile Pro Pro Val Pro Asp Asp Lys Phe Gln Asp Leu Val Asp

65 70 75 80

Ala Val Arg Thr Glu Lys Gly Phe Leu Leu Leu Ala Ser Leu Arg Gln

85 90 95

Met Lys Lys Thr Arg Gly Thr Leu Leu Ala Leu Glu Arg Lys Asp His

100 105 110

Ser Gly Gln Val Phe Ser Val Val Ser Asn Gly Lys Ala Gly Thr Leu

115 120 125

Asp Leu Ser Leu Thr Val Gln Gly Lys Gln His Val Val Ser Val Glu

130 135 140

Glu Ala Leu Leu Ala Thr Gly Gln Trp Lys Ser Ile Thr Leu Phe Val

145 150 155 160

Gln Glu Asp Arg Ala Gln Leu Tyr Ile Asp Cys Glu Lys Met Glu Asn

165 170 175

Ala Glu Leu Asp Val Pro Ile Gln Ser Val Phe Thr Arg Asp Leu Ala

180 185 190

Ser Ile Ala Arg Leu Arg Ile Ala Lys Gly Gly Val Asn Asp Asn Phe

195 200 205

Gln Gly Val Leu Gln Asn Val Arg Phe Val Phe Gly Thr Thr Pro Glu

210 215 220

Asp Ile Leu Arg Asn Lys Gly Cys Ser Ser Ser Thr Ser Val Leu Leu

225 230 235 240

Thr Leu Asp Asn Asn Val Val Asn Gly Ser Ser Pro Ala Ile Arg Thr

245 250 255

Asn Tyr Ile Gly His Lys Thr Lys Asp Leu Gln Ala Ile Cys Gly Ile

260 265 270

Ser Cys Asp Glu Leu Ser Ser Met Val Leu Glu Leu Arg Gly Leu Arg

275 280 285

Thr Ile Val Thr Thr Leu Gln Asp Ser Ile Arg Lys Val Thr Glu Glu

290 295 300

Asn Lys Glu Leu Ala Asn Glu Leu Arg Arg Pro Pro Leu Cys Tyr His

305 310 315 320

Asn Gly Val Gln Tyr Arg Asn Asn Glu Glu Trp Thr Val Asp Ser Cys

325 330 335

Thr Glu Cys His Cys Gln Asn Ser Val Thr Ile Cys Lys Lys Val Ser

340 345 350

Cys Pro Ile Met Pro Cys Ser Asn Ala Thr Val Pro Asp Gly Glu Cys

355 360 365

Cys Pro Arg Cys Trp Pro Ser Asp Ser Ala Asp Asp Gly Trp Ser Pro

370 375 380

Trp Ser Glu Trp Thr Ser Cys Ser Thr Ser Cys Gly Asn Gly Ile Gln

385 390 395 400

Gln Arg Gly Arg Ser Cys Asp Ser Leu Asn Asn Arg Cys Glu Gly Ser

405 410 415

Ser Val Gln Thr Arg Thr Cys His Ile Gln Glu Cys Asp Lys Arg Phe

420 425 430

Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser

435 440 445

Val Thr Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser

450 455 460

Pro Ser Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu

465 470 475 480

Thr Lys Ala Cys Lys Lys Asp Ala Cys Pro Ile Asn Gly Gly Trp Gly

485 490 495

Pro Trp Ser Pro Trp Asp Ile Cys Ser Val Thr Cys Gly Gly Gly Val

500 505 510

Gln Lys Arg Ser Arg Leu Cys Asn Asn Pro Thr Pro Gln Phe Gly Gly

515 520 525

Lys Asp Cys Val Gly Asp Val Thr Glu Asn Gln Ile Cys Asn Lys Gln

530 535 540

Asp Cys Pro Ile Asp Gly Cys Leu Ser Asn Pro Cys Phe Ala Gly Val

545 550 555 560

Lys Cys Thr Ser Tyr Pro Asp Gly Ser Trp Lys Cys Gly Ala Cys Pro

565 570 575

Pro Gly Tyr Ser Gly Asn Gly Ile Gln Cys Thr Asp Val Asp Glu Cys

580 585 590

Lys Glu Val Pro Asp Ala Cys Phe Asn His Asn Gly Glu His Arg Cys

595 600 605

Glu Asn Thr Asp Pro Gly Tyr Asn Cys Leu Pro Cys Pro Pro Arg Phe

610 615 620

Thr Gly Ser Gln Pro Phe Gly Gln Gly Val Glu His Ala Thr Ala Asn

625 630 635 640

Lys Gln Val Cys Lys Pro Arg Asn Pro Cys Thr Asp Gly Thr His Asp

645 650 655

Cys Asn Lys Asn Ala Lys Cys Asn Tyr Leu Gly His Tyr Ser Asp Pro

660 665 670

Met Tyr Arg Cys Glu Cys Lys Pro Gly Tyr Ala Gly Asn Gly Ile Ile

675 680 685

Cys Gly Glu Asp Thr Asp Leu Asp Gly Trp Pro Asn Glu Asn Leu Val

690 695 700

Cys Val Ala Asn Ala Thr Tyr His Cys Lys Lys Asp Asn Cys Pro Asn

705 710 715 720

Leu Pro Asn Ser Gly Gln Glu Asp Tyr Asp Lys Asp Gly Ile Gly Asp

725 730 735

Ala Cys Asp Asp Asp Asp Asp Asn Asp Lys Ile Pro Asp Asp Arg Asp

740 745 750

Asn Cys Pro Phe His Tyr Asn Pro Ala Gln Tyr Asp Tyr Asp Arg Asp

755 760 765

Asp Val Gly Asp Arg Cys Asp Asn Cys Pro Tyr Asn His Asn Pro Asp

770 775 780

Gln Ala Asp Thr Asp Asn Asn Gly Glu Gly Asp Ala Cys Ala Ala Asp

785 790 795 800

Ile Asp Gly Asp Gly Ile Leu Asn Glu Arg Asp Asn Cys Gln Tyr Val

805 810 815

Tyr Asn Val Asp Gln Arg Asp Thr Asp Met Asp Gly Val Gly Asp Gln

820 825 830

Cys Asp Asn Cys Pro Leu Glu His Asn Pro Asp Gln Leu Asp Ser Asp

835 840 845

Ser Asp Arg Ile Gly Asp Thr Cys Asp Asn Asn Gln Asp Ile Asp Glu

850 855 860

Asp Gly His Gln Asn Asn Leu Asp Asn Cys Pro Tyr Val Pro Asn Ala

865 870 875 880

Asn Gln Ala Asp His Asp Lys Asp Gly Lys Gly Asp Ala Cys Asp His

885 890 895

Asp Asp Asp Asn Asp Gly Ile Pro Asp Asp Lys Asp Asn Cys Arg Leu

900 905 910

Val Pro Asn Pro Asp Gln Lys Asp Ser Asp Gly Asp Gly Arg Gly Asp

915 920 925

Ala Cys Lys Asp Asp Phe Asp His Asp Ser Val Pro Asp Ile Asp Asp

930 935 940

Ile Cys Pro Glu Asn Val Asp Ile Ser Glu Thr Asp Phe Arg Arg Phe

945 950 955 960

Gln Met Ile Pro Leu Asp Pro Lys Gly Thr Ser Gln Asn Asp Pro Asn

965 970 975

Trp Val Val Arg His Gln Gly Lys Glu Leu Val Gln Thr Val Asn Cys

980 985 990

Asp Pro Gly Leu Ala Val Gly Tyr Asp Glu Phe Asn Ala Val Asp Phe

995 1000 1005

Ser Gly Thr Phe Phe Ile Asn Thr Glu Arg Asp Asp Asp Tyr Ala

1010 1015 1020

Gly Phe Val Phe Gly Tyr Gln Ser Ser Ser Arg Phe Tyr Val Val

1025 1030 1035

Met Trp Lys Gln Val Thr Gln Ser Tyr Trp Asp Thr Asn Pro Thr

1040 1045 1050

Arg Ala Gln Gly Tyr Ser Gly Leu Ser Val Lys Val Val Asn Ser

1055 1060 1065

Thr Thr Gly Pro Gly Glu His Leu Arg Asn Ala Leu Trp His Thr

1070 1075 1080

Gly Asn Thr Pro Gly Gln Val Arg Thr Leu Trp His Asp Pro Arg

1085 1090 1095

His Ile Gly Trp Lys Asp Phe Thr Ala Tyr Arg Trp Arg Leu Ser

1100 1105 1110

His Arg Pro Lys Thr Gly Phe Ile Arg Val Val Met Tyr Glu Gly

1115 1120 1125

Lys Lys Ile Met Ala Asp Ser Gly Pro Ile Tyr Asp Lys Thr Tyr

1130 1135 1140

Ala Gly Gly Arg Leu Gly Leu Phe Val Phe Ser Gln Glu Met Val

1145 1150 1155

Phe Phe Ser Asp Leu Lys Tyr Glu Cys Arg Asp Pro

1160 1165 1170

<210>29

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>29

gaccatgctg cctgctctga cac 23

<210>30

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>30

cacccgcctc cttgctctcc 20

<210>31

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>31

gggggagacc acaccacctg ct 22

<210>32

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>32

ttggacctcg gctccttgct gttc 24

<210>33

<211>5495

<212>DNA

＜213〉mice (Mus musculus)

<220>

<221>misc_feature

<222>(1)..(5495)

＜223〉n=G, A, T, or C

<400>33

cttatctttc tcctttatta acggttgctg ttgtatccat aactcaattc caaaggatat 60

aaaccttaac atatagatat aattttgtgt accttctatg aaacagcatt aaagcaaaga 120

agttcaaata gaaagactgg cttagttatt attaactaag agatgctagt gagttctaaa 180

ttaataccat ttaaaattta taatttgcag aattaccacc accaccacca ctcagcccag 240

gaaaagttac aaagaactgg ctgtaccatt tgtttgtttt cctccttttt agagttcttt 300

tatttatgtg tgagtgaatg ccatgtactt gtggatgcag aggctgtcag attccttgca 360

gctggagtaa cagacagttg tgagctactt atagtactag aactaagatc ctatggaaga 420

gcagcgagtg ccactaactg ctgagccacc tctccagccc atttctttat ttttcaatga 480

acaaataata agcagtccta tgtgacatgc ttctaaagca aaagatataa tatttagtat 540

tatatacatt aataataaaa tacattatct tctaagaatt gaagtctcaa ctatgaaaat 600

cagcagttct ctgtcagaga agcccaagcg cttccacgca tgcttggaga gggggttaag 660

ctttcgccta cccactgctc tgttcctctt cagtgaggag ggtttttgta cagccagaca 720

gtggagtact accactgtgg tggacgttcg gtggaggcac caagctggaa atcaaacgta 780

agtagaatcc aaagtctctt tcttccgttg tctatgtctg tggcttctat gtctaaaaat 840

gatgtataaa atcttactct gaaaccagat tctggcactc tccaaggcaa agatacagag 900

taactccgta agcaaagctg ggaataggct agacatgttc tctggagaat gaatgccagt 960

gtaataatta acacaagtga tagtttcaga aatgctcaaa gaagcagggt agcctgccct 1020

agacaaacct ttactcggtg ctcagaccat gctcagtttt tgtatggggg ttgagtgaag 1080

ggacaccagt gtgtgtacac gttcggaggg gggaccaagc tggaaataaa acgtaagtag 1140

tcttctcaac tcttgttcac taagtctaac cttgttaagt tgttctttgt tgtgtgtttt 1200

tcttaaggag atttcaggga tttagcaaat tccattctca gatcaggtgt taaggaggga 1260

aaactgtccc acaagaggtt ggaatgattt tcaggctaaa ttttaggctt tctaaaccaa 1320

agtaactaaa ctaggggaag agggataatt gtctacctag ggagggtttt gtggaggtaa 1380

agttaaaata aatcactgta aatcacattc agtgatggga ccagactgga aataaaacct 1440

aagtacattt ttgctcaact gcttgtgaag ttttggtccc attgtgtcct ttgtatgagt 1500

ttgtggtgta cattagataa atgaactatt ccttgtaacc caaaacttaa atagaagaga 1560

accaaaaatc tagctactgt acaagctgag caaacagact gacctcatgt cagatttgtg 1620

ggagaaatga gaaaggaaca gtttttctct gaacttagcc tatctaactg gatcgcctca 1680

ggcaggtttt tgtaaagggg ggcgcagtga tatgaatcac tgtgattcac gttcggctcg 1740

gggacaaagt tggaaataaa acgtaagtag acttttgctc atttacttgt gacgttttgg 1800

ttctgtttgg gtaacttgtg tgaatttgtg acattttggc taaatgagcc attcctggca 1860

acctgtgcat caatagaaga tcccccagaa aagagtcagt gtgaaagctg agcgaaaaac 1920

tcgtcttagg cttctgagac cagttttgta aggggaatgt agaagaaaga gctgggcttt 1980

tcctctgaat ttggcccatc tagttggact ggcttcacag gcaggttttt gtagagaggg 2040

gcatgtcata gtcctcactg tggctcacgt tcggtgctgg gaccaagctg gagctgaaac 2100

gtaagtacac ttttctcatc tttttttatg tgtaagacac aggttttcat gttaggagtt 2160

aaagtcagtt cagaaaatct tgagaaaatg gagagggctc attatcagtt gacgtggcat 2220

acagtgtcag attttctgtt tatcaagcta gtgagattag gggcaaaaag aggctttagt 2280

tgagaggaaa gtaattaata ctatggtcac catccaagag attggatcgg agaataagca 2340

tgagtagtta ttgagatctg ggtctgactg caggtagcgt ggtcttctag acgtttaagt 2400

gggagatttg gaggggatga ggaatgaagg aacttcagga tagaaaaggg ctgaagtcaa 2460

gttcagctcc taaaatggat gtgggagcaa actttgaaga taaactgaat gacccagagg 2520

atgaaacagc gcagatcaaa gaggggccta gagctctgag aagagaagga gactcatccg 2580

tgttgagttt ccacaagtac tgtcttgagt tttgcaataa aagtgggata gcagagttga 2640

gtgtnagccg tanagtatac tctcttttgt ctcctaagat ttttatgact acaaaaatca 2700

gtagtatgtc ctgaaataat cattaagctg tttgaaagta tgactgcttg ccatgtagat 2760

accatggctt gctgaatgat cagaagaggt gtgactctta ttctaaaatt tgtcacaaaa 2820

tgtcaaaatg agagactctg taggaacgag tcccttgaca gacagctgca aggggttttt 2880

ttcctttgtc tcatttctac atgaaagtaa atttgaaatg atcntttttt attataagag 2940

tagaaataca gttgggtttg aactatatgt tttaatnggc cncacggttt tgtaagacat 3000

ttggtccttt gttttcccag ttattactcg attgtaattt tatatcgcca gcantggtct 3060

gaaacggtnn nnnncgcaac ctcttcgttt actaactggg tgaccttcgg ctgtgccagc 3120

catttggcgt tcaccctgcc gcnggccnat gagaaccccc gcggtagnnc ccttgctccg 3180

cgggaaccac tttcctgagg acacagtgat aggaacagag ccactaatct gaagagaaca 3240

gagatgtgac agactacact aatgtgagaa aaacaaggaa agggtgactt attggagatt 3300

tcagaaataa aatgcattta ttattatatt cccttattta atttctattg ggaattagaa 3360

agggcataaa ctgctttatc cagtgttata ttaaaagctt aatgtatata atcttttaga 3420

ggtaaaatct acagccagca aaagtcatgg taaatattct ttgactgaac tctcactaaa 3480

ctcctctaaa ttatatgtca tattaactgg ttaaattaat ataaatttgt gacatgacct 3540

taactggtta ggtaggatat ttttcttcat gcaaaaatat gactaataat aatttagcac 3600

aaaaatattt cccaatactt taattctgtg atagaaaaat gtttaactca gctactataa 3660

tcccataatt ttgaaaacta tttattagct tttgtgtttg acccttccct gccaaaggca 3720

actatttaag gaccctttaa aactcttgaa actactttag agtcattaag ttatttaacc 3780

acttttaatt actttaaaat gatgtcaatt cccttttaac tattaattta ttttaagggg 3840

ggaaaggctg ctcataattc tattgttttt cttggtaaag aactctcagt ttctgtttta 3900

ctacctctgt cacccaagag ttggcatctc aacagagggg actttccgag agccatctgg 3960

cagttgctta agatcagaag tgaagtctgc cagttcctcc taggcaggtg gcccagatta 4020

cagttgacct gttctggtgt ggctaaaaat tgtcccatgt ggttacaaac cattagacca 4080

gggtctgatg aattgctcag aatatttctg gacacccaaa tacagaccct ggcttaaggc 4140

ctgtccatac agtaggttta gcttggctac accaaaggaa gccatacaga ggctaatatc 4200

agagtattct tggaagagac aggagaaaat gaaagccagt ttctgctctt accttatgtg 4260

cttgtgttca gactcccaaa catcaggagt gtcagtaaac tggtctgaat ctctgtctga 4320

agcatggaac tgaaaagaat gtagtttcag ggaagaaagg caatagaagg aagcctgaga 4380

atatcttcaa agggtcagac tcnnnaattt actttctaaa gaagtagcta ggaactaggg 4440

aataacttag aaacaacaag attgtatata tgtgcatcct ggcccattgt tccttatctg 4500

tagggataag cgtgcttttt tgtgtgttgt atataacata actgtttaca cataatacac 4560

tgaaatggag cccttccttg ttacttcata ccatcctctg tgcttccttc ctcaggggct 4620

gatgctgcac caactgtatc catcttccca ccatccagtg agcagttaac atctggaggt 4680

gcctcagtcg tgtgcttctt gaacaacttc taccccaaag acatcaatgt caagtggaag 4740

attgatggca gtgaacgaca aaatggcgtc ctgaacagtt ggactgatca ggacagcaaa 4800

gacagcacct acagcatgag cagcaccctc acgttgacca aggacgagta tgaacgacat 4860

aacagctata cctgtgaggc cactcacaag acatcaactt cacccattgt caagagcttc 4920

aacaggaatg agtgttagag acaaaggtcc tgagacgcca ccaccagctc cccagctcca 4980

tcctatcttc ccttctaagg tcttggaggc ttccccacaa gcgacctacc actgttgcgg 5040

tgctccaaac ctcctcccca cctccttctc ctcctcctcc ctttccttgc cttttatcat 5100

gctaatattt gcagaaaata ttcaataaag tgagtctttg cacttgagat ctctgtcttt 5160

cttactaaat ggtagtaatc agttgttttt ccagttacct gggtttctct tctaaagaag 5220

ttnaatgttt agttgccctg aaatccacca cacttaaagg ataaataaaa ccctccactt 5280

gccctggttg gctgtccact acatggcagt cctttctaag gttcacgagt actattcatg 5340

gcttatttct ctggccatgg taggtttgag gaggcatacc tcctagtttt cttcccctaa 5400

gtcgtcaaag tcctgaaggg ggacagtctt tacaagcaca tgttctgnnc tgattcaacc 5460

tacccagtaa acttggcgaa gcagtagcat catta 5495

<210>34

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>34

atctcgagga accactttcc tgaggacaca gtgatagg 38

<210>35

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>35

atgaattcct aacactcatt cctgttgaag ctcttgac 38

<210>36

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>36

atgaattcag acaaaggtcc tgagacgcca cc 32

<210>37

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>37

atggatcctc gagtcgactg gatttcaggg caactaaaca tt 42

<210>38

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>38

atgaattcgc ccctctccct cccccccccc ta 32

<210>39

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>39

atgaattcgt cgacttgtgg caagcttatc atcgtgtt 38

<210>40

<211>8

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>40

agtcgaca 8

<210>41

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>41

aatttgtcga ctgc 14

<210>42

<211>798

<212>DNA

＜213〉mice (Mus musculus)

<400>42

tggtgattat tcagagtagt tttagatgag tgcatcttca tgaatctcca gcccatattc 60

tcccatgtgt ttatcagtta agaactgact agactctatc ttgctatttg catattacat 120

tttcagtaac cacaaatatc tcacagttgg tttatagcaa agtacttatg agaatagtag 180

taattagcta gggaccaaag ttcaaagaca aaatggattt tcaagtgcag attttcagct 240

tcctgctaat cagtgcctca ggtaacagag ggcagggaat ttgagatcag aatccaacca 300

aaattatttt ccctggggaa tttgagtcta aaatacagtt ttttcatttt ctttcatctg 360

aatgttgggt ggtataaaat tatttttgta tctctatttc tactaatccc tctctctcta 420

ttttgctttt ttctagtcat actgtccaga ggacaaattg ttctcaccca gtctccagca 480

atcatgtctg catctccagg ggagaaggtc accatgacct gcagtgccag ctcaagtgta 540

agttacatgt actggtacca gcagaagcca ggatcctccc ccagactcct gatttatgac 600

acatccaacc tggcttctgg agtccctgtt cgcttcagtg gcagtgggtc tgggacctct 660

tactctctca caatcagccg aatggaggct gaagatgctg ccacttatta ctgccagcag 720

tggagtagtt acccacccac acagtgatac agactggaac aaaaaccctc taagtcctta 780

gggtctagct acttcctc 798

<210>43

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>43

cccaagcttt ggtgattatt cagagtagtt ttagatgagt gcat 44

<210>44

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>44

acgcgtcgac tttgtctttg aactttggtc cctagctaat tacta 45

<210>45

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>45

acgcgtcgac gcggccggcc gcgctagcag acaaaggtcc tgagacgcca ccaccagctc 60

ccc 63

<210>46

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>46

gaagatctca agtgcaaaga ctcactttat tgaatatttt ctg 43

<210>47

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>47

ggaattcaga caaaggtcct gagacgccac caccagctcc cc 42

<210>48

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>48

ccaagcttgc ctcctcaaac ctaccatggc ccagagaaat aag 43

<210>49

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>49

ataagaatgc ggccgcctca gagcaaatgg gttctacagg cctaacaacc t 51

<210>50

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>50

ccggaattcc taacactcat tcctgttgaa gctcttgaca atgg 44

<210>51

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>51

acgcgtcgac ccacatgcgg cttgggctgt gtgt 34

<210>52

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>52

acgcgtcgac gtcgacctag gcaggccctg 30

<210>53

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>53

ctgactagac tctatcttgc 20

<210>54

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>54

ccacggagac cactcgctca tt 22

<210>55

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>55

cacccctagg tcaatattgg ccattagc 28

<210>56

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>56

cacccctagg taggcatccc cagcatgc 28

<210>57

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>57

caccatgcgg cttgggctgt ctc 23

<210>58

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>58

ggcaggccct gcagatgtga gtg 23

<210>59

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>59

gatcaagggg aaacagcaga ggcggatcag 30

<210>60

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>60

ctgatccgcc tctgctgttt ccccttgatc 30

<210>61

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>61

caccgctagc ctcgagaatt cacgcgtg 28

<210>62

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>62

gctgatggtg aggtgcgtc 19

<210>63

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>63

atcagtgccg aggggagcca g 21

<210>64

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>64

gccctctaga gcggcaggcc ctgcagatg 29

<210>65

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>65

ccggctagcc accatggcgc aatgtgaaat g 31

<210>66

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>66

ccatgcggcc gccctcctca ctgtgcacct 30

<210>67

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉GIPF peptide

<400>67

Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln

1 5 10 15

<210>68

<211>798

<212>DNA

＜213〉mice (Mus musculus)

<220>

<221>CDS

<222>(61)..(795)

<400>68

atgcggcttg ggctgtgcgt ggtggccctg gttctgagct ggacacacat cgccgtgggc 60

agc cgg ggg atc aag ggc aag aga cag agg cgg arc agt gct gag ggg 108

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc caa gcc tgc gcc aag ggc tgt gag ctc tgt tca gaa gtc aac ggt 156

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tcg ccc aag ctc ttc att ctg ctg gag agg aac gac 204

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ctg ccg tcc tgc cca cct gga tac ttt 252

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gat gcc cgc aac ccc gac atg aac aaa tgc atc aaa tgc aag atc gag 300

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cac aac ttc tgc acc aag tgt cag gag 348

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Gln Glu

85 90 95

gcc ttg tac tta cac aag ggc cgc tgc tat cca gcc tgc cct gag ggc 396

Ala Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tct aca gcc gct aac agc acc atg gag tgc ggc agt cct gca caa tgt 444

Ser Thr Ala Ala Asn Ser Thr Met Glu Cys Gly Ser Pro Ala Gln Cys

115 120 125

gaa atg agc gag tgg tcc ccg tgg gga ccc tgc tcc aag aag agg aag 492

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Arg Lys

130 135 140

ctg tgc ggt ttc cgg aag gga tcg gaa gag cgg aca cgc aga gtg ctc 540

Leu Cys Gly Phe Arg Lys Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

cat gct ccc ggg gga gac cac acc acc tgc tcc gac acc aaa gag acc 588

His Ala Pro Gly Gly Asp His Thr Thr Cys Ser Asp Thr Lys Glu Thr

165 170 175

cgc aag tgt acc gtg cgc agg acg ccc tgc cca gag ggg cag aag agg 636

Arg Lys Cys Thr Val Arg Arg Thr Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

agg aag ggg ggc cag ggc cgg agg gag aat gcc aac agg cat ccg gcc 684

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg His Pro Ala

195 200 205

agg aag aac agc aag gag ccg agg tcc aac tct cgg aga cac aaa ggg 732

Arg Lys Asn Ser Lys Glu Pro Arg Ser Asn Ser Arg Arg His Lys Gly

210 215 220

caa cag cag cca cag cca ggg aca aca ggg cca ctc aca tca gta gga 780

Gln Gln Gln Pro Gln Pro Gly Thr Thr Gly Pro Leu Thr Ser Val Gly

225 230 235 240

cct acc tgg gca cag tga 798

Pro Thr Trp Ala Gln

245

<210>69

<211>245

<212>PRT

＜213〉mice (Mus musculus)

<400>69

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Gln Glu

85 90 95

Ala Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Thr Ala Ala Asn Ser Thr Met Glu Cys Gly Ser Pro Ala Gln Cys

115 120 125

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Arg Lys

130 135 140

Leu Cys Gly Phe Arg Lys Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

His Ala Pro Gly Gly Asp His Thr Thr Cys Ser Asp Thr Lys Glu Thr

165 170 175

Arg Lys Cys Thr Val Arg Arg Thr Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg His Pro Ala

195 200 205

Arg Lys Asn Ser Lys Glu Pro Arg Ser Asn Ser Arg Arg His Lys Gly

210 215 220

Gln Gln Gln Pro Gln Pro Gly Thr Thr Gly Pro Leu Thr Ser Val Gly

225 230 235 240

Pro Thr Trp Ala Gln

245

<210>70

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>70

gtgtgaggtc cacggaaac 19

<210>71

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>71

ggtgcaaaga catagccaga 20

<210>72

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>72

gcaaacaaca caggggtgta 20

<210>73

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>73

ggcaggtctg tgactgatgt 20

<210>74

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>74

tatgcagaac tgcgatttcc 20

<210>75

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>75

ccaatttgtc tcctatctgt gg 22

<210>76

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>76

tgctccatga ggagacacc 19

<210>77

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>77

cctgcctctt ttccacaga 19

<210>78

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>78

caacgttccc ttcaagacac 20

<210>79

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>79

cgcttgtcct cgttcatct 19

<210>80

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>80

ctcaacaccg gaatttttga 20

<210>81

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>81

aattgcattt cgaaggaagg 20

<210>82

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>82

gtagattcgg gcaagtccac cac 23

<210>83

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>83

ttcccatctc agcagcctcc tt 22

<210>84

<211>105

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(105)

<400>84

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag 105

Cys Leu Lys

35

<210>85

<211>35

<212>PRT

＜213〉people (Homo sapiens)

<400>85

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys

35

<210>86

<211>180

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(180)

<400>86

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca 180

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

50 55 60

<210>87

<211>60

<212>PRT

＜213〉people (Homo sapiens)

<400>87

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

50 55 60

<210>88

<211>270

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(270)

<400>88

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc 270

His Cys Glu Ala Cys Phe Ser His Asn Phe

85 90

<210>89

<211>90

<212>PRT

＜213〉people (Homo sapiens)

<400>89

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe

85 90

<210>90

<211>360

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(360)

<400>90

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag 288

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc 336

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tcc tca gct gcc aat ggc acc atg 360

Ser Ser Ala Ala Asn Gly Thr Met

115 120

<210>91

<211>120

<212>PRT

＜213〉people (Homo sapiens)

<400>91

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Ser Ala Ala Asn Gly Thr Met

115 120

<210>92

<211>450

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(450)

<400>92

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag 288

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc 336

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tcc tca gct gcc aat ggc acc atg gag tgc agt agt cct gcg caa tgt 384

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

gaa atg agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag 432

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

ctc tgt ggt ttc cgg agg 450

Leu Cys Gly Phe Arg Arg

145 150

<210>93

<211>150

<212>PRT

＜213〉people (Homo sapiens)

<400>93

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

Leu Cys Gly Phe Arg Arg

145 150

<210>94

<211>540

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(540)

<400>94

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag 288

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc 336

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tcc tca gct gcc aat ggc acc atg gag tgc agt agt cct gcg caa tgt 384

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

gaa atg agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag 432

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg aca cgc agg gtg cta 480

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

cat gcc cct gtg ggg gac cat gct gcc tgc tct gac acc aag gag acc 528

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

cgg agg tgc aca 540

Arg Arg Cys Thr

180

<210>95

<211>180

<212>PRT

＜213〉people (Homo sapiens)

<400>95

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

Arg Arg Cys Thr

180

<210>96

<211>630

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(630)

<400>96

agc cgg ggg atc aag ggg aaa agg cag agg cgg atc agt gcc gag ggg 48

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc aac ggc 96

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg aac gac 144

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc 192

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag 240

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag 288

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc 336

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

tcc tca gct gcc aat ggc acc atg gag tgc agt agt cct gcg caa tgt 384

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

gaa atg agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag 432

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg aca cgc agg gtg cta 480

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

cat gcc cct gtg ggg gac cat gct gcc tgc tct gac acc aag gag acc 528

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

cgg agg tgc aca gtg agg aga gtg ccg tgt cct gag ggg cag aag agg 576

Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

agg aag gga ggc cag ggc cgg cgg gag aat gcc aac agg aac ctg gcc 624

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala

195 200 205

agg aag 630

Arg Lys

210

<210>97

<211>210

<212>PRT

＜213〉people (Homo sapiens)

<400>97

Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile Ser Ala Glu Gly

1 5 10 15

Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val Asn Gly

20 25 30

Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp

35 40 45

Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe

50 55 60

Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu

65 70 75 80

His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu

85 90 95

Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly

100 105 110

Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys

115 120 125

Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln

130 135 140

Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu

145 150 155 160

His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr

165 170 175

Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg

180 185 190

Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala

195 200 205

Arg Lys

210

<210>98

<211>615

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(615)

<400>98

aag ctg ttc atc ctg ctg gag agg aac gac atc cgc cag gtg ggc gtc 48

Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val

1 5 10 15

tgc ttg ccg tcc tgc cca cct gga tac ttc gac gcc cgc aac ccc gac 96

Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp

20 25 30

atg aac aag tgc atc aaa tgc aag atc gag cac tgt gag gcc tgc ttc 144

Met Asn Lys Cys Ile Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe

35 40 45

agc cat aac ttc tgc acc aag tgt aag gag ggc ttg tac ctg cac aag 192

Ser His Asn Phe Cys Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys

50 55 60

ggc cgc tgc tat cca gct tgt ccc gag ggc tcc tca gct gcc aat ggc 240

Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly

65 70 75 80

acc atg gag tgc agt agt cct gcg caa tgt gaa atg agc gag tgg tct 288

Thr Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser

85 90 95

ccg tgg ggg ccc tgc tcc aag aag cag cag ctc tgt ggt ttc cgg agg 336

Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg

100 105 110

ggc tcc gag gag cgg aca cgc agg gtg cta cat gcc cct gtg ggg gac 384

Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp

115 120 125

cat gct gcc tgc tct gac acc aag gag acc cgg agg tgc aca gtg agg 432

His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg

130 135 140

aga gtg ccg tgt cct gag ggg cag aag agg agg aag gga ggc cag ggc 480

Arg Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly

145 150 155 160

cgg cgg gag aat gcc aac agg aac ctg gcc agg aag gag agc aag gag 528

Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu

165 170 175

gcg ggt gct ggc tct cga aga cgc aag ggg cag caa cag cag cag cag 576

Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln

180 185 190

caa ggg aca gtg ggg cca ctc aca tct gca ggg cct gcc 615

Gln Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

195 200 205

<210>99

<211>205

<212>PRT

＜213〉people (Homo sapiens)

<400>99

Lys Leu Phe Ile Leu Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val

1 5 10 15

Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp

20 25 30

Met Asn Lys Cys Ile Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe

35 40 45

Ser His Asn Phe Cys Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys

50 55 60

Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly

65 70 75 80

Thr Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser

85 90 95

Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg

100 105 110

Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp

115 120 125

His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg

130 135 140

Arg Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly

145 150 155 160

Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu

165 170 175

Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln

180 185 190

Gln Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

195 200 205

<210>100

<211>432

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(432)

<400>100

ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc tcc tca gct 48

Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala

1 5 10 15

gcc aat ggc acc atg gag tgc agt agt cct gcg caa tgt gaa atg agc 96

Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser

20 25 30

gag tgg tct ccg tgg ggg ccc tgc tcc aag aag cag cag ctc tgt ggt 144

Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly

35 40 45

ttc cgg agg ggc tcc gag gag cgg aca cgc agg gtg cta cat gcc cct 192

Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro

50 55 60

gtg ggg gac cat gct gcc tgc tct gac acc aag gag acc cgg agg tgc 240

Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys

65 70 75 80

aca gtg agg aga gtg ccg tgt cct gag ggg cag aag agg agg aag gga 288

Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly

85 90 95

ggc cag ggc cgg cgg gag aat gcc aac agg aac ctg gcc agg aag gag 336

Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu

100 105 110

agc aag gag gcg ggt gct ggc tct cga aga cgc aag ggg cag caa cag 384

Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln

115 120 125

cag cag cag caa ggg aca gtg ggg cca ctc aca tct gca ggg cct gcc 432

Gln Gln Gln Gln Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

130 135 140

<210>101

<211>144

<212>PRT

＜213〉people (Homo sapiens)

<400>101

Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala

1 5 10 15

Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala Gln Cys Glu Met Ser

20 25 30

Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly

35 40 45

Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro

50 55 60

Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys

65 70 75 80

Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly

85 90 95

Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu

100 105 110

Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln

115 120 125

Gln Gln Gln Gln Gly Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

130 135 140

<210>102

<211>366

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(366)

<400>102

tgc agt agt cct gcg caa tgt gaa atg agc gag tgg tct ccg tgg ggg 48

Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly

1 5 10 15

ccc tgc tcc aag aag cag cag ctc tgt ggt ttc cgg agg ggc tcc gag 96

Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu

20 25 30

gag cgg aca cgc agg gtg cta cat gcc cct gtg ggg gac cat gct gcc 144

Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala

35 40 45

tgc tct gac acc aag gag acc cgg agg tgc aca gtg agg aga gtg ccg 192

Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro

50 55 60

tgt cct gag ggg cag aag agg agg aag gga ggc cag ggc cgg cgg gag 240

Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu

65 70 75 80

aat gcc aac agg aac ctg gcc agg aag gag agc aag gag gcg ggt gct 288

Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala

85 90 95

ggc tct cga aga cgc aag ggg cag caa cag cag cag cag caa ggg aca 336

Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr

100 105 110

gtg ggg cca ctc aca tct gca ggg cct gcc 366

Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

115 120

<210>103

<211>122

<212>PRT

＜213〉people (Homo sapiens)

<400>103

Cys Ser Ser Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly

1 5 10 15

Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu

20 25 30

Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala

35 40 45

Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro

50 55 60

Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu

65 70 75 80

Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala

85 90 95

Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr

100 105 110

Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

115 120

<210>104

<211>228

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(1)..(228)

<400>104

cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga tac ttc gac 48

Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp

1 5 10 15

gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag atc gag cac 96

Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu His

20 25 30

tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt aag gag ggc 144

Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu Gly

35 40 45

ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc gag ggc tcc 192

Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser

50 55 60

tca gct gcc aat ggc acc atg gag tgc agt agt cct 228

Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro

65 70 75

<210>105

<211>76

<212>PRT

＜213〉people (Homo sapiens)

<400>105

Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly Tyr Phe Asp

1 5 10 15

Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys Ile Glu His

20 25 30

Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys Lys Glu Gly

35 40 45

Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro Glu Gly Ser

50 55 60

Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro

65 70 75

<210>106

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>106

caccgctagc agccggggga tcaagg 26

<210>107

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>107

cacgcggccg ctcttgaggc agccgttg 28

<210>108

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>108

cacgcggccg cttgggcagg acggcaagc 29

<210>109

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>109

cacgcggccg ctgaagttat ggctgaagca g 31

<210>110

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>110

cacgcggccg ctcatggtgc cattggcagc 30

<210>111

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>111

cacgcggccg ctcctccgga aaccacagag 30

<210>112

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>112

cacgcggccg cttgtgcacc tccgggtctc 30

<210>113

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>113

cacgcggccg ctcttcctgg ccaggttcc 29

<210>114

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>114

caccgctagc aagctgttca tcctgctgg 29

<210>115

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>115

cacgcggccg ctggcaggcc ctgcagatg 29

<210>116

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>116

caccgctagc ctgcacaagg gccgctg 27

<210>117

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>117

caccgctagc tgcagtagtc ctgcgca 27

<210>118

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>118

caccgctagc cgccaggtgg gcgtctg 27

<210>119

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>119

cacgcggccg ctaggactac tgcactcca 29

<210>120

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>120

gatcagcagc tggaacaaac acag 24

<210>121

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>121

tgcacaatca gtcaatcaac agagc 25

<210>122

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>122

ggcggatccc tgagttggag gccagtttgg 30

<210>123

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide 123

<400>123

gctctagacc atggtggacg agcctagagg agaaggcat 39

<210>124

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>124

ccgctcgagg catgcggctt gggctgtgtg tggtggccct g 41

<210>125

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>125

gctctagaag atctctaggc aggccctgca gatgtgagtg gccc 44

<210>126

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>126

aattcggatc cggcgcgcc 19

<210>127

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>127

gatcggcgcg ccggatccg 19

<210>128

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>128

ggccgtttaa acataacttc gtataatgta tgctatacga agttatc 47

<210>129

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>129

tcgagataac ttcgtatagc atacattata cgaagttatg tttaaac 47

<210>130

<211>41

<212>DNA

＜213〉forward primer

<220>

＜223〉oligonucleotide

<400>130

cgggatccgt ttaaacctgt gccttctagt tgccagccat c 41

<210>131

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>131

cggatatccc atagagccca ccgcatcccc agc 33

<210>132

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>132

taaccgcgat cgcggccgg 19

<210>133

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>133

ccgcgatcgc ccttaat 17

<210>134

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>134

ctaacgttac tggccgaagc 20

<210>135

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>135

attatcatcg tgtttttcaa aggaa 25

<210>136

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>136

gctctgacac caaggagacc 20

<210>137

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>137

ccctaggaat gctcgtcaag 20

<210>138

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>138

caggagcctc acccttcg 18

<210>139

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>139

acgccgaggt gcttgccc 18

<210>140

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>140

caccatggag aaggccgggg cccac 25

<210>141

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>141

atcatacttg gcaggtttct ccagg 25

<210>142

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>142

cgggatcccc atggctcctc tcggatacct cttagtgct 39

<210>143

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>143

gctctagagt ttaaacctac ttgcaggtgt gcacgtcata g 41

<210>144

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>144

tcgagtcgcg acaccggcgg gcgcgccc 28

<210>145

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>145

tcgagggcgc gcccgccggt gtcgcgac 28

<210>146

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>146

ggccgcttaa ttaaggccgg ccgtcgacg 29

<210>147

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>147

aattcgtcga cggccggcct taattaagc 29

<210>148

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>148

ttggcgcgcc ctccctagga ctgcagttga gctcagattt ga 42

<210>149

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>149

ccgctcgagt cttactgtct cagcaacaat aatataaaca gggg 44

<210>150

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>150

ataagaatgc ggccgcaaag ctggtgggtt aagactatct cgtgaagtg 49

<210>151

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>151

acgcgtcgac tcacaggttg gtccctctct gtgtgtggtt gctgt 45

<210>152

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>152

tcttactaga gttctcacta gctct 25

<210>153

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>153

ggaaccaaag aatgaggaag ctgtt 25

<210>154

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>154

ggccaggcgc gccttgc 17

<210>155

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<223>olignucleotide

<400>155

ggccgcaagg cgcgcct 17

<210>156

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>156

taagggctag ctagggccgg 20

<210>157

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>157

ccctagctag cccttaat 18

<210>158

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>158

tcgagttaac 10

<210>159

<211>10

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>159

agctgttaac 10

<210>160

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>160

agctgtcgac ttaattaagg ccggccg 27

<210>161

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>161

ctagcggccg gccttaatta agtcgac 27

<210>162

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>162

tcgacttaat taaggccggc cctagctagc a 31

<210>163

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>163

agcttgctag ctagggccgg ccttaattaa g 31

<210>164

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>164

ccttaattaa agttatgtgt cctagagggc tgcaaactca agatc 45

<210>165

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>165

ttggccggcc ttggcgccag tggaacctgg aatgataaac acaaagatta ttg 53

<210>166

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>166

aagcgtcgac caccatgcgg cttgggctgt gtg 33

<210>167

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>167

atggccggcc ctacatggtg ccattggcag 30

<210>168

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>168

agccggggga tcaaggggaa aaggcagagg 30

<210>169

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>169

atggccggcc ctacatggtg ccattggcag 30

<210>170

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>170

acgcgtcgac caccatgata ttccgagtca gtgc 34

<210>171

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>171

ggccggccct aggcaggccc tgcagatgtg agtgg 35

<210>172

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>172

atgatattcc gagtcagtgc cgaggggagc cag 33

<210>173

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>173

ggccggccct aggcaggccc tgcagatgtg agtgg 35

<210>174

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>174

atcaagggga aaaggcagag 20

<210>175

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400>175

cgcttgtggg gaagcctcca agacc 25

<210>176

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400>176

ataacttctg caccaagtgt aagga 25

<210>177

<211>2333

<212>DNA

＜213〉people (Homo sapiens)

<220>

<221>CDS

<222>(219)..(926)

<400>177

aggcgagccg ggcgcccagg acagtcccgc agcgggcggg tgagcgggcc gcgccctcgc 60

ccctcccggg cctgcccccg tcgcgactgg cagcacgaag ctgagattgt ggtttcctgg 120

tgattcaggt gggagtgggc cagaagatca ccgctggcaa ggactgggtt ggtttttgga 180

gtagtccctg ctgacgtgac aaaaagatct ctcatatg ata ttc cga gtc agt gcc 236

Ile Phe Arg Val Ser Ala

1 5

gag ggg agc cag gcc tgt gcc aaa ggc tgt gag ctc tgc tct gaa gtc 284

Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser Glu Val

10 15 20

aac ggc tgc ctc aag tgc tca ccc aag ctg ttc atc ctg ctg gag agg 332

Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu Glu Arg

25 30 35

aac gac atc cgc cag gtg ggc gtc tgc ttg ccg tcc tgc cca cct gga 380

Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro Pro Gly

40 45 50

tac ttc gac gcc cgc aac ccc gac atg aac aag tgc atc aaa tgc aag 428

Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys Cys Lys

55 60 65 70

atc gag cac tgt gag gcc tgc ttc agc cat aac ttc tgc acc aag tgt 476

Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr Lys Cys

75 80 85

aag gag ggc ttg tac ctg cac aag ggc cgc tgc tat cca gct tgt ccc 524

Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala Cys Pro

90 95 100

gag ggc tcc tca gct gcc aat ggc acc atg gag tgc agt agt cct gcg 572

Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser Pro Ala

105 110 115

caa tgt gaa gtg agc gag tgg tct ccg tgg ggg ccc tgc tcc aag aag 620

Gln Cys Glu Val Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys Lys

120 125 130

cag cag ctc tgt ggt ttc cgg agg ggc tcc gag gag cgg aca cgc agg 668

Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg Arg

135 140 145 150

gtg cta cat gcc cct gtg ggg gac cat gct gcc tgc tct gac acc aag 716

Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr Lys

155 160 165

gag acc cgg agg tgc aca gtg agg aga gtg ccg tgt cct gag ggg cag 764

Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly Gln

170 175 180

aag agg agg aag gga ggc cag ggc cgg cgg gag aat gcc aac agg aac 812

Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg Asn

185 190 195

ctg gcc agg aag gag agc aag gag gcg ggt gct ggc tct cga aga cgc 860

Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg Arg Arg

200 205 210

aag ggg cag caa cag cag cag cag caa ggg aca gtg ggg cca ctc aca 908

Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro Leu Thr

215 220 225 230

tct gca ggg cct gcc tag ggacactgtc cagcctccag gcccatgcag 956

Ser Ala Gly Pro Ala

235

aaagagttca gtgctactct gcgtgattca agctttcctg aactggaacg tcgggggcaa 1016

agcatacaca cacactccaa tccatccatg catacacaga cacaagacac acacgctcaa 1076

acccctgtcc acatatacaa ccatacatac ttgcacatgt gtgttcatgt acacacgcag 1136

acacagacac cacacacaca catacacaca cacacacaca cacacacctg aggccaccag 1196

aagacacttc catccctcgg gcccagcagt acacacttgg tttccagagc tcccagtgga 1256

catgtcagag acaacacttc ccagcatctg agaccaaact gcagagggga gccttctgga 1316

gaagctgctg ggatcggacc agccactgtg gcagatggga gccaagcttg aggactgctg 1376

gtgacctggg aagaaacctt cttcccatcc tgttcagcac tcccagctgt gtgactttat 1436

cgttggagag tattgttacc cttccaggat acatatcagg gttaacctga ctttgaaaac 1496

tgcttaaagg tttatttcaa attaaaacaa aaaaatcaac gacagcagta gacacaggca 1556

ccacattcct ttgcagggtg tgagggtttg gcgaggtatg cgtaggagca agaagggaca 1616

gggaatttca agagacccca aatagcctgc tcagtagagg gtcatgcaga caaggaagaa 1676

aacttagggg ctgctctgac ggtggtaaac aggctgtcta tatccttgtt actcagagca 1736

tggcccggca gcagtgttgt cacagggcag cttgttagga atgagaatct caggtctcat 1796

tccagacctg gtgagccaga gtctaaattt taagattcct gatgattggc atgttaccca 1856

aatttgagaa gtgctgctgt aattcccctt aaaggacggg agaaagggcc ccggccatct 1916

tgcagcagga gggattctgg tcagctataa aggaggactt tccatctggg agaggcagaa 1976

tctatatact gaagggctag tggcactgcc aggggaaggg agtgcgtagg cttccagtga 2036

tggttgggga caatcctgcc caaaggcagg gcagtggatg gaataactcc ttgtggcatt 2096

ctgaagtgtg tgccaggctc tggactaggt gctaggtttc cagggaggag ccaaacacgg 2156

gccttgctct tgtggagctt agaggttggt ggggaagaaa ataggcatgc accaaggaat 2216

cgtacaaaca catatataac tacaaaagga tggtgccaag ggcaggtgac cactggcatc 2276

tatgcttagc tatgaaagtg aataaagcag aataaaaata aaatactttc tctcagg 2333

<210>178

<211>235

<212>PRT

＜213〉people (Homo sapiens)

<400>178

Ile Phe Arg Val Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys

1 5 10 15

Glu Leu Cys Ser Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu

20 25 30

Phe Ile Leu Leu Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu

35 40 45

Pro Ser Cys Pro Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn

50 55 60

Lys Cys Ile Lys Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His

65 70 75 80

Asn Phe Cys Thr Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg

85 90 95

Cys Tyr Pro Ala Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met

100 105 110

Glu Cys Ser Ser Pro Ala Gln Cys Glu Val Ser Glu Trp Ser Pro Trp

115 120 125

Gly Pro Cys Ser Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser

130 135 140

Glu Glu Arg Thr Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala

145 150 155 160

Ala Cys Ser Asp Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val

165 170 175

Pro Cys Pro Glu Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg

180 185 190

Glu Asn Ala Asn Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly

195 200 205

Ala Gly Ser Arg Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly

210 215 220

Thr Val Gly Pro Leu Thr Ser Ala Gly Pro Ala

225 230 235

Claims (52)

1. compositions, it comprises the GIPF polypeptide for the treatment of effective dose, its fragment, or its analog and pharmaceutically useful carrier.

2. Pharmaceutical composition, it comprises polypeptide and the pharmaceutically useful carrier that contains the GIPF bioactive fragment.

3. the Pharmaceutical composition of claim 2, wherein GIPF is people GIPF.

4. the Pharmaceutical composition of claim 2, wherein polypeptide comprises the bioactive fragment of SEQ ID NO:4 polypeptide.

5. Pharmaceutical composition, it comprises the polypeptide of the polypeptide fragment that contains SEQ ID NO:4, wherein polypeptide fragment comprises and is selected from following aminoacid sequence: SEQ ID NO:10,12,14,16,18,85,87,89,91,93,95,97,99,101,103,105 and 178.

6. any one Pharmaceutical composition of claim 1 to 5, wherein polypeptide is glycosylated.

7. any one Pharmaceutical composition of claim 1 to 5, wherein polypeptide is not glycosylated.

8. any one Pharmaceutical composition of claim 1 to 6, wherein polypeptide stimulates epithelial cell proliferation.

9. the Pharmaceutical composition of claim 1, it comprises the polypeptide that contains the aminoacid sequence identical with SEQ ID NO:4 aminoacid sequence at least 80%.

10. stimulate the method for epithelial cell proliferation on the experimenter, it comprises that giving the experimenter contains the GIPF polypeptide, the compositions of its fragment or its analog and carrier.

11. a Therapeutic Method, it comprises the compositions that contains GIPF polypeptide and pharmaceutically useful carrier that need give mammalian subject treatment effective dose by it.

12. the treatment mucositis, inflammatory bowel disease, or the method for short bowel syndrome, it comprises the compositions that contains GIPF polypeptide and pharmaceutically useful carrier that need give mammalian subject treatment effective dose by it.

13. the method for epithelial cell proliferation in the stimulation patient's gastrointestinal tract, wherein method comprises the compositions that contains GIPF polypeptide and pharmaceutically useful carrier for the treatment of effective dose.

14. the method for claim 13, epithelial cell proliferation in its moderate stimulation esophagus.

15. the method for claim 13, epithelial cell proliferation in its moderate stimulation small intestinal.

16. the method for claim 13, epithelial cell proliferation in its moderate stimulation large intestine.

17. the method for claim 13, epithelial cell proliferation in its moderate stimulation stomach.

Be lining in the method for destroying the patient of risk to small part gastrointestinal epithelial cell 18. be used for the treatment of to have, wherein this method comprises the compositions that contains GIPF polypeptide and pharmaceutically useful carrier for the treatment of effective dose.

19. the method for claim 18, wherein the patient carries out maybe will carrying out radiotherapy.

20. the method for claim 18, wherein the patient carries out maybe will carrying out chemotherapy.

21. treatment has lived through radiotherapy or chemotherapeutical patient's method, it comprises the compositions that contains GIPF polypeptide and pharmaceutically useful carrier for the treatment of effective dose.

22. claim 10-13,18 and 21 any one methods, wherein mammalian subject is human.

23. an adenovirus vector, it comprises the gene of the coding GIPF that operability ground links to each other with expression control sequenc.

24. Pharmaceutical composition, it comprises the adenovirus vector of claim 23.

25. stimulate the method for epithelial cell proliferation on the experimenter, it comprises the Pharmaceutical composition that gives this experimenter's claim 24.

26. transgenosis construct, it comprises coding GIPF proteic nucleic acid, wherein this nucleic acid by operability be connected in the transcriptional regulatory sequences that instructs it in the B-cell, to express.

27. the transgenosis construct of claim 26, it comprises the promoter of B-cell-specific.

28. transgenic mouse, it produces the natural human GIPF albumen of the level that can detect in the B-cell, wherein this transgenic mouse proteic nucleic acid stability of GIPF of will encoding is incorporated in its genome, is connected in to operability the transcriptional regulatory sequences that instructs it to express in the B-cell.

29. the transgenic mouse of claim 28, wherein this transcriptional regulatory sequences comprises B-cell promoter.

30. identify the method for drug candidate, this Drug therapy mucositis wherein, inflammatory bowel disease or short bowel syndrome, this method comprises:

(a) give test-compound to transgenic mouse, wherein compare, the enterocyte propagation that express recombinant GIPF polypeptide and performance increase in this transgenic mouse B-cell with the same mouse of the propagation of express recombinant GIPF polypeptide enterocyte not; With

(b) estimate the influence of this test-compound to enterocyte propagation, the test-compound that wherein will increase enterocyte propagation is accredited as and is used for the treatment of mucositis, the drug candidate of inflammatory bowel disease or short bowel syndrome.

31. the method for claim 30, its midgut epithelial cell is a pit cell.

32. it comprises isolating polynucleotide and is selected from SEQ ID NO:9,11,13,15,17,84,86,88,90,92,94,96,98,100,102 and 104 nucleotide.

33. coding has the isolating polynucleotide of biologically active polypeptide, these polynucleotide have and the about sequence homogeneity more than 95% of the polynucleotide of claim 32.

34. isolating polypeptide, it comprises and is selected from SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101,103 and 105 aminoacid sequence.

35. isolating polypeptide, it comprises and be selected from SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101,103 aminoacid sequences identical with 105 aminoacid sequence at least 95%.

36. expression vector, it is connected in with comprising operability and is selected from SEQ ID NO:5,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102 and the expression regulation element of 104 polynucleotide sequences.

37. with SEQ ID NO:5,9,11,13,15,17,84,86,88,90,92,94,96,98,100,102,104 or 177 the polynucleotide conversion or the host cell of transfection.

38. the conversion of claim 37 or the host cell of transfection, wherein this cell be protokaryon or eucaryon.

Comprise SEQ ID NO:12 39. produce, 14,16,18,85,87,89,91,93,95,97,99,101, the method for the polypeptide of 103 or 105 aminoacid sequence, this method comprises:

(a) be suitable under the condition of express polypeptide the cell of culture of isolated in culture medium, wherein this cell comprises coding SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101, the nucleic acid molecules of 103 or 105 polypeptide; With

(b) the described polypeptide of purification from cell or culture medium.

40. produce the method for Pharmaceutical composition, wherein Pharmaceutical composition comprises and contains SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101, and the polypeptide of 103,105 or 178 aminoacid sequences, this method comprises

(a) be suitable under the condition of express polypeptide the cell of culture of isolated in culture medium, wherein this cell comprises coding SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101, the nucleic acid molecules of 103,105 or 178 polypeptide;

(b) the described polypeptide of purification from described cell or culture medium; With

(c) polypeptide and the pharmaceutically useful carrier of purification are united.

41. produce the method for Pharmaceutical composition, wherein Pharmaceutical composition comprises and contains SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101, and the polypeptide of 103,105 or 178 aminoacid sequences, this method comprises

(a) contain SEQ ID NO:12,14,16,18,85,87,89,91,93,95,97,99,101, the polypeptide of 103,105 or 178 aminoacid sequence synthetic comprising;

(b) the described polypeptide of purification; With

(c) polypeptide and the pharmaceutically useful carrier of purification are united.

42. comprise coding GIPF proteic expression of nucleic acids vector construct, wherein with the nucleic acid operability be connected in the transcription regulating nucleotide sequence that instructs GIPF albumen in enterocyte, to express.

43. transgenic mouse, but it produces the GIPF albumen of detection level in enterocyte, wherein this transgenic mouse proteic nucleic acid stability of GIPF of will encoding is incorporated in its genome, wherein encode the proteic nucleotide sequence of GIPF by operability be connected in the transcriptional regulatory sequences that instructs GIPF albumen in enterocyte, to express.

44. comprise coding GIPF albumen and the proteic expression of nucleic acids vector construct of Wnt3a, be connected in to wherein said nucleic acid operability the transcription regulating nucleotide sequence that instructs it in enterocyte, to express.

45. transgenic mouse, but it produces the natural GIPF and the Wnt3a albumen of detection level in enterocyte, wherein this transgenic mouse proteic nucleic acid stability of GIPF of will having encoded is incorporated in its genome, and operability be connected in the transcriptional regulatory sequences that instructs GIPF albumen in enterocyte, to express.

46. the transgenic mouse of claim 43 or 45, wherein said Mus performance enterectasis.

47. be used for the treatment of the Pharmaceutical composition of the claim 1 of mucositis.

48. be used for the treatment of the Pharmaceutical composition of the claim 1 of inflammatory bowel disease.

49. be used for the treatment of the Pharmaceutical composition of the claim 1 of short bowel syndrome.

50. be used for the treatment of the Pharmaceutical composition of the claim 1 of the gastrointestinal tract infringement that causes by chemotherapy or radiotherapy.

51. the method for claim 13, epithelial hypertrophy in its moderate stimulation oral cavity.

52. comprise the polypeptide of the variant of SEQ ID NO:4, wherein be substituted in the valine of the 50th amino acids with isoleucine.

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