CN1369561A - Cell factor CK-HA and its variant with hematogenesis promoting and immunoregulating functions - Google Patents

Abstract

A novel cell factor CK-HA and its variant CKB, the nucleic acid sequence and amino acid sequence of CK-HA (or B), the carrier of CH-HA (or B), the host cell for expressing CK-HA (or B), the process for preparing polypeptide CK-HA (or B) by genetic engineering, the composite medicine containing them, and the application of said cell factor in treating diseases of hemopoietic system and immune system are disclosed.

Description

CK-HA and varient thereof with hematopoietic stimulation and immunoregulation effect

The present invention relates to a kind of new cytokine and the nucleic acid coding sequence and the aminoacid sequence of varient thereof, carry the carrier of this cytokine nucleotide sequence, express the host cell of this cytokine, the pharmaceutical composition that contains this cytokine, and the purposes of cytokine in the pharmaceutical composition of preparation treatment disease of hematopoietic system and disease of immune system.

Cytokine by the synthetic justacrine of body inner cell, participate in the proliferation and differentiation of various kinds of cell, the general name of the micromolecule polypeptide that in the physiology of body and pathologic process, plays a significant role.Cytokine comprises interleukin-, G CFS, Interferon, rabbit, tumour necrosis factor, somatomedin and chemokine etc.Different physiological roles such as cytokine has: they can regulate body's immunological function, participate in propagation and the differentiation of hemopoietic stem cell, promote vascular endothelial cell proliferation and new vessel generation, also significant aspect antitumor and anti-microbial infection.In recent years, continuous development along with Protocols in Molecular Biology, the structure function of people's pair cell factor and acceptor thereof, the signal conduction and the expression regulation of cytokine have had more deep understanding, it is clinical to utilize the cytokine of genetic engineering technique production reorganization or its antagonist to be used for as medicine, and obtains good efficacy.For example marrow sample hemopoietic progenitor cell supressor (MPIF-1) as the heavy dose that the hematopoiesis protective material is used to tumour put, in the chemotherapy; be expected to become biotechnology medicine (the Marshall A of a new generation; HGS Launches " first " genomics product in clinic.NatBiotechnol 1998,16:129).This prompting cytokine has broad application prospects in the treatment of difficult and complicated illness such as tumour, hematopoietic disorder disease, autoimmune disorder.

The nucleotide sequence of CK-HA (Chemokine Homologue 1 is called for short CK-HA) gene and known does not have tangible homology.The inventor in protokaryon and eukaryotic cell, successfully expressed this polynucleotide encoding CK-HA, B polypeptide.

First purpose of the present invention provides the polynucleotide sequence of Codocyte factor CK-HA, B.

Second purpose of the present invention provides the carrier that has CK-HA, B nucleotide sequence.

The 3rd purpose of the present invention provides carrier transfection, conversion through carrying CK-HA, B nucleotide sequence or the host cell of transduceing.

The 4th purpose of the present invention provides the method for producing CK-HA, B mature polypeptide with genetic engineering means.

The 5th purpose of the present invention provides the aminoacid sequence of CK-HA, B.

The 6th purpose of the present invention provides the pharmaceutical composition that contains CK-HA, B.

The 7th purpose of the present invention provides CK-HA, B and has purposes in the pharmaceutical composition of hemopoinesis stimulating activity and immunoregulation effect in preparation.

Technical scheme of the present invention is as follows: the nucleotide sequence that the inventor obtains from the cDNA library, by expressed sequence tag (Expression Sequence Tag, be called for short EST) analyze (ttp: //www.tigenm.it), obtained the cDNA sequence of total length, in design, downstream primer P1 (5 '-GCG ATC CAA AGA GCG CGT-3 ') and P2 (5 '-TCT CGA TAA AAG CAG CAG CCC-3 ') increase from the cDNA library of normal adult, separation also clones CK-HA, the gene of B (Chemokine Homologue A, B is hereinafter to be referred as CK-HA, B).

According to a first aspect of the invention, the invention discloses CK-HA and nucleotide sequence CK-HB.CK-HA and CK-HB encode respectively and have 149 amino acid and 94 amino acid whose mature polypeptides, respectively shown in SEQ ID NO:2 and SEQ ID NO:4.

The nucleotide sequence of CK-HA shown in SEQ ID NO:1,603 Nucleotide of total length, the coding region is a 1-450 position Nucleotide.The nucleotide sequence of CK-HB shown in SEQ ID NO:3,587 Nucleotide of total length, the coding region is a 1-285 position Nucleotide.Except that 16 Nucleotide of disappearance, other sequence of CK-HB (SEQ ID NO:3) is identical with the sequence (SEQ ID NO:1) of CK-HA, 16 Nucleotide of disappearance are equivalent to the 280-295 position of SEQ ID NO:1, and the appearance of disappearance causes occurring terminator codon in advance among the SEQ ID NO:3.So SEQ ID NO:3 can regard the varient of SEQ ID NO:1 as, the generation of difference may be that different mRNA shearings causes.

Nucleotide sequence of the present invention can be RNA, cDNA, genomic dna and synthetic DNA, and DNA can be two strands or single stranded form, and single stranded DNA can be coding strand or noncoding strand.

The nucleotide sequence of CK-HA, B preferably provides with unpack format, isolated nucleic acid sequences can include only the encoding sequence of mature polypeptide, the encoding sequence and the additional code sequence that also can comprise mature polypeptide, the encoding sequence and the non-coding sequence that can also comprise mature polypeptide, for example intron, encoding sequence 5 ' or the non-coding sequence of 3 ' end etc.The encoding sequence of CK-HA, B can be entirely identical to the encoding sequence shown in SEQ ID NO:1 or the SEQ IDNO:3.Owing to the degeneracy of genetic code, it can be different from the sequence shown in SEQ ID NO:1 or the SEQ ID NO:3 in addition.

The invention further relates to coding with shown in the SEQ ID NO:2 more than peptide have nucleic acid fragment, analogue and derivative, and the varient of same acid sequence.Varient can be the different varients of shearing varient or non-natural existence of naturally occurring allelic variation body, mRNA.Varient also can be deletion mutation body, replacement varient and adding (insertion) varient that coding and polypeptide shown in the SEQID NO:2 have the polypeptide of same or similar biologic activity.Said allelic variant can have Nucleotide and replace, inserts or add, but do not change in fact coded more than the alternative form of polynucleotide of function of peptide.The coded product of nucleic acid variation varient can be identical with CK-HA of the present invention on function, similar or different.

The small segment of full-length gene of the present invention can be used as hybridization probe, separates full length sequence reaches high homology with CK-HA of the present invention, B gene nucleotide sequence from the cDNA library.Probe generally has 30 bases at least, also can contain the base more than 50.Can identify the cDNA clone who is equivalent to the overall length transducer and contains the genomic clone of complete genome with probe.Method comprises according to the known dna sequence synthetic oligonucleotide probe, and under hybridization conditions, use the oligonucleotide contain with the mark of the dna sequence dna of gene complementation of the present invention to hybridize with people cDNA genomic dna or RNA storehouse, therefrom isolate in the library and the needed nucleotide sequence of probe hybridization.

The gene order that the invention still further relates to CK-HA, B has 80% at least, and is better 90%, the polynucleotide sequence of best 95% homology.Be particularly related under stringent condition the polynucleotide with above-mentioned CK-HA, B gene recombination, said " stringent condition " means the prerequisite that hybridization takes place is to possess 95% homology between sequence at least.Such sequence can be natural existence or artificial the generation, can comprise the allelic variation body of CK-HA, B nucleotide sequence, also can comprise disappearance, insertion and the displacement of base in the CKLFH1 nucleotide sequence.The polypeptide of such sequence encoding can be identical with CK-HA of the present invention, B on function, similar or different, but preferably coding and CK-HA, the essentially identical polypeptide of B biologic activity.

According to a second aspect of the invention, the invention discloses the carrier that carries above-mentioned polynucleotide, said polynucleotide can be inserted into the various suitable expression vectors that are used for expressing this polypeptide.Such carrier comprises that karyomit(e), non-chromosome are originated or the synthetic dna sequence dna, the for example derivative of SV40, bacterial plasmid, phage DNA, yeast plasmid, phage (by plasmid and phage DNA molectron deutero-carrier) and virus (as baculovirus, vaccinia virus, adenovirus, domestic animal poxvirus) DNA, condition is that these carriers can duplicate in selected host cell and survive.

Available various known method is inserted into suitable dna sequence dna in the suitable restriction endonuclease site of carrier.Being inserted into dna sequence dna in the expression vector, to be operably connected to suitable expression control sequenc be promotor, synthetic to instruct mRNA.The example of such promotor comprises RSV, HIV, CMV or SV40 promotor, intestinal bacteria lac or trp promotor, phage PL promotor and other promotors that controlling gene is expressed in protokaryon or eukaryotic cell or its virus.

In addition, expression vector can contain ribosome bind site and the transcription terminator that starts translation, and contain one or more selected marker genes, to provide by the phenotypic characteristic selected of the host cell of conversion, as be applicable to eukaryotic neomycin resistance or dihydrofolate reductase gene, or be applicable to penbritin or tetracycline resistance gene in the intestinal bacteria.

In case of necessity, for the DNA that improves code book invention polypeptide transcribes efficient in higher eucaryotic cells, can in carrier, insert enhancer sequence.Enhanser generally is to contain 10-300 base pair approximately and act on promotor to strengthen the cis-acting elements that DNA transcribes.In addition, also can instruct translation product excretory leader sequence (secretion signal) in pericentral siphon or extracellular substratum in case of necessity in one of insertion between promotor and the downstream configurations sequence.Perhaps, can import the allogeneic dna sequence of encoding fusion protein matter as required, said fusion rotein can comprise that is given a required feature, as is used for stable not held identification polypeptide by the N product of expression or that simplify its purification step.

Specifically, but be applicable to that the commercially available expression vector of prokaryotic cell prokaryocyte generally all has selection marker and cellular replication initial point, have bacterium promotors such as lacI, T7, λ PL and trp, and other genetic elements of known cloning vector pBR322 (ATCC37017).Commercially available carrier like this comprises pGEM (Promega) and pKK223-3 (Pharmacia).Can select to be derived from the suitable carrier of pBR322 according to selected suitable promotor and structural gene sequence to be expressed.Be applicable to that eukaryotic carrier has eukaryotic cell promotor such as CMV, SV40 etc., such carrier comprises pQE-9 (Qiagen), pD10, pNH18A (Stratagene), pKK233-3, pDR540, pRIT5 (Pharmacia), and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL (Pharmacia).

In a word; the present invention relates to comprise the recombinant chou of CK-HA and CK-HB nucleotide sequence; recombinant chou comprises the various carriers that inserted nucleotide sequence of the present invention forward or backwards; as plasmid, virion or phage, and in adjusting sequence such as promoter sequence and the enhancer sequence that it is suitable that the upstream of said polynucleotide sequence has been operably connected.Mammalian expression vector can contain replication origin, transcription termination sequence, and 5 ' flank non-coding sequence.Can use the dna sequence dna in the shearing that is derived from SV40 and polyadenylation site that necessary genetic elements is provided.

According to a third aspect of the present invention, the invention provides cloning vector or expression vector transduction, conversion or transfection appropriate host cell through carrying polynucleotide of the present invention, methods known in the art comprise calcium chloride infection protocol, liposome transfection method, electroporation or microprojectile bombardment methods etc.In order to activate promotor, to select transformant or the required CK-HA that increases, B gene, can in the conventional nutritional medium of suitably modifying, cultivate by transduction, transfection or transformed host cells.Culture condition such as employed temperature, pH generally all are by the decision of the host cell of selected expression specified protein in the cultivation, and these conditions all are well known to those skilled in the art.

Can use any appropriate host cell to express polynucleotide of the present invention, suitably host's example has: bacterial cell such as intestinal bacteria, genus bacillus, streptomycete etc.; Low eukaryotic cell such as the yeast cell of waiting; Insect cell such as fruit bat and fortunatus cell; Higher eucaryotic cells such as mammalian cell such as CHO, COS cell.The example of mammalian expression system comprises the human cell line, as Hela, 293 and U937 clone, and COS, CHO and bhk cell system.

The 4th aspect of the present invention discloses with DNA recombinant technology production the present invention has the method for the polypeptide of immunoregulatory activity and hematopoietic cell stimulating activity.At transformed host cell and after being grown into suitable cell density,, continue cultivation then with appropriate means (inducing) evoked promoter as temperature variation or chemical speciality by transformed host cells.After cultivation is finished, available centrifuging collecting cell, and with any known method, as freeze-thaw method, supersound process method, N,O-Diacetylmuramidase dissolution method or mechanical crushing method smudge cells.Can reclaim from the host cell culture and purifying CK-HA of the present invention, B and varient polypeptide thereof with various known methods, these methods comprise ammonium sulfate or ethanol precipitation, acid extraction method, ultrafiltration process, ion exchange chromatography, phosphoric acid fiber chromatography, hydrophobic interaction chromatography method, gel filtration method, affinity chromatography and high pressure fluid column chromatography.

Can biological engineering method routinely produce polypeptide product by the coding of the recombinant DNA sequence in the host cell.Also can use conventional peptide synthesizer chemosynthesis polypeptide of the present invention.Perhaps can use the mRNA that is derived from DNA construct of the present invention, in cell free translation system, produce required protein.

According to a fifth aspect of the present invention, the invention provides the aminoacid sequence of CK-HA, B mature polypeptide, CK-HA, B have multi-form mature polypeptide, have 149 Aa and 94 Aa respectively as SEQ ID NO:2 and SEQ ID NO:4.

Utilize RT-PCR technology for detection CK-HA, B expression, find that CK-HA, B have special express spectra, in various kinds of cell, all have low abundance to express at different tissues, but in people's testis library special high expression level, and different varient forms is arranged, as CK-HB.To clone and 94 amino acid of its coding of sequential analysis proof of varient gene, called after CKLF-HB, this varient are likely the different shear-forms of CK-HA gene.

Amino acid sequence analysis shows that CK-HA, B and other known cytokine do not have homology.With the aminoacid sequence that the Prosite computer software analysis is inferred, do not find tangible transmembrane domains sequence, there are not DNA binding site and N-glycosylation site.The 22-23 amino acids of the N end of SignalP V2.0 software analysis proof CK-HA contains natural signals peptide cleavage site.The activation analysis proof CK-HA of eukaryotic cell expression supernatant liquor, B albumen can be secreted into the extracellular, belong to secreted protein.The constitutional features that contains Cys-Cys (CC structure) in the amino acid structure of SEQ ID NO:2, and SEQ ID NO:4 does not have the CC structure.

Polypeptide of the present invention can be natural generation, chemosynthesis or be produced by protokaryon or eukaryotic cell with the DNA recombinant technology.The polypeptide that preferred reorganization produces.

According to a sixth aspect of the invention, CK-HA of the present invention, B and activeconstituents thereof can mix with pharmaceutical compositions mutually with one or more pharmaceutically acceptable carriers or vehicle.Can pharmaceutical composition be made multiple formulation according to the needs of therapeutic purpose, route of administration, for example solution, liposome agent, microcapsule and other sustained release preparations.The example of carrier or vehicle comprise physiological saline, etc. ooze the combination of glucose solution, buffer saline, glycerine, ethanol and above-mentioned solution.Can in composition, add as required and assist: for example synergistic compound be arranged with CK-HA of the present invention, B; And for example human serum albumin, low molecular weight peptide, amino acid (as glycine or Methionin) and metallic cation are (as Zn ²⁺, Mn ²⁺, Mg ²⁺And Ca ²⁺) wait protein protective agent; Stablizers such as polyoxyethylene glycol, carboxymethyl cellulose, polyglycine, gsh; Proteinase inhibitor and free-radical scavengers; And under the situation of mucous membrane or local skin administration, add skin penetrants such as dimethyl sulfoxide (DMSO) or laurocapram.

For the convenience on using be convenient to keep the biologic activity of the special primary activity composition of each composition of composition, can be with the special packaged form that makes medicine box of drug regimen of the present invention, such medicine box can comprise one or more containers that one or more compositions of pharmaceutical composition of the present invention are housed, and indicates the use of relevant medicine or the lot number and the relevant information of sale by the specified form of pharmacy administration of government on each container.

Can pass through the outer approach of conventional route, particularly gi tract and use administration of the present invention.The effective dosage ranges of pharmaceutical composition of the present invention can be from several micrograms to several milligrams/kg body weight/sky, but should be determined by the clinician according to factors such as age of the character of disease to be treated or pathological state and severity, patient, body weight, general health situation administering modes at the concrete dosage of each given patient.

Also can express polypeptide of the present invention in vivo, promptly use these polypeptide with so-called " gene therapy " method according to the present invention.For example, can handle patient's cell in the outer-gene through engineering approaches, and then cell that will be engineered imports in the patient body that desire treats with said polypeptide with the polynucleotide of code book invention polypeptide.For example can use the RNA counter-transcription-ing virus particle that contains code book invention polypeptide to come transfection and engineered said cell of expressing polypeptide of the present invention in vivo.Can the production cell that generation contains the counter-transcription-ing virus particle of code book invention polypeptide be come into operation in the patient body by currently known methods, carry out genetically engineered transformation and express said polypeptide in vivo with pair cell in vivo.

Above-mentioned these methods and be used for these methods retrovirus, be included in carry intravital suitable promotor, comprise promotor and be in nucleotide coding sequence more than the suitable promotor control down carrier, be used for the transfection package cell line forming the retroviral plasmid vector of production clone, and suitable packing cell that can be transfected all is known in the field of gene.

Can by various known methods with the carrier of the nucleic acid coding sequence that carries polypeptide of the present invention transfection packing cell or target cell in the external or body.These methods comprise but are not only limited to electroporation, microparticle bombardment (for example using the Biolistic device), liposome transfection method, and really calcium phosphate precipitation method or these methods unite use.In addition, also retrovirus vector can be wrapped in the liposome, perhaps be coupled on the liposome, and then be transported in the suitable target cell of host.

In a word, can produce the infectious retrovirus carrier granule of the nucleic acid coding sequence comprise polypeptide of the present invention, use such carrier granule host eukaryotic cell of in external or body, transduceing then by production clone.The eukaryotic cell of being transduceed will be expressed the nucleotide sequence of the said polypeptide of coding.The eukaryotic cell that can be changeed comprises but is not only limited to embryonic stem cell, embryo cells and becomes human cancer cell, and hemopoietic stem cell, liver cell, inoblast, sarcoplast, mesenchymal cell, epithelial cell and epidermic cell.

The invention still further relates to and use polynucleotide of the present invention, detect the gene of mutant form and express deficiency or express excessive disease that is caused or pathological state because of CK-HA, B in the body with diagnosis as diagnostic reagent.

Available various technology detects the individuality that carries CK-HA, B transgenation on dna level.Can be from patient's body fluid, as obtaining being used for the nucleic acid of diagnostic test in blood, urine, saliva or live body or the postmortem material.Can use gene DNA or RNA directly to detect, detect again after also can using PCR method (Saiki et al., Nature 324:163-166,1986) DNA amplification.For example, can use the sudden change of identifying and analyze corresponding gene in the material to be checked with the nucleic acid complementary PCR primer of code book invention polypeptide, as comparing with normal genotype and changing to determine the disappearance or the insertion of gene to be checked according to the size of amplified production.Can be with DNA and radiolabeled CK-HA, B and the varient RNA or the antisense dna sequence hybridization of amplification, to identify point mutation.

Available direct dna sequencing method is analyzed with reference to the sequence difference between gene and the mutator gene.In addition, the dna fragmentation that also can utilize the clone detects specific dna fragmentation as probe and in conjunction with PCR method with hypersensitivity and specificity, for example can unite the strand PCR product or the single-stranded template molecule that use sequencing primer and produced by amplification PCR products.

Can be at the gel that contains or do not contain denaturing agent, the electrophoretic mobility that particularly detects DNA in the high resolving power gel is finished the genetics test based on dna sequence dna difference.Can on sex change methane amide gradient gel, distinguish not homotactic dna fragmentation (as referring to Myers et al., Science 230:1242,1985).In addition, also available nucleic acid enzyme protection detection method or chemical cracking method (as referring to Cotton et al., PNAS, USA, 85:4397-4401,1985) disclose the sequence change of privileged site.

The invention still further relates to the diagnostic test method that CK-HA, B in the various tissues and varient protein level thereof change that detects.It is well known by persons skilled in the art being used for detecting CK-HA, the B of the interior sample of host-derived body and the detection method of varient protein level thereof, and comprises radioimmunology, competitive combined techniques, Western engram analysis method and enzyme-linked immunosorbent assay (ELISA).Wherein preferable methods is the ELISA method.

Polynucleotide sequence of the present invention identifies it also is valuable for karyomit(e).This sequence can be positioned the chromosomal specific position of one or two people specifically and can hybridize with it.According to the present invention, this bright DNA is carried out first important step that chromosome mapping connects these sequences and disease related gene.

Can prepare the PCR primer to carry out the chromosome mapping of sequence from cDNA.3 ' translation district to gene carries out Computer Analysis, thereby crosses over an above exon in the genomic dna and add primer in the amplification procedure to select rapidly.Use these primers to contain the chromosomal somatic cell hybrid of one or two people then with the screening of PCR method.Have only those hybrids that contain corresponding to the Human genome of primer just to produce the fragment of amplification.It is a kind of facilitated method of specifying the position of specific DNA on specific karyomit(e) that somatic cell hybrid is carried out the PCR mapping.Using according to the present invention under the situation of same Oligonucleolide primers, available one group derives from specific chromosomal fragment or big genomic clone colony finishes inferior location by similarity method.And then cDNA clone is carried out accurate chromosomal localization (referring to Verma et al., Humanchromosomes:A Manual of Basic Techniques, Pergamon Press, NY (1988)) with fluorescent in situ hybridization method (FISH).

In case finished the accurate chromosomal localization mapping of sequence, the physical location and the genetic map data of this sequence can have been connected.Identify that by linkage analysis (the common heredity of the adjacent gene of physics) mapping is positioned gene on the same chromosomal region and the relation between the disease then.Then, can determine impaired or the difference of the cDNA between injured individual or genomic dna not.If in some or all injured individual, observe sudden change, and do not observe this sudden change in the normal individual, just then this sudden change is likely paathogenic factor.

According to a seventh aspect of the present invention, the invention discloses CK-HA, the purposes of B in the pharmaceutical composition of preparation treatment hemopoietic system and disease of immune system.The biologic activity experiment shows CK-HA of the present invention, B polypeptide as a kind of secretion small-molecule substance, and people and bone marrow cells in mice are had obviously short proliferation function, and mouse thymus and splenocyte are also had the obvious stimulation effect, sees embodiment six for details.

In order more to be expressly understood essence of the present invention, referring now to following drawings and Examples it is made an explanation, the purpose of drawings and Examples does not just limit the present invention in any way for explanation.

Fig. 1 is the structure synoptic diagram of prokaryotic expression carrier pMTY4-CKHA, B.

Fig. 2 is the result that CK-HA expresses in prokaryotic cell prokaryocyte.

Fig. 3 has shown the proliferation function of the transfection supernatant of CK-HA, B to the fetus medullary cell.

Fig. 4 has shown CK-HA, the B transfection supernatant short proliferation function to mouse chest cell.

Fig. 5 has shown the proliferation function of the transfection supernatant of CK-HA, B to mouse boosting cell.

Embodiment one, CK-HA, B full length cDNA cloneization

1. analysis of biological information:

Utilize expressed sequence tag (the Expression Sequence Tag of CKLF-1 sequence to the people, abbreviation EST) carries out data retrieval and homology relatively, 19 people's est sequences that low homology is arranged have been found, carry out the EST splicing by ESTAssemBly machine (network address is http://www.tigenm.it), form contig (contig) and carry out the BLAST splicing once more, find complete coding framework.

2.CK-HA, the B full length cDNA cloneization:

Design of primers: splicing obtains according to EST CK-HA, B full length cDNA sequence, it is as follows to design special primer: upstream primer P1:5 '-GCG ATC CAA AGA GCG CGT-3 ', downstream primer P2:5 '-TCTCGA TAA AAG CAG CAG CCC-3 ' pcr amplification: the testis strand cDNA library of personnel selection is that template is carried out pcr amplification.The PCR product downcuts the PCR band after the agarose gel electrophoresis of TAE preparation separates, add 45 ℃ of-55 ℃ of wash-outs of NaI (6M) of 3 times of volumes, add granulated glass sphere suspension solution 10 μ l, of short duration centrifugation granulated glass sphere-DNA mixture is washed 3 times with washing lotion, adds 45 ℃ of-50 ℃ of eluted dnas of H2O.

3.PCR product cloning is to pGEM-T Easy carrier:

The PGEM-Teasy carrier is purchased the company in Promega.The PCR product of purifying is connected to the pGEM-Teasy carrier, connects product and transform XL-1 Blue bacterium, with blue hickie color reaction screening positive clone.

4. the extraction of plasmid and purifying:

The order-checking plasmid carries out purifying with the Tip-20 Mini-preparation test kit of Qiagen company, sequencing kit is available from Pharmacia company, and the specification sheets (seeing StandardAnnealing of Primer to DouBle-Strand Template for details) that provides according to company utilizes ALF Express II dna sequence analysis instrument to carry out sequencing.

Result: utilize the CKLF-1 sequence people's est database retrieval splicing to be obtained the human cDNA sequence of CK-HA, B total length 700Bp.Through RT-PCR increase its coding region and sequential analysis, find that its encoding part is new sequence, shown in SEQ ID NO:1 and SEQ ID NO:3.SEQ ID NO:3 (587 Nucleotide of total length) lacks 16 Nucleotide than SEQ ID NO:1 (603 Nucleotide of total length), and it is the special shape of SEQ ID NO:1.The initial sub-ATG week edge sequence basic symbols of CK-HA, B is closed Kozak rule (GCGATGG) by retrieval, and except that the difference of 16 Nucleotide, the sequence of SEQ ID NO:3 and SEQ ID NO:1 is identical.This difference may be that shear histories different in the same tissue forms.

SEQ ID NO:1 and SEQ ID NO:3 encode respectively and have 149 amino acid and 94 amino acid whose mature polypeptides, respectively shown in SEQ ID NO:2 and SEQ ID NO:4.Aminoacid sequence carries out analysis revealed CK-HA, B and other known cytokine does not have homology.With the aminoacid sequence that the Prosite computer software analysis is inferred, do not find tangible transmembrane domains sequence, there are not DNA binding site and N-glycosylation site.The N end 22-23 amino acids of SignalP V2.0 software analysis proof CK-HA contains natural signals peptide cleavage site.Contain the Cys-Cys constitutional features of (being called for short the CC structure) in the amino acid structure of SEQ ID NO:2, SEQ ID NO:4 does not then possess the CC structure.

Embodiment two, construction of prokaryotic expression vector

As shown in Figure 1, select the pGEM-T plasmid of CK-HA, the insertion of B fragment forward, with upstream and downstream primer amplification CK-HA, the B fragment of CK-HA, B, amplification condition is the same.SEQ ID NO:1 is used to increase.Remove the unnecessary VITAMIN B4 " A " of PCR product 3 ' end with the Klenow fragment earlier, cut with the NotI enzyme again, reclaiming the purifying rear clone goes in the pMTY4 carrier (being made up by this chamber) that the StuI-NotI enzyme is cut, plasmid pMTY4-CK-HA, the B that makes up can be in intestinal bacteria the fusion rotein of abduction delivering MS2 and CK-HA, B, between the two by the polypeptide connection at band zymoplasm point of contact.PMTY4-CKHA, B cut through EcoR I enzyme and discharge CK-HA, B fragment, the sequencing that carries out same as above, and sequencing result is entirely true.

Embodiment three, the CK-HA expression in prokaryotic cell prokaryocyte

(C1857 endA, thi hsdR) are the C600 bacterium that derives to intestinal bacteria pop2136, and the temperature sensitive λ aporepressor of encoding is available from German Bao Ling Man.PMTY4-CK-HA is 30 ℃ of normal cultivations in the pop2136 bacterial strain, 42 ℃ of abduction deliverings.Centrifugal results are induced bacterium, and with the resuspended bacterium of ultrasonic damping fluid of about 1/10 original volume, ultrasonic degradation bacterium, fusion rotein MS2-CK-HA is with the inclusion body formal representation, centrifugal results inclusion body.Inclusion body is at 8M urea, 14mM β-ME, and 37 ℃ of sex change are 30 minutes among the 50mM Tris-HCl (pH8.0).By 1: 10 dilution refolding, 4 ℃ were spent the night, and renaturation is carried out zymoplasm simultaneously and cut.Utilize the heparin affinity chromatography post to carry out protein purification.

As shown in Figure 2, the fusion rotein MS2-CK-HA of fusion protein expression vector pMTY4-CK-HA is efficiently expressed in intestinal bacteria, expression amount accounts for 20% of tropina, and albumen is cut the heparin affinity chromatography column purification through sex change renaturation zymoplasm, and purity can reach (Fig. 2) more than 90%.

The structure of embodiment four, eukaryon expression plasmid

Plasmid pCI is available from Promega company, and pcDNA3 is available from Invitrogen company, and plasmid pcDI is that this chamber makes up, and is a carrier for expression of eukaryon that obtains after the BglI-KpnI fragment of pcDNA3 is replaced with the BglI-KpnI fragment of pCI.As shown in Figure 3, cut with EcoR I enzyme and to discharge CK-HA, B fragment, be connected to it after EcoR I enzyme is cut and handle with CIP after the pCDI carrier in, made up pCDI-CK-HA, B carrier.

Embodiment five, CK-HA, B change eukaryotic cell over to

The 293rd, HEKC, COS-7 is the green monkey kidney cell that SV40 transforms, cell cultures is in RPM1 1640 substratum that contain 10% foetal calf serum.

Transfection experiment: utilize the efficient transfection reagent of SuperFect (Qiagen company), with the pCDI of purifying, pCDI-CK-HA, B plasmid transfection COS-7 cell, in six orifice plates with 1 * 10 ⁵/ 1.5ml substratum repopulating cell.37 ℃ of CO ₂Cultivated 18-24 hour in the incubator, get 2.0 μ g plasmids and add in 100 μ l, 1640 substratum, get 10 μ l SuperFect transfection reagents, add in the DNA mixed solution mixing.It is complete 1640 to get 600 μ l, adds in the transfection reaction mixture.Said mixture is added in the cell, cultivated 2-3 hour.Sucking-off transfection reaction mixture.Add complete 1640 1.5ml, 37 ℃, 5%CO ₂Cultivated 48-72 hour.

Stably express clone's acquisition: the stably express clone is behind plasmid transfection 293 cells 72 hours, adds G418 in substratum, and G418 content rises to high density gradually from lower concentration, to 500 μ g/ml, grows cell clone after two weeks.

The proliferation function of embodiment six, CK-HA, B pair cell

1.CK-HA, B is to the short proliferation function of bone marrow cells in mice

Bone marrow cells in mice behind the lysed erythrocyte is taped against in the 96 porocyte culture plates, negative control group adds the normal COS-7 cell conditioned medium of cultivating 72 hours of 10 μ l, and experimental group respectively adds the original content transfection supernatant of CK-HA, B and pCDI respective amount, does 6 multiple holes for every group, 37 ℃, 5%CO ₂After cultivating for 2 weeks, add the MTT of 10 μ l 10mg/ml in culture hole, cell continues to spend the night after cultivation added lysate after 6 hours.Measured the absorbance value of 570nm wavelength in second day with microplate reader.Found that CK-HA, B transfection supernatant can obviously promote the propagation of bone marrow cells in mice, the pCDI contrast does not have obviously effect.

2.CK-HA, B is to the short proliferation function of people's low density medullary cell

Low density medullary cell after the separation and purification is adjusted into 2 * 10 ⁶/ ml gets 90 μ l and is taped against 96 porocyte culture plates, and negative control group adds normal 29 3 cell conditioned mediums of cultivating of 10 μ l, and experimental group adds 10 μ l, 293 cell transfecting supernatants, 37 ℃ of 5%CO ₂Cultivate 2 weeks added 10 μ l 10mg/ml in culture hole MTT, add lysate after 6 hours and spend the night, the absorbance value of second day survey 570nm wavelength.Found that CK-HA, B can obviously promote the propagation of human fetal medullary cell.

3.CK-HA, B detects the effect of mouse chest cell and splenocyte

Get normal BALB/C mice 20g, get spleen, thymus gland respectively, obtain mouse thymus and splenocyte, adjusting concentration is 2 * 10 ⁶/ ml gets 90 μ l and is taped against 96 porocyte culture plates, and negative control group adds normal 293 cells and supernatant or the 10 μ l Tris-HCl (50mM pH8.0) that cultivate of 10 μ l; Positive group respectively adds the transfection supernatant of respective amount, pCDI empty carrier, pCDI-CK-HA, the prokaryotic expression protein pMTY-CK-HA of pCDI-CK-HB and purifying, 37 ℃ of 5%CO ₂Cultivated for 2 weeks, add 10 μ l 10mg/ml MTT in nutrient solution, cell spends the night after continue cultivating and adding lysate after 6 hours, measures the absorbance value of 570 wavelength in second day with microplate reader.Found that CK-HA, B can promote the propagation of mouse chest cell and splenocyte.

The information of sequence table SEQ ID NO:1

(i) sequence signature:

(A) length: 603 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) sequence description: SEQ ID NO:1

3 9 15 21 27 33 39 45

| | | | | | | |

1 ATG?TTG?AAG?ATC?CTG?AGA?CTG?AGT?CTT?ATC?TTA?GGA?GCA?TTA?GCT

46 TGT?TTC?ATC?ATC?ACC?CAA?GCC?AAT?GAG?TCA?TTT?ATA?ACA?ATC?ACA

91 AGT?CTG?GAA?ATC?TGC?ATT?GTC?GTT?TTT?TTT?ATT?CTA?ATA?TAT?GTG

136 CTA?ACC?CTT?CAC?CAC?TTG?CTG?ACC?TAT?TTA?CAT?TGG?CCC?TTA?CTT

181 GAT?CTT?ACC?AAC?AGT?ATC?ATT?ACA?GCT?GTG?TTC?CTT?TCA?GTA?GTT

226 GCC?ATC?TTG?GCC?ATG?CAA?GAA?AAG?AAA?AGA?AGG?CAT?TTA?CTC?TAT

271 GTC?GGG?GGG?TCC?CTG?TGT?CTC?ACA?GCG?GTA?ATC?GTG?TGT?TGC?ATC

316 GAT?GCG?TTT?GTG?GTC?ACC?ACG?AAG?ATG?AGG?ACC?AAC?TTG?AAA?AGA

361 TTC?CTG?GGA?GTC?GAA?GTT?GAA?AGG?AAG?CTT?TCC?CCC?GCC?AAG?GAC

406 GCC?TAC?CCC?GAA?ACC?GGC?CCC?GAC?GCC?CCG?CAG?AGG?CCC?GCC?TGA

451 AGC?CAG?CCC?GGC?GCC?CTA?GCA?GAT?GCA?CGT?GTC?TGT?CGA?ATC?GCT

496 GCC?TCC?GAG?CCC?ACC?CCC?GAG?CTC?GCA?TGC?TGT?CAC?CCA?TTC?CAG

541 CCT?AAA?TGT?GAC?CAT?AAA?ATT?AGG?GCT?GCT?GCT?TTT?ATC?GAG?AAA

The information of 586 AAA AAA AAA AAA AAA AAASEQ ID NO:2

(i) sequence signature:

(A) length: 149 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: protein

(iii) sequence description: SEQ ID NO:2

5 10 15

| | |

1?Met?Leu?Lys?Ile?Leu?Arg?Leu?Ser?Leu?Ile?Leu?Gly?Ala?Leu?Ala

16?Cys?Phe?Ile?Ile?Thr?Gln?Ala?Asn?Glu?Ser?Phe?Ile?Thr?Ile?Thr

31?Ser?Leu?Glu?Ile?Cys?Ile?Val?Val?Phe?Phe?Ile?Leu?Ile?Tyr?Val

46?Leu?Thr?Leu?His?His?Leu?Leu?Thr?Tyr?Leu?His?Trp?Pro?Leu?Leu

61?Asp?Leu?Thr?Asn?Ser?Ile?Ile?Thr?Ala?Val?Phe?Leu?Ser?Val?Val

76?Ala?Ile?Leu?Ala?Met?Gln?Glu?Lys?Lys?Arg?Arg?His?Leu?Leu?Tyr

91?Val?Gly?Gly?Ser?Leu?Cys?Leu?Thr?Ala?Val?Ile?Val?Cys?Cys?Ile

106?Asp?Ala?Phe?Val?Val?Thr?Thr?Lys?Met?Arg?Thr?Asn?Leu?Lys?Arg

121?Phe?Leu?Gly?Val?Glu?Val?Glu?Arg?Lys?Leu?Ser?Pro?Ala?Lys?Asp

136 Ala Tyr Pro Glu Thr Gly Pro Asp Ala Pro Gln Arg Pro Ala--the information sequence features of-SEQ ID NO:3:

(A) length: 587 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) sequence description: SEQ ID NO:3

3 9 15 21 27 33 39 45

| | | | | | | |

1 ATG?TTG?AAG?ATC?CTG?AGA?CTG?AGT?CTT?ATC?TTA?GGA?GCA?TTA?GCT

46 TGT?TTC?ATC?ATC?ACC?CAA?GCC?AAT?GAG?TCA?TTT?ATA?ACA?ATC?ACA

91 AGT?CTG?GAA?ATC?TGC?ATT?GTC?GTT?TTT?TTT?ATT?CTA?ATA?TAT?GTG

136 CTA?ACC?CTT?CAC?CAC?TTG?CTG?ACC?TAT?TTA?CAT?TGG?CCC?TTA?CTT

181 GAT?CTT?ACC?AAC?AGT?ATC?ATT?ACA?GCT?GTG?TTC?CTT?TCA?GTA?GTT

226 GCC?ATC?TTG?GCC?ATG?CAA?GAA?AAG?AAA?AGA?AGG?CAT?TTA?CTC?TAT

271 GTC?GGG?GGG?CGG?TAA?TCG?TGT?GTT?GCA?TCG?ATG?CGT?TTG?TGG?TCA

316 CCA?CGA?AGA?TGA?GGA?CCA?ACT?TGA?AAA?GAT?TCC?TGG?GAG?TCG?AAG

361 TTG?AAA?GGA?AGC?TTT?CCC?CCG?CCA?AGG?ACG?CCT?ACC?CCG?AAA?CCG

406 GCC?CCG?ACG?CCC?CGC?AGA?GGC?CCG?CCT?GAA?GCC?AGC?CCG?GCG?CCC

451 TAG?CAG?ATG?CAC?GTG?TCT?GTC?GAA?TCG?CTG?CCT?CCG?AGC?CCA?CCC

496 CCG?AGC?TCG?CAT?GCT?GTC?ACC?CAT?TCC?AGC?CTA?AAT?GTG?ACC?ATA

541 AAA?TTA?GGG?CTG?CTG?CTT?TTA?TCG?AGA?AAA?AAA?AAA?AAA?AAA?AAA

The information of 586 AASEQ ID NO:4

(i) sequence signature:

(E) length: 94 amino acid

(F) type: amino acid

(G) chain: strand

(H) topological framework: linearity

(ii) molecule type: protein

(iii) sequence description: SEQ ID NO:4

5 10 15

| | |

1?Met?Leu?Lys?Ile?Leu?Arg?Leu?Ser?Leu?Ile?Leu?Gly?Ala?Leu?Ala

16?Cys?Phe?Ile?Ile?Thr?Gln?Ala?Asn?Glu?Ser?Phe?Ile?Thr?Ile?Thr

31?Ser?Leu?Glu?Ile?Cys?Ile?Val?Val?Phe?Phe?Ile?Leu?Ile?Tyr?Val

46?Leu?Thr?Leu?His?His?Leu?Leu?Thr?Tyr?Leu?His?Trp?Pro?Leu?Leu

61?Asp?Leu?Thr?Asn?Ser?Ile?Ile?Thr?Ala?Val?Phe?Leu?Ser?Val?Val

76?Ala?Ile?Leu?Ala?Met?Gln?Glu?Lys?Lys?Arg?Arg?His?Leu?Leu?Tyr

91?Val?Gly?Gly?Arg

Claims (10)

1. one section isolating polynucleotide sequence, it comprises a member who is selected from as next group:

The polynucleotide sequence of (1) one section shown in SEQ ID NO:2 polypeptide of can encoding;

The polynucleotide sequence of (2) one sections shown in SEQ ID NO:4 polypeptide of can encoding;

Can hybridize with (1) or (2), and have the polynucleotide sequence of at least 80% homology for (3) one sections with them;

(4) one sections polynucleotide sequences, this sequence are the nucleic acid fragments of (1), (2) or (3).

2. nucleotide sequence as claimed in claim 1, polynucleotide wherein are DNA.

3. nucleotide sequence as claimed in claim 1, polynucleotide wherein are RNA.

4. nucleotide sequence as claimed in claim 1, polynucleotide wherein are genomic dnas.

5. carrier, this carrier comprises the nucleotide sequence of claim 1.

6. host cell, this host cell is through carrier conversion, the transfection of claim 5 or transduceed.

7. method of producing polypeptide with genetic engineering means, this method are included in the polypeptide of expressing in the host cell of claim 6 by described dna encoding.

8. a peptide species, it comprises a member who is selected from as next group:

(1) a kind of polypeptide with aminoacid sequence shown in SEQ ID NO:2;

(2) a kind of polypeptide with aminoacid sequence shown in SEQ ID NO:4;

(3) polypeptide fragment of a kind of (1) or (2) described polypeptide.

9. pharmaceutical composition, described pharmaceutical composition comprises the described polypeptide of the claim 8 of effective dose or its activeconstituents, and one or more pharmaceutically acceptable carrier or vehicle.

10. the purposes of polypeptide as claimed in claim 8 in the pharmaceutical composition of preparation treatment hemopoietic system and disease of immune system.

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