CN101595125A

CN101595125A - Avian derived erythropoietin

Info

Publication number: CN101595125A
Application number: CNA2007800391304A
Authority: CN
Inventors: R·D·伊瓦瑞; G·刘; J·C·拉普; J·A·莫里斯; A·J·哈维
Original assignee: University of Georgia Research Foundation Inc UGARF; Synageva Biopharma Corp
Current assignee: University of Georgia Research Foundation Inc UGARF; Synageva Biopharma Corp
Priority date: 2006-11-09
Filing date: 2007-10-10
Publication date: 2009-12-02

Abstract

Has an erythropoietin that bird N-connects the glycosylation pattern be connected with O-available from what transgenic avian was laid eggs.

Description

Avian derived erythropoietin

Related application data

60/918,504 the right of priority of submitting to 16,60/877,601 and 2007 on the March that the application requires to submit on November 9th, 2006 U.S. Provisional Patent Application is submitted to number on December 28th, 60/857,896,2006.

Invention field

The present invention relates to exogenous genetic material is imported the bird cell and exogenous genetic material is expressed in the bird cell.The present invention be more particularly directed to transgenic avian, comprise chicken, quail and turkey, also relate to produce and contain exogenous protein in the birds, beasts and eggs, as the bird of medical protein.

Background

Much natural and synthetic protein has been used for diagnosis and therapeutic application; A lot of other protein are in exploitation or clinical experimental stage.Existing method for producing protein comprises and separating from natural origin and recombinant production in bacterium and mammalian cell.Yet because the complicacy of these method for producing protein and expensive, people are developing other method.For example, reported the method for in the milk of pig, sheep, goat and ox, producing exogenous protein.These methods have several limitation, comprise original animal and produce long-time passage number between the transgenic animal colony, a large amount of management and medical treatment cost, and because changes of expression level due to the position effect of transgenic insert locus in the genome.Also utilizing milling and Fructus Hordei Germinatus explained hereafter protein of barley and rye.Yet posttranslational modification of plant and vertebrate posttranslational modification are different, and this difference often exerts a decisive influence to the function of exogenous protein such as medical protein.

The same with galactophore biological reactor as tissue culture, the uterine tube of bird also may be as bio-reactor.Modifying the bird genetic material makes high-level secretory exogenous protein in the uterine tube and the method in the birds, beasts and eggs of being packaged in successfully make to produce a large amount of protein cheaply.This method has several advantages: a) reduced passage number (24 week) and can set up transgenosis colony fast by artificial insemination; B) expand the scale of production to satisfy product demand by increasing group size easily; C) expressed protein can experience posttranslational modification; 4) can feed and collect egg automatically; D) egg white is natural aseptic; And e) thus because the high technology cost that reduced of proteinic concentration in the egg white.

Bird comprises the existing well description of the reproductive system of chicken.The egg of hen is formed by secreting on yolk which floor when the uterine tube.The generation of egg starts from the formation of big yolk in the hen ovary.Unfertilized then ovocyte is positioned yolk sac top.Behind ovary ovulation or the release yolk, ovocyte enters Fallopian funnel, if then fertilization herein of sperm.Move to the uterine tube protein excretion portion (magnum) that tubular gland cell is arranged then.These gland cells can be secreted egg white protein in protein excretion portion chamber, comprise Protalbinic acid, N,O-Diacetylmuramidase, ovomucoid, conalbumin and ovomucin, and these are proteins deposited on fowl embryo and yolk in the chamber.

The Protalbinic acid genes encoding a kind of in uterine tube protein excretion portion tubular gland cell the protein (Beato, Cell 56:335-344 (1989)) of the 45kD of specifically expressing.Protalbinic acid is the abundantest egg white protein of content, account for more than 50% of gross protein that tubular gland cell produces, or each big A level egg has 4 gram protein (Gilbert approximately, " albumin of egg and formation thereof ", Physiology and Biochemistryof the Domestic Fowl, Bell and Freeman chief editor, academic press (Academic Press), London, New York, 1291-1329 page or leaf).1291-1329)。Protalbinic acid gene and each flanking region 20kb thereof above sequence (Lai etc., Proc.Natl.Acad.Sci.USA 75:2205-2209 (1978) have been cloned and have analyzed; Gannon etc., Nature 278:428-424 (1979); Roop etc., Cell19:63-68 (1980); With Royal etc., Nature 279:125-132 (1975)).

Regulation and control to the Protalbinic acid gene have given very big concern.This gene response steroid hormone, for example oestrogenic hormon, glucocorticosteroid and progestin, these hormones can induce each the tubular gland cell accumulation of prematurity chick to produce about 70,000 Protalbinic acid mRNA transcript, with induce each tubular gland cell 100 of ripe bird inlay, 000 ovalbumin mRNA transcript (Palmiter, J.Biol.Chem.248:8260-8270 (1973); Palmiter, Cell 4:189-197 (1975)).DNA enzyme hypersensitivity in the tubular gland cell of transfection analyzes and promotor-reporter gene test determines that the required sequence of Protalbinic acid genetic expression is contained in the zone of 7.4kb.This 5 ' flanking region contain four therefrom mental arithmetic rise apart from transcription initiation site-0.25 ,-0.8 ,-3.2 and-the DNA enzyme I hypersensitive site of 6.0kb.These sites be called HS-I ,-II ,-III and-IV.The change of chromatin Structure has been reflected in these zones, and relevant with Protalbinic acid expression of gene specificity in the oviduct cell (Kaye etc., EMBO3:1137-1144 (1984)).HS-II and-hypersensitivity of III is an estrogen-induced, has supported these zones to have effect (viewpoint) in the hormone induction of Protalbinic acid genetic expression.

HS-I and-II is that the steroidal of Protalbinic acid genetic transcription is induced necessary, in the tubular gland cell of transplanting, a 1.4kb who contains 5 ' district of these elements partly is enough to drive the ovalbumin expression (Sanders and McKnight, Biochemistry 27:6550-6557 (1988)) that steroidal relies on.6550-6557(1988))。HS-I is called Negative Acknowledgment element (" NRE "), because it contains several negative regulatory elements (Haekers etc., Mol.Endo.9:1113-1126 (1995)) that Protalbinic acid is expressed that can suppress when lacking hormone.Protein factor is incorporated into these elements, comprises the factor that some are only found in uterine tube nuclear, points out these factors that effect is arranged in tissue specific expression.HS-II is called the response element (" SDRE ") that steroidal relies on, and is essential because it is transcribed startup steroidal inductive." it can be in conjunction with protein that is called Chirp-I or protein complex.Chirp-is subjected to estrogen-induced and conversion (Dean etc., Mol.Cell.Biol.16:2015-2024 (1996)) rapidly in the presence of hexahydroaniline.The experiment that the tubular gland cell culture systems that utilization is transplanted is carried out has determined that a cover is additional with the factor of steroidal dependence mode in conjunction with SDRE, comprises that NF κ category-B is like the factor (Nordstrom etc., J.Biol.Chem.268:13193-13202 (1993); Schweers and Sanders, J.Biol.Chem.266:10490-10497 (1991)).10490-10497(1991))。

About HS-III and-function of IV is understood few.HS-III comprises functional oestrogenic hormon response element, makes ovalbumin proximal promoter or allogeneic promoter have estrogen-induced when it and estrogen receptor cDNA cotransfection are gone into the HeLa cell.These Notes of Key Datas, HS-III may play functional effect in the overall regulation and control of Protalbinic acid gene.Understand seldom about the function of HS-IV, only know that it does not contain functional oestrogenic hormon response element (Kato etc., Cell 68:731-742 (1992)).731-742(1992))。

By importing exogenous genetic material and/or interrupting specific gene and modify the eukaryotic gene group and caused the very big interest of people.Proved that some eukaryotic cell may be the good host who produces exogenous eukaryotic protein.Import some proteinic gene of coding and also may produce new phenotype with higher economic worth.In addition, its proteinic foreign gene that can not produce of hereditary defect type cell expressing can be made, some genetic diseasess can be treated by importing.At last, the animal gene group is modified, help the fundamental research of gene function, and may finally import the gene that is used for the treatment of disease, perhaps improve the phenotype of animal by inserting or remove genetic material.

Realized mammiferous transgenosis with several diverse ways.First, comprise in mouse, pig, goat, sheep and the ox Mammals, the protokaryon of zygote is gone in the transgenosis microinjection, then zygote is put in the intrauterine of replace-conceive parent, development of fertilized ova becomes its kind to carry genetically modified original animal in being therein.Carry with the transgenosis of engineering method transformation and to contain specific regulating and controlling sequence and instruct exogenous protein expression promoter in specific cell type.Because transgenosis be insert at random genomic, so transgenosis is inserted the decline that the position effect of genomic locus may cause transgene expression level to some extent.This method also requires promotor is identified, so that instruct in required cell type the necessary sequence of express transgenic to be determined and be included in (Hogan etc. in the transgene carrier, mice embryonic operation (Manipulating the Mouse Embryo), press of cold spring harbor laboratory (ColdSpring Harbor Laboratory), New York (1988)).

Producing the genetically modified second method of animal is that target gene interrupts, and in this method, will contain side joint has the targeting vector of the target gene sequences of selectable marker gene to import dried (" the ES ") cell of embryo.By homologous recombination, this targeting vector has substituted position or the insertion sequence inner expression that stop target gene product of target gene sequences on karyomit(e).Select the ES cell clone that contains the gene that correctly interrupts, then it is injected into early stage blastocyst and produces the original animal of mosaic, contain this transgenosis in the original animal kind of the some of them system.If this transgenosis has been deleted the target gene seat, then this target gene seat is substituted by the foreign DNA in the transgene carrier, this foreign DNA contains coding and is used for selecting to cultivate DNA through the selected marker of transfection ES cell, also may contain the proteinic insertion of encoding exogenous and substitute deleted gene, so that the dna sequence dna of this exogenous gene expression of target gene promoters driven (United States Patent (USP) 5,464,764 and 5,487,992 (M.P.Capecchi and K.R.Thomas)).The limitation of this method is that the ES cell can not obtain, and comprises goat, ox, sheep and pig in a lot of animals.And when deleted gene when being essential for the existence of this biology or cellular type or normal development, this method is infeasible.

Recently, the genetically modified development of bird has made us modify the bird genome.The replication defect type retrovirus can be injected into and produce kind in the chicken blastodisc subgerminal cavity of newly laying eggs is transgenic chicken (U.S. Patent number 5,162,215; Bosselman etc., Science 243:533-534 (1989); Thoraval etc., Transgenic Research 4:369-36 (1995)).The retroviral nucleic acid that contains foreign gene can be inserted at random in the karyomit(e) of embryonic cell and produce transgenic animal, carry transgenosis in the some of them transgenic animal kind system.Existing description utilizes the insulator element in this insertion fusion gene construction 5 ' or 3 ' district to overcome the position effect (Chim etc., Cell 74:504-514 (1993)) of inserting the site.

In another approach, the transgenosis microinjection is gone into the stable energy of generation in the zygote blastodisc this gene is passed to the F1 original fowl (Love etc., Bio/Technology 12:60-63 (1994)) of transgenosis in generation.Yet this method has some not enough.Must sacrifice hen for collecting zygote, the umber of the original fowl of transgenosis is low, and the ovum of injection need spend a large amount of labor force's vitro culture in the replace-conceive shell.

In another approach, the blastoderm cells that will contain the primordial germ cells (" PGC ") of supposition downcuts from the donor ovum, with the transgenosis transfection and import the subgerminal cavity of acceptor embryo.The donorcells of this transfection is mixed acceptor embryo generation transgenosis embryo, contain this transgenosis in the kind system of expection some of them embryo.Transgenosis is inserted chromosomal foci at random by non-homogeneous reorganization.Yet, also do not produce the original bird of transgenosis with this method.

Lui, Poult.Sci.68:999-1010 (1995) is with the intrinsic gene of part of the targeting vector deletion cultured chick embryo dish cell that contains vitellogenin gene side joint dna sequence dna.Yet, do not prove that also these cells help to form kind of system and therefore produce the transgenosis embryo.In addition, when deleted gene is essential for the existence or the normal development of this biology or cellular type, this method is infeasible.

Therefore as seen, need a kind of foreign DNA that operability can be connected with suitable promotor to import in the bird genome to realize the method for foreign gene effective expression.And, need to create can be in uterine tube expression alien gene and the exogenous protein of expressing is secreted into the transgenic avian that the kind system in the egg modifies.

Nineteen fifty-seven, it received an acclaim as a kind of important anti-virus formulation when finding Interferon, rabbit.In the later stage seventies, Interferon, rabbit begins to combine with recombinant DNA technology.Today, Interferon, rabbit is the symbol of the complicacy of cancer bioprocess and patience that tackles this complicacy and persistence value.

The aberrant gene that causes cancer comprises at least three types: first kind is oncogene, promotes distinctive misgrowth of cancer and division when it changes.The second, tumor suppressor gene can not be controlled this misgrowth and division when it changes.The 3rd, DNA-repair gene can not be repaired carcinogenic sudden change when it changes.The investigator infers nearly 30-40 kind tumor suppressor gene in the body, and coding produces protein respectively.These protein may be subjected to for example control of Rb (retinoblastoma at first is associated with it) and p53 (relevant with a lot of different tumours) of " master " tumor suppressor protein matter.Laboratory evidence prompting only makes one of these tumor suppressor genes be returned to the aggressive that normal function just can significantly reduce malignant tumour.

When finding Interferon, rabbit growth capable of inhibiting cell, evoked the very big interest of scientists to Interferon, rabbit.In addition, find that also Interferon, rabbit has some positive effect to immunity system.Think that now Interferon, rabbit and tumor suppressor protein matter are similar: it can suppress the especially growth of malignant cell of cell; Prevent the effect of many oncogenes and somatomedin; Different with the other biological preparation, it can suppress for the very crucial cell migration of metastasis of cancer process.

Cell-cell communication depends in the tissue all structural constituents function of bringing into normal play, and information is by following structural constituent transmission: matrix, cytolemma, cytoskeleton and cell self.In cancer, intercellular network of communication is damaged.If cytoskeleton is destroyed, information just can not be transferred in the nuclear, nuclear beginning dysfunction.Because nuclear is the position that oncogene or tumor suppressor gene are opened or closed, this dysfunction can cause malignant tumour.When this thing happens, cell began irregular growth and does not break up.They also may begin to move and bother other cell.It is believed that Interferon, rabbit may be collaborative with other extracellulars or cellular material, restore balance and homeostasis, guarantee that information correctly transmits.Interferon, rabbit suppresses growth, suppresses migration, strengthens the ability of cell to environmental response by adhesion molecule.It also corrects the defective and the damage of cytoskeleton.Found that Interferon, rabbit can prevent neovascularization, the initial step of neovascularization is that malignant growth is very necessary.And it can prevent fibrosis, and fibrosis is to stimulating a lot of dissimilar cells to promote a kind of reaction of injury due to the cells growth, (Kathryn L.Hale, Oncolog, Interferon, rabbit: a kind of evolution of biotherapy, the biological new view of pair cell).

Interferon, rabbit is released in blood flow or the intercellular liquid when zooblast is subjected to producing when virus is invaded, and induces healthy cell to produce and resists the enzyme that infects.Since a lot of years, the supply of the human interferon that is used to study is subjected to the restriction of expensive extractive technique.Yet 1980, can obtain this protein of greater amount (being this proteinic recombinant forms) by genetic engineering.Scientists is also determined can produce three kinds of different types of interference elements in the body, is called α, β and IFN-.Originally think that Interferon, rabbit is the height species specificity, but it is now know that, various Interferon, rabbit may have different field of activities in other species.(α-IFN) has been approved for hairy cell leukemia and therapy for hepatitis C to interferon-alpha.Find that also α-IFN is effective to the chronic hepatitis B of liver cancer and liver cirrhosis major cause and reproductive tract wart and blood and marrow cancer that some are rare.The nasal spray that contains α-IFN can provide some protection of the flu that rhinovirus causes.People α-IFN belongs to an extracellular signal transferrin matter family, has antiviral, antiproliferative and immunoregulatory activity.The IFN-alpha protein is by the multigene family coding of 13 genomic constitutions of cluster on No. 9 karyomit(e)s of people.Most of IFN-α genes are expressed on the mRNA level by celestial platform (Sendai) virus induction in the white corpuscle.In addition, also find to produce at least the protein of nine kinds of different subtypes at protein level.Express the biological significance of several similar IFN-alpha proteins and also do not know, yet, think that they have pattern inequality on the dosage aspect antiviral, growth-inhibiting and the suicide cell-stimulating activity.Now, two kinds of IFN-α variants, IFN-α 2a and IFN-α 2b are by recombinant technology mass production and putting goods on the market as medicine in intestinal bacteria.

Different with natural IFN-α, found that the IFN-alpha production of these reorganization has immunogenicity in some patient, this may be because due to the non-natural form of IFN-alpha protein.Therefore,, not only need to identify the IFN-alpha hypotype and the variant of expressing in normal people's white corpuscle, and need their possible posttranslational modifications of signature analysis (Nyman etc. (1998) Eur.J.Biochem.253:485-493) for the development of IFN-α medicine.

Nyman etc. (on seeing) have studied natural humanIFN-'s glycosylation.They find have two to be glycosylated in nine kinds of hypotypes that Sendai virus induces the back white corpuscle to produce, and are called IFN-α 14c and IFN-α 2b, and are consistent with former research.IFN-α 14 is unique IFN-alpha hypotypes that potential N-glycosylation site is arranged, and described potential glycosylation site is Asn2 and Asn72, but in fact has only Asn72 that glycosylation takes place.The O-glycosylation takes place in Threonine 106 (Thr106) position in IFN-α 2.Interesting is that other IFN-alpha hypotype does not contain Thr in this position.In this research, releases such as Nyman with separated these oligonucleotide chains and digested the structure of having analyzed them with mass spectroscopy and specificity Glycosylase.IFN-α 2b and IFN-α 14c all resolve to three peaks in RPLC (RP-HPLC).The all components of IFN-α 2b of RP-HPLC generation are done EFI ion massspectrum (ESI-MS) analysis show that their molecular weight is different, point out these components to represent different sugared shapes.The mass spectroscopy of the O-glycan that each component discharges has confirmed this point.According to estimates, IFN-α 2b contains have an appointment 20% core 2 type pentasaccharides, about 50% pair of saliva acidifying and 30% single saliva acidifying core 1 type glycan.The data of Nyman etc. and the former glycosylated Partial Feature of IFN-α 2b consistent (Adolf etc., (1991) Biochem.J.276:511-518).Glycosylated effect also is not very clear among IFN-α 14c and the IFN-α 2b.According to (on seeing) such as Nyman, hydrocarbon chain is inessential for its biological activity, but glycosylation may influence this proteinic pharmacokinetics and stability.

The protein that has 15 kinds of functional gene coding IFN-α families in the human genome at least.Generally in 90% scope, these molecules structurally are closely related amino acid sequence similarity.The IFN-alpha protein contains 166 amino acid (except the IFN-α 2, it has 165 amino acid), and feature is to contain four conservative cysteine residues that form two disulfide linkage.All IFN-α classes be characterized as slightly acidic, lack the glycosylation recognition site (except the IFN-α 14, it contains the glycosylation recognition site that asparagine links to each other) that asparagine links to each other.Known have three kinds of IFN-α 2 variants, and they are different with 34 upper amino acids at the 23rd: IFN-α 2a (Lys-23, His-34), IFN-α 2b (Arg-23, His-34) and IFN-α 2c (Arg-23, Arg-34).Think that IFN-α 2a and IFN-α 2c are the allele variants of IFN-α 2b.Referring to (1993) J.Interferon Res. such as Gewert the 13rd volume, 227-231 page or leaf.Estimate that the fine difference of different types of IFN-α 2 on aminoacids content can not influence the glycosylation of this Interferon, rabbit.In other words, the glycosylation pattern of expectation IFN-α 2a, 2b and 2c is basic identical.Two kinds of other people IFN kinds, promptly IFN-ω 1 and IFN-β are that N-is glycosylated, and be farther with IFN-α relation.IFN-α ,-β ,-ω is generically and collectively referred to as I class IFN, they can in conjunction with identical high-affinity cell-membrane receptor (Adolf etc., (1991), Biochem.J.276:511-518).

Adolf etc. (on seeing) utilize the specificity of monoclonal antibody to separate from human leukocyte IFN and have obtained natural IFN-α 2.They have obtained having IFN-α 2 protein of the antiviral activity purity 95% of expectation by immunoaffinity chromatography.Analyze natural IFN-α 2 with reversed-phase HPLC and show that this natural protein can be distinguished as two kinds of compositions, IFN-α 2 height that the wetting ability of these two kinds of compositions all produces than intestinal bacteria.SDS/PAGE shows that this protein also is heterogeneous, can produce three bands on molecular mass, the electrophoretic mobility of all bands all is lower than the protein that equal intestinal bacteria produce.

Adolf etc. (on seeing) infer that also natural IFN-α 2 carries the saccharide residue that O-connects.With the polypeptide-sugared bond rupture of alkali cutting supposition, the protein of generation is homogeneous and identical with the molecular weight of recombinant protein, has confirmed their hypothesis.After the proteolysis fracture, separate and analyze the fragment of generation, the protein of natural and reorganization is done further to compare, make them determine a kind of candidate's glycopeptide.The sequential analysis of this polypeptide has identified that Thr-106 is the O-glycosylation site.The aminoacid sequence of IFN-α 2 kinds that all have been delivered shows that relatively this threonine residues is that IFN-α 2 is exclusive.(107) are glycine, Isoleucine or L-glutamic acid on the corresponding position in other all IFN protein.

IFN-α 2 goods that intestinal bacteria produce lack the O-glycosylation and have been medicine at a lot of national registrations.Yet, lack the immunogenicity that glycosylation may influence the IFN-α 2 that treats the intestinal bacteria generation of using.Studies show that among the patient that 16 are accepted the macrophage colony stimulating factor of recombinant human granulocyte that yeast produces, 4 have produced this proteinic antibody.Interesting is, finds that these antibody capables are with the glycosylation protection that be connected by O-but the epi-position reaction (Adolf etc. are on seeing) that exposes in this recombinant factor.

Similarly, described the antibody of having induced at recombination bacillus coli IFN-α 2 after patient's long-term treatment, by inference, natural IFN-α 2 is than reorganization IFN-α 2 proteinic immunogenicities low (Galton etc., (1989) Lancet 2:572-573).

Need to produce treatment or medicinal protein,, comprise improving one's methods of Interferon, rabbit, G-CSF and erythropoietin as antibody and cytokine.

Summary of the invention

The invention provides and be used for the stable bird genome that imports of exogenous nucleic acid sequences to express carrier and the method that exogenous array changes the bird phenotype or produces desired protein.Specifically, produced in uterine tube and to express exogenous array and to make exogenous protein such as medical protein deposits to transgenic avian in the egg.The present invention includes the birds, beasts and eggs that contain this class exogenous protein.The present invention also provides the new form of therapeutic protein (as human cell's factor), is included in effective expression in the transgenic avian uterine tube and deposits to Interferon, rabbit, G-CSF, G-MCSF and erythropoietin in the birds, beasts and eggs.

In one aspect, the present invention relates to protein (as, human protein), the cytokine that produces as bird.One concrete aspect, the present invention relates to have a kind of erythropoietin (erythropoietin that produces as, poultry) of glycosylation pattern, wherein said erythropoietin is available from the avian cell of transgenic chicken, transgenosis quail or transgenosis turkey.The present invention also comprises human protein, comprises the isolated or purified form that bird produces and appears at cytokine in the pharmaceutical composition, as erythropoietin.Can separate the recombinant protein of the present invention that comprises erythropoietin by the method that the those of ordinary skill in protein purification field is understood easily.It is well-known in the art that the prescription that is used to produce pharmaceutical composition is formed.In one embodiment, the protein of the present invention that comprises erythropoietin has available from poultry or bird oviduct cell, for example the glycosylation pattern of tubular gland cell (as the tubular gland cell of chicken).

One aspect of the present invention is provided at the method that produces exogenous protein in the bird particular organization.Exogenous protein can be expressed in bird uterine tube, blood and/or other cells and tissue.In one embodiment, transgenosis is imported in embryo's blastoderm cells, for example near the blastoderm cells of X phase, produce transgenic avian, thereby in the tubular gland cell of uterine tube protein excretion portion, express protein of interest matter, be secreted in the chamber, be deposited in the egg white of hard shelled egg.So the transgenic avian that produces can carry transgenosis in its kind is.Therefore, can change foreign gene over to bird for this dual mode of bird offspring the foreign gene stable delivery in Mendelian's mode by foreign gene manually being imported the neutralization of bird embryonic cell.

Present invention resides in the method that produces exogenous protein in the bird uterine tube.This method can comprise, the first step provides and contains encoding sequence and operability is connected in the promotor of this encoding sequence so that promotor can influence the carrier that this nucleic acid is expressed in the bird uterine tube.Next step can produce transgenic cell and/or tissue, wherein, described carrier is imported fowl embryo blastoderm cells among fresh separated, that cultivate or the embryo, makes this carrier sequence insert (for example inserting at random) fowl genome.At last, can be created in the ripe transgenic avian of expressing exogenous protein in the uterine tube by genetically modified cell and/or tissue.The exogenous protein of expressing in uterine tube also is secreted into fallopian tube lumen and is deposited on the ovum of hard shelled egg, and as in the egg white time, also available this method is produced and contained exogenous protein, as the birds, beasts and eggs of medical protein (as cytokine).

On the one hand, carrier is inserted avian-based can randomly comprise blastoderm cells, then these injection cells are gone into the subgerminal cavity under the acceptor blastodisc with DNA transfection embryo because of the process that produces transgenic chicken in the group chromosome.The used carrier of this method can contain and is blended in outer source coding sequence and and guides this encoding sequence expression promoter in the uterine tube tubular gland cell.

Another aspect of the present invention, replication defect type by carrying the transgenosis genetic code between 5 ' and the 3 ' LTR that is used in retroviral vector or replication competent type retroviral particle transduction embryo blastoderm cells insert at random karyomit(e) and produce transgenic avian.For example, can utilize and contain avian leukosis virus (ALV) retroviral vector or murine leukemia virus (MLV) retroviral vector of pNLB plasmid that modified containing is inserted into the foreign gene in promotor section downstream.The RNA of the modified retroviral vector of used pack in virion copies the infecting embryo blastodisc, grows to be transgenic avian.Perhaps, the helper that can produce the retrovirus transducing particles is delivered to embryo's blastodisc.

Another aspect of the present invention provides a kind of relevant carrier that makes this encoding sequence expression promoter in the bird uterine tube on encoding sequence and operation and the position that contains.This class carrier includes but not limited to: avian leukosis virus (ALV) retroviral vector, murine leukemia virus (MLV) retroviral vector and lentiviral vectors.In addition, this carrier can be the nucleotide sequence that comprises the LTR of avian leukosis viruses (ALV) retroviral vector, murine leukemia virus (MLV) reverse transcription carrier or lentiviral vectors.This promotor can fully drive the effective expression of this encoding sequence in the bird uterine tube.This encoding sequence coding can be deposited on the exogenous protein in the hard shelled egg egg white.Equally, this encoding sequence encoding exogenous protein, the protein that produces of transgenosis poultry for example, as interferon alpha 2 b (TPD IFN-α 2b), and the erythropoietin (TPD EPO) that produces of transgenosis poultry and the granulocyte colony-stimulating factor (TPD G-CSF) of transgenosis poultry generation.In one embodiment, used carrier contains and is particularly suited for exogenous protein expression promoter in bird and birds, beasts and eggs in the inventive method.Like this, can in the uterine tube of transgenic avian and blood and in the egg white of birds, beasts and eggs, express source coding sequence in addition.Described promotor includes but not limited to, cytomegalovirus (CMV) promotor, MDOT promotor, rous sarcoma virus (RSV) promotor, beta-actin promotor (as the avian beta-actin promotor), murine leukemia virus (MLV) promotor, MuMTV (MMTV) promotor, Protalbinic acid promotor, N,O-Diacetylmuramidase promotor, conalbumin promotor, ovomucoid promotor, ovomucin promotor and ovotransferrin promotor.Randomly, this promotor can be a section of at least a promoter region, for example, a section of the promoter region of Protalbinic acid promotor, N,O-Diacetylmuramidase promotor, conalbumin promotor, ovomucoid promotor, ovomucin promotor and ovotransferrin.In one embodiment, this promotor can be the combination or the fusions of following one or more promotors, or the fusions of the part of following one or more promotors, these promotors comprise for example promotor of Protalbinic acid, N,O-Diacetylmuramidase, conalbumin, ovomucoid, ovomucin and ovotransferrin.

One aspect of the present invention comprises with the brachymemma of Protalbinic acid promotor and/or with the crucial controlling element compression of Protalbinic acid promotor makes it be retained in the required sequence of expression in the uterine tube protein excretion portion tubular gland cell, makes it enough little and be easy to mix carrier simultaneously.For example, can utilize a section of Protalbinic acid promoter region.This section comprises 5 ' flank region of ovalbumin gene.The total length of Protalbinic acid promotor section is 0.88kb-7.4kb, preferred 0.88kb-1.4kb.Preferred this section contains the steroid class dependency controlling element and the negative regulatory element of ovalbumin gene.Randomly, this section also comprises 5 ' non-translational region (5 ' UTR) residue of Protalbinic acid gene.Perhaps, this promotor is a section of lysozyme gene, conalbumin gene, ovomucoid gene, ovomucin gene and ovotransferrin gene promoter area.An example of this type of promotor is the MDOT promotor that synthetic contains ovomucoid (MD) promotor and ovotransferrin (OT) promoter element.

Another aspect of the present invention, be integrated into the genomic carrier of bird and contain the constitutive promoter (for example, cytomegalovirus (CMV) promotor, rous sarcoma virus (RSV) promotor, murine leukemia virus (MLV) promotor) that is connected in outer source coding sequence in the operation.Perhaps, can use for example mouse breast tumor (MMTV) promotor of non-constitutive promoter.

Other aspects of the present invention are provided in the genetic material of its germ line tissue and carry genetically modified transgenic avian.Say that more specifically this transgenosis comprises promotor relevant with foreign gene on foreign gene and operation and the position to express this external source gene.Described foreign gene can be expressed in the uterine tube of transgenic poultry and blood.This foreign gene encoding exogenous protein as medical protein, comprises cytokine, as TPD IFN-α (as IFN-α 2), TPD EPO and TPD G-CSF.This exogenous protein can be deposited in the egg white of hard shelled egg.

Other one side of the present invention provides that to contain for this bird be ectogenic proteinic birds, beasts and eggs.Utilize the present invention that exogenous protein is expressed in oviduct cell, be secreted into uterine tube protein excretion portion chamber and be deposited in the egg white of birds, beasts and eggs.The protein mass that wraps in the birds, beasts and eggs can reach more than every egg 1 gram.Described exogenous protein includes but not limited to: TPD IFN-α 2b, TPD EPO and TPD G-CSF.

Other one side of the present invention provides isolating polynucleotide sequence, it comprises the optimum coding sequence of human interferon-alpha 2b (IFN-α 2b), the reorganization transgenosis poultry of the interferon-' alpha ' 2b (TPDIFN-α 2b) that the transgenosis of promptly encoding poultry the produces interferon-' alpha ' 2b encoding sequence of deriving.The present invention also comprises the protein of the isolating TPD of comprising IFN-α 2b peptide sequence, and wherein this proteinic Thr-106 position is by N-acetyl-GalN, semi-lactosi, N-acetyl-glycosamine, sialic acid and its combination O-glycosylation.

The present invention also imagines a kind of pharmaceutical composition of the TPD of comprising IFN-α 2b peptide sequence, protein wherein in the Thr-106 position by N-acetyl-GalN, semi-lactosi, N-acetyl-glycosamine, sialic acid and its combination O-glycosylation.

One aspect of the present invention provides the encoding sequence of the exogenous protein of generation as described herein, and wherein this encoding sequence is according to bird, carries out codon optimized as expression in the chicken.Can be at least a by what express in the bird cell (as chicken cell), preferably more than one proteinic codons make and are used for determining codon optimized scheme.For example, can determine that codon uses by the nucleotide sequence of Protalbinic acid, N,O-Diacetylmuramidase, ovomucin and ovotransferrin of coding chicken.For example, can pass through the BACKTRANSLATE program of Wisconsin Package (Wisconsin software package) 9.1 editions (the Genetics Computer Group Inc. of state of Wisconsin Madison (genetics calculating group company)), utilize and to carry out codon optimized to the dna sequence dna of exogenous protein by the codon use table of Hongyuan chicken (Gallus gallus) Protalbinic acid, N,O-Diacetylmuramidase, ovomucoid and ovotransferrin compiling.

One aspect of the present invention provides a kind of isolating polynucleotide sequence, it comprises human erythropoietin (EPO) optimum coding sequence, the reorganization transgenosis poultry of the erythropoietin (TPD EPO) that the transgenosis of promptly encoding poultry the produces erythropoietin encoding sequence of deriving.

The present invention provides a kind of carrier that can in bird uterine tube express the promotor of this first and second encoding sequence relevant with this first and second encoding sequence on first and second encoding sequences and operation and the position that contain on the other hand.Aspect this, this carrier can comprise internal ribosome entry site (IRES) element between this first and second encoding sequence, wherein, the first encoding sequence proteins encoded X, the second encoding sequence proteins encoded Y, one or both in albumin X and the protein Y are deposited in the ovum (as egg white) of hard shelled egg.

For example, albumin X can be the light chain (LC) of monoclonal antibody, and protein Y can be the heavy chain (HC) of monoclonal antibody.Perhaps, the second encoding sequence encoded protein (for example, enzyme) can be carried out posttranslational modification to the first encoding sequence encoded protein.This carrier randomly can contain other encoding sequence and other IRES element, by the IRES element each encoding sequence in the carrier and other encoding sequences is separated.Use other example of considering to be used for IRES of the present invention referring to the U.S. Patent application of for example submitting on January 31st, 2,005 11/047,184, it is for referencial use to include its full content in this paper.

The invention still further relates to produce and contain protein such as medical protein, comprise the method for monoclonal antibody, enzyme and other proteinic birds, beasts and eggs.This method can comprise provides the carrier that contains promotor, encoding sequence and at least one IRES element; Produce transgenic cell or tissue by this carrier being imported bird embryo blastoderm cells, wherein, this carrier sequence is inserted the bird genome at random; Produce sophisticated transgenic avian by transgenic cell or tissue.So the transgenic avian that produces can be expressed described encoding sequence in its uterine tube, the protein secreting that produces is gone in the fallopian tube lumen, thereby be deposited in the egg white of hard shelled egg.In addition, the present invention includes the offspring of this transgenic avian that output contains the birds, beasts and eggs of this recombinant protein.Usually, this offspring's all cells all contains this transgenosis basically, and perhaps this offspring's all cells does not all contain this transgenosis.

An importance of the present invention relates to and contains exogenous peptide or protein, includes but not limited to the bird hard shelled egg (as the chicken hard shelled egg) of medical protein.This exogenous peptide or protein can be by the transgenes encodings of transgenic avian.In one embodiment, this exogenous peptide or protein (as medical protein) are glycosylated proteins.This albumen can have consumption to exist.In one embodiment, this proteic content is about 0.01 microgram/hard shelled egg-Yue 1 gram/hard shelled egg.In one embodiment, this proteic content is about 1 microgram/hard shelled egg-Yue 1 gram/hard shelled egg.For example, this proteic content is about 10 micrograms/hard shelled egg-Yue 1 gram/hard shelled egg (for example about 10 micrograms/hard shelled egg-Yue 400 milligrams/hard shelled egg).

In one embodiment, exogenous protein of the present invention is present in as exogenous medicinal albumen in the egg white of this egg.In one embodiment, this proteic content is about 1 nanograms/milliliter egg white-Yue 0.2 grams per milliliter egg white.For example, this proteic content is about 0.1 mcg/ml egg white-Yue 0.2 grams per milliliter egg white (for example this proteic content is about 1 mcg/ml egg white-Yue 100 mg/ml egg whites).In one embodiment, this proteic content is about 1 mcg/ml egg white-Yue 50 mg/ml egg whites.For example, this proteic content is about 1 mcg/ml egg white-Yue 10 mg/ml egg whites (for example this proteic content is about 1 mcg/ml egg white-Yue 1 mg/ml egg white).In one embodiment, protein content is greater than 0.1 μ g/ milliliter egg white.In one embodiment, protein content is greater than 0.5 μ g/ milliliter egg white.In one embodiment, protein content is greater than 1 μ g/ milliliter egg white.In one embodiment, protein content is greater than 1.5 μ g/ milliliter egg whites.

The present invention relates to produce and contain any useful proteins matter, comprise the hard shelled egg of one or more medical proteins.This proteinoid includes but not limited to: the part of hormone, immunoglobulin (Ig) or immunoglobulin (Ig), cytokine (as GM-CSF, G-CSF, erythropoietin and Interferon, rabbit) and CTLA4.The present invention also comprises producing and contains fusion rotein, includes but not limited to the hard shelled egg of the fusion rotein of certain part of immunoglobulin (Ig) or immunoglobulin (Ig) and some useful peptide sequence.In one embodiment, the invention provides the hard shelled egg that generation contains antibody Fc fragment.For example, this egg can contain Fc-CTLA4 fusion rotein of the present invention.

The bird that is produced by the blastoderm cells of introducing carrier is G0 generation, can be described as " initiator ".Original bird generally is to contain respectively to insert genetically modified chimaeric animals.In other words, have only some cells to contain this transgenosis in the G0 transgenic avian.G0 generation also is this genetically modified hemizygote generally.G0 is for breeding with the non-transgenic animal, and the G1 transgenic progeny of generation also is this genetically modified hemizygote, and the cell of all these birds all contains this transgenosis basically.G1 hemizygote offspring can breed with the non-transgenic animal, produces G2 hemizygote offspring, or breeding produces the G2 offspring that transgenosis is isozygotied mutually.Basically all transgenic positive bird cells derived from the G1 offspring all contain this transgenosis.In one embodiment, can breed from the hemizygote G2 offspring of same pedigree and produce the G3 offspring that this transgenosis is isozygotied.In one embodiment, hemizygote G0 animal breeds mutually and produces the homozygote G1 offspring that each zooblast contains two transgenosis copies.These are the example of some useful breeding method, and the present invention considers any useful breeding method, the method as known to persons of ordinary skill in the art of adopting.

One aspect of the present invention relates to containing and has the glycosylation pattern that poultry produces, the proteinic composition that produces by the present invention of the glycosylation pattern that produces as chicken.One aspect of the present invention relates to containing and has the glycosylation pattern that bird produces, the proteinic composition that produces by the present invention of the glycosylation pattern that produces as chicken.For example, the present invention includes glycosylation pattern, the medical protein of one or more glycosylation patterns as described herein with poultry generation.The present invention also comprises having the glycosylation pattern that poultry produces, the human protein of one or more glycosylation patterns as described herein.

One aspect of the present invention comprises the G-CSF of the glycosylation pattern with poultry generation, i.e. the G-CSF or the TPD G-CSF of transgenosis poultry generation.One aspect of the present invention comprises the G-CSF with transgenic avian deutero-glycosylation pattern, i.e. transgenic avian deutero-G-CSF.In one embodiment, this glycosylation pattern is different from the G-CSF that produces in people's cell and/or the Chinese hamster ovary celI.In other words, said composition contains and has the G-CSF molecule that does not have this sugar chain or glycosylation structure on the G-CSF that obtains in poultry or avian derived sugar chain (being the glycosylation structure) and people's cell and/or the Chinese hamster ovary celI.Yet said composition also can contain the identical G-CSF molecule of G-CSF that obtains in glycosylation structure and Chinese hamster ovary celI and/or the people's cell.The glycosylation of the human G-CSF that Chinese hamster ovary celI produces is included this paper Holloway for referencial use in referring to full content, C.J., European J.of Cancer (1994), 30A volume, pS2-S6; Full content is included this paper (1988) J.Biochem. such as Oheda for referencial use in, the 103rd volume, and 544-546 page or leaf and full content are included this paper (1994) Glycobiology such as Andersen for referencial use in, the 4th volume, 459-467 page or leaf.As if the glycosylation structure of structure shown in the embodiment 20 such as A and the G G-CSF that may produce with the Chinese hamster ovary celI of report is same or similar.In one embodiment, the glycosylation pattern of the G-CSF that produces according to the present invention is different from the G-CSF that produces in the mammalian cell.

In one embodiment, the invention provides isolating G-CSF.In other words, contained G-CSF can be isolating G-CSF in the said composition.For example, this G-CSF separates with egg white.Isolating G-CSF can be the G-CSF molecule with different glycosylation structure, and perhaps isolating G-CSF can be the independent isolating G-CSF molecule that has only a kind of concrete glycosylation structure.

In one embodiment, the G-CSF of the present composition is present in the hard shelled egg.For example, G-CSF can be present in the egg white of hard shelled egg that transgenic avian of the present invention produces.In other words, in one embodiment, the present invention relates to contain birds, beasts and eggs (as the egg) egg white of G-CSF of the present invention.In one embodiment, the content of G-CSF surpasses about 1 mcg/ml egg white in the egg white.For example, the content of G-CSF can be greater than about 2 mcg/ml egg whites (for example about 2 micrograms-200 of content mcg/ml egg white).

Of the present invention one concrete aspect, bird, as G-CSF in the oviduct cell of chicken glycosylation takes place.For example, G-CSF can produce in oviduct cell and by glycosylation.In one embodiment, G-CSF in tubular gland cell by glycosylation (for example G-CSF in tubular gland cell, produce and by glycosylation).

Think G-CSF glycosylation on Threonine 133.Yet, the invention is not restricted to the glycosylation on any specific site of G-CSF molecule.

G-CSF of the present invention generally is a human G-CSF.In one embodiment, ripe G-CSF contains aminoacid sequence shown in Figure 18 C.

In one embodiment, the present composition comprises with following structure glycosylated G-CSF molecule:

The present invention also is particularly related to the composition that comprises the G-CSF molecule with one of these specific glycosylation structures.This based composition also can comprise one or more G-CSF molecules with one or more other glycosylation structures.

In other words, in one embodiment, The present invention be more particularly directed to comprise the composition of G-CSF molecule with following structure:

And relate to the composition that comprises G-CSF molecule with following structure:

SA-Gal-NAcGal-；

Gal-NAcGal-，

Wherein, the Gal=semi-lactosi,

NAcGal=N-acetyl-GalN,

NAcGlu=N-acetyl-glycosamine and

The SA=sialic acid

The invention still further relates to the method that improves patient's white blood cell count(WBC), described method comprises the G-CSF according to the present invention's generation that gives the patient treatment significant quantity.The treatment significant quantity generally is patient's white blood cell count(WBC) to be improved the G-CSF consumption of aequum.

One aspect of the present invention relates to and contains EPO, i.e. the composition of the EPO molecule that produces according to the present invention.In a useful especially embodiment, described EPO is a purifying or isolating.For example, described EPO produces the hard shelled egg content from transgenic avian to take out.In a useful especially embodiment, described EPO is people EPO.In one embodiment, EPO of the present invention has the glycosylation pattern of the EPO that produces in the bird oviduct cell.The present invention relates to the composition that contains the EPO with certain glycosylation pattern on the other hand, and described glycosylation pattern is different from the glycosylation pattern of the EPO of people's cell or Chinese hamster ovary celI generation, and described EPO produces in the oviduct cell of chicken.One aspect of the present invention provides and contains the composition that separates EPO (as people EPO), and described EPO has bird or poultry deutero-glycosylation pattern.For example, said composition can contain according to the present invention bird, for example produce in the chicken, and by the mixture of the isolating EPO molecule of egg white.In a useful embodiment, the composition that contains EPO is a pharmaceutical preparation.

In one embodiment, the oligosaccharides on the EPO of the present invention does not contain Fucose.In another embodiment, EPO of the present invention last about 90% or more N-connect oligosaccharides and do not contain Fucose.In another embodiment, EPO of the present invention last about 80% or more N-connect oligosaccharides and do not contain Fucose.In another embodiment, EPO of the present invention last about 70% or more N-connect oligosaccharides and do not contain Fucose.In another embodiment, EPO of the present invention last about 60% or more N-connect oligosaccharides and do not contain Fucose.In another embodiment, EPO of the present invention last about 50% or more N-connect oligosaccharides and do not contain Fucose.

In one embodiment, EPO of the present invention last about 95% or more N-connect oligosaccharides and do not contain sialic acid.In another embodiment, EPO of the present invention last about 90% or more N-connect oligosaccharides and do not contain sialic acid.In another embodiment, EPO of the present invention last about 80% or more N-connect oligosaccharides and do not contain sialic acid.In another embodiment, EPO of the present invention go up greater than about 70% or more N-connect oligosaccharides and do not contain sialic acid.In another embodiment, EPO of the present invention last about 60% or more N-connect oligosaccharides and do not contain sialic acid.In another embodiment, EPO of the present invention last about 50% or more N-connect oligosaccharides and do not contain sialic acid.

In one embodiment, EPO of the present invention last about 95% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.In another embodiment, EPO of the present invention last about 90% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.In another embodiment, EPO of the present invention last about 80% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.In another embodiment, EPO of the present invention last about 70% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.In another embodiment, EPO of the present invention last about 60% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.In another embodiment, EPO of the present invention last about 50% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.

In one embodiment, the structure type of the connection of the N-on the EPO molecule of the present invention oligosaccharides does not contain Fucose basically.In another embodiment, on the EPO molecule of the present invention about 90% or the more N-structure type that connects oligosaccharides do not contain Fucose.For example, if 20 kinds of oligosaccharide structure types are arranged, 18 kinds or more kinds of structure type do not contain Fucose so.In another embodiment, on the EPO molecule of the present invention about 80% or the more N-structure type that connects oligosaccharides do not contain Fucose.In another embodiment, on the EPO molecule of the present invention about 70% or the more N-structure type that connects oligosaccharides do not contain Fucose.In another embodiment, on the EPO molecule of the present invention about 60% or the more N-structure type that connects oligosaccharides do not contain Fucose.In another embodiment, on the EPO molecule of the present invention about 50% or the more N-structure type that connects oligosaccharides do not contain Fucose.

In one embodiment, the structure type of the connection of the N-on the EPO molecule of the present invention oligosaccharides does not contain sialic acid basically.In another embodiment, on the EPO molecule of the present invention about 90% or the more N-structure type that connects oligosaccharides do not contain sialic acid.For example, if 20 kinds of oligosaccharide structure types are arranged, 18 kinds or more kinds of structure type do not contain sialic acid so.In another embodiment, on the EPO molecule of the present invention about 80% or the more N-structure type that connects oligosaccharides do not contain sialic acid.In another embodiment, on the EPO molecule of the present invention about 70% or the more N-structure type that connects oligosaccharides do not contain sialic acid.In another embodiment, on the EPO molecule of the present invention about 60% or the more N-structure type that connects oligosaccharides do not contain sialic acid.In another embodiment, on the EPO molecule of the present invention about 50% or the more N-structure type that connects oligosaccharides do not contain sialic acid.

In one embodiment, the structure type of the connection of the N-on the EPO molecule of the present invention oligosaccharides all contains terminal N-acetyl-glucosamine basically.In another embodiment, on the EPO molecule of the present invention about 90% or the more N-structure type that connects oligosaccharides contain terminal N-acetyl-glucosamine.For example, if 20 kinds of oligosaccharide structure types are arranged, 18 kinds or more kinds of structure type contain terminal N-acetyl-glucosamine so.In another embodiment, on the EPO molecule of the present invention about 80% or the more N-structure type that connects oligosaccharides contain terminal N-acetyl-glucosamine.In another embodiment, on the EPO molecule of the present invention about 70% or the more N-structure type that connects oligosaccharides contain terminal N-acetyl-glucosamine.In another embodiment, on the EPO molecule of the present invention about 60% or the more N-structure type that connects oligosaccharides contain terminal N-acetyl-glucosamine.In another embodiment, on the EPO molecule of the present invention about 50% or the more N-structure type that connects oligosaccharides contain terminal N-acetyl-glucosamine.

In one aspect, the present invention relates to, as the EPO of transgenic chicken acquisition by the genetically modified transgenic avian that contains the EPO that encodes.In one embodiment, EPO is the bird oviduct cell, for example, produces in the tubular gland cell.In one embodiment, EPO is included in hard shelled egg, for example, and in the hard shelled eggs that bird produced such as chicken.For example, EPO may be present in the content of complete hard shelled egg.In a useful especially embodiment, EPO of the present invention is people EPO.

In one aspect, the present invention relates to contain separative EPO molecule, as the composition of people EPO molecule, wherein said EPO is that the genetically modified bird that contains this EPO of coding produces.In one embodiment, described EPO is the middle generation of the oviduct cell (as tubular gland cell) of transgenic avian (as transgenic chicken), and described EPO is isolating by the egg white of transgenic avian.In one embodiment, EPO of the present invention has the aminoacid sequence shown in Figure 19 A.Consider that described EPO is that N-is glycosylated and/or O-is glycosylated.In one embodiment, described EPO is birds, and is glycosylated in the oviduct cell (as tubular gland) as chicken.

In one aspect, the present invention relates to a kind of composition, as pharmaceutical preparation, it contains the separation EPO with avian derived glycosylation pattern, for example people EPO.In one aspect, the present invention relates to a kind of composition, as pharmaceutical preparation, it contains and has poultry derive the separation EPO of glycosylation pattern, for example people EPO.In one aspect, the present invention relates to a kind of composition, as pharmaceutical preparation, it contains the separation EPO that produces according to the present invention, for example people EPO.In one embodiment, the glycosylation pattern that EPO had in the present composition is different from the EPO that produces in the mammalian cell.In one embodiment, the glycosylation pattern that EPO had in the present composition is different from the EPO that produces in Chinese hamster ovary celI and the people's cell.In one embodiment, EPO of the present invention is connected in one or more N-connection oligosaccharide structure (as shown in figure 21) as herein described.In one embodiment, EPO of the present invention is connected in one or more O-connection oligosaccharide structure (as shown in figure 20) as herein described.

One aspect of the present invention relates to the method for the treatment of the patient, and described method comprises the EPO available from transgenic avian that gives the patient treatment significant quantity.In one embodiment, the treatment significant quantity is patient's red blood cell count(RBC) to be improved the consumption of required numerical value.Consider that can utilize the EPO treatment chronic nephropathy that produces according to the present invention, for example, tissue can't be kept the situation that erythropoietin produces.

One aspect of the present invention relates to the composition that contains separative glycosylated human protein molecule, described human protein molecule produces in the transgenic avian uterine tube, described transgenic avian (as transgenic chicken) contains the transgenosis of this human protein of encoding, and described human protein contains the chicken deutero-oligosaccharides that can not appear at usually on the human protein.In one embodiment, described human protein is connected in one or more N-connection oligosaccharide structure (as shown in figure 21) as herein described.In one embodiment, described human protein is connected in one or more O-connection oligosaccharide structure (as shown in figure 20) as herein described.

In one embodiment, the present invention relates to produce the isolating protein molecule of (as described herein) in the transgenic chicken uterine tube, described transgenic chicken contains the transgenosis of this protein molecule of encoding, and described protein molecule contains chicken deutero-oligosaccharides.For example, described protein molecule can be following proteinic glycosylation form: GM-CSF, interferon beta, fusion rotein, the CTLA4-Fc fusion rotein, tethelin, cytokine, works (structural), Interferon, rabbit, N,O-Diacetylmuramidase, beta-casein, albumin, α-1 antitrypsin, Antithrombin III, collagen, Factor IX, factors IX, factor X (etc.), fibrinogen, lactoferrin, PROTEIN C, tissue plasminogen activator (tPA), tethelin and Chymotrypsin, immunoglobulin (Ig), antibody, immunotoxin, Factor IX, the Factor IX of β-Jie Gou territory disappearance, factor VIIa, factors IX, antithrombotics; R-hirudin, alteplase, tpa, reteplase, tpa, disappearance is 3 in tpa-5 the structural domain, Regular Insulin, Insulin lispro, aspartame's Regular Insulin (insulin aspart), Lantus, Recent Development of Long-acting Insulin Analogs, glucagon, tsh, follitropin-β, fsh, pdgh, inf-β 1b, ifn-β la, ifn-γ 1b, il-2, il-11, hbsag, ospa, dornase alfa-DNA enzyme, the β glucocerebrosidase, tnf-α, il-2-diphtheria toxin fusion rotein, tnfr-lgg fragment fusion rotein, the La Luoni enzyme, the DNA enzyme, Ah method's Saite (alefacept), tositumomab, the mouse monoclonal antibody, Ah coming organizes monoclonal antibody (alemtuzumab), rasburicase, Ah add'sing carbohydrase β, teriparatide, parathyroid hormone derivative, adalimumab (lgg1), Kineret, the bio-modification agent, Nesiritide, people β-type natriuretic peptide (hbnp), G CFS, pegvisomant, the growth hormone receptor antagonist, the albumen c of recombination activation, Ao Mazuo monoclonal antibody (omalizumab), ige (lge) blocking-up thing, ibritumomab tiuxetan, ACTH, glucagon, Somatostatin, tethelin, thymosin, Rat parathyroid hormone 1-34, pigment hormone, somatomedin, lutropin, chorionic gona dotropin, hypothalamic releasing factor, etanercept, antidiuretic hormone, prolactin antagonist and thyrotropic hormone, immunoglobulin polypeptides, immunoglobulin polypeptides D district, immunoglobulin polypeptides J district, immunoglobulin polypeptides C district, light chain immunoglobulin, heavy chain immunoglobulin, immunoglobulin heavy chain variable region, immunoglobulin light chain variable region and joint peptide.It will be understood by those skilled in the art that usually not glycosylated protein can be through engineered and contain glycosylated glycosylation site in the bird system.In one embodiment, described isolated protein is connected in one or more N-connection oligosaccharide structure (as shown in figure 21) as herein described.In one embodiment, described isolated protein is connected in one or more O-connection oligosaccharide structure (as shown in figure 20) as herein described.

The special feature of considering (as composition, glycosylation structure) also can be used for other concrete albumen that produces according to the present invention as herein described among some protein described herein such as the EPO.

The present invention also comprises the method for preparation glycosylated protein as herein described such as erythropoietin, described method comprises produces the genetically modified transgenic avian that contains coded protein (as erythropoietin), and wherein said protein pack is in the hard shelled egg that this bird produced.The egg that contains described protein (as erythropoietin) that also comprises bird and produced.

The present invention also provides the useful proteins matter that contains independent type molecule, the composition of proteinic separating mixture as described herein, one or more contained protein molecules of wherein said mixture are connected with the specific oligosaccharides structure, specifically are the oligosaccharide structures that can be produced by transgenic avian as herein described.For example, the invention provides the EPO molecule, as the separating mixture of people EPO molecule (as the EPO of SEQ ID NO:50), it contains in order to the following glycosylated EPO molecule of one or more structures:

With

With

With

With

With

With

With

With

With

Gal

NacGlu

Sialic acid

Seminose

With

Gal

NacGlu

Sialic acid

NacGal

And Figure 20 and other each oligosaccharide structure shown in Figure 21.

Any useful combination of feature described herein all is included in the scope of the present invention, as long as can find out that from context, this specification sheets and those of ordinary skills' knowledge contained feature can be not conflicting in these combinations.

With reference to following description details, when taking into consideration, can obviously find out other purpose of the present invention and aspect with the accompanying drawing of brief description.

Brief Description Of Drawings

Figure 1A and 1B show the Protalbinic acid promoter expression vector, and this carrier contains the Protalbinic acid promoter fragment and encoding sequence is the gene X of encoding exogenous protein X.X represents interested any foreign gene or exogenous protein.

Fig. 2 A, 2B, 2C and 2D show retroviral vector of the present invention, and this carrier contains the Protalbinic acid promotor and encoding sequence is the gene X of encoding exogenous protein X.X represents interested any foreign gene or exogenous protein.

Fig. 2 E shows that amplification is used for inserting the method for 2A and 2B carrier foreign gene.

Fig. 2 F shows retroviral vector, and this carrier contains the control encoding sequence, the Protalbinic acid promotor that gene X expresses.This carrier also comprises internal ribosome entry site (IRES) element, so that second encoding sequence, gene Y can express.X and Y represent interested any gene.

Fig. 3 A and 3B show the synoptic diagram of ALV deutero-carrier pNLB and pNLB-CMV-BL respectively.Because NLB is not order-checking fully also, so the measurement of bp (base pair) is data (Cosset etc., 1991 from having delivered; Thoraval etc., 1995) and this paper data estimation of discussing.Because this two carriers will occur when being integrated into the genome of chicken, so shown them.

Fig. 4 A and 4B show the amount of β-Nei Xiananmei (lactamase) in the serum of chimeric and transgenic chicken.Among Fig. 4 A, hatch measured in back 8 months with biological activity lactamase concentration in the G0 chicken serum of NLB-CMV-BL transgenosis transduction.Show generation, sex and wing circle number.Measured the lactamase serum-concentration of 6 to 7 months G1 transgenic chickens in hatching back.Arrow is represented from the G1 chicken of cock 2395 breeding generations.In Fig. 4 B, measured the lactamase serum-concentration of G1 and G2 transgenic chicken.Arrow is represented from the G2 chicken of hen 5657 or cock 4133 breeding generations.Identical among

chicken

4133,5308 and 5657 sample and Fig. 4 A.Collect 3 to 60 days the sample in G2 chicken hatching back of 5657 breedings.The G2 chicken of collecting 4133 breedings hatches back 3 months sample.

Fig. 5, show have hen 5657 (Fig. 5 A) or cock 4133 (Fig. 5 B) with the pedigree of the chicken of transgenosis locus.The 2395th, carry the cock of a plurality of transgenosis locus.With the mating of 2395 and non-transgenic hens, produced 3 and respectively carried genetically modified offspring in its genomic unique site.For simplicity, transgenic progeny and the non-transgenic offspring who does not show expression data omitted from pedigree.With following symbolic representation wing circle number: zero hen; The cock; ● carry the genetically modified hen of NLB-CMV-BL; ■ carries the genetically modified cock of NLB-CMV-BL.

Fig. 6 shows β-Nei Xiananmei (lactamase) amount in hen 5657 and the offspring's egg white thereof.Among Fig. 6 A, detected 5657 and the transgenic progeny egg white in active lactamase.Contrast is taken from untreated hen, and even producing chicken (clutchmate) is the non-transgenic G2 chicken that breeds from hen 5657.Collect egg in March, 2000.Arrow is represented from the G2 chicken of hen 5657 breedings.Among Fig. 6 B, the egg white sample that will carry the G2 transgenosis hen of a copy transgenosis (hemizygote) compares with the egg white sample that carries the G3 hen 6978 of two copies transgenosis (isozygotying).Collect egg February calendar year 2001.The left side has been indicated from generation to generation and wing circle number.

Fig. 7 shows from the G2 of cock 4133 breedings and the β-Nei Xiananmei (lactamase) the G3 hen egg.Among Fig. 7 A, detected from the active lactamase of four representative hemizygote transgenosis hen egg whites of cock 4133 breedings.The time of collecting egg is respectively in October, 1999, in March, 2000 and February calendar year 2001, collects back one month every hen at every turn and detects 4 eggs at least.The egg white of the hen of being untreated is represented in contrast.Wing circle number has been indicated on the left side.Calculate the mean value of four hens in each period.Among Fig. 7 B, egg white of hemizygote G2 transgenosis hen and the egg white of hemizygote and homozygote transgenosis G3 hen are made comparisons.Collect egg February calendar year 2001.Generation of every hen and transgenosis copy number are listed in the field.Carry the mean concns of the hen of one or two copy and see this figure bottom.

Fig. 8 A and 8B show the pNLB-CMV-IFN carrier and the pNLB-MDOT-EPO carrier that is used in chicken expressing promoting erythropoietin (EPO) that are used for expressing chicken IFN-α 2b respectively.

Fig. 9, the new glycosylation pattern of the interferon-' alpha ' 2b (TPD IFN-α 2b) that demonstration transgenosis poultry produces comprises all 6 bands.

Figure 10 shows the comparison of the interferon-' alpha ' 2b (PBL IFN-α 2b or natural hIFN) and the interferon-' alpha ' 2b (TPD IFN-α 2b or egg white hIFN) that the transgenosis poultry produces of human peripheral leucocytes generation.

Figure 11 A, the synthetic nucleic acid sequence (cDNA, residue 1-498) of the human interferon-alpha 2b of display optimization (IFN-α 2b), TPD IFN-α 2b (SEQ ID NO:1) promptly recombinates.1). Figure 11 B, the synthetic nucleic acid sequence (residue 1-165) (SEQID NO:2) of the interferon-' alpha ' 2b (TPD IFN-α 2b) that demonstration transgenosis poultry produces.

Figure 12 A, the synthetic nucleic acid sequence of the human erythropoietin of display optimization (EPO) (cDNA, residue 1-579), the TPD EPO that promptly recombinates (SEQ ID NO:3).Figure 12 B, the synthetic nucleic acid sequence (residue 1-193) (SEQ IDNO:4) of the erythropoietin (TPD EPO) that the demonstration transgenic poultry produces.4). (, seeing NCBI accession number NP_000790 again) for natural people EPO.

Figure 13 shows the synthetic MDOT promotor that is connected with IFN-MM CDS.The MDOT promotor comprises-251 to+29bp (ovotransferrin promotor) (see NCBI accession number Y00497, M11862 and X01205) the element of avian ovomucoid gene-435bp to-166bp (ovomucoid promotor) (seeing NCBI accession number J00894) and chicken conalbumin gene.

Figure 14 provides the brief summary of main egg white protein.

Figure 15 A and 15D show the pCMV-LC-emcvIRES-HC carrier, wherein, for detecting the expression of monoclonal antibody, make the light chain (LC) and the heavy chain (HC) of this carrier energy expressing human monoclonal antibody by the IRES that places encephalomyocarditis virus (EMCV).By comparison, Figure 15 B and 15C show independently carrier pCMV-HC and pCMV-LC respectively, and wherein, these carriers also are used to detect the expression of monoclonal antibody.

Figure 16 shows the urgent base of knob that silver dyes

The SDS PAGE of (swimming lane A) and TPD G-CSF (swimming lane B).

Figure 17 demonstration is compared with the human G-CSF of bacterial derivation, and the absolute neutrophil leucocyte counting (ANC) of TPD G-CSF increases in 14 days.

The nucleotide sequence of the aminoacid sequence of Figure 18 A (SEQ ID NO:39) code displaying Figure 18 B.Show to be included in corresponding to Figure 18 B (SEQ ID NO:40) of NCBI accession number NP 7577373 and be cut during the emiocytosis and form the G-CSF aminoacid sequence of the natural signals sequence of ripe G-CSF.Figure 18 C (SEQ ID NO:41) shows the proteic aminoacid sequence of ripe G-CSF that produces by the present invention.

Figure 19 A shows the nucleotide coding sequence that is used for producing at transgenic avian 165 amino acid form of erythropoietin.Figure 19 B is presented at the aminoacid sequence of 165 amino acid form of the erythropoietin that produces in the transgenic avian.

Figure 20 shows that the representative O-of the erythropoietin that produces according to the present invention of mensuration connects the glycosylation structure.

Figure 21 A and Figure 21 B show that the representative N-of the erythropoietin of measuring that produces according to the present invention is connected the glycosylation structure.Sugar can be connected in any sugar in the braces shown in the braces of one group of saccharide residue front was represented.For example, in structure E-n, shown in be connected in sialic galactose molecule and can be connected in any in five kinds of terminal N-acetyl-glucosamines.Shown that also the supposition in the structure connects, as known in the art.Consider, be designated as in each structure of C-n, E-n, F-n and H-n that two terminal NAcGlu residues that are connected in a seminose can be 2,6-is connected in seminose, rather than 2,4 are connected in seminose.

Figure 22 shows the external activity of the transgenic chicken deutero-EPO of purifying.ED50＝0.44ng/ml。

Detailed Description Of The Invention

This paper proposes implication and the scope that following definition is used for setting forth and clearly describing the used various terms of the present invention.

" nucleic acid or polynucleotide sequence " includes but not limited to, Eukaryotic mRNA, cDNA, genomic DNA and synthetic DNA and RNA sequence comprise natural nucleoside base adenine, guanine, cytimidine, thymine and uracil. This term also comprises the sequence that contains one or more modified bases.

Term used herein " bird " refers to any kind of Aves classification, subspecies or ancestor, such as but not limited to chicken, turkey, duck, goose, quail, pheasant, parrot, sparrow, hawk, crow and ratite, comprises ostrich, emu and cassowary. The various known strain that this term comprises Hongyuan chicken (Gallus gallus) or chicken (for example, white leghorn, brownly come Hangzhoupro chicken, speckle chukar (Barred-Rock), Su Saikesi chicken (Sussex), New Hampshire chicken, Rhode Island chicken, Australorp, Minorca, A Mu chicken (Amrox), California ash chicken), and the strain of turkey, pheasant, quail, duck, ostrich and other poultry usually raised with commercial size. Also comprise all stages of development, comprise the independent bird organism of embryo and the fetal state.

" therapeutic protein " or " medical protein " comprises the amino acid sequence of all or part of formation medicine.

" coded sequence " or " ORFs " refers to that suitable regulating and controlling sequence control is lower can be transcribed and translate (if DNA) or translate polynucleotides or the nucleotide sequence that (if mRNA) becomes polypeptide when placing in external or body. The border of coded sequence is defined by the terminal translation initiation codon of 5 ' (amino) and terminal translation stop codon of 3 ' (carboxyl). Transcription terminator generally is positioned at 3 ' end of coded sequence. But 5 ' and/or 3 ' end side joint of coded sequence has non-translational region.

" extron " refers to the part of gene, when genetic transcription is nuclear during transcript, this part after introne or intervening sequence are removed in nuclear splicing with the formal representation of cytoplasm mRNA.

Nucleic acid " control sequence " or " regulating and controlling sequence " refer to promoter sequence, translation initiation and terminator codon, ribosome bind site, polyadenylic acid signal, transcription terminator, upstream regulatory region, enhancer etc., they for the particular host cell transcription and the translation certain given coded sequence be essential and sufficient. Be fit to eukaryotic control sequence promoter, polyadenylic acid signal and enhancer are for example arranged. Not necessarily existing these all control sequences only need exist in the recombinant vector transcribes and translates essential and sufficient control sequence for required gene.

" operability or operability are connected in " refers to bring into play the coding of required function and the configuration of control sequence. Therefore, the operability control sequence that is connected in coded sequence can affect the expression of this coded sequence. In cell, the coded sequence operability is connected in or is controlled by transcription regulatory region, is incorporated into promoter sequence at this district's archaeal dna polymerase, coded sequence is transcribed into the mRNA that can translate into protein. Control sequence can be adjoined with coded sequence, as long as they can bring into play the expression of functional guidance coded sequence. Therefore, for example, when the transcription sequence that interleaves untranslated is present between promoter sequence and the coded sequence, still can think this promoter sequence and this coded sequence " operability is connected ".

For nucleotide sequence for example coded sequence and control sequence, term " allos " and " external source " refer under the normal condition not sequence relevant with certain zone of recombination to construct thing or specific chromosomal foci, and/or not relevant with specific cells sequence under the normal condition. Therefore, " external source " zone of nucleic acid construct thing is in other nucleic acid molecules of not finding under nature with its associated or the section identified of the nucleic acid that links to each other with this molecule. For example, the outer source region of construction can comprise with nature under the coded sequence that links to each other of the sequence of associated company not. Another example of outer source coding sequence is that coded sequence itself is the undiscovered construction of occurring in nature (for example, the synthetic sequence that contains the codon different from natural gene). Similarly, with this host cell that non-existent construction under certain host cell normal condition transforms, also think for the purpose of the present invention external source.

The implication of term used herein " oligosaccharides ", " oligosaccharide structure ", " glycosylation pattern " and " glycosylation structure " is basic identical, all refers to be formed and be connected in by saccharide residue one or more structures of glycosylated protein.

" exogenous proteins " used herein refers to not be present under the nature protein in particular organization or the cell, heterogenous expression construction or transgene expression product are this protein, or the protein that does not exist with certain quantification under particular organization or cell nature. The exogenous proteins of egg is usually non-existent protein in this egg. For example, the exogenous proteins of egg can be the protein that is present in because of the expression of coded sequence in the transgenosis of the animal of laying eggs in this egg.

" endogenous gene " refers to gene relevant with specific cells under the abiogenous normal condition or fragment.

" EPO " refers to " erythropoietin(EPO) ", and these two terms are used interchangeably in whole specification.

Expression product as herein described can comprise the protein material with definite chemical constitution. Yet accurate structure depends on several factors, especially for the very general chemical modification of protein. For example, because all protein all contains ionogenic amino and carboxylic group, so can obtain the protein of acidity or alkaline salt forms or neutral form. For example, available glycan molecule (glycosylation) or other chemical method comprise such as with covalent bond or the ions binding such as fat, phosphoric acid, acetyl group, often produce the one-level amino acid sequence by the chemically derived method that is connected with sugar. These modifications can occur in external or the body, and the latter is that host cell is undertaken by translating rear system of processing. These modifications can increase or fall low molecular biologically active, and the molecule of these chemical modifications is also included within the scope of the present invention.

The additive method of clone, amplification, expression and purifying it will be apparent to those skilled in the art that. Exemplary process is seen Sambrook, Fritsch and Maniatis, molecular cloning: lab guide, second edition, publishing house of cold spring harbor laboratory (Cold Spring Harbor Laboratory) (1989).

" carrier " refers to comprise the polynucleotides of strand, two strands, ring-type or super coiled DNA or RNA. Typical carriers can comprise that following operability is connected in suitable location so that the element that functional gene is expressed: origin of replication, promoter, enhancer, 5 ' RNA targeting sequencing, ribosome bind site, nucleic acid box (Nucleic acid cassette), termination site and polyadenylation site and selected marker sequence. When concrete the application, can omit one or more these class components. The nucleic acid box can comprise the restriction site that inserts for nucleotide sequence to be expressed. In functional carrier, the nucleic acid box comprises the nucleotide sequence to be expressed that contains translation initiation site and termination site. Optionally comprise introne in the construction, for example be positioned at 5 ' of coded sequence. Will make specific coded sequence and suitable regulating and controlling sequence be arranged in this carrier during carrier construction, coded sequence is answered so that coded sequence is transcribed under " control " of regulating and controlling sequence or regulating and controlling sequence with respect to position and the direction of regulating and controlling sequence. For realizing this purpose, may need the sequence of the specific proteins of interest matter of encoding is modified. For example, it is linked to each other may needing in some cases to modify this sequence with control sequence with correct direction, or make it keep reading frame. Before insertion vector with control sequence with are connected regulating and controlling sequence and are connected with coded sequence. Perhaps, in the expression vector with the restriction site of coded sequence Direct Cloning in the suitable reading frame that contains under the regulation and control of control sequence and this control sequence.

" promoter " thus be to be combined with RNA polymerase the site that promotor gene transcribes on the DNA. In some embodiment, by adding or deleting sequence and modify promoter, perhaps comprise that with other sequence compositions natural and composition sequence and these two kinds of sequences substitute promoter. Many eukaryotic promoters contain two types recognition sequence: TATA frame and upstream promoter element. The former is positioned at the transcription initiation site upstream, participate in the guide RNA polymerase and transcribe in correct position startup, and as if the latter has determined transcription rate, is positioned at TATA box upstream. Enhancer also can promote transcribing of continuous promoter, but a lot of enhancer is only brought into play function in the specific cells type. A lot of enhancers/promoters elements derive from virus, for example, the SV40 promoter, cytomegalovirus (CMV) promoter, rous sarcoma virus (RSV) promoter and murine leukemia virus (MLV) promoter all have activity in a lot of cellular types, be called as " all in type " promoter. Perhaps, also can adopt for example mouse mammary tumor virus (MMTV) promoter of non-constitutive promoter among the present invention. The nucleotide sequence that inserts cloning site can contain any opening code-reading frame of the polypeptide of interest of encoding, condition is when this coded sequence is encoded interested polypeptide, and it should not have to hinder the correct mRNA molecule of generation and/or produces cryptic splice site aberrant splicing or unusual mRNA molecule.

Term " poultry derives " refers to composition or the material by poultry generation or acquisition. " poultry " refers to can be used as the bird of cattle breeding, includes but not limited to: chicken, duck, turkey, quail and ratite. For example, " poultry derives " can refer to that chicken derives, turkey derives and/or quail derives.

" marker gene " refer to encode cell of making correct transfection is identified gene with the protein that separates. Suitable marker gene includes, but are not limited to green, yellow and blue fluorescent protein gene (being respectively GFP, YFP, BFP). Other suitable marker gene comprise thymidine kinase (tk), dihyrofolate reductase (DHFR) and aminoglycoside phosphotransferase (APH) gene. The latter introduces the resistance to aminoglycoside antibiotics such as kanamycins, neomycin and gentamicin sulphate. Chloramphenicol acetyltransferase (CAT), beta-lactamase, the beta galactosidase (gene of β-gal) of these and other marker gene for example can being encoded, be integrated into main nucleic acid box with the gene of expressing desired protein, perhaps selected marker may be included in independently cotransfection on the carrier.

" reporter " is the marker gene of reporting cytoactive by the existence of its encoding proteins.

" reverse transcription particle " or " transducing particles " refer to the virus that non-viral DNA or RNA transduction maybe can be able to be copied such as the replication defect type in the cell.

Term " conversion ", " transduction " and " transfection " all refer to polynucleotides are introduced in the bird blastoderm cells. " PE section " is to contain the infundibulum tubae uterinae of tubular gland cell of the egg white protein that can synthesize and secrete birds, beasts and eggs and the part between the gorge (Magnum).

" MDOT promoter " used herein is activated synthetic promoter in fallopian tubal PE section but not in the tubular gland cell of other tissue. MDOT comprises the element (Figure 13) of ovomucoid (MD) and ovotransferrins (OT) promoter.

In " coded sequence of optimization ", use term " optimization ", wherein, can will be used for design at the most frequently used codon of each specific amino acids of egg white protein ovalbumin, lysozyme, ovomucoid and ovotransferrins and be inserted into the polynucleotide sequence of human interferon-alpha 2b (IFN-α 2b) of the optimization of carrier of the present invention. More specifically, the dna sequence dna of the human interferon-alpha 2b of optimization can utilize the codon utilization rate table of Hongyuan chicken ovalbumin, lysozyme, ovomucoid and ovotransferrins compiling to create by the BACKTRANSLATE program of winconsin software kit 9.1 editions (the science of heredity calculating group company of Wisconsin State Madison) according to hen fallopian tubal optimization Codon usage rate. For example, in these four kinds of egg white proteins, four kinds of alanine Codon usage percentage are GCU 34%, GCC 31%, GCA 26%, GCG 8%. Therefore, can be with the codon of GCU as most of alanine in the humanIFN-α 2b coded sequence of optimizing. Carrier with the humanIFN-α 2b gene that contains code optimization produces transgenic poultry, the IFN-α 2b (TPD IFN-α 2b) that it can express transgenic fowl generation in tissue and ovum. Similarly, with said method design other coded sequence albumen according to the present invention's generation, such as erythropoietin (EPO) or other oroteins.

By method of the present invention, transgenosis can be imported bird embryo blastoderm cells, be created in the inhereditary material of its germ line tissue and carry this genetically modified transgenic chicken, transgenosis turkey, transgenosis quail and other birds. Blastoderm cells is typical VII-XII phase cell, or the cell that is equal to mutually with it, is the cell of nearly X phase in one embodiment. Can be used for cell of the present invention and comprise embryonic genital cell (EG), embryonic stem cell (ES) and primordial germ cells (PGC). Embryo's blastoderm cells can be new that separate, maintain in the culture or among the embryo.

This paper has described the carrier that can be used for implementing the inventive method. Available these carriers are with the stable bird genome that imports of outer source coding sequence. Perhaps, available these carriers are for example produced exogenous proteins in the bird oviduct tissue in bird particular organization. Also can be with in the method for these carriers for the production of the birds, beasts and eggs that contain exogenous proteins. In one embodiment, introduce before the blastoderm cells coded sequence and promoter all 5 ' and 3 ' LTR between. In one embodiment, described carrier is retroviral vector, and coded sequence and promoter all are positioned between 5 ' and the 3 ' LTR of retroviral vector. In a useful embodiment, this LTR or retroviral vector derive from avian leukosis virus (ALV), murine leukemia poison (MLV) or slow virus.

In one embodiment, this carrier comprises the signal coding sequence that operability is connected in coded sequence, thereby this signal peptide will instruct the exogenous proteins of this vector expression to be secreted in the egg white of hard shelled egg after translating in cell. This carrier can comprise marker gene, and wherein said marker gene operability is connected in promoter.

In some cases, with carrier of the present invention import embryo's blastoderm cells be with new that separate or cultivate in embryo's blastodisc carry out. Then, generally be that transgenic cell is injected in the subgerminal cavity under the acceptor blastodisc of egg. Yet in some cases, also carrier directly can be sent into the cell of blastodisc embryo.

In an embodiment of the invention, be used for the transfection blastoderm cells and produce stable integration entering the genomic carrier of fowl and containing relevant promoter on certain coded sequence and operation and the position, in the tubular gland cell of bird fallopian tubal PE section, to express this coded sequence, wherein, a kind of exogenous proteins that is deposited in the hard shelled egg egg white of this coded sequence coding. Randomly, promoter can be one of the ovalbumin promoter region enough big section to instruct coded sequence in tubular gland cell, to express. The present invention includes with the brachymemma of ovalbumin promoter and/or with the crucial controlling element compression of ovalbumin promoter, make it be retained in the required sequence of expression in the fallopian tubal PE section tubular gland cell, make simultaneously it enough little and be easy to and vector integration. In one embodiment, can utilize a section of ovalbumin promoter region. This section comprises 5 ' flank region of ovalbumin gene. The total length of ovalbumin promoter section is 0.88kb-7.4kb, preferred 0.88kb-1.4kb. Preferred this section contains steroid class dependence controlling element and the negative regulatory element of ovalbumin gene. Randomly, this section also comprises 5 ' non-translational region (5 ' UTR) residue of ovalbumin gene. Therefore, this promoter can derive from the promoter region (Figure 14) of ovalbumin gene, lysozyme gene, conalbumin gene, ovomucoid gene, ovotransferrins gene or ovomucin gene. An example of this type of promoter is the synthetic property MDOT promoter (Figure 13) that comprises ovomucoid promoter and ovotransferrins promoter element. But this promoter also can be not exclusively to be specific to fallopian tubal PE section to a great extent, for example the lysozyme promoter. This promoter also can be MuMTV (MMTV) promoter. Perhaps, this promoter can be constitutive promoter (for example, cytomegalovirus (CMV) promoter, rous sarcoma virus (RSV) promoter, murine leukemia virus (MLV) promoter etc.). In a preferred embodiment of the present invention, this promoter is cytomegalovirus (CMV) promoter, MDOT promoter, rous sarcoma virus (RSV) promoter, murine leukemia virus (MLV) promoter, MuMTV (MMTV) promoter, ovalbumin promoter, lysozyme promoter, conalbumin promoter, ovomucoid promoter, ovomucin promoter and ovotransferrins promoter. Randomly, this promoter can be at least one section of promoter region, for example a section of ovalbumin promoter, lysozyme promoter, conalbumin promoter, ovomucoid promoter, ovomucin promoter and ovotransferrins promoter region. In one embodiment, this promoter is the CMV promoter.

Figure 1A and 1B have illustrated the example of ovalbumin promoter expression vector. Gene X is the coded sequence of encoding exogenous protein. Curved arrow represents transcription initiation site. In one embodiment, this carrier contains the 5 ' flanking sequence (Figure 1A) of ovalbumin gene 1.4kb. " 1.4kb promoter " sequence is corresponding to the sequence that begins to extend to about 9 residues the ovalbumin gene 5 ' non-translational region from the ovalbumin genetic transcription initiation site about 1.4kb in upstream among Figure 1A. The long section of this about 1.4kb contains two crucial controlling elements, steroid class dependence controlling element (SDRE) and negative regulatory element (NRE). Why being called NRE is because it contains the negative regulatory element of several gene expressions capable of blocking when lacking hormone (for example estrogen). A shorter 0.88kb section also can contain this two kinds of elements. In another embodiment, this carrier contains the 5 ' flanking sequence of the about 7.4kb of ovalbumin gene and contains two add ons (HS-III and HS-IV), known one of them contain the functional areas (Figure 1B) that this gene is induced by estrogen. A shorter 6kb section also contains this four elements, optionally is used for the present invention.

Each carrier that is used for random integration of the present invention preferably comprises the chicken beta-globin locus element of at least one 1.2kb, and this element can make gene neither activate also non-inactivation in the genomic site of insertion. In one embodiment, two insulator elements are joined an end of ovalbumin gene constructs. In the beta-globin locus, this insulator element effect is the gene that stops upstream, far-end locuscontrol region (LCR) activated beads GFP district, and shown the position effect that can overcome in the transgenosis fly, shown that they can prevent from inserting positive-effect and the negative effect in site. Only 5 ' or 3 ' end at this gene just needs insulator element, because transgenosis is to integrate with the multi-copy in tandem form effectively to have produced the gene that a series of flanks are connected with adjacent genetically modified insulator. In another embodiment, insulator element is not connected in this carrier but and this carrier cotransfection. In this case, described carrier and element enter genomic process by random integration and are connected in series in cell.

Randomly, each carrier also can comprise marker gene to identify and the enrichment cell clone of stable integration expression vector. With can drive various types of cells type high level expression all in the expression of this marker gene of promoters driven. In an embodiment of the invention, this marker gene is the human interferon (gene) that is subjected to the lysozyme promoters driven. In another embodiment, to be subjected to toad EF-1-α (ef-1 α) promoter (Johnson and Krieg, Gene 147:223-26 (1994)) green fluorescent protein (GFP) reporter gene (Zolotukhin etc., J.Virol 70:4646-4654 (1995)) that drives. Toad ef-1 α promoter is a kind of strong promoter that can all express in various types of cells. GFP contain the sudden change that can strengthen fluorescence and by humanization or modification so that its codon meets the codon handling characteristics of people's gene. Because the codon of bird uses and the people is basic identical, thus the humanization form of this gene also can be in the bird blastoderm cells high expressed. In other optional embodiments, this marker gene operability be connected in HSV tk, CMV, beta-actin or RSV all in promoter one.

Although the codon of people and bird uses and to match finely, with the gene of invertebrate during as the coded sequence in the transgenosis, thereby the gene order that can modify invertebrate makes the similar of codon use and people and bird to change suitable codon.

Can adopt any several method transfection blastoderm cells known to those of ordinary skills. At first with nucleic acid and polylysine or thereby cation lipid is mixed helps the carrier transfered cell to promote carrier to pass cell membrane. Yet, preferably carry for example liposome or viral with the carrier transfered cell of fortune carrier by adopting. Be used for the virus of carrier importing blastoderm cells of the present invention is included, but not limited to retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus and vaccinia virus.

In a kind of method of transfection blastoderm cells, with the retroviral vector of packing this carrier is transported into embryo's blastoderm cells so that this vector integration enters the bird genome.

The another kind of method that embryo's blastoderm cells in the embryo transports the retrovirus transducing particles is to transport into blastodisc with producing retroviral auxiliary cell.

Being used for the genomic retrovirus of transgenosis radom insertion bird is replication defect type avian leukosis viruses (ALV), replication defect type murine leukemia virus (MLV) or slow virus. In order to produce suitable retroviral vector, with a zone of ovalbumin promoter and 5 ' and 3 ' long terminal the repetition between (LTR) to modify the pNLB carrier of one or more foreign gene insertion reverse transcription virus gene group. The present invention considers that any coded sequence that is arranged in the activated promoter of tubular gland cell downstream will express at tubular gland cell. For example, because the expression of ovalbumin promoters driven ovalbumin and in the fallopian tubal tubular gland cell, activity is arranged, so the ovalbumin promoter can be expressed in fallopian tubal PE section tubular gland cell. Although when in the fallopian tubal tubular gland cell of cultivating, detecting, find that the ovalbumin promoter of 7.4kb produces the highest active construction, yet preferably the ovalbumin promoter shortened when being used for retroviral vector. In one embodiment, described retroviral vector contains the 1.4kb fragment of ovalbumin promoter; 0.88kb section also is enough.

Any carrier of the present invention also can be chosen the coded sequence that comprises signal peptide wantonly, and this signal peptide can instruct the protein that coded sequence is expressed in the carrier to secrete out from the fallopian tubal tubular gland cell. Of the present invention this enlarged the scope of utilizing the inventive method can be deposited on the exogenous proteins of birds, beasts and eggs on the one hand effectively. When a kind of exogenous proteins can not be secreted, can modify the carrier that contains its coded sequence and make it contain the dna sequence dna of the 60bp code signal peptide of lysozyme gene. Can be with in the dna sequence dna insertion vector of this signal peptide of coding and be located at the N end of this dna encoding protein.

The example of the retroviral vector construct thing that Fig. 2 A-2D explanation is suitable. This vector construct is inserted in the bird genome with 5 ' and 3 ' flank LTR. Neo is neomycin phosphotransferase gene. Curved arrow represents transcription initiation site. Fig. 2 A and 2B show LTR and the fallopian tubal transcript of the sequence that contains encoding muramidase signal peptide (LSP), and Fig. 2 C and 2D show the transcript that does not contain this sequence. The retroviral vector strategy has two parts. Any protein clone that contains the eukaryotic signal peptide can be entered shown in Fig. 2 B and the 2D in the carrier. Usually any protein clone that can not secrete can be entered carrier shown in Fig. 2 A and the 2B being secreted by tubular gland cell.

Fig. 2 E explanation is cloned into exogenous proteins the strategy of lysozyme signal peptide carrier. Contain the gene that can make amplification inserts restriction enzyme site in the plasmid behind double digestion oligonucleotide as primer with a pair of, utilize the PCR amplification coded sequence, the copy of gene X. 5 ' and 3 ' oligonucleotide contains respectively Bsu36I and Xba1 restriction site.

Another aspect of the present invention is included in and utilizes internal ribosome entry site (IRES) with translation two or more protein (embodiment 15) from bicistronic mRNA or polycistronic mRNA in any carrier of the present invention. The IRES unit is blended in 5 ' end of one or more other coded sequences, the end of original coded sequence in the insertion vector then, thus make these coded sequences be separated from each other (Fig. 2 F, 15A and 15D) by IRES. According to this one side of the present invention, be conducive to the posttranslational modification of product, because the endonuclease capable of a coded sequence coding is modified the product of another coded sequence. For example, the collagen of first coded sequence coding may be by enzyme hydroxylating and the activation of second coded sequence coding. In the retroviral vector example of Fig. 2 F, internal ribosome entry site (IRES) element is positioned between two outer source coding sequences (gene X and gene Y). IRES allows albumin X and protein Y to be translated by same transcript, and transcribing by promoter such as ovalbumin promoter of this transcript instructed. Curved arrow represents transcription initiation site. It is the highest in the tubular gland cell that the expression of gene X coded protein is expected at, this cell specific expression but do not secrete. The protein of gene Y coding is specifically expressing in tubular gland cell also, but is effectively secreted because of it, so protein Y is packed in the egg. In the retroviral vector example of Figure 15 A and 15D, owing to be provided with the IRES of encephalomyocarditis virus (EMCV), a carrier pCMV-LC-emcvIRES-HC can express light chain (LC) and the heavy chain (HC) of human monoclonal antibodies. Available CMV promoters driven is transcribed. (see again (1997) " by containing the replicative-competent retrovirus carrier high level expression foreign gene of internal ribosome entry site " (High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site) Gene 202:23-29 such as Murakami; Chen etc., (1999) " generation of more effective bird Replication defective retroviral vector and design " (Production and design of more effective avian replication-incompetent retroviral vectors) Dev.Biol.214:370-384; Noel etc. (2000) " SF by genetic modification is carried monoclonal antibody to systemic sustained " (Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts) J.Invest.Dermatol.115:740-745).

Another aspect of the present invention, the coded sequence of used carrier all contains a 3 ' non-translational region (3 ' UTR) with the stability of the RNA of assurance generation in any method of the present invention. When adding 3 ' UTR to retroviral vector, this promoter, gene X and the direction of 3 ' UTR in construction must be reversed, so that the adding of 3 ' UTR does not affect transcribing of full length genomic rna. In one embodiment, this 3 ' UTR can be 3 ' UTR of ovalbumin or lysozyme gene, or any 3 ' UTR of function is arranged in PE section cell, such as the zone in late period of SV40.

In another embodiment of the present invention, with the coded sequence of constitutive promoter (for example CMV) express transgenic in bird fallopian tubal PE section. In this case, express the PE section that is not limited to; In its hetero-organization (for example, blood) of bird, expression is arranged also. Utilize this transgenosis that contains constitutive promoter and coded sequence, be particularly suitable for realizing or kinesin matter in fallopian tubal expression and be secreted into subsequently in the egg white and (see Fig. 8 A, the construction that drives take CMV is as example, as express the pNLB-CMV-IFN carrier of IFN-α 2b in chicken).

Fig. 3 A shows the carrier pNLB based on replication defect type avian leukosis virus (ALV), a kind ofly is suitable for carrier of the present invention. In the pNLB carrier, with the lacZ gene substitution of neomycin resistance gene (Neo) and the β-galactosidase of encoding the genome of most of ALV. Fig. 3 B shows carrier pNLB-CMV-BL, wherein, replaces the lacZ gene with CMV promoter and beta-lactamase coded sequence (β-La or BL). In specific embodiment (embodiment 1, sees lower), reported this Vector construction. By CMV promoter expression beta-lactamase, and in its 3 ' LTR (LTR), polyadenylic acid signal (pA) is arranged. The beta-lactam zymoprotein contains the natural signals peptide; Therefore, it is present in blood and the egg white.

With pNLB-CMV-BL carrier transduction bird embryo (embodiment 2, see lower). The egg white of every ovum of hen of the stable transduction that produces contains the active beta-lactamase (

embodiment

2 and 3 sees lower) up to the secretion of 60 micrograms (μ g).

The pNLB-MDOT-EPO carrier that Fig. 8 A and 8B display respectively are used for expressing the pNLB-CMV-IFN carrier of interferon-' alpha ' 2b (IFN-α 2b) and are used for expressing promoting erythropoietin(EPO) (EPO). These two kinds of exogenous proteins (EPO, IFN) can be expressed in bird, preferably chicken and turkey.

The pNLB-MDOT-EPO carrier substitutes the BL coded sequence and creates (embodiment 10, see lower) with the EPO coded sequence. In one embodiment, adopt the synthetic promoter that is called MDOT to drive the expression of EPO. MDOT contains ovomucoid and ovotransferrins promoter element. Can optimize according to the hen fallopian tubal codon use of the dna sequence dna of people EPO, by the BACKTRANSLATE program of winconsin software kit 9.1 editions (the science of heredity calculating group company of state of Wisconsin Madison), utilize the codon by Hongyuan chicken ovalbumin, lysozyme, ovomucoid and ovotransferrins compiling to create with table. Synthesize the EPO dna sequence dna and it is cloned into carrier and produce plasmid pNLB-MDOT-EPO (having another name called pAVIJCRA145.27.2.2). In one embodiment, produced the transducing particles of this carrier, these transducing particles of titration are to determine can be used for the suitable concn of embryonal vaccination. Then transducing particles are injected egg, incubated egg is 21 days thereafter.

The available chick blood serum sample that is collected in a rear week of hatching is ELISA and is tested to measure for example level of EPO of exogenous proteins. Select cock to be used for mating, wherein filter out and contain the genetically modified G of EPO in the sperm₀Cock. Preferably make cock and the mating of non-transgenic hen that transgene level is the highest in the seminal fluid sample by artificial insemination. This genetically modified existence in the screening blood DNA sample. Often can find that some chicks are genetically modified (G₁Fowl). Detect exist (for example ELISA test) of people EPO in the chick serum. Also can detect G₁The existence of people EPO in the egg white of hen egg. EPO among the present invention in the egg (that is, deriving from the people EPO coded sequence of optimization) has biologically active (embodiment 11).

Similarly, substitute the BL coded sequence with the IFN coded sequence and can obtain pNLB-CMV-IFN carrier (Fig. 8 A) (embodiment 12, see lower). In one embodiment, driving IFN with composing type cytomegalovirus promoter (CMV) expresses. More specifically, with cytomegalovirus promoter immediate early promoter/enhancer and the poly-A site control of SV40 IFN coded sequence. Fig. 8 A explanation is used for bird, for example expresses the pNLB-CMV-IFN of IFN in chicken and the turkey. Created the Optimized Coding Based sequence of humanIFN-α 2b, wherein, be used in egg white protein ovalbumin, lysozyme, ovomucoid and the ovotransferrins each specific amino acids the codon of normal usefulness design the humanIFN-α 2b sequence of inserting in the carrier of the present invention. More specifically, the codon of the humanIFN-α 2b dna sequence dna (Figure 11 A) of optimizing uses and can according to the fallopian tubal optimization of hen, utilize the codon of Hongyuan chicken ovalbumin, lysozyme, ovomucoid and ovotransferrins compiling to create with table by BACKTRANSLATE program (on seeing). For example, in these four kinds of egg white proteins, four kinds of alanine Codon usage percentage are GCU 34%, GCC 31%, GCA 26%, GCG 8%. Therefore, can be with the codon of GCU as most of alanine in the humanIFN-α 2b sequence of optimizing. The carrier on the basis of the available humanIFN-α 2b sequence that contains optimization is created in the transgenic avian of expressing TPD IFN-α 2b in its tissue and the egg.

Produce transducing particles and the suitable concn (embodiment 2, see lower) of these transducing particles of titration to determine can be used for embryonal vaccination of this carrier. So just produced chimeric bird (see again embodiment 13, see lower). According to Speksnijder technology (U.S. Patent number 5,897,998) birds, beasts and eggs are put in the nest, transducing particles are injected egg. Inject egg incubation after about 21 days. Gather the chick blood serum sample in a rear week of hatching and measure the wherein level of hIFN (for example ELISA test). With EPO similar (on seeing), select cock to be used for mating. For containing the genetically modified G0 cock of IFN in the screening seminal fluid, extract the DNA of cock seminal fluid sample. By artificial insemination, make transgene level is the highest in the seminal fluid sample G0 cock and the mating of non-transgenic hen. This genetically modified existence in the screening blood DNA sample. Detect exist (for example the testing with ELISA) of hIFN in the serum of transgenosis cock. If confirm to have this exogenous proteins, with the seminal fluid artificial insemination non-transgenic hen of transgenosis cock. The offspring of certain ratio will be contained this transgenosis (for example, greater than 50%). When having IFN (namely deriving from the people IFN coded sequence of optimization) in the egg of the present invention, detect the biologically active of IFN. Similar with EPO, this class egg usually contains and has bioactive IFN, such as TPD IFN-α 2b (Figure 11 B).

The invention provides the method for in the bird fallopian tubal, producing exogenous proteins and producing the egg that contains exogenous proteins, the method also comprises an additional subsequent step, makes vector integration enter the bird genome thereby suitable carrier namely is provided and this carrier is imported embryo's blastoderm cells. This subsequent step comprises that the transgenosis blastoderm cells that produces from preceding step obtains ripe transgenic avian. Randomly, produce ripe transgenic avian from blastoderm cells and the transgenosis blastoderm cells is transferred to the embryo and this embryo is reached full growth optional comprising, thereby along with the described cellular integration of this embryo's growth enters bird. Make then the chick growth and maturity of generation. In one embodiment, directly with the carrier transfection among the embryo or transduction blastoderm cells (embodiment 2). Make the embryonic development of generation, make the chick maturation.

In both cases, the transgenic avian of transgenosis blastoderm cells generation is called original fowl. Some original fowl carries transgenosis in its fallopian tubal PE section tubular gland cell, these fowl will be in its fallopian tubal the exogenous proteins of express transgenic coding. Except fallopian tubal, exogenous proteins also may be expressed (for example, blood) in its hetero-organization. If exogenous proteins contains suitable burst, it will be secreted in the egg white of fallopian tube lumen and egg. Some original fowl is the original fowl (embodiment 8 and 9) of kind of system. Planting the original fowl of system is to carry genetically modified original fowl in the inhereditary material of its germ line tissue, and they also can carry the transgenosis of expressing exogenous proteins in fallopian tubal PE section tubular gland cell. Therefore, according to the present invention, transgenic avian will contain the tubular gland cell of expressing exogenous proteins, and the offspring of transgenic poultry also will be contained the fallopian tubal PE section tubular gland cell of expressing exogenous proteins. Perhaps, offspring fowl can be expressed this exogenous gene expression and certain phenotype (embodiment 6, table 2) of determining in the particular organization of fowl. In one embodiment of the present invention, described transgenic poultry is chicken or turkey.

Available high yield low cost of the present invention is expressed desired protein, comprises the protein of the medicine, diagnosticum and the domestic animal feed additive that can be used as the human and animal. For example, the present invention includes the egg that the transgenic avian that contains this albumen in the transgenic avian that produces this albumen and the egg white produces. The present invention considers for the production of any desired protein, comprises medical protein, wherein need to the coded sequence of this albumen be introduced in the oviduct cell according to the present invention. In fact, the all proteins that produces according to allos of the present invention that detects up to now comprises that interferon alpha 2 b, GM-CSF, interferon beta, erythropoietin(EPO), G-CSF, CTLA4-Fc fusion and beta-lactamase all successfully produce with methods described herein.

Especially interested is production human protein as described herein. Consider people's form of producing each albumen described herein (people's form is arranged) according to the present invention.

The protein that consideration is produced with methods described herein includes but not limited to: fusion, growth hormone, cell factor, structural proteins and enzyme comprise human growth hormone (HGH), interferon, lysozyme and beta-casein, albumin, α-1 antitrypsin, Antithrombin III, collagen, Factor IX, IX, X (etc.), fibrinogen, insulin, lactoferrin, PROTEIN C, erythropoietin(EPO) (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), tectotype plasminogen activator (tPA), somatotropin and chymotrypsin. Also can according to immunoglobulin (Ig) and the antibody of generation modification described herein, comprise the immunotoxin that is incorporated into the human tumor cells surface antigen and destroys them.

Can include but not limited to according to other object lesson of the therapeutic protein of production described herein: Factor IX, factor VIIa, the factors IX of Factor IX, b-domain disappearance, anti-coagulants; Hirudin, Alteplase, tpa, Reteplase, tpa, disappearance is 3 in tpa-5 the domain, insulin, insulin lispro, aspartame's insulin, insulin glargine, Recent Development of Long-acting Insulin Analogs, hgh, glucagons, tsh, follicle-stimulating hormone-β, fsh, gm-csf, pdgh, ifn α 2, ifn α 2a, ifn α 2b, inf-α, inf-β 1b, ifn-β 1a, ifn-γ 1b, il-2, il-11, hbsag, ospa, the mouse monoclonal antibody of anti-t-lymphocyte antigen, the mouse monoclonal antibody of anti-tag-72, tumor-associated glycoprotein, fab fragment derived from the chimeric monoclonal antibody of antiplatelet surface receptor gpII (b)/III (a), the mouse monoantibody segment of antitumor related antigen ca125, the mouse monoantibody segment of anti-human cancer embryoantigen cea, the mouse monoantibody segment of anti-Autopsy Cases globulin, the mouse monoantibody segment of antitumor surface antigen psma, the mouse monoantibody segment of anti-hmw-maa (fab/fab2 mixture), the mouse monoantibody segment (fab) of anti--cancer associated antigen, the monoantibody segment of anti-nca 90 (fab), surface granulocyte NCA, the chimeric monoclonal antibody of the cd20 antigen of finding on the anti-b lymphocytic cell surface, the humanization monoclonal antibody of anti-il2 receptor alpha chain, the chimeric monoclonal antibody of anti-il2 receptor alpha chain, the chimeric monoclonal antibody of anti-tnf-α, the humanization monoclonal antibody of the lip-deep epi-position of anti respiratory syncytial virus, the humanization monoclonal antibody of anti-her2, the human epidermal growth factor receptor 2, people's monoclonal antibody of anti-cell keratin tumor associated antigen, anti--ctla4, the chimeric monoclonal antibody of anti-b lymphocyte cd 20 surface antigens, Dornase Alfa-DNA enzyme, the β glucocerebrosidase, tnf-α, il-2-diphtheria toxin fusion, tnfr-lgg segment composition albumen, the La Luoni enzyme, the DNA enzyme, Ah method's Saite (alefacept), A Fadabeiting (colony stimulating factor), tositumomab, the mouse monoclonal antibody, Ah coming organizes monoclonal antibody, rasburicase, Ah add'sing carbohydrase β, Teriparatide, parathyroid hormone derivative, adalimumab (lgg1), anakinra, the bio-modification agent, Nesiritide, people β type natriuretic peptide (hbnp), colony stimulating factor, pegvisomant, the growth hormone receptor antagonist, the albumen c of recombination activation, the Ao Mazuo monoclonal antibody, ige (lge) blocking agent, ibritumomab tiuxetan, ACTH, glucagons, somatostatin, growth hormone, thymosin extrasin, parathyroid hormone, pigment hormone, somatomedin, erythropoietin(EPO), lutropin, HCG, hypothalamic releasing factor, Etanercept, antidiuretic hormone, prolactin and thyrotropic hormone.

The present invention includes the generation multimeric protein, comprise immunoglobulin (Ig), such as the method for antibody and its Fab. Therefore, in an embodiment of the invention, this multimeric protein is immunoglobulin (Ig), and wherein first second heterologous polypeptide is respectively heavy chain immunoglobulin and light chain.

In some embodiments, the immunoglobulin polypeptides of the transcript unit of at least one expression vector coding can be the heavy chain immunoglobulin polypeptide that contains variable region or its variant, also can comprise D district, J district, C district or its combination. The immunoglobulin polypeptides of expression vector codes also can be the light chain immunoglobulin polypeptide that contains variable region or its variant, also can comprise J district and C district. The present invention also relates to the multiple immunoglobulin (Ig) zone by same animal species or multiple speciation, these species include but not limited to people, mouse, rat, rabbit and chicken. In some embodiments, antibody is people's antibody or humanized antibody.

In other embodiments, the immunoglobulin polypeptides of at least a expression vector codes comprises immunoglobulin heavy chain variable region, immunoglobulin light chain variable region and joint peptide, thus form can selective binding antigen single-chain antibody.

The example of the therapeutic antibodies that available the inventive method produces includes but not limited to: He Saiting^TM(Herceptin) (California Genentech company (Genetech, CA)), it is that the humanization that is used for the treatment of the metastatic breast cancer patient resists-the HER2 monoclonal antibody; REOPRO^TM(Abciximab) (Sen Tuoke company (Centocor)), it is the antibody of glycoprotein iib/iiia acceptor on the blood platelet, is used for prevention and forms blood clot; ZENAPAX^TM(daclizumab) (company of Roche Group of Switzerland (Roche Pharmaceuticals, Switzerland)), it is that the inhibitive ability of immunity humanization resists-the CD25 monoclonal antibody, is used for the prophylaxis of acute kidney transplantation exclusion reaction; PANOREX^TM, it is mouse-anti-17-IA cell surface antigen IgG2a antibody (GlaxoSmithKline PLC/Sen Tuoke (Glaxo Wellcome/Centocor)); BEC2, it is anti--idiotype (GD3 epi-position) IgG antibody (because of cloning system company (ImClone System)); IMC-C225, it is chimeric anti-EGFR IgG antibody (because of cloning system company); Wei Taxin (VITAXIN^TM), it is that humanization resists-α V β 3 alpha 2 integrin antibodies (using molecular evolution/Midi Buddhist nun Miao (Applied Molecular Evolution/MedImmune)); Alemtuzumab (Campath); Alemtuzumab 1H/LDP-03, it is humanized anti-CD 52 IgG1 antibody (Liu Ke Saite company (Leukosite)); Smart M195, it is that humanization resists-CD33 IgG antibody (protein design laboratory/Ka Nibai (Protein Design Lab/Kanebo)); Rituximab^TM, it is chimeric anti-CD 20 IgG1 antibody (IDEC pharmacy/Genentech (IDEC Pharm/Genentech), Luo Shi/outstanding person's topic gram (Roche/Zettyaku)); LYMPHOCIDE^TM, it is that humanization resists-CD22 IgG antibody (immune medical science company (Immunomedics)); ICM3, it is that humanization resists-ICAM3 antibody (ICOS drugmaker (ICOS Pharm)); IDEC-114, it is that Primate resists-CD80 antibody (IDEC drugmaker/Mitsubishi (IDEC Pharm/Mitsubishi)); ZEVALIN^TM, it is radiolabeled mouse anti-CD 20 antibodies (IDEC/Schering AG); IDEC-131, it is humanization anti-CD40L antibodies (IDEC/ Ethiopia company (Eisai)); IDEC-151, it is the long source anti-CD 4 antibodies (IDEC) of spirit; IDEC-152, it is that the long sourceization of spirit resists-CD23 antibody (IDEC/Seikagaku); SMART resists-CD3, and it is that humanization resists-CD3 IgG (protein design laboratory); 5G1.1 it is that humanization resists-complement factor 5 (CS) antibody (the emerging drugmaker of Ah Lai (Alexion Pharm)); D2E7, it is humanization anti-TNF-Alpha antibodies (CATIBASF); CDP870, it is humanization anti-TNF-α Fab fragment (Sai Er Imtech (Celltech)); IDEC-151, it is that the long sourceization of spirit resists-CD4 IgG1 antibody (but IDEC drugmaker/history BiCheng Co., Ltd (SmithKline Beecham)); MDX-CD4, it is that the people resists-CD4 IgG antibody (U.S.A reaches Simon Rex company/Ethiopia company/Ji Ma company (Medarex/Eisai/Genmab)); CDP571, it is humanization anti-TNF-α IgG4 antibody (Sai Er Imtech); LDP-02, it is that humanization resists-α 4 β 7 antibody (Liu Ke Saite company/Genentech company); OrthoClone OKT4A, it is that humanization resists-CD4IgG antibody (Ao Sen biotech company (Ortho Biotech)); ANTOVA^TM, it is humanization anti-CD 40 L IgG antibody (hundred collection company (Biogen)); ANTEGREN^TM, it is that humanization resists-VLA-4 IgG antibody (Elan Co.,Ltd (Elan)); CAT-152, it is that the people resists-TGF-β₂Antibody (cambridge antibody scientific ﹠ technical corporation (Cambridge Ab Tech)); Cetuximab (BMS), it is that monoclonal resists-EGF acceptor (EGFr) antibody; Bevacizumab (Genentech company), it is the anti-VEGF human monoclonal antibodies; Infliximab (Sen Tuoke company (Centocore), JJ), it is chimeric (mouse and the people) monoclonal antibody that is used for the treatment of autoimmunity disease; WAY-CMA 676 (Wyeth (Wyeth)), it is the monoclonal antibody for chemotherapy; With draw the Buddhist nun to organize monoclonal antibody (Ranibizumab) (Genentech company), it is chimeric (mouse and the people) monoclonal antibody that is used for the treatment of macular degeneration.

On the one hand, the present invention relates to the G-CSF that poultry or bird produce. On the one hand, the present invention relates to have the G-CSF (TPD G-CSF) of the glycosylation pattern that poultry produces, wherein this G-CSF is available from avian cell, such as the avian cell of chicken, quail or turkey. The present invention also comprises human protein, comprises cell factor, and the G-CSF of the isolated or purified form that produces such as poultry also comprises human protein, comprises cell factor, the G-CSF of the pharmaceutical compositions that produces such as poultry. Can separate the protein that comprises G-CSF by the method that the those of ordinary skill in protein purification field is understood easily. Prescription composition for generation of pharmaceutical composition is well-known in the art.

The present invention includes therapeutic protein or medical protein that the transgenosis poultry produces, it has the glycosylation pattern derived from the poultry generation of bird. For example, the present invention includes the interferon-' alpha ' 2 (TPD IFN-α 2) that is produced by bird. TPD IFN-α 2b (such as species type b) shows among the interferon-' alpha ' 2b (PBL IFN-α 2b) that human peripheral leucocytes produces under the normal condition non-existent new glycosylation pattern and contains new sugared shape that (

band

4 and 5 is disaccharides that α-Gal extends; See Fig. 9). TPD IFN-α 2b also contains the sugared structure that in chicken than the O-of in people more effective generation be connected similar with people PBL IFN-α 2b.

The present invention considers the separation polynucleotides of the optimization polynucleotide sequence of the protein that contains generation as described herein. For example, the present invention includes the coded sequence of the bird optimization of humanIFN-α 2b, the interferon-' alpha ' 2b (TPD IFN-α 2b) (SEQ ID NO:1) that the transgenosis of namely recombinating poultry produces. The humanIFN-α 2b coded sequence of optimizing comprises 498 nucleic acid and 165 amino acid (seeing SEQ ID NO:1 and Figure 11 A). Similarly, natural humanIFN-α 2b coded sequence comprises 498 nucleotides (NCBI accession number AF405539 and GI:15487989) and 165 amino acid (NCBI accession number AAL01040 and GI:15487990). Can utilize in egg white protein ovalbumin, lysozyme, ovomucoid and ovotransferrins each specific amino acids the codon of normal usefulness come the human interferon-alpha 2b coded sequence of design optimization and be inserted into carrier of the present invention. More specifically, the codon of the dna sequence dna of the human interferon-alpha 2b that optimizes uses and can optimize according to the hen fallopian tubal, by the BACKTRANSLATE program of winconsin software kit 9.1 editions (the science of heredity calculating group company of Wisconsin State Madison), utilize the codon of Hongyuan chicken ovalbumin, lysozyme, ovomucoid and ovotransferrins compiling to create with table. For example, in these four kinds of egg white proteins, four kinds of alanine Codon usage percentage are GCU 34%, GCC 31%, GCA 26%, GCG 8%. Therefore, can be with the codon of GCU as most of alanine in the humanIFN-α 2b coded sequence of optimizing. The available carrier that contains the humanIFN-α 2b gene of optimization is created in the transgenic avian of expressing TPD IFN-α 2b in its tissue and the egg.

Such as (vide infra) as described in the embodiment 13, in chicken, produce TPD IFN-α 2b. Yet, also can in turkey and other avian species such as quail, produce TPD IFN-α 2b. In a preferred embodiment of the present invention, in chicken and turkey and their hard shelled egg, express TPD IFN-α 2b. Hydrocarbon analysis (embodiment 14, vide infra) comprises that monose analysis and FACE analyze sugar composition or the new glycosylation pattern that has disclosed this protein. Like this, TPD IFN-α 2b demonstration contains following monosaccharide residue: N-acetyl-galactosamine (NAcGal), galactolipin (Gal), N-acetyl-aminoglucose (NAcGlu) and sialic acid (SA). Yet, the glycosylation that does not have N to connect among the TPD IFN-α 2b. On the contrary, TPD IFN-α 2b is glycosylated at the O-of Thr-106 place. This type of glycosylation is similar to humanIFN-α 2, and wherein 106 Thr residue is unique residue of IFN-α 2. Similar to natural IFN-α, TPD IFN-α 2b does not contain mannose residue. FACE analyzes and has shown 6 bands (Fig. 9) that represent various saccharide residues, and wherein

band

1,2,3 is respectively (Figure 10) of not saliva acidifying, single saliva acidifying and two saliva acidifyings. It is α 2-3-galactolipin (Gal) and α 2-6-N-acetyl-galactosamine (NAcGal) that sialic acid (SA) connects. Band 6 represents the not tetrose of saliva acidifying.

Band

4 and 5 is the (disaccharide that α-Gal) extends of undiscovered α-galactolipin in people PBL IFN-α 2b or natural human IFN (natural hIFN). Figure 10 shows the comparison of TPD IFN-α 2b (egg white hIFN) and people PBL IFN-α 2b (natural hIFN).

Band

3 and 4 among the TPD IFN-α 2b, band 4 and be with 5 between band (seeing lower) is arranged time.

The present invention imagines peptide sequence (SEQ ID NO:2) (seeing again Figure 11 B) and the pharmaceutical composition thereof of a kind of separation of TPD IFN-α 2b, wherein this protein in the Thr-106 position by one or more sugared structure O-glycosylations as follows as herein described:

(i)Gal-NAcGal-

(ii)SA-Gal-NAcGal-

(v)Gal-Gal-NAcGal-

Wherein, the Gal=galactolipin,

NAcGal=N-acetyl-galactosamine,

NAcGlu=N-acetyl-aminoglucose and

The SA=sialic acid

In a preferred embodiment of the present invention, percentage is as follows:

(i) Gal-NAcGal-about 20%

(ii) SA-Gal-NAcGal-about 29%

(v) Gal-Gal-NAcGal-about 7%

Wherein, the Gal=galactolipin,

NAcGal=N-acetyl-galactosamine,

NAcGlu=N-acetyl-aminoglucose and

The SA=sialic acid

Band

3 and 4, band 4 and be with 5 between band is arranged time, in TPD IFN-α 2b, account for 17%.

In one embodiment, the present invention relates to have the human protein of the glycosylation pattern that poultry derives. In one embodiment, the glycosylation pattern that described poultry derives is available from bird oviduct cell, for example tubular gland cell. For example, glycosylation pattern disclosed herein is presented on the human protein that produces in the chicken salpingo cell according to the present invention.

In one embodiment, the present invention relates to bird, the human G-CSF with glycosylation pattern that poultry derives that for example produces in chicken, turkey and the quail (such as the bird oviduct cell). Show ripe hG-CSF amino acid sequence among Figure 18 C. Show the nucleotide sequence for generation of G-CSF described herein among Figure 18 A and the NCBI accession number NM 172219. Also consider to use according to bird (such as chicken) codon to produce G-CSF and the other oroteins that produces according to the present invention with the nucleotide sequence of optimizing, such as human protein.

The present invention includes the egg that contains G-CSF molecule of the present invention and the bird (such as chicken, turkey and quail) that produces this kind egg, described G-CSF molecule comprises and shows the glycosylation structure under one or more:

Wherein, the Gal=galactolipin,

NAcGal=N-acetyl-galactosamine,

NAcGlu=N-acetyl-aminoglucose and

The SA=sialic acid

In one embodiment, the present invention includes the mixture of G-CSF molecule, wherein this mixture comprises and has the G-CSF molecule that is selected from one or more glycosylation structure among structure A, structure B, structure C, structure D, structure E, structure F and the structure G.The present invention also comprises the mixture of G-CSF molecule, wherein this mixture comprises and has the G-CSF molecule that is selected from one or more glycosylation structure among structure A, structure B, structure C, structure D, structure E, structure F and the structure G, described mixture is an isolated or purified, for example the egg that is produced by the present invention or egg white purifying.The present invention also comprises the mixture of G-CSF molecule, and wherein this mixture comprises the G-CSF molecule with two kinds, three kinds, four kinds, five kinds or six kinds following structures: structure A, structure B, structure C, structure D, structure E, structure F and structure G.The present invention also comprises the mixture of G-CSF molecule, wherein this mixture comprises the G-CSF molecule with two kinds, three kinds, four kinds, five kinds or six kinds following structures: structure A, structure B, structure C, structure D, structure E, structure F and structure G, this molecule is an isolated or purified, for example the egg that is produced by the present invention or egg white purifying.

The present invention also comprises a kind of G-CSF molecule that comprises structure A.The present invention also comprises a kind of G-CSF molecule that comprises structure B.The present invention also comprises a kind of G-CSF molecule that comprises structure C.The present invention also comprises a kind of G-CSF molecule that comprises structure D.The present invention also comprises a kind of G-CSF molecule that comprises structure E.The present invention also comprises a kind of G-CSF molecule that comprises structure F.The present invention also comprises a kind of G-CSF molecule that comprises structure G.In one embodiment, described a kind of G-CSF molecule appears in the G-CSF molecule mixture, and this mixture may be the G-CSF molecule mixture of isolated or purified, and for example, the egg or the egg white purifying that are produced by the present invention obtain this mixture.In one embodiment, various G-CSF molecules are isolated or purifieds, for example according to (for example by embodiment 20 described HPLC) of purifying described herein.

The specifically described relevant G-CSF of this paper also can be applicable to various other protein of producing according to the present invention and its corresponding poultry glycosylation structure of deriving as the embodiment of the present invention of G-CSF molecule mixture and a kind of G-CSF molecule (preceding two paragraghs).

As described in embodiment 22 and 23, produce the transgenic chicken that the egg that produces contains EPO.In addition, production method of the chicken of the production EPO of second kind of strain and embodiment 22 and 23 described basic identical, difference is to use different production clone, as (including its full content in this paper by reference) as described on May 5th, the 2007 laid-open U.S. Patents publication number 2007/0077650.Among CMV promotor of the chicken of the production EPO of second kind of strain of this kind and the LTR as if disappearance is arranged, because producing the level of EPO in the egg white of gained transgenic chicken improves, of the Application No. 11/880,838 that submit to 14 days July in 2007 by reference its full content being included in this paper.Utilize this EPO high-yielding strain to obtain to be used to carry out the EPO that oligosaccharides is analyzed.

Can be by any useful method, for example the method known to the those of ordinary skill of protein purification field is by the protein of egg white purifying transgenic poultry generation of the present invention.For example, can be by the EPO of the method known to the those of ordinary skill of protein purification field by the transgenic poultry generation of the present invention of egg white purifying.Embodiment 24 has described the example of the method for the EPO in the purifying egg white.

Prove that the erythropoietin that produces in the chicken (hEPO) contains the sugar chain that sugar chain that O-connects is connected with three N-.Prove that O-connects glycosylation and appears on the Ser-126 of EPO, N-connects glycosylation and appears on Asn-24, Asn-38 and the Asn-83.The aminoacid sequence of the sophisticated erythropoietin that produces according to the present invention is seen Figure 19 B.The human nucleotide sequence of coding EPO is seen Figure 19 A.Also considering to use according to bird (as chicken) codon uses the nucleotide sequence of optimizing to produce EPO of the present invention and other protein.

Measure the representative glycosylation structure of erythropoietin of the present invention, seen embodiment 25 and Figure 20 and 21.Specifically, through identifying, there are B-n, D-n, F-n, H-n, J-n, L-n, N-n, O-n, P-n and Q-n on the avian derived EPO.In addition, evidence show and one or more oligosaccharide structures A-n, C-n, E-n, G-n, I-n, K-n and M-n also may occur on the EPO.In addition, data show to have second kind of form of Q-n, wherein only occur four in five terminal NAcGlu residues.Second kind of precursor forms that form can be Q-n of Q-n.

The present invention includes egg and the egg white that contains erythropoietin molecule of the present invention and produce this kind egg and the bird (as chicken, turkey and quail) of this kind of generation egg white, described erythropoietin molecule comprises one or more glycosylation structures as herein described.

In one embodiment, the present invention includes the mixture of erythropoietin molecule, wherein said mixture comprise have O-connect the glycosylation structure erythropoietin molecule (as, one or more erythropoietin molecules), described O-connection glycosylation structure is selected from structure A-o, structure B-o or structure C-o.Though O-connects the existence that the glycosylation analysis has confirmed A-o, B-o and C-o up to now; But also consider to have structure D-o, structure E-o, structure F-o and structure G-o on the poultry deutero-people EPO.Shortcoming, the main O-on the avian derived EPO connects oligosaccharides C-o seemingly.

The present invention is also included within has the EPO that N-connects the glycosylation structure on three sites, wherein the structure on three sites is selected from A-n, B-n, C-n, D-n, E-n, F-n, G-n, H-n, I-n, J-n, K-n, L-n, M-n, N-n, O-n, P-n or Q-n respectively.

The present invention also comprise erythropoietin molecule (as, more than one erythropoietin molecules) mixture, some of them or all erythropoietin molecules have one or more glycosylation structures that are selected from down group: structure A-o, structure B-o, structure C-o, structure A-n, structure B-n, structure C-n, structure D-n, structure E-n, structure F-n, structure G-n, structure H-n, structure I-n, structure J-n, structure K-n, structure L-n, structure M-n, structure N-n, structure O-n, structure P-n, structure Q-n.In one embodiment, the mixture of erythropoietin molecule is a purifying or isolating, is for example separated by egg, perhaps egg white purifying or the separation that is produced by transgenic poultry.

The present invention also comprises the independent erythropoietin molecule that comprises structure A-o.The present invention also comprises the independent erythropoietin molecule that comprises structure B-o.The present invention also comprises the independent erythropoietin molecule that comprises structure C-o.The present invention also comprises the independent erythropoietin molecule that comprises structure A-n.The present invention also comprises the independent erythropoietin molecule that comprises structure B-n.The present invention also comprises the independent erythropoietin molecule that comprises structure C-n.The present invention also comprises the independent erythropoietin molecule that comprises structure D-n.The present invention also comprises the independent erythropoietin molecule that comprises structure E-n.The present invention also comprises the independent erythropoietin molecule that comprises structure F-n.The present invention also comprises the independent erythropoietin molecule that comprises structure G-n.The present invention also comprises the independent erythropoietin molecule that comprises structure H-n.The present invention also comprises the independent erythropoietin molecule that comprises structure I-n.The present invention also comprises the independent erythropoietin molecule that comprises structure J-n.The present invention also comprises the independent erythropoietin molecule that comprises structure K-n.The present invention also comprises the independent erythropoietin molecule that comprises structure L-n.The present invention also comprises the independent erythropoietin molecule that comprises structure M-n.The present invention also comprises the independent erythropoietin molecule that comprises structure N-n.The present invention also comprises the independent erythropoietin molecule that comprises structure O-n.The present invention also comprises the independent erythropoietin molecule that comprises structure P-n.The present invention also comprises the independent erythropoietin molecule that comprises structure Q-n.In one embodiment, at transgenic poultry, as independent erythropoietin molecule as described in existing in the erythropoietin molecule mixture that produces in the transgenic chicken.In one embodiment, have described independent erythropoietin molecule in the erythropoietin molecule mixture of isolated or purified, for example, this mixture is the egg produced by transgenic poultry or egg white isolated or purified.In one embodiment, described independent erythropoietin molecule is an isolated or purified.

The present invention includes the glycosylation of one of Asn-24, Asn-38 and Asn-83 glycosylation site each personal A-n, B-n, C-n, D-n, E-n, F-n, G-n, H-n, I-n, J-n, K-n, L-n, M-n, N-n, O-n, P-n and Q-n structure, Ser-126 A-o, B-o or the glycosylated exemplary EPO molecule of C-o.

The peptide prod that the avian derived EPO of the present invention is obtained through proteolysis digestion carries out MALDI-TOF-MS to be analyzed and shows, it is basic identical that three N-connect the oligosaccharide structure that occurs on the glycosylation site.That is, as if on the EPO molecule three N-connect ratio that each N-that exists on the glycosylation site connects oligosaccharides about equally.This shows that the glycosylated foreign protein of other N-that produces according to the present invention may have similar N-connection glycosylation pattern.In addition, data presentation, three N-connection site are respectively extensively glycosylated, and each site is surpassing 95%, may surpass 98%, and it is glycosylated for example surpassing on 99% time.The erythropoietin of being analyzed is that transgenic chicken produces, and described transgenic chicken comprises coding and cuts away the transgenosis that contains the aminoacid sequence of 165 amino acid whose human proteins behind the signal peptide.Yet, estimate that the nucleotides sequence with regard to the EPO of 166 amino acid form of encoding is listed in regard to the EPO that produces in the transgenic chicken, the oligosaccharides fill-in (complement) on described 166 amino acid whose protein is identical with 165 amino acid whose protein.

The N-that is connected in people EPO that transgenic chicken produces connects oligosaccharides and contains a small amount of terminal sialic acid part.That is, has only small amount of N-connection oligosaccharide structure by terminal saliva acidifying.This is opposite with the people EPO that produces among the EPO that produces and the CHO in people's cell, and N-connects oligosaccharide structure by terminal saliva acidifying widely among these EPO.In addition, the N-of the EPO that transgenic chicken produces connects on the oligosaccharide structure and extensively has terminal N-acetyl-glucosamine (NAcGlu), but then really not so in the people EPO that EPO that people's cell produces and Chinese hamster ovary celI produce.In addition, the N-of the EPO of transgenic chicken generation connects on the oligosaccharide structure and does not have Fucose.Yet all of the people EPO that EPO that produces at people's cell and Chinese hamster ovary celI produce or most of N-are connected and have Fucose on the oligosaccharide structure.

Consider, can connect the glycosylation textural association on the erythropoietin.For example, available A-o, A-n, B-n and C-n, or A-o, B-n, C-n and D-n, or A-o, D-n, E-n and F-n, or A-o, E-n, F-n and G-n, or B-o, A-n, D-n and H-n, or B-o, E-n, F-n and G-n, or B-o, A-n, A-n and A-n, or C-o, D-n, D-n and C-n, or C-o, F-n, G-n and H-n, or C-o, A-n, B-n and C-n, or C-o, A-n, B-n and H-n, or C-o, A-n, B-n and E-n, or C-o, A-n, B-n and H-n, or other this class combination glycosylation erythropoietin molecule.

Carry out in bird though should be understood that the method for being reported for preparing the present composition, the present composition is not limited thereto.For example, some glycosylated protein molecule of the present invention can be other organism, as the transgenosis fish, and transgene mammal such as transgenic goat, or produce in transgenic plant such as tobacco and the duckweed (Lemna minor).

Also consider, can on the another kind of protein of the present invention, present in the glycosylation structure that presents on a kind of protein of the present invention.For example, the glycosylation structure that is presented on the TPD G-CSF also may be presented on TPDGM-CSF, TPD IFN and/or other TPD protein.In another embodiment, consider that the glycosylation structure that is presented on the TPD IFN α 2 can be presented on TPD G-CSF, TPD GM-CSF and/or other transgenosis poultry deutero-(TPD) protein.The present invention also considers the human protein that has one or more TPD glycosylation structures described herein usually especially.

The present invention also considers, will may be favourable according to the protein PEGization of generation described herein, and is of the U.S. Patent application 11/584,832 (by with reference to including its full content in this paper) submitting on October 23rd, 2006.

Though when being used for the treatment of, the therapeutic protein that the present invention produces can the prototype administration, preferably the part as pharmaceutical preparation gives this therapeutic protein.

Therefore, the present invention also provides and has contained poultry deutero-glycosylation therapeutic protein or its pharmaceutically acceptable derivates and one or more pharmaceutically acceptable carriers and other therapeutic chosen wantonly and/or the pharmaceutical preparation of preventative composition, and the method that gives this pharmaceutical preparation.Described carrier must be " acceptable ", this means that other composition of it and said preparation is compatible, and harmless to the recipient.The method (as dosage, administration frequency and the treatment time length of pharmaceutical protein) with medicine composite for curing patient of the present invention is determined in the standard method that can utilize doctor with art technology to know,

Pharmaceutical preparation comprises the preparation that is fit to oral, rectum, intranasal, part (comprising mucous membrane and hypogloeeis), vagina or parenteral administration.Pharmaceutical preparation comprises suitable drug administration by injection, comprises the preparation of intramuscular, subcutaneous and intravenous administration.Pharmaceutical preparation also comprises by sucking or be blown into the preparation of administration.In the time of suitably, said preparation can be prepared into independently dose unit easily, can prepare by any method that pharmaceutical field is known.The method of producing pharmaceutical preparation generally comprises following steps: therapeutic protein and liquid vehicle or solid carrier in small, broken bits or the two are put together, and (if necessary) makes this formed product make required preparation then.

The pharmaceutical preparation that is applicable to oral administration can be made the powder agent of the activeconstituents that contains predetermined amount separately or the individual of granule, solution, suspensoid or emulsion form easily, as capsule, cachet or tablet.This activeconstituents also can be made into bolus, electuary or patch.The tablet of oral administration and capsule can contain conventional excipients, as tackiness agent, weighting agent, lubricant, disintegrating agent or wetting agent.Can carry out dressing to tablet according to well known method.Oral liquid can be the form of (for example) water-based or oiliness suspensoid, solution, emulsion, syrup or elixir, perhaps can be made into to face the drying products of rebuilding with preceding water or other suitable carriers.This class I liquid I preparation can contain conventional additive, as suspending agent, emulsifying agent, non-aqueous carrier (eating is with oily) or sanitas.

Therapeutic protein of the present invention also can be mixed with the form of gi tract external administration (for example injection, as inject or continuous infusion), and can be made into unit dosage and be contained in ampoule, pre-filled syringe, small volume infusion bottle or add in the multi-dose container of sanitas.Can or inject this therapeutic protein by (for example) subcutaneous injection, intramuscular injection and intravenous fluids.

This therapeutic protein can be the form such as the suspension agent in oiliness or the aqueous carrier, solution or emulsion, can contain prescription reagent, as suspending agent, stablizer and/or dispersion agent.Consider that also this therapeutic protein can be sterile solid and face the powder type of rebuilding with suitable carriers such as aseptic apirogen water with preceding through aseptic separation or solution through what freeze-drying obtained.

When being used for epidermis outward, the therapeutic protein that the present invention produces can be mixed with ointment, ointment or lotion, or transdermal patch.Available water-based or oil binder add suitable thickening and/or gelifying agent and prepare ointment and ointment.Available water-based or oil binder preparation lotion also contain one or more emulsifying agents, stablizer, dispersion agent, suspension agent, thickening material or tinting material usually.

The preparation that is fit to the topical administration oral cavity comprises and contains activeconstituents and flavouring base material, normally the lozenge of sucrose and gum arabic or tragacanth; The pastille that contains activeconstituents and inertia base-material such as gelatin and glycerine or sucrose and gum arabic; And the collutory that contains activeconstituents and suitable liquid carrier.

Carrier is that the pharmaceutical preparation of the suitable rectal administration of solid most preferably is unitary dose suppository.Suitable carrier comprises theobroma oil and this area other material commonly used, and by active compound is mixed with carrier softening or that melt, cooling forming in mould can form suppository easily then.

The preparation that is fit to vagina administration can be made into vaginal suppository, tampon agent, ointment, gelifying agent, patch, foaming agent or sprays.

With regard to intranasal administration, but therapeutic protein of the present invention can be used as liquid spray or dispersed powders agent, or the drops form.

Can use the water-based or the non-aqueous base-material that also contain one or more dispersion agents, solubilizing agent or suspending agent to prepare drops.By pressurized tank delivering liquid sprays easily.

During inhalation, can send therapeutic protein of the present invention easily by insufflator, atomizer or pressurized tank or other mode of sending aerosol spray easily.Pressurized tank can comprise suitable propelling agent, as Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can send metered amount by valve, to determine dose unit.

Suck or when being blown into administration, therapeutic protein of the present invention can be the form of dry powder compositions, for example this compound and suitable powder binder such as the powdered mixture of lactose or starch.Powder composition can be made unit dosage, be contained in (for example) capsule or the cartridge case, perhaps (as) in gelatin or the Blister Package, can give this powder by assisting of sucker or insufflator.

When needing, above-mentioned preparation is configured to realize the slowly-releasing of activeconstituents.

Pharmaceutical composition of the present invention also can contain other activeconstituents, as biocide or sanitas.

In an object lesson, to be used for pharmaceutical preparation according to the people EPO of generation described herein (may by PEGization), wherein every 1mL contains the 0.05mg Polysorbate 80, and is formulated into pH 6.2 ± 0.2 with 2.12mg one hypophosphite monohydrate sodium dihydrogen, 0.66mg disodium hydrogen phosphate,anhydrous and 8.18mg sodium-chlor and water for injection.In another object lesson, to be used for pharmaceutical preparation according to the human interferon-alpha of generation described herein, described pharmaceutical preparation contains 7.5mg/ml sodium-chlor, 1.8mg/ml Sodium phosphate dibasic, 1.3mg/ml SODIUM PHOSPHATE, MONOBASIC, 0.1mg/ml two ethylenediamine hydrate tetraacethyl disodiums, 0.7mg/ml and tells

80 and the 1.5mg/ml meta-cresol.In another object lesson, will be used for pharmaceutical preparation according to the human G-CSF of generation described herein, described pharmaceutical preparation contains 0.82mg/ml sodium-acetate, 2.8 μ l/ml Glacial acetic acid, 50mg/ml mannitol and 0.04mg/ml and tells

80.

In addition, consider that also therapeutic protein of the present invention can unite use with other therapeutical agent.

The present composition or compound can be used for treating various diseases.For example, those skilled in the art become known for treating the treatment plan of numerous disease, and these schemes are utilized the therapeutic protein available from cell culture (as Chinese hamster ovary celI).The present invention considers to utilize having the poultry therapeutic protein that produces in the bird system of glycosylation pattern of deriving and treats this class disease.In other words, the present invention considers that the therapeutic protein utilize the present invention to produce treats the known disease that can treat with conventional treatment protein easily.For example, the epo treatment human diseases that can utilize the present invention to produce, as anemia and kidney disease (maybe can be by giving other illness of EPO treatment of the present invention as chronic renal failure), the G-CSF that can use the present invention to produce treats the cancer patients, as this area is understood.

Usually, dosage depends on known facts, as the type of recipient's age, healthy state and body weight, concurrent treatment, therapeutic frequency etc.Usually, the dosage of activeconstituents is about 0.0001-10 mg/kg body weight.Can determine that dosage, administration frequency and treatment time are at interval accurately by the doctor of the administration knowledge of knowing each therapeutic protein.

Following specific embodiment is for setting forth the present invention rather than limitation of the scope of the invention.

Embodiment 1

Vector construction

Use the expression cassette of forming by cytomegalovirus (CMV) promotor and reporter gene β-Nei Xiananmei to substitute the lacZ gene of the carrier pNLB (Cosset etc., 1991) of replication defect type avian leukosis virus (ALV).Fig. 3 A and 3B illustrate pNLB and pNLB-CMV-BL vector construct respectively.

In order effectively to substitute the lacZ gene of pNLB with transgenosis, at first created a middle adapter plasmid, the pNLB-adapter.PNLB ApaI/ApaI fragment (Cosset etc. with passivation, J.Virol.65:3388-94 (1991)) (5 ' ApaI residue is positioned at lacZ upstream 289bp among the pNLB, and 3 ' ApaI residue is positioned at 3 ' end of 3 ' LTR and Gag section) pBluescriptK (-) KpnI/SacI site of being inserted into passivation obtains the pNLB-adapter.With the pCMV-B MluI/XbaI section (Moore etc. that fill and lead up, Anal.Biochem.247:203-9 (1997)) the KpnI/NdeI site of the pNLB-adapter of insertion passivation, with CMV promotor and BL gene substitution lacZ (in pNLB, the KpnI residue is positioned at lacZ upstream 67bp, the NdeI residue is positioned at lacZ terminator codon upstream 100bp), like this, obtained pNLB-adapter-CMV-BL.In order to create pNLB-CMV-BL, insert the HindIII/BlpI insertion fragment (containing lacZ) of fragment replacement pNLB with the HindIII/BlpI of pNLB-adapter-CMV-BL.Can produce the subclone of rearrangement because unknown reason directly connects into the blunt end fragment HindIII/BlpI site great majority of pNLB, so this two step clone is necessary.

Embodiment 2

The original fowl group's of pNLB-CMV-BL establishment

Sentas and Isoldes are cultivated in F10 (Ji Buke company) substratum that contains 5% new-born calf serum (Ji Buke company (Gibco)), 1% chicken serum (Ji Buke company), 50 μ g/ml phleomycin (the triumphant laboratory company (Cayla Laboratories) of drawing) and 50 μ g/ml Totomycin (Sigma company (Sigma)).As generation transducing particles as described in (1993) such as Cosset that are incorporated herein by reference, but following exception.(derive from embodiment 1, as above) transfection 9 * 10 for retroviral vector pNLB-CMV-BL ⁵Sentas continues collection virus 6-16 hour and filtration in fresh culture two days later.310 in 3 100mm flat boards that final concentration is 4 μ g/ml polybrenes have been added with all substratum transduction ⁶Isoldes.Next in one day, this substratum is replaced by the substratum that contains 50 μ g/ml phleomycin, 50 μ g/ml Totomycin and 200 μ g/ml G418 (Sigma company).After 10-12 days, separate one G418 resistance colony and transfer in 24 orifice plates.After 7-10 days, carry out G418 then by transduction Sentas and screen and measure tiring of each clone.Generally, there are 2 to tire and be 1-3x10 among 60 clones ⁵As include this paper Allioli for referencial use etc. in, and Dev.Biol.165:30-7 (1994) is described, these colonies that increase, and virus is concentrated into 2-7x10 ⁶β-Nei Xiananmei in the cell culture medium that detection NLB-CMV-BL transducing particles are transduceed has been proved conclusively the integrity of CMV-BL expression cassette.

Transduction vector pNLB-CMV-BL is injected in 546 SPF white leghorns that do not hatch (White Leghorn) embryo's the subgerminal cavity, has wherein hatched 126 chickens, detect these chickens and whether in blood, secrete β-Nei Xiananmei (lactamase).For measuring the concentration of active lactamase in the unknown sample, adopted dynamic colorimetric method, in the method, a kind of purple substrate PADAC is converted into yellow compound by the lactamase specificity.By monitoring standard OD in the reaction times _570nmThe activity of reduction quantitative assay lactamase, make comparisons with the typical curve of different levels purifying lactamase (being called " lactamase test ").Also can by the observation test sample spend the night the back or after several days purple change xanchromatic into and mark to determine whether there is lactamase in the sample (" lactamase that spends the night test ").Back one method is applicable to the very low-level lactamase of detection or screens a large amount of samples.One to around during age, detect the existence of lactamase in the chicken serum sample.Have in 27 little chicken serums very low-level can only be in the lactamase test of spending the night detected lactamase, after these chicken maturations, no longer detect lactamase.Shown in following table 1 and Fig. 4 A, after hatching, also had the serum lactamase level of other 9 chickens (3 heros, 6 is female) in six to seven months at 11.9-173.4ng/ml.

Table 1: in NLB-CMV-BL-transduction chicken, express β-Nei Xiananmei

¹NA: do not have

²Contrast is available from the hen of being untreated.

³Represent the mean value of 5 to 20 eggs

Embodiment 3

The expression of β-Nei Xiananmei in G0 hen egg white

57 pullets with the transduction of pNLB-CMV-BL retroviral vector grow sexual maturity, detect the active β-Nei Xiananmei (lactamase) of every hen egg white when 8 monthly ages.In 57 chickens, have 6 lactamase levels remarkable, scope is 56.3 to 250.0ng/ml (table 1 is on seeing).Even after PADAC and sample are cultivated several days, still do not have in this group can detect lactamase in the egg whites of other hens.In 24 injection hens of stand-in and egg white, do not detect lactamase with 42 hens that do not carry the genetically modified NLB carrier transduction of lactamase.In initial trial after 6 months, have still can detect stable lactamase in the egg white of 6 chickens of expression and express (table 1 is on seeing).

Carry out with anti-beta-lactamase antibody all having detected β-Nei Xiananmei in the egg whites of Western trace test these 6 hens at all.This egg white lactamase is identical with the bacteriogenic lactamase size of the purifying that is used as standard substance.With Western analyze in the egg white detected amount and zymetology test detected consistent, show the egg white lactamase be greatly have bioactive.Even 4 ℃ store some months after, the lactamase that produces in the hen egg white can loss of activity yet, molecular weight does not change yet.This discovery makes the egg that contains lactamase can store the longer time up to analysis.

Embodiment 4

The genetically modified kind of β-Nei Xiananmei system transmits and the serum expression in G1 and the G2 transgenic chicken

The sperm DNA from 56 G0 cocks is collected in extracting, select quantitative PCR detection wherein to significant three chickens of sperm DNA transfer gene content level come mating.These cocks are same three the highest (cocks 2395,2421 and 2428) of β-Nei Xiananmei in its blood (lactamase) level.Cock 2395 has produced 3 G1 transgenic progeny (in 422 offsprings), and other two are not producing transgenic progeny among 630 offsprings altogether.Three G1 transgenic chickens blood DNA is separately done the Southern analysis confirmed that transgenosis is complete and is incorporated into unique site at random.Hatching back 6.0-11 is during week, and G1

transgenic chicken

5308,5657 and 4133 serum contain the lactamase of 0.03,2.0 and 6.0 μ g/ml respectively.When these chickens being detected at the 6-7 monthly age, the level of lactamase has dropped to 0.03,1.1 and 5.0 μ g/ml (Fig. 4 A) again.

Hen 5657 and cock 4133 and the mating of non-transgenic chicken are obtained the transgenosis offspring of partly isozygotying.Fig. 5 shows the transgenic chicken of breeding from cock 4133 or hen 5657 and offspring's pedigree thereof.Also the breeding transgene cock 5308, but the lactamase concentration in its offspring's serum and the egg white is very low or detect less than.After hatching, measured active lactamase concentration in the G2 transgenic chicken serum of selecting at random in 3-90 days.From five G2 transgenic chickens that hen 5657 breeds, active lactamase concentration is all in 1.9-2.3 μ g/ml (compared to the expression of parental generation 1.1 μ g/ml, Fig. 4 B).All samples are all collected in identical period, therefore, because the lactamase concentration of hen 5657 correspondingly descended, expect lactamase concentration ratio parental generation height in its offspring's serum after its maturation.Similarly, the transgenic chicken serum lactamase concentration of five random chooses of breeding from cock 4133 is all to the similar of its parental generation but be higher than parental generation (Fig. 4 B).

Embodiment 5

The expression of β-Nei Xiananmei in transgenosis hen egg white

Contain the active β-Nei Xiananmei (lactamase) (Fig. 6 A) of 130ng in every milliliter of egg white of the egg of G1 hen 5657.Lactamase concentration is higher in some eggs of primiparity, reaches the plateau that can stablize at least nine months then.The lactamase concentration similar to parental generation (Fig. 6 A) of the transgenosis hen egg that hen 5657 and non-transgenic cock breed.Through quantitative PCR and Southern analyzing and testing, the hen 6978 that G2 hen 8617 and equal G2 cock 8839 breed is transgenosis homozygotes.Just as was expected, and lactamase concentration is higher than its hemizygote parental generation twice (Fig. 6 B) nearly in the egg of chicken 6978.Do not analyze other the G3 hen that breeds by hen 5657 because hen 6978 is them connect produce in the chicken unique female.Importantly, should note

hen

8867,8868 and 8869 egg be separated by 11 months that collect and have similar lactamase concentration (Fig. 6 A and 6B), another shows that the expression level in the egg white is consistent in the spawning time.

Cock 4133 and the mating of non-transgenic hen are obtained hemizygote G2 hen.Analyzed 15 transgenosis hens, all contained the lactamase of concentration range in the egg white at 0.47-1.34 μ g/ml.Fig. 7 A shows four representational hens.When detecting after 6 months, average expression level drops to 0.8 μ g/ml (Fig. 7 A) from about 1.0 μ g/ml.Expression level height in initial egg, level reduces in the some months thereafter.Then, lactamase concentration keeps constant in the egg.

G2 hen 8150 and 8191 hybridization of equal G2 cock produce hemizygote and homozygote G3 hen.Expressed the lactamase (Fig. 7 B) of concentration range in all G3 hen egg whites at 0.52-1.65 μ g/ml.The isozygoty average expression level of hen of G3 is higher by 47% than hemizygote G2 and G3 hen.The amount difference of lactamase very big (Fig. 7 A and 7B) in G2 that cock 4133 and offspring thereof breed and the G3 hen egg is though the lactamase level in any given hen egg is constant relatively in this group.Expection is for homozygous genotype, and the average expression of lactamase doubles.The Western engram analysis confirms that transgenosis correctly produces complete lactamase in the egg of G2 transgenic chicken.The detected lactamase level of detected lactamase level of Western trace and enzyme test alive is closely related, shows most of biologically active of egg white lactamase.Therefore, successfully utilize retroviral vector in chicken, to carry out the stable and expression reliably of transgenosis.

Can detect lactamase in the yolk deposition but than low in the egg white.Seven G2 that analyzed cock 4133 and be or G3 hen, the concentration range of lactamase is at 107-375ng/ml or be about 20% of concentration in the egg white in the yolk.Yolk of certain given hen and egg white lactamase level do not have dependency (Harvey etc., " expression of exogenous protein in the transgenic chicken egg white " (Expression of exogenousprotein in egg white of transgenic chickens) (in April, 2000) Nat.Biotechnol.20:396-399).

Embodiment 6

The generation of original rooster

Carry out the pNLB-CMV-BL transduction with the white leghorn zygote of new product.7-10 microlitre concentrated granular is injected in the subgerminal cavity of the egg of windowing, hatches chick behind the closed window.546 eggs have been injected.Extract blood DNA and adopt the probe-primer that detects neomycin resistance gene right, analyze this genetically modified existence by the Taqman test.As shown in table 2 below, in all chicks, can detect transgenosis among about 25% the blood DNA.

Embodiment 7

Genetically modified kind is to transmit

Identify that with the neo resistant gene that Taqman detects in the sperm DNA candidate G0 that is used for mating is male.It is male to identify three G0, and wherein the NLB-CMV-BL transgenosis all is higher than background level in the sperm DNA of every chicken.All G0 cocks that transgenosis in the sperm is positive and the mating of non-transgenic hen are to identify complete genetically modified G1 offspring.

For NLB-CMV-BL, bred 1026 chicks respectively, every kind of transgenosis has obtained three G1 chicks (table 2 is on seeing).All G1 offsprings are from the highest male of transgene level in the sperm DNA, and every male chick of breeding out equal number.

Embodiment 8

The Southern of G1 and G2 analyzes

For the integration and the integrity of carrier sequence that confirm to insert, G1 and the genetically modified DNA of G2 are carried out the Southern engram analysis.With HindIII digestion blood DNA, itself and neo resistance are visited the hybridization of resistance pin with the linker fragment (Fig. 3 B) of detection pNLB-CMV-BL carrier inside HindIII site formation and the genomic locus of integration site flank.Carry every linker fragment that all contains unique length in 3 G1 chickens of NLB-CMV-BL, show that transgenosis has been incorporated into three different genomic locus.G1 and the mating of non-transgenic hen are obtained hemizygote G2.Shown in table 2 (on seeing), the Mendelian who integrates as single transgenosis separate desired, the offspring 50.8% who carries the G1 cock of NLB-CMV-BL is genetically modified.The Southern of the DNA of G2 offspring HindIII digestion analyzes, and detects and the big or small similar linker fragment of their transgenosis parental generation, shows that transgenosis is by complete transmission.

Embodiment 9

Screening transgenosis homozygote G3 offspring

Be to obtain transgenosis homozygote transgenic chicken, the G2 that same loci is integrated with NLB-CMV-BL chicken (for example, same offspring that G1 the is male) hybridization that narrows.Bred two groups of chickens: first group is to derive from G1 4133 male hen and cock, and second group derives from G1 5657 hens.Change resistant gene with the neo resistance among the typical curve quantitative assay G3 offspring of Taqman test.Analyzing the genomic dna be defined as hemizygous G1 transgenosis 4133 cocks of transgenosis through Southern and make up this typical curve with known quantity.The scope of the total copy of this typical curve transgenosis is 10 ³-1.6x10 ⁴Or each diploid gene group 0.2-3.1 transgenosis copy.Because exponential phase reactive component be unconfined, so amplification efficiency is very high and given copy number has been provided repeatably value.Differ at copy number between each typical curve of 2 times repeatably round-robin difference is arranged.

Be to determine the number of G3 offspring transfer allele gene, DNA amplification and with standard substance relatively.The DNA of non-transgenic chicken does not increase.According to the transgenosis allelotrope figure that chicken draws that isozygotys, it is initial than the allelotrope Zao circulation of chicken that narrows to increase.This sequential detection program energy establishing criteria curve and threshold cycle number (Ct) calculate allelic number in the unknown DNA sample, show at the amplification figure of the threshold cycle place of number sample and significantly increase.Data are shown in following table 3.

For checking Taqman copy number is analyzed, pass through the DNA of the selected chicken of Southern engram analysis because of complementary probe in detecting 0.9kb fragment with the DNA of PstI digestion with neo resistance base resistance.Selecting to detect small segment is because less DNA is transferred to the easier quantification of film from gel.The strength of signal of this 0.9kb band is well corresponding with the copy number that Taqman tests detected G3 transgenic chicken.With the copy number of other 18 G3 transgenic chickens of Southern engram analysis and Taqman measure consistent.Be divided into and analysed 33 offsprings of 4133 pedigrees, wherein 9 (27.3%) are not genetically modified, and 16 (48.5%) are hemizygotes, and 8 (24.2%) are homozygotes.5657 pedigrees are divided into has analysed 10 offsprings, and wherein 5 (50.0%) are not genetically modified, and 1 (10.0%) is hemizygote, and 4 (40.0%) is homozygote.The viewed non-transgenic of 4133 pedigree G3 offsprings, hemizygote and homozygous ratio statistics go up the x that uses with expection ²1: 2: 1 ratio that test (P≤0.05) is measured does not have significant difference.The offspring of 5657 pedigrees does not have the separation of expection, but this may be the offspring's that detects the few reason (Harvey etc., " carrying out the continuous production of transgenic chicken with replication defect type retroviral vector and high flux screening program " (Consistent production of transgenic chickens using replicationdeficient retroviral vectors and high-throughput screening procedures) _ (in February, 2002) Poultry science 81:202-212) of number.

Table 3: measure the G3 offspring transfer gene copy number that the G2 transgenic chicken is bred

¹Std number: standard No.; NTC: no template contrast.

²Ct: threshold cycle number; Fluorescent is significantly higher than the cycle number of background.

³Mean number is divided by 5100 copy numbers that are rounded up to a determined every diploid gene group of decimal then.

⁴NA: do not have.

Embodiment 10

The vector construction of pNLB-MDOT-EPO carrier

Described according to this specification sheets embodiment 1 (vector construction), the BL encoding sequence is replaced with EPO encoding sequence (Fig. 8 B), created the pNLB-MDOT-EPO carrier.With MDOT replaced C MV promotor (Figure 13).MDOT is the synthetic promoter that contains ovomucoid (MD) and ovotransferrin (TO) promoter element.(the pNLB-MDOT-EPO carrier also claims pAVIJCR-A145.27.2.2).

Can use according to the codon that the hen uterine tube is optimized, by the BACKTRANSLATE program of winconsin software package 9.1 editions (the genetics calculating group company of state of Wisconsin Madison), utilize the dna sequence dna of showing founder EPO by the codon use of Hongyuan chicken ovalbumin, N,O-Diacetylmuramidase, ovomucoid and ovotransferrin compiling.Synthetic this dna sequence dna and by the dna integration technology company of Iowa carat Weir (Integrated DNA Technologies, Coralville IA) go into this sequence clone among pCRII-TOPO (hero company) 3 ' the protruding end T with the form of regulation.With HindIII and Fse I the EPO encoding sequence is downcut from pEpoMM then,, be connected in the pCMV-IFNMM of the alkaline phosphatase treatment of HindIII and Fse I digestion with 0.8% agarose-TAE gel-purified.The plasmid that produces is the pAVIJCR-A137.43.2.2 that contains the EPO encoding sequence that is subjected to cytomegalovirus immediate early promoter/enhanser and SV40 poly-A site control.With Nco I and Fse I digested plasmid pAVIJCR-A137.43.2.2, with correct fragment be connected with the pMDOTIFN fragment that Fse I enzyme is cut with Nco I, obtain containing the pAVIJCR-A137.87.2.1 of the EPO that is subjected to the MDOT promoters driven.For the EPO encoding sequence with the control of MDOT promotor is cloned into the NLB retroviral plasmid, with Kpn I and Fse I digested plasmid pALVMDOTIFN and pAVIJCR-A137.87.2.1.The correct dna fragmentation of purifying on 0.8% agarose-TAE gel connects these fragments then and is transformed into DH5 α cell.The plasmid that obtains is pNLB-MDOT-EPO (also claiming pAVIJCR-A145.27.2.2).

Embodiment 11

Express the transgenic chicken of EPO and the generation of transgenosis G1 chicken fully

As NLB-CMV-BL (seeing embodiment 2), prepare the NLB-MDOT-EPO transducing particles.According to Speksnijder technology (U.S. Patent number 5,897,998) 300 white leghorn eggs are windowed, then every about 7x10 of egg injection ⁴Individual transducing particles.Injection back incubated egg 21 days is measured the people EPO level of hatching in the chick serum sample of gathering in one week of back with EPO ELISA.

For containing the genetically modified G0 cock of EPO in the screening sperm, extract the DNA of cock seminal fluid sample with Chelex-100 extraction process (Walsh etc., 1991).Using the NEO-probe 1 (5 '-CCTCTCCACCCAAGCGGCCG-3 ') of " neo forward primer-1 " (5 '-TGGATTGCACGCAGGTTCT-3 ') and " neo anti-phase primer-1 " (5 '-GTGCCCAGTCATAGCCGAAT-3 ') primer and FAM mark to go up at 7700 sequenators (Pa Jinaierma company (Perkin Elmer)) then carries out the DNA sample

Analysis is to detect transgenosis.8 G0 cocks that transgene level in the seminal fluid sample is the highest are by artificial insemination and the mating of non-transgenic SPAFAS (white leghorn) hen.Use as mentioned above

Analyze genetically modified existence in the screening blood DNA sample.

In 1,054 offspring, find that 16 chicks are genetically modified (G1 fowl).With the existence of people EPO in the EPO ELISA detection chicken serum, EPO is about 70 nanograms/milliliter (ng/ml).With the existence of people EPO in the egg white of EPO ELISA detection G1 hen egg, find to contain the about 70ng/ml of people EPO.Find when detecting that when going up in people EPO response clone (HCD57 mouse erythroid cells) EPO (the optimization encoding sequence of derived from human EPO) that exists in the egg has biological activity with cell culture test.

Embodiment 12

The vector construction of pNLB-CMV-IFN

Described according to embodiment 1, created pNLB-CMV-IFN carrier (Fig. 8 A) with the BL encoding sequence in the IFN encoding sequence alternate embodiment 1.

Created the encoding sequence of optimizing, wherein, design preferred human interferon-alpha 2b (IFN-α 2b) encoding sequence with the highest codon of each specific amino acids rate of utilization in egg white protein Protalbinic acid, N,O-Diacetylmuramidase, ovomucoid and the ovotransferrin, and be inserted into carrier of the present invention.More specifically, the dna sequence dna of the human interferon-alpha 2b that optimizes can use according to the codon that the hen uterine tube is optimized, and utilizes the codon of Hongyuan chicken ovalbumin, N,O-Diacetylmuramidase, ovomucoid and ovotransferrin compiling to use counting rate meter to create by the BACKTRANSLATE program of winconsin software package 9.1 editions (the genetics calculating group company of Wisconsin State Madison).For example, in these four kinds of egg white proteins, the percentage that uses of four kinds of L-Ala codons is GCU 34%, GCC 31%, GCA 26%, GCG 8%.Therefore, can be with the codon of GCU as most of L-Ala in the humanIFN-2b encoding sequence of optimizing.Carrier with the humanIFN-2b gene that contains code optimization produces transgenic poultry, the interferon-' alpha ' 2b (TPD IFN-α 2b) that it can express transgenic fowl generation in tissue and ovum.

(department of La Jolla, California looks into column foot company (Stratagene, LaJolla, CA)) with 20 following circulations: 94 ℃ 1 minute with the pfu polysaccharase by PCR; 50 ℃ 30 seconds; Template and the primer tasteless nucleotide listed in the following table 4 with 72 ℃ of amplifications in 1 minute 10 seconds.With the polyacrylamide-TBE gel-purified PCR product of " push and soak into " method (Maniatis etc., 1982) from 12%, then this PCR product is mixed, in the amplified reaction of only making primer with IFN-1 and IFN-8 as template (seeing Table 4).The PCR product that produces is cut with HindIII and Xba I enzyme, from 2% agarose-TAE gel-purified, be connected into the pBluescript KS (department looks into column foot company) that cuts alkaline phosphatase treatment with HindIII and Xba I enzyme then, produce plasmid pBluKSP-IFNMagMax.(the Pa Jinaierma company in Foster city, California (Perkin-Elmer, Foster City, Calif)) goes up with general T7 or T3 primer, by the sequence of two chains of cycle sequencing mensuration at ABI PRISM377DNA sequenator.With transforming gene site-directed mutagenesis test kit (Transformer Site-Directed the Mutagenesis Kit) ((Clotech of clone Imtech of California Palo Alto, Palo Alto, Calif)), by the rite-directed mutagenesis correction derived from the sudden change among the pBluKSP-IFN of original oligonucleotide template.Then with HindIII and Xba I pBluKSP-IFN cutting-out IFN encoding sequence, from 0.8% agarose-TAE gel-purified and be connected into HindIII and Xba I enzyme is cut the pCMV-BetaLa-3B-dH of alkaline phosphatase treatment from revising.The plasmid that produces is the pCMV-IFN that contains the IFN encoding sequence that is subjected to cytomegalovirus immediate early promoter/enhanser and SV40 poly-A site control.For the IFN encoding sequence with CMV promotor/enhanser control is cloned into the NLB retroviral vector, earlier cut pCMV-IFN with Cla I and Xba I enzyme, two terminal Klenow fragment ((New England BioLabs of New England Biolabs, Inc. (US) Massachusetts, United States of America of Massachusetts Bei Fuli then with the DNA polymerases, Beverly, Mass)) fill and lead up.The pNLB-adapter is cut with NdeI and Kpn I enzyme, made two ends be passivity with T4 archaeal dna polymerase (New England Biolabs, Inc. (US) Massachusetts, United States of America).The correct dna fragmentation of purifying on 0.8% agarose-TAE gel connects these fragments then and is transformed into DH5 α cell.The plasmid that produces is pNLB-adapter-CMV-IFN.Then with this plasmid with Mlu I enzyme partially digested, the correct fragment gel-purified of cutting Blp I.PNLB-CMV-EGFP is cut with Mlu I and Blp I enzyme, use alkaline phosphatase treatment and gel-purified then.The Mlu I/Blp I part fragment of pNLB-adapter-CMV-IFN is connected with the big fragment of cutting derived from pNLB-CMV-EGFP Mlu I/Blp I enzyme, produces pNLB-CMV-IFN.

Table 4

Template	Template sequence	Primer 1	The sequence of primer 1	Primer 2	The sequence of primer 2
Template	Template sequence	Primer 1	The sequence of primer 1	Primer 2	The sequence of primer 2	IFN-A SEQ ID NO：8	5’ATGGCTTTGACCTTTGCCTTACT GGTGGCTCTCCTGGTGCTGAGCTG CAAGAGCAGCTGCTCTGTGGGCT GCGATCTGCCTCA3’	IFN-1 SEQ ID NO：9	5’CCCAAGCTTT CACCATGGCTT TGACCTTTGCCT T3’	IFN-2 SEQ ID NO：10	5’CTGTGGGT CTGAGGCAG AT3’
IFN-B SEQ ID NO：11	5’GACCCACAGCCTGGGCAGCAGG AGGACCCTGATGCTGCTGGCTCA GATGAGGAGAATCAGCCTGTTTA GCTGCCTGAAGGATAGGCACGAT TTTGGCTTT3’	IFN-2b SEQ ID NO：12	5’ATCTGCCTCA GACCCACAG3’	IFN-3b SEQ ID NO：13	5’AACTCCTC TTGAGGAAA GCCAAAATC 3’	IFN-A SEQ ID NO：8		IFN-1 SEQ ID NO：9	5’CCCAAGCTTT CACCATGGCTT TGACCTTTGCCT T3’	IFN-2 SEQ ID NO：10	5’CTGTGGGT CTGAGGCAG AT3’
IFN-B SEQ ID NO：11		IFN-2b SEQ ID NO：12	5’ATCTGCCTCA GACCCACAG3’	IFN-3b SEQ ID NO：13	5’AACTCCTC TTGAGGAAA GCCAAAATC 3’	IFN-C SEQ ID NO：14	5’CTCAAGAGGAGTTTGGCAACCA GTTTCAGAAGGCTGAGACCATCC CTGTGCTGCACGAGATG3’	IFN-3c SEQ ID NO：15	5’GATTTTGGCT TTCCTCAAGAG GAGTT3’	IFN-4 SEQ ID NO：16	5’ATCTGCTG GATCATCTC GTGC3’
IFN-D SEQ ID NO：14	5’ATCCAGCAGATCTTTAACCTGT TTAGCACCAAGGATAGCAGCGCT GCTTGGGATGAGACCCTGCTGGA TAAGTTTTACACCGAGCTGTACCA GCA3’	IFN-4b SEQ ID NO：18	5’GCACGAGATG ATCCAGCAGAT 3’	IFN-5 SEQ ID NO：19	5’ATCGTTCA GCTGCTGGT ACA3’	IFN-C SEQ ID NO：14		IFN-3c SEQ ID NO：15	5’GATTTTGGCT TTCCTCAAGAG GAGTT3’	IFN-4 SEQ ID NO：16	5’ATCTGCTG GATCATCTC GTGC3’
IFN-D SEQ ID NO：14		IFN-4b SEQ ID NO：18	5’GCACGAGATG ATCCAGCAGAT 3’	IFN-5 SEQ ID NO：19	5’ATCGTTCA GCTGCTGGT ACA3’	IFN-E SEQ ID NO：20	5’GCTGAACGATCTGGAGGCTTGC GTGATCCAGGGCGTGGGCGTGAC CGAGACCCCTCTGATGAAGGAGG ATAGCATCCT3’	IFN-5b SEQ ID NO：21	5’TGTACCAGCA GCTGAACGAT 3’	IFN-6 SEQ ID NO：22	5’CCTCACAG CCAGGATGC TAT3’
IFN-F SEQ ID NO：23	5’GGCTGTGAGGAAGTACTTTCAG AGGATCACCCTGTACCTGAAGGA GAAGAAGTACAGCCCTTGCGCTT GGGAAGTCGTGAGGG3’	IFN-6b SEQ ID NO：24	5’ATAGCATCCT GGCTGTGAGG 3’	IFN-7 SEQ ID NO：25	5’ATGATCTC AGCCCTCAC GAC3’	IFN-E SEQ ID NO：20		IFN-5b SEQ ID NO：21	5’TGTACCAGCA GCTGAACGAT 3’	IFN-6 SEQ ID NO：22	5’CCTCACAG CCAGGATGC TAT3’
IFN-F SEQ ID NO：23		IFN-6b SEQ ID NO：24	5’ATAGCATCCT GGCTGTGAGG 3’	IFN-7 SEQ ID NO：25	5’ATGATCTC AGCCCTCAC GAC3’	IFN-G SEQ ID NO：26	5’CTGAGATCATGAGGAGCTTTAG CCTGAGCACCAACCTGCAAGAGA GCTTGAGGTCTAAGGAGTAA3′	IFN-7b SEQ ID NO：27	5’GTCGTGAGGG CTGAGATCAT 3’	IFN-8 SEQ ID NO：28	5’TGCTCTAG ACTTTTTACT CCTTAGACC TCAAGCTCT 3’

Embodiment 13

Express the transgenic chicken of IFN and the generation of transgenosis G1 chicken fully

Program according to embodiment 2 prepares the pNLB-CMV-IFN transducing particles.According to Speksnijder technology (U.S. Patent number 5,897,998) 300 Bai Laihang (strain 0) egg is windowed, then every about 7x10 of egg injection ⁴Individual transducing particles.Injection back incubated egg 21 days is by the people IFN level in the chick serum sample of gathering in one week of IFN ELISA test determination hatching back.

For containing the genetically modified G0 cock of IFN in the screening sperm, extract the DNA of cock seminal fluid sample with Chelex-100 extraction process (Walsh etc., 1991).Use then " neo forward primer-1 " (5 '-TGGATTGCACGCAGGTTCT-3 '; SEQ ID NO:5) and the NEO-probe 1 of " neo reverse primer-1 " (5 '-GTGCCCAGTCATAGCCGAAT-3 ') primer and FAM mark (5 '-CCTCTCCACCCAAGCGGCCG-3 ') go up at 7700 sequenators (Pa Jinaierma company (Perkin Elmer)) the DNA sample carried out Taqman ^TMAnalysis is to detect transgenosis.3 G0 cocks that transgene level in the seminal fluid sample is the highest are by artificial insemination and the mating of non-transgenic SPAFAS (white leghorn) hen.

Use as mentioned above Analyze the genetically modified existence of screening blood DNA sample.In 1,597 offspring, find that 1 cock is transgenic chicken (having another name called " Ao Fei " (Alphie)).Detect the existence of hIFN in the luxuriant and rich with fragrance serum difficult to understand with hIFN ELISA, hIFN is 200ng/ml.

With seminal fluid difficult to understand luxuriant and rich with fragrance to the artificial insemination of non-transgenic SPAFAS (white leghorn) hen.With Analyzing and testing to 202 106 (about 52%) offsprings that merely hit are contained transgenosis.These are bred the result and follow the Mendelian inheritance pattern, show that Ao Fei is genetically modified.

Embodiment 14

The glycan analysis of the interferon alpha 2 b (TPD IFN-α 2b) that the transgenosis poultry produces

It (is among the TPD IFN α-2b) a kind of new glycosylation pattern to be arranged that experimental evidence discloses the interferon-' alpha ' 2b that poultry produces.Find TPD IFN α-2b contain the interferon-' alpha ' 2b that human peripheral leucocytes produces under the normal circumstances (PBL IFN α-2b) or among the natural human interferon-' alpha ' 2b (natural hIFN) non-existent two kinds of new sugared shapes (

band

4 and 5 is disaccharides that α-Gal extends; See Fig. 9).Find that also TPD IFN α-2b contains with people PBL IFN α-2b is similar but O-more effective generation in chicken than in the people is connected sugared structure.

HumanIFN-2b encoding sequence (embodiment 12, on seeing) is optimized, produces reorganization IFN-α 2b encoding sequence.In chicken, produce TPD IFN-α 2b (embodiment 13, on seeing) then.Glycan analysis comprises that monose analysis and FACE analysis have disclosed this proteinic sugar and formed or new glycosylation pattern.Like this, TPDIFN-α 2b demonstration contains following monosaccharide residue: N-acetyl-GalN (NAcGal), semi-lactosi (Gal), N-acetyl-glycosamine (NAcGlu) and sialic acid (SA).In TPD IFN-α 2b, do not find the glycosylation that N-connects, but in the O-glycosylation of Thr-106 position.Such glycosylation is similar to humanIFN-'s 2, and wherein, 106 Thr residue is that IFN-α 2 is exclusive.On the contrary, find that TPD IFN-α 2b is glycosylated at the O-of Thr-106 place.This type of glycosylation is similar to humanIFN-2, and wherein 106 Thr residue is unique residue of IFN-α 2.In addition, find that TPD IFN-α 2b does not have mannose residue.FACE analyzes and has shown 6 bands (Fig. 9) of representing various saccharide residues, and wherein band 1,2,3 is respectively not saliva acidifying, single saliva acidifying and two saliva acidifying (Figure 10).It is α 2-3-semi-lactosi (Gal) and α 2-6-N-acetyl-GalN (NAcGal) that sialic acid (SA) connects.Band 6 is represented not saliva acidifying tetrose.Band 4 and 5 is the (disaccharide that α-Gal) extends of undiscovered α-semi-lactosi in people PBL IFN-α 2b.Figure 10 shows the comparison of TPD IFN-α 2b (egg white hIFN) and people PBL IFN-α 2b (natural hIFN).Band 3 and 4 among the TPD IFN-α 2b, band 4 and be with 5 between band (as follows) is arranged time.

Find this protein in the Thr-106 site by specific residue O-glycosylation as described below:

(i)Gal-NAcGal-

(ii)SA-Gal-NAcGal-

(v)Gal-Gal-NAcGal-

Wherein, the Gal=semi-lactosi,

NAcGal=N-acetyl-GalN,

NAcGlu=N-acetyl-glycosamine and

The SA=sialic acid.

Per-cent is as follows:

(i) Gal-NAcGal-about 20%

(ii) SA-Gal-NAcGal-about 29%

(v) Gal-Gal-NAcGal-about 7%

Band

Embodiment 15

In the bird cell with EMCV IRES by plasmid transfection and retrovirus transduction carrying out MAb Expression

Express light chain and the heavy chain of human monoclonal antibodies from single carrier pCMV-LC-emcvIRES-HC by the IRES (see again Jang etc., a section of (1988) encephalomyocarditis virus RNA 5 ' non-translational region instructs inside to enter ribosomes (A segment of the 5 ' nontranslated regionof encephalomyocarditis virus RNA directs internal entry of riboxomes duringin vitro translation) J.Virol.62:2636-2643) that encephalomyocarditis virus (EMCV) is set in the In Vitro Translation process between light chain (LC) and heavy chain (HC) coded sequence. Transcribe with the CMV promoters driven.

Expression for the monoclonal antibody of measuring two separate carrier; The LC that will link to each other with the CMV promoter or HC cotransfection enter LMH/2 á cell, and this cell is estrogen response chicken liver cell system (seeing again (1990) such as Binder apolipoprotein II and the expression of vitellogenin II gene in estrogen response chicken liver cell system of transfection " endogenous and " (Expression of endogenous and transfectedapolipoprotein II and vitellogenin II genes in an estrogen responsive chickenliver cell line) Mol.Endocrinol.4:201-208). The cotransfection of pCMV-LC and pCMV-HC has produced the monoclonal antibody that is determined as 392ng/ml through MAb ELISA, and the pCMV-LC-emcvIRES-HC transfection has produced the monoclonal antibody of 185ng/ml.

With the retroviral vector of CMV-LC-emcv-HC box insertion, produce pL-CMV-LC-emcvIRES-HC-RN-BG based on Moroni (Moloney) murine leukemia virus (MLV).Parent line LMH cell with LMH/2 á (is seen (1987) such as Kawaguchi again, the foundation of a kind of chicken gizzard cancerous cell line LMH and evaluation (Establishment and characterization of a chicken hepatocellularcarcinoma cell line, LMH), Cancer Res.47:4460-4464) as target cell, because they do not have neomycin resistance.With pL-CMV-LC-emcvIRES-HC-RN-BG retroviral vector transduction LMH cell, with the Xin Meisu screening and go down to posterity several weeks.Screen with parental generation MLV carrier LXRN transduction LMH cell and with Xin Meisu independently.The substratum of LXRN cell is the monoclonal antibody feminine gender, and the substratum of L-CMV-LC-emcvIRES-HC-RN-BG-transducer cell contains the 22ng/ml monoclonal antibody.

Embodiment 16

Express the transgenic chicken of monoclonal antibody and the generation of transgenosis G1 chicken fully

CMV-LC-emcv-HC box with embodiment 15 replaces the CMV-BL box of the pNLB-CMV-BL of embodiment 1 to obtain the pNLB-CMV-LC-emcv-HC carrier.

Program according to embodiment 2 prepares the pNLB-CMV-LC-emcv-HC transducing particles.According to Speksnijder technology (U.S. Patent number 5,897,998) 300 Bai Laihang (strain 0) egg is windowed, then every about 7x10 of egg injection ⁴Individual transducing particles.Injection back incubated egg 21 days is measured the people of hatching in the chick serum sample of gathering in one week of back with ELISA Monoclonal antibodyLevel.

With

Contain this genetically modified G0 cock in the Analysis and Identification sperm.3 G0 cocks that transgene level in the seminal fluid sample is the highest are by artificial insemination and the mating of non-transgenic SPAFAS (white leghorn) hen.

Screening surpasses 1000 offsprings, finds that 10 above chicks contain this transgenosis (G1 bird).Detecting with ELISA should in the chick serum Monoclonal antibodyExistence.Finding should Monoclonal antibodyContent in every milliliter of serum is greater than 10 μ g.Also be somebody's turn to do in the egg white with ELISA detection G1 hen institute raw egg Monoclonal antibodyExistence, find that content in every milliliter of egg white is greater than 10 μ g.

Embodiment 17

The structure of pNLB-CMV-hG-CSF

This vector construction process can be with the IFN coding region of the pNLB-CMV-IFN carrier of the effective alternate embodiment 12 of encoding sequence of G-CSF.With primer 5 ' GCSF (ggggggaagctttcaccatggctggacctgcca; SEQ ID NO:32) and 3 ' GCSF (actagacttttcagggctgggcaaggtggcg; SEQ ID NO:33) by pORF9-hG-CSFb (catalog number (Cat.No.) porf-hgcsfb, (the Invivogen of hero company in San Diego, California, San Diego, CA)) amplification hG-CSF ORF (Filgrastim's open reading frame) obtains the PCR product of 642 bases (bp).For the pNLB-CMV-hG-CSF construction that contains the G-CSF encoding sequence 3 ' sequence identical with 3 ' sequence among pNLB-CMV-IFN α-2b is provided, with primer 5 ' GCSF-NLB (ccagccctgaaaagtctagtatggggattggtg; SEQ ID NO:34) and 3 ' GCSF-NLB (gggggggctcagctggaattccgcc; SEQ ID NO:35) the 86bp fragment of pNLB-CMV-IFN α-2b that pcr amplification is adjacent with INF encoding sequence 3 ' end.Mix this two kinds of PCR products (642bp and 86bp), utilize primer 5 ' GCSF and 3 ' GCSF-NLB they to be merged by pcr amplification.According to manufacturer's specification sheets this PCR product cloning is arrived

In the plasmid vector (hero company (Invitrogen)), electroporation produces pFusion-hG-CSF-NLB to DH5 α-E cell.With Hind III and Blp I digestion pFusion-hG-CSF-NLB, this 690bp G-CSF fragment of gel-purified.By the IFN α-2b encoding sequence among digestion removal pNLB-CMV-IFN α-2b of Blp I.Reconnect this carrier then, select to lack the IFN coding and insert segmental clone, produce pNLB-CMV-δ hIFN α-2b.With Blp I digestion pNLB-CMV-δ hIFN α-2b, partly digest this 8732bp Blp I-Hind III carrier segments of gel-purified with Hind III.This 8732bp fragment is connected in the 690bp Hind III/Blp I G-CSF fragment, produces pNLB-CMV-G-CSF.By sequence verification G-CSF ORF.

Embodiment 18

The generation of the transgenic chicken of expressing human granulocyte colony-stimulating factor (hG-CSF)

As preparing the NLB-CMV-hG-CSF transducing particles about NLB-CMV-BL described among the embodiment 2.Embryo with 277 X phase eggs of 7 μ l NLB-CMV-hG-CSF transducing particles injection (tires and is 2.1x10 ⁷-6.9x10 ⁷).Hatch 86 chicks, raise to sexual maturity.60 are on average separated according to sex for G-CSF male chick after tested; 30 hens, 30 cocks.Detect the existence of hG-CSF in 21 hen egg whites with ELISA.Contain the hG-CSF albumen of significance level in the egg white of 5 hens of discovery, its concentration is 0.05-0.5 μ g/ml.

With the DNA in Chelex-100 extraction process (Walsh etc., 1991) the extraction cock seminal fluid sample.Use forward primer SJ-G-CSF (cagagcttcctgctcaagtgctta then; SEQ ID NO:36) and reverse primer SJ-G-CSF (ttgtaggtggcacacagcttct; SEQ ID NO:37) and SJ-G-CSF probe (agcaagtgaggaagatccagggcg; SEQ ID NO:38) upward the DNA sample is carried out Taqman at 7700 sequenators (Pa Jinaierma company) ^TMAnalyze, to detect this transgenosis.With transgene level is the highest in the seminal fluid sample cock by artificial insemination and the mating of non-transgenic SPAFAS (white leghorn) hen.

Use Taqman as mentioned above ^TMAnalyze the genetically modified existence of screening blood DNA sample.In 2264 offsprings, find that 13 G1 are transgenic animal, serum is the G-CSF positive serum separately, contains in ELISA measures wherein a hen (XGF498) serum and contains 5.6 μ g/ml G-CSF in have an appointment 136.5ng/ml G-CSF, the egg white.

The G1 cock (QGF910 and DD9027) and the hybridization of non-transgenic hen of two identical with the XGF498 strain (thereby having identical transgenosis to insert same position in the genome), the egg that female offspring produces of generation contains poultry deutero-G-CSF.G-CSF by QGF910 and DD9027 offspring's egg egg white purifying milligram magnitude.Measure the representative glycosylation structural pattern of poultry deutero-G-CSF by the G-CSF of acquisition as described in embodiment 20.

Embodiment 19

The product of the transgenic chicken of expressing human cytotoxic lymphocyte antigen 4-Fc fusion rotein (CTLA4-Fc) Give birth to

As include Application No. 11/047,184 described structure pNLB-1.8OM-CTLA4Fc and the pNLB-3.9OM-CTLA4Fc that this paper submits at 31 days January in 2005 for referencial use in.As described in pNLB-CMV-BL, produce pNLB-1.8OM-CTLA4Fc and pNLB-3.9OM-CTLA4Fc transducing particles as embodiment 2.(tire and be about 4x10 with 7 μ l pNLB-1.8OM-CTLA4Fc transducing particles ⁶) 193 white leghorn eggs of injection, hatched 72 chicks.(tire and be about 4x10 with 7 μ l pNLB-3.9OM-CTLA4Fc transducing particles ⁶) 199 white leghorn eggs of injection, hatched 20 chicks.

Utilize ELISA to measure the existence of CTLA4-Fc in 30 hen egg whites that produce with the pNLB-1.8OM-CTLA4Fc particle.Contain the CTLA4-Fc of conspicuous level in the egg white of a hen of discovery, its mean level (ML) is 0.132 μ g/ml (having detected 5 eggs).

Utilize ELISA to measure the existence of CTLA4-Fc in 7 hen egg whites that produce with the pNLB-3.9OM-CTLA4Fc particle.Contain the CTLA4-Fc albumen of conspicuous level in the egg white of two hens of discovery, the mean level (ML) of a hen is 0.164 μ g/ml (having detected 5 eggs), and the mean level (ML) of second positive hen is 0.123 μ g/ml (having detected 5 eggs).

Embodiment 20

The glycan analysis of transgenosis poultry deutero-G-CSF

Adopt following analytical technology well known to those skilled in the art to determine TPD G-CSF oligosaccharide structure.After the release of peptide main chain, oligosaccharides is carried out MALDI-TOF-MS (lining matter assisted laser desorption and ionization-flight time mass spectrum is learned) analyze and ESI MS/MS (electron spray ionisation tandem mass spectrum) analysis.The O that discharges on the protein by chemical process connects oligosaccharides, and is permethylated with the NaOH method, and this method is included in anhydrous DMSO and exists down and the methyl-iodide reaction, uses chloroform extraction, analyzes then.Complete glycosylation G-CSF is carried out direct mass spectroscopy.Also polysaccharide structures is analyzed with HPLC.Briefly say, after discharging by protein main chain, separate this structure with HPLC.With the sample of the various polysaccharide materials of some enzymic digestion, analyze this digestion product with HPLC as is known in the art then, to determine its structure.

Show the structure of measuring below.What is interesting is that structure C and structure D may be the precursor forms that shows structure E down.(but the invention is not restricted to this) according to estimates, the lifetime of structure A on poultry deutero-glycosylation G-CSF is about 20%-40%, the lifetime of structure B on poultry deutero-glycosylation G-CSF is about 5%-25%, the lifetime of structure C on poultry deutero-glycosylation G-CSF is about 10%-20%, the lifetime of structure D on poultry deutero-glycosylation G-CSF is about 5%-15%, the lifetime of structure E on poultry deutero-glycosylation G-CSF is about 1%-5%, the lifetime of structure F on poultry deutero-glycosylation G-CSF is about 10%-25%, and the lifetime of structure G on poultry deutero-glycosylation G-CSF is about 20%-30%.

With GC/MS (gas chromatography-mass spectrography) to being mixed with arabitol (interior mark), with those skilled in the art's currently known methods be hydrolyzed, the poultry deutero-G-CSF of N acetylize and TMS derivatize carries out the monose analysis.The standard mixture of derivatize sample and similar deutero-sugar is made comparisons.Mix ketone deoxidation nonanone saccharic acid, freeze-drying, hydrolysis, desalination and again after the freeze-drying, G-CSF carries out the sialic acid analysis to the poultry deutero-.Utilize suitable standard substance analytic sample in Dionex BioLC system.These analytical proofs exist semi-lactosi, glucose, N-acetylgalactosamine, N-acetyl-glucosamine and sialic acid (N-n acetylneuraminic acid n), and are as shown in table 5.Data in the table 5 have replaced HPAEC-PAD and have analyzed the preliminary data that produces, and there is the N-acetyl-glucosamine of higher percentage ratio in the HPAEC-PAD assay determination.

Table 5

TPD G-CSF

Detected detected the rubbing of rubbing of monose

That number that number/milligram

Semi-lactosi 4.5 34.5

N-acetylgalactosamine 2.9 22.2

N-acetyl-glucosamine 0.9 57.3

Sialic acid 6.0 46.0

To hydrolysis among the TFA and in boron deuterate sodium the glycan sample that methylates in advance of reductive poultry deutero-G-CSF connect analysis.Add three times methyl alcohol: Glacial acetic acid (9: 1), the acetic anhydride acetylize is also used in freeze-drying then, thereby removes boric acid.Behind the chloroform extraction purifying, detect this sample with GC/MS.Also examination criteria product mixture under the same conditions.The connection of measuring is as follows:

I. the sialic acid connection is that 2-3 is connected in semi-lactosi, and 2-6 is connected in N-acetylgalactosamine

Ii. the semi-lactosi connection is that 2-3 is connected in N-acetylgalactosamine, and 2-4 is connected in N-acetyl-glucosamine

It is that 2-6 is connected in N-acetylgalactosamine that the iii.N-acetylglucosamine connects

Embodiment 21

The body outer cell proliferation activity of poultry deutero-G-CSF (TPD G-CSF)

External biological activity with NFS-60 cell proliferation experiment proof TPD G-CSF.Briefly say, cultivate the NFS-60 cell with the growth medium that contains GM-CSF.The culture that results are converged, washing, and single growth medium of using is with cell density 10 ⁵Individual cells/well is inoculated.Human G-CSF with growth medium serial dilution TPD G-CSF and bacterial derivation (is that knob is compeled base

), add in triplicate in each hole.By 3-(4,5-dimethylthiazole-2-yl)-2, the metabolism reduction reaction of 5-phenylbenzene bromination tetrazolium (MTT) is measured cell proliferation, and uses the spectrophotometric standard measure.By relatively knob is urgent

ED with the avian derived G-CSF of purifying ₅₀, determine that the ratio of avian derived G-CSF is lived.After measured, the ratio of the TPD G-CSF G-CSF knob that is higher than bacterial derivation far away alive is compeled in 14 days

(not glycosylated G-CSF).Referring to Figure 17.

Embodiment 22

The structure of pNLB-CMV-Des-Arg166-EPO

With Hind III and EcoRI digestion embodiment 12 described pNLB-CMV-IFN, so as with under show EPO encoding sequence and alternative hIFN α 2 encoding sequences of signal peptide (SEQ ID NO:42) and signal coding sequence.Because have a plurality of EcoRI and Hind III site in the carrier, so adopt the auxiliary restriction endonuclease of RecA-(RARE) cutting method to cut required site.Following oligonucleotide is used for the RARE step:

PnlbEcoRI3805 rare (5 '-GAC TCC TGG AGC CCG TCA GTA TCGGCG GAA TTC CAG CTG AGC GCC GGT CGC TAC CAT TAC-3 ') (SEQID NO:43) and

PnlbHinD III3172 rare (5 '-TAA TAC GAC TCA CTA TAG GGA GACCGG AAG CTT TCA CCA TGG CTT TGA CCT TTG CCT TAC-3 ') (SEQ IDNO:44).

Obtain the also linearized vector of gel-purified 8740bp.

As described below, prepare the EPO inset by overlapping PCR.Utilize Pfu polysaccharase and following primer amplification to be cloned into synthetic EPO sequence in the standard cloning vector, to produce a PCR product: 5 ' pNLB/Epo (5 '-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3 ') and (SEQ ID NO:45) and pNLB/3 ' Epo (5 '-TCCCCATACTAGACTTTTTACCTATCGCCGGTC-3 ') (SEQ ID NO:46).Utilize the zone of Pfu polysaccharase and following primer amplification pNLB-CMV-hIFN α-2b, to produce the 2nd PCR product: 3 ' Epo/pNLB (5 '-ACCGGCGATAGGTAAAAAGTCTAGTATGGG-3 ') and (SEQ ID NO:47) and pNLB/SapI (5 '-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3 ') (SEQ ID NO:48).Mix this two kinds of PCR products, increase once more with following primer: 5 ' pNLB/Epo (5 '-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3 ') and (SEQ ID NO:45) and pNLB/SapI (5 '-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3 ') (SEQ IDNO:48).

With the PCR product that Hind III and Eco RI digestion are merged, gel-purified 633bp fragment.Connect 8740bp and 633bp fragment to produce pNLB-CMV-EPO.

EPO 1-synthetic EPO sequence (610nt)

AAGCTTTCACCATGGGCGTGCACGAGTGCCCTGCTTGGCTGTGGCTGC

TCTTGAGCCTGCTCAGCCTGCCTCTGGGCCTGCCTGTGCTGGGCGCTC

CTCCAAGGCTGATCTGCGATAGCAGGGTGCTGGAGAGGTACCTGCTG

GAGGCTAAGGAGGCTGAGAACATCACCACCGGCTGCGCTGAGCACTG

CAGCCTGAACGAGAACATCACCGTGCCTGATACCAAGGTGAACTTTT

ACGCTTGGAAGAGGATGGAGGTGGGCCAGCAGGCTGTGGAGGTGTG

GCAGGGCCTGGCTCTGCTGAGCGAGGCTGTGCTGAGGGGCCAGGCTC

TGCTGGTGAACAGCTCTCAGCCTTGGGAGCCTCTGCAGCTGCACGTGG

ATAAGGCTGTGAGCGGCCTGAGAAGCCTGACCACCCTGCTGAGGGCT

CTGAGGGCTCAGAAGGAGGCTATCAGCCCTCCAGATGCTGCAAGCGC

TGCCCCTCTGAGGACCATCACCGCTGATACCTTTAGGAAGCTGTTTAG

GGTGTACAGCAACTTTCTGAGGGGCAAGCTGAAGCTGTACACCGGCG

AGGCTTGCAGGACCGGCGATAGGTAAAAAGGCCGGCCGAGCTC(SEQID NO：42)

Produce the EPO encoding sequence of the EPO of removed 165 amino acid form of coding termination codon (encode 166 arginine).The 179bp zone of synthetic pNLB-CMV-EPO, this zone is corresponding to extending to the EcoRI site that is positioned at EPO terminator codon downstream from the Eco 47III site that is positioned at the EPO encoding sequence among the pNLB-CMV-EPO; Eliminate terminal arginine codon (166) so that aspartic acid (amino acid/11 65) becomes the end amino acid codon, produce 176bp Eco47III/EcoRI fragment.Integrated dna technique (the IntegratedDNA Technologies of company by Iowa carat Weir, Coralville, Iowa 52241) synthetic this fragment, and be cloned into pDRIVE the carrier ((Qiagen of Kai Jie company, Inc)) in, produce pDRIVE-des-Arg166-EPO.176bpEco 47III/EcoRI fragment subclone in the Eco47III/EcoRI site of pNLB-CMV-EPO, is produced pNLB-CMV-Des-Arg 166-EPO.

Substantially as described in the embodiment 2, prepare transducing particles by pNLB-CMV-Des-Arg166-EPO.

Embodiment 23

The generation of the transgenic chicken of expressing human erythropoietin

1234 white leghorn eggs are windowed, inject transducing particles (substantially as embodiment 2 as described in).334 egg incubations.With the DNA in Chelex-100 extraction process (Walsh etc., 1991) the extraction cock seminal fluid sample.Then, upward the DNA sample is carried out Taqman at 7700 sequential detection instrument (Pa Jin Elmer Co., Ltd (Perkin Elmer)) ^TMAnalyze, to detect transgenosis.The G0 cock of 7 hatchings is the NLB-CMV-EPO transgenic positive after testing.Make three to produce 1190 offsprings after testing for the chimeric kind of NLB-CMV-EPO transgenic positive is transgenosis cock and the female mating of non-transgenic by artificial insemination, wherein 14 is that the transgenic positive kind is transgenosis G1.Measure through ELISA, the G1 kind is to contain the 0.4-1.9 μ g/ml EPO that has an appointment in the egg white of the female or egg that its offspring produces of transgenosis.

Embodiment 24

The purifying of transgenosis poultry deutero-EPO

With the long-pending 50mM sodium-acetate of triploid, the egg white that the transgenic chicken of generation EPO is laid eggs in the pH 4.6 dilution uterine tubes mixes also and filters, and is loaded into then on the agarose cationic exchange coloum.With the 50mM sodium-acetate that contains 100mM NaCl, after pH 5.0 washes post, with the identical acetate buffer wash-out EPO that contains 500mM NaCl and 0.05% polysorbas20.The EPO that elutes on the agarose column is loaded on the phenyl sepharose hydrophobic interaction chromatography post.Use 2M NaCl, 50mM Tris-HCl, pH 7.2,0.05% these posts of polysorbas20 balance.After loading above-mentioned prepared product, utilize same buffer to wash post.Wash with water then.Use 30%IPA wash-out EPO subsequently.Then the EPO prepared product is put on the reversed-phase HPLC post, the ethanolic soln wash-out EPO of the 0.1% Tricholroacetic Acid preparation that increases progressively with concentration.Be about the wash-out peak value that reached EPO at 53% o'clock at alcohol concn.Utilize diafiltration to concentrate final EPO prepared product, and use the 0.1M sodium phosphate buffer, pH 7.0 replace solvents.

Embodiment 25

The glycan analysis of transgenosis poultry deutero-erythropoietin

The following analytical technology that adopts those of ordinary skills to know is measured the oligosaccharide structure of avian derived people EPO.

The O-that discharges on the protein by chemical process connects oligosaccharides, and the N-that discharges on the protein by enzymatic means connects oligosaccharides.After the release, it is permethylated to make O-connect the oligosaccharides that is connected with N-with the NaOH method, and described method is included among the anhydrous DMSO reacts with methyl-iodide, uses chloroform extraction then, and analyzes.Use the HPLC isolating construction.

Behind release of peptide main chain and purifying, as is known in the art oligosaccharides is carried out MALDI-TOF-MS (lining matter assisted laser desorption and ionization-flight time mass spectrum is learned) and analyze and ESIMS/MS (electron spray ionisation tandem mass spectrum) analysis.Also use the sample of the various polysaccharide materials of some enzymic digestion, analyze this digestion product with HPLC as is known in the art then.

Identify that O-as follows connects the oligosaccharide structure that is connected with N-.The connection analysis that structure is carried out discloses Figure 20 and being connected shown in 21.

The EPO structure that N-connects is as follows.

Gal

NacGlu

Sialic acid Seminose

The EPO structure that O-connects is as follows.

(i) SA-Gal-NAcGal structure A-o

Structure B-o

Structure C-o

Structure D-o

Structure E-o

(vi) Gal-NAcGal structure F-o

Structure G-o

Wherein, the Gal=semi-lactosi,

NAcGal=N-acetyl-GalN,

NAcGlu=N-acetyl-glycosamine and

The SA=sialic acid.

Embodiment 26

The glycan analysis of transgenosis poultry deutero-EPO

By GC/MS (gas chromatography-mass spectrography) EPO available from transgenic chicken is carried out the monose analysis.In sample, mix arabitol (interior mark), with method known to those skilled in the art hydrolysis, N-acetylize and TMS derivatize.The standard mixture of derivatize sample and similar deutero-sugar is made comparisons.Mix ketone deoxidation nonanone saccharic acid, freeze-drying, hydrolysis, desalination and again after the freeze-drying, EPO is carried out the sialic acid analysis.Utilize suitable standard substance analytic sample in Dionex BioLC system.Table 6 has shown the monose detection by quantitative result of EPO.In the monose analysis, also detect wood sugar, Fucose and the glucose of trace contamination.Replace HPAEC-PAD to analyze the raw data that produces with data shown in the table 6.

Table 6

Detected nmole

Monose nmole number/milligram sample

Seminose 49 245

Semi-lactosi 16 80

N-acetylgalactosamine 6.0 30

N-acetyl-glucosamine 91 455

Sialic acid 4.7 24

Embodiment 27

The body outer cell proliferation activity of TPD people EPO

Utilize the TF-1 cell proliferation experiment to detect the extracorporeal biology activity of poultry deutero-people EPO.Detect two the different samples (SP1 post: 130mM NaCl and 250mM NaCl) of representative by two kinds of components of initial ion-exchange purification step recovery.The cell-proliferation activity of these two kinds of components is basic identical, proves that subsequently glycosylated erythropoietin contained in these two kinds of components is also basic identical.Briefly say, cultivate the TF-1 cell with the growth medium that contains GM-CSF (2ng/ml).The culture that is paved with of results, washing is inoculated in the growth medium that does not contain GM-CSF that (each hole is 0.32cm in each holes of standard 96 orifice plates ²), cell density is 10 ⁴Individual cells/well.With the avian derived EPO of growth medium serial dilution, add in triplicate in the different holes.After 5 days, by 3-(4,5-dimethylthiazole-2-yl)-2, the metabolism reduction reaction of 5-phenylbenzene bromination tetrazolium (MTT) is measured cell proliferation, and uses the spectrophotometric standard measure.The external activity of purifying EPO is seen Figure 22.

All documents that above specification sheets is quoted (for example, United States Patent (USP), U.S. Patent application, publication) all form with reference are incorporated herein.Is conspicuous to various improvement of the present invention and change to persons skilled in the art, does not exceed scope of the present invention and design.Though in conjunction with concrete preferred embodiment the present invention is described, should be appreciated that, as claimed in claim, the present invention's these specific embodiments that is limited within reason.In fact, the conspicuous various improvement that are used to implement mode of the present invention all drop in the scope of following claim to persons skilled in the art.

Sequence table

＜110〉R.D. Yi Warui (Ivarie, Robert D.)

G. Liu (Liu, Guodong)

J.C. draw general (Rapp, Jeffrey C.)

J.A. not in this (Morris, Julie A.)

A.J. breathe out dimension (Harvey, Alex J.)

＜120〉avian derived erythropoietin

<130>AVI-000CIP5PCT

<160>50

＜170〉PatentIn is 3.1 editions

<210>1

<211>498

<212>DNA

＜213〉artificial sequence

<220>

＜223〉transgenosis poultry deutero-IFN α 2b CDS

<400>1

tgcgatctgc ctcagaccca cagcctgggc agcaggagga ccctgatgct gctggctcag 60

atgaggagaa tcagcctgtt tagctgcctg aaggataggc acgattttgg ctttcctcaa 120

gaggagtttg gcaaccagtt tcagaaggct gagaccatcc ctgtgctgca cgagatgatc 180

cagcagatct ttaacctgtt tagcaccaag gatagcagcg ctgcttggga tgagaccctg 240

ctggataagt tttacaccga gctgtaccag cagctgaacg atctggaggc ttgcgtgatc 300

cagggcgtgg gcgtgaccga gacccctctg atgaaggagg atagcatcct ggctgtgagg 360

aagtactttc agaggatcac cctgtacctg aaggagaaga agtacagccc ctgcgcttgg 420

gaagtcgtga gggctgagat catgaggagc tttagcctga gcaccaacct gcaagagagc 480

ttgaggtcta aggagtaa 498

<210>2

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉transgenosis poultry deutero-IFN α 2b

<400>2

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu

165

<210>3

<211>579

<212>DNA

＜213〉artificial sequence

<220>

＜223〉transgenosis poultry deutero-EPO CDS

<400>3

atgggcgtgc acgagtgccc tgcttggctg tggctgctct tgagcctgct cagcctgcct 60

ctgggcctgc ctgtgctggg cgctcctcca aggctgatct gcgatagcag ggtgctggag 120

aggtacctgc tggaggctaa ggaggctgag aacatcacca ccggctgcgc tgagcactgc 180

agcctgaacg agaacatcac cgtgcctgat accaaggtga acttttacgc ttggaagagg 240

atggaggtgg gccagcaggc tgtggaggtg tggcagggcc tggctctgct gagcgaggct 300

gtgctgaggg gccaggctct gctggtgaac agctctcagc cttgggagcc tctgcagctg 360

cacgtggata aggctgtgag cggcctgaga agcctgacca ccctgctgag ggctctgggc 420

gctcagaagg aggctatcag ccctccagat gctgcaagcg ctgcccctct gaggaccatc 480

accgctgata cctttaggaa gctgtttagg gtgtacagca actttctgag gggcaagctg 540

aagctgtaca ccggcgaggc ttgcaggacc ggcgatagg 579

<210>4

<211>193

<212>PRT

＜213〉artificial sequence

<220>

＜223〉transgenosis poultry deutero-EPO

<400>4

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1 5 10 15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

20 25 30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

35 40 45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

50 55 60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65 70 75 80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

85 90 95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

100 105 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

115 120 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu

130 135 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145 150 155 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

165 170 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

180 185 190

Arg

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉neo forward primer-1

<400>5

tggattgcac gcaggttct 19

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉neo reverse primer-1

<400>6

gtgcccagtc atagccgaat 20

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the NEO-probe 1

<400>7

cctctccacc caagcggccg 20

<210>8

<211>83

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-A primer

<400>8

atggctttga cctttgcctt actggtggct ctcctggtgc tgagctgcaa gagcagctgc 60

tctgtgggct gcgatctgcc tca 83

<210>9

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-1 primer

<400>9

cccaagcttt caccatggct ttgacctttg cctt 34

<210>10

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-2 primer

<400>10

ctgtgggtct gaggcagat 19

<210>11

<211>100

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-B primer

<400>11

gacccacagc ctgggcagca ggaggaccct gatgctgctg gctcagatga ggagaatcag 60

cctgtttagc tgcctgaagg ataggcacga ttttggcttt 100

<210>12

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-2b primer

<400>12

atctgcctca gacccacag 19

<210>13

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-3b primer

<400>13

aactcctctt gaggaaagcc aaaatc 26

<210>14

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-C primer

<400>14

ctcaagagga gtttggcaac cagtttcaga aggctgagac catccctgtg ctgcacgaga 60

tg 62

<210>15

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-3c primer

<400>15

gattttggct ttcctcaaga ggagtt 26

<210>16

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-4 primer

<400>16

atctgctgga tcatctcgtg c 21

<210>17

<211>95

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-D primer

<400>17

atccagcaga tctttaacct gtttagcacc aaggatagca gcgctgcttg ggatgagacc 60

ctgctggata agttttacac cgagctgtac cagca 95

<210>18

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-4b primer

<400>18

gcacgagatg atccagcaga t 21

<210>19

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-5 primer

<400>19

atcgttcagc tgctggtaca 20

<210>20

<211>78

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-E primer

<400>20

gctgaacgat ctggaggctt gcgtgatcca gggcgtgggc gtgaccgaga cccctctgat 60

gaaggaggat agcatcct 78

<210>21

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-5b primer

<400>21

tgtaccagca gctgaacgat 20

<210>22

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-6 primer

<400>22

cctcacagcc aggatgctat 20

<210>23

<211>83

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-F primer

<400>23

ggctgtgagg aagtactttc agaggatcac cctgtacctg aaggagaaga agtacagccc 60

ttgcgcttgg gaagtcgtga ggg 83

<210>24

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-6b primer

<400>24

atagcatcct ggctgtgagg 20

<210>25

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-7 primer

<400>25

atgatctcag ccctcacgac 20

<210>26

<211>65

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-G primer

<400>26

ctgagatcat gaggagcttt agcctgagca ccaacctgca agagagcttg aggtctaagg 60

agtaa 65

<210>27

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-7b primer

<400>27

gtcgtgaggg ctgagatcat 20

<210>28

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IFN-8 primer

<400>28

tgctctagac tttttactcc ttagacctca agctct 36

<210>29

<211>69

<212>DNA

＜213〉artificial sequence

<220>

＜223〉N,O-Diacetylmuramidase signal sequence

<400>29

ccaccatggg gtctttgcta atcttggtgc tttgcttcct gccgctagct gccttagggc 60

cctctagag 69

<210>30

<211>671

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be connected in the MDOT promotor of IFN-MM CDS

<400>30

atcgataggt accgggcccc ccctcgaggt gaatatccaa gaatgcagaa ctgcatggaa 60

agcagagctg caggcacgat ggtgctgagc cttagctgct tcctgctggg agatgtggat 120

gcagagacga atgaaggacc tgtcccttac tcccctcagc attctgtgct atttagggtt 180

ctaccagagt ccttaagagg tttttttttt ttttggtcca aaagtctgtt tgtttggttt 240

tgaccactga gagcatgtga cacttgtctc aagctattaa ccaagtgtcc agccaaaatc 300

gatgtcacaa cttgggaatt ttccatttga agccccttgc aaaaacaaag agcaccttgc 360

ctgctccagc tcctggctgt gaagggtttt ggtgccaaag agtgaaaggc ttcctaaaaa 420

tgggctgagc cggggaaggg gggcaacttg ggggctattg agaaacaagg aaggacaaac 480

agcgttaggt cattgcttct gcaaacacag ccagggctgc tcctctataa aaggggaaga 540

aagaggctcc gcagccatca cagacccaga ggggacggtc tgtgaatcaa gctttcacca 600

tggctttgac ctttgcctta ctggtggctc tcctggtgct gagctgcaag agcagctgct 660

cgtgggttgc g 671

<210>31

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉be connected in the MDOT promotor of IFN-MM CDS

<400>31

Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys

1 5 10 15

Lys Ser Ser Cys Ser Trp Val Ala

20

<210>32

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5 ' GCSF primer

<400>32

ggggggaagc tttcaccatg gctggacctg cca 33

<210>33

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉3 ' GCSF primer

<400>33

actagacttt tcagggctgg gcaaggtggc g 31

<210>34

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5 ' GCSF-NLB primer

<400>34

ccagccctga aaagtctagt atggggattg gtg 33

<210>35

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉3 ' 6CSF-NLB primer

<400>35

gggggggctc agctggaatt ccgcc 25

<210>36

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SJ-G-CSF primer

<400>36

cagagcttcc tgctcaagtg ctta 24

<210>37

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SJ-G-CSF reverse primer

<400>37

ttgtaggtgg cacacagctt ct 22

<210>38

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SJ-G-CSF probe

<400>38

agcaagtgag gaagatccag ggcg 24

<210>39

<211>612

<212>DNA

＜213〉artificial sequence

<220>

＜223〉G-CSF nucleotide coding sequence

<400>39

atggctggac ctgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg 60

cacagtgcac tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc 120

cagagcttcc tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg 180

ctccaggaga agctgtgtgc cacctacaag ctgtgccacc ccgaggagct ggtgctgctc 240

ggacactctc tgggcatccc ctgggctccc ctgagcagct gccccagcca ggccctgcag 300

ctggcaggct gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag 360

gccctggaag ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc 420

gccgactttg ccaccaccat ctggcagcag atggaagaac tgggaatggc ccctgccctg 480

cagcccaccc agggtgccat gccggccttc gcctctgctt tccagcgccg ggcaggaggg 540

gtcctagttg cctcccatct gcagagcttc ctggaggtgt cgtaccgcgt tctacgccac 600

cttgcccagc cc 612

<210>40

<211>204

<212>PRT

＜213〉artificial sequence

<220>

＜223〉G-CSF and signal peptide

<400>40

Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln

1 5 10 15

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro

20 25 30

Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu

35 40 45

Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys

50 55 60

Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu

65 70 75 80

Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser

85 90 95

Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu

100 105 110

Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu

115 120 125

Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala

130 135 140

Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu

145 150 155 160

Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg

165 170 175

Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu

180 185 190

Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro

195 200

<210>41

<211>174

<212>PRT

＜213〉artificial sequence

<220>

＜223〉G-CSF mature peptide

<400>41

Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys

1 5 10 15

Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln

20 25 30

Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val

35 40 45

Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys

50 55 60

Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser

65 70 75 80

Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser

85 90 95

Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp

100 105 110

Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro

115 120 125

Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe

130 135 140

Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe

145 150 155 160

Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro

165 170

<210>42

<211>610

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic EPO sequence

<400>42

aagctttcac catgggcgtg cacgagtgcc ctgcttggct gtggctgctc ttgagcctgc 60

tcagcctgcc tctgggcctg cctgtgctgg gcgctcctcc aaggctgatc tgcgatagca 120

gggtgctgga gaggtacctg ctggaggcta aggaggctga gaacatcacc accggctgcg 180

ctgagcactg cagcctgaac gagaacatca ccgtgcctga taccaaggtg aacttttacg 240

cttggaagag gatggaggtg ggccagcagg ctgtggaggt gtggcagggc ctggctctgc 300

tgagcgaggc tgtgctgagg ggccaggctc tgctggtgaa cagctctcag ccttgggagc 360

ctctgcagct gcacgtggat aaggctgtga gcggcctgag aagcctgacc accctgctga 420

gggctctgag ggctcagaag gaggctatca gccctccaga tgctgcaagc gctgcccctc 480

tgaggaccat caccgctgat acctttagga agctgtttag ggtgtacagc aactttctga 540

ggggcaagct gaagctgtac accggcgagg cttgcaggac cggcgatagg taaaaaggcc 600

ggccgagctc 610

<210>43

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉pnlbEcoRI3805 is rare

<400>43

gactcctgga gcccgtcagt atcggcggaa ttccagctga gcgccggtcg ctaccattac 60

<210>44

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉pnlbHinD III3172 is rare

<400>44

taatacgact cactataggg agaccggaag ctttcaccat ggctttgacc tttgccttac 60

<210>45

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<223>5’pNLB/Epo

<400>45

ggggggaagc tttcaccatg ggcgtgcacg ag 32

<210>46

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<223>pNLB/3’Epo

<400>46

tccccatact agacttttta cctatcgccg gtc 33

<210>47

<211>30

<212>DNA

＜213〉artificial sequence

<220>

<223>3’Epo/pNLB

<400>47

accggcgata ggtaaaaagt ctagtatggg 30

<210>48

<211>35

<212>DNA

＜213〉artificial sequence

<220>

<223>pNLB/SapI

<400>48

gggggggctc ttctcagctg gaattccgcc gatac 35

<210>49

<211>495

<212>DNA

＜213〉artificial sequence

<220>

＜223〉nucleotide coding sequence of ripe EPO

<400>49

gctcctccaa ggctgatctg cgatagcagg gtgctggaga ggtacctgct ggaggctaag 60

gaggctgaga acatcaccac cggctgcgct gagcactgca gcctgaacga gaacatcacc 120

gtgcctgata ccaaggtgaa cttttacgct tggaagagga tggaggtggg ccagcaggct 180

gtggaggtgt ggcagggcct ggctctgctg agcgaggctg tgctgagggg ccaggctctg 240

ctggtgaaca gctctcagcc ttgggagcct ctgcagctgc acgtggataa ggctgtgagc 300

ggcctgagaa gcctgaccac cctgctgagg gctctgggcg ctcagaagga ggctatcagc 360

cctccagatg ctgcaagcgc tgcccctctg aggaccatca ccgctgatac ctttaggaag 420

ctgtttaggg tgtacagcaa ctttctgagg ggcaagctga agctgtacac cggcgaggct 480

tgcaggaccg gcgat 495

<210>50

<211>165

<212>PRT

＜213〉artificial sequence

<220>

＜223〉aminoacid sequence of ripe EPO

<400>50

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

1 5 10 15

Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

20 25 30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

65 70 75 80

Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

85 90 95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

100 105 110

Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

115 120 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

130 135 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala

145 150 155 160

Cys Arg Thr Gly Asp

165

Claims

1. composition that comprises isolating people EPO, described people EPO contains avian derived oligosaccharide structure.

2. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 50% does not contain sialic acid.

3. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 60% does not contain sialic acid.

4. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 70% does not contain sialic acid.

5. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 80% does not contain sialic acid.

6. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 90% does not contain sialic acid.

7. composition as claimed in claim 1 is characterized in that, described N connects in the oligosaccharide structure type and do not contain sialic acid greater than about 50%.

8. composition as claimed in claim 1 is characterized in that, described EPO go up greater than about 50% or more N-connect oligosaccharides and contain terminal N-acetyl-glucosamine.

9. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 60% contains terminal N-acetyl-glucosamine.

10. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 70% contains terminal N-acetyl-glucosamine.

11. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 80% contains terminal N-acetyl-glucosamine.

12. composition as claimed in claim 1 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 90% contains terminal N-acetyl-glucosamine.

13. composition as claimed in claim 1 is characterized in that, described EPO last about 50% or more N-connect the oligosaccharide structure type and contain terminal N-acetyl-glucosamine.

14. the N-connection oligosaccharides that a composition that comprises glycosylation EPO, wherein said EPO are gone up greater than about 80% does not contain Fucose.

15. composition as claimed in claim 14 is characterized in that, the N-connection oligosaccharides that described EPO goes up greater than about 90% does not contain Fucose.

16. composition as claimed in claim 14 is characterized in that, described EPO is available from the genetically modified transgenic avian that contains the described EPO that encodes.

17. composition as claimed in claim 14 is characterized in that, described EPO is available from the genetically modified transgenic chicken that contains the described EPO that encodes.

18. composition as claimed in claim 14 is characterized in that, described EPO produces in the bird oviduct cell.

19. composition as claimed in claim 14 is characterized in that, described EPO appears in the hard shelled egg.

20. composition as claimed in claim 14 is characterized in that, described EPO is people EPO.

21. a composition that comprises isolating EPO molecule, wherein said EPO are to produce in the transgenic chicken oviduct cell, and are separated by the egg white of transgenic chicken, described transgenic chicken contains the transgenosis of the described EPO that encodes.

22. composition as claimed in claim 21 is characterized in that, described EPO is people EPO.

23. composition as claimed in claim 21 is characterized in that, described oviduct cell is a tubular gland cell.

24. composition as claimed in claim 21 is characterized in that, described EPO is included in the hard shelled egg.

25. composition as claimed in claim 21 is characterized in that, described EPO is that N-is glycosylated.

26. composition as claimed in claim 21 is characterized in that, described EPO is that O-is glycosylated.

27. composition as claimed in claim 21 is characterized in that, described composition is a pharmaceutical preparation.

28. composition as claimed in claim 21 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

29. a composition that comprises isolating EPO, described EPO have chicken deutero-glycosylation pattern.

30. composition as claimed in claim 29 is characterized in that, described EPO is people EPO.

31. composition as claimed in claim 29 is characterized in that, described EPO produces in oviduct cell.

32. composition as claimed in claim 29 is characterized in that, described EPO is included in the hard shelled egg.

33. composition as claimed in claim 29 is characterized in that, described EPO is glycosylated in the chicken salpingo cell.

34. composition as claimed in claim 29 is characterized in that, described oviduct cell is a tubular gland cell.

35. composition as claimed in claim 29 is characterized in that, described composition is a pharmaceutical preparation.

36. composition as claimed in claim 29 is characterized in that, described glycosylation pattern is different from the glycosylation pattern of the EPO that produces in Chinese hamster ovary celI and the people's cell.

37. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu Sialic acid

Seminose.

38. separating mixture as claimed in claim 37 is characterized in that, described EPO is available from hard shelled egg.

39. separating mixture as claimed in claim 37 is characterized in that, described EPO is that N-is glycosylated.

40. separating mixture as claimed in claim 37 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

41. separating mixture as claimed in claim 37 is characterized in that, described EPO is in pharmaceutical preparation.

42. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

Seminose.

43. separating mixture as claimed in claim 42 is characterized in that, described EPO is present in the hard shelled egg.

44. separating mixture as claimed in claim 42 is characterized in that, described EPO is that N-is glycosylated.

45. separating mixture as claimed in claim 42 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

46. separating mixture as claimed in claim 42 is characterized in that, described EPO is in pharmaceutical preparation.

47. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

Seminose.

48. separating mixture as claimed in claim 47 is characterized in that, described EPO is present in the hard shelled egg.

49. separating mixture as claimed in claim 47 is characterized in that, described EPO is that N-is glycosylated.

50. separating mixture as claimed in claim 47 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

51. separating mixture as claimed in claim 47 is characterized in that, described EPO is in pharmaceutical preparation.

52. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

Seminose.

53. separating mixture as claimed in claim 52 is characterized in that, described EPO is present in the hard shelled egg.

54. separating mixture as claimed in claim 52 is characterized in that, described EPO is that N-is glycosylated.

55. separating mixture as claimed in claim 52 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

56. separating mixture as claimed in claim 52 is characterized in that, described EPO is in pharmaceutical preparation.

57. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu Sialic acid

Seminose.

58. separating mixture as claimed in claim 57 is characterized in that, described EPO is present in the hard shelled egg.

59. separating mixture as claimed in claim 57 is characterized in that, described EPO is that N-is glycosylated.

60. separating mixture as claimed in claim 57 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

61. separating mixture as claimed in claim 57 is characterized in that, described EPO is in pharmaceutical preparation.

62. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal NacGlu

Sialic acid

Seminose.

63. separating mixture as claimed in claim 62 is characterized in that, described EPO is present in the hard shelled egg.

64. separating mixture as claimed in claim 62 is characterized in that, described EPO is that N-is glycosylated.

65. separating mixture as claimed in claim 62 is characterized in that, described EPO has aminoacid sequence SEQ ID NO:50.

66. separating mixture as claimed in claim 62 is characterized in that, described EPO is in pharmaceutical preparation.

67. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal NacGlu

Sialic acid

Seminose.

68., it is characterized in that described EPO is present in the hard shelled egg as the described separating mixture of claim 67.

69., it is characterized in that described EPO is that N-is glycosylated as the described separating mixture of claim 67.

70., it is characterized in that described EPO has aminoacid sequence SEQ ID NO:50 as the described separating mixture of claim 67.

71., it is characterized in that described EPO is in pharmaceutical preparation as the described separating mixture of claim 67.

72. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid Seminose.

73., it is characterized in that described EPO is present in the hard shelled egg as the described separating mixture of claim 72.

74., it is characterized in that described EPO is that N-is glycosylated as the described separating mixture of claim 72.

75., it is characterized in that described EPO has aminoacid sequence SEQ ID NO:50 as the described separating mixture of claim 72.

76., it is characterized in that described EPO is in pharmaceutical preparation as the described separating mixture of claim 72.

77. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

Seminose.

78., it is characterized in that described EPO is present in the hard shelled egg as the described separating mixture of claim 77.

79., it is characterized in that described EPO is that N-is glycosylated as the described separating mixture of claim 77.

80., it is characterized in that described EPO has aminoacid sequence SEQ ID NO:50 as the described separating mixture of claim 77.

81., it is characterized in that described EPO is in pharmaceutical preparation as the described separating mixture of claim 77.

82. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

Seminose.

83., it is characterized in that described EPO is present in the hard shelled egg as the described separating mixture of claim 82.

84., it is characterized in that described EPO is that N-is glycosylated as the described separating mixture of claim 82.

85., it is characterized in that described EPO has aminoacid sequence SEQ ID NO:50 as the described separating mixture of claim 82.

86., it is characterized in that described EPO is in pharmaceutical preparation as the described separating mixture of claim 82.

87. the separating mixture of an EPO molecule, it comprises with the glycosylated EPO molecule of following structure:

Gal

NacGlu

Sialic acid

NacGal.

88., it is characterized in that described EPO is present in the hard shelled egg as the described separating mixture of claim 87.

89., it is characterized in that described EPO is that O-is glycosylated as the described separating mixture of claim 87.

90., it is characterized in that described EPO has aminoacid sequence SEQ ID NO:50 as the described separating mixture of claim 87.

91., it is characterized in that described EPO is in pharmaceutical preparation as the described separating mixture of claim 87.

92. a composition that comprises the EPO with glycosylation pattern, wherein said EPO produces in the bird oviduct cell.

93. a composition that comprises the EPO with glycosylation pattern, wherein said glycosylation pattern are different from the glycosylation pattern of the EPO of people's cell or Chinese hamster ovary celI generation, and described EPO produces in the oviduct cell of chicken.

94., it is characterized in that described EPO is isolating as the described composition of claim 93.

95., it is characterized in that described oviduct cell is a tubular gland cell as the described composition of claim 93.

96. a method for the treatment of the patient, described method comprise the EPO available from transgenic avian that gives the patient treatment significant quantity.

97., it is characterized in that described treatment significant quantity is patient's red blood cell count(RBC) to be improved the consumption of required numerical value as the described method of claim 96.

98. composition that contains separative glycosylated human protein molecule, described human protein molecule produces in the transgenic chicken uterine tube, described transgenic chicken contains the transgenosis of this human protein molecule of encoding, and described protein molecule contains the chicken deutero-oligosaccharides that can not appear at usually on this human protein.

99. composition that contains separative protein molecule, described protein molecule produces in the transgenic chicken uterine tube, described transgenic chicken contains the transgenosis of this protein molecule of encoding, described protein molecule contains chicken deutero-oligosaccharides and is selected from down group: EPO, G-CSF, GM-CSF, interferon beta, fusion rotein, the CTLA4-Fc fusion rotein, tethelin, cytokine, works, Interferon, rabbit, N,O-Diacetylmuramidase, beta-casein, albumin, α-1 antitrypsin, Antithrombin III, collagen, Factor IX, factors IX, factor X (etc.), fibrinogen, lactoferrin, PROTEIN C, tissue plasminogen activator (tPA), tethelin and Chymotrypsin, immunoglobulin (Ig), antibody, immunotoxin, Factor IX, the Factor IX of b-structural domain disappearance, factor VIIa, factors IX, antithrombotics; R-hirudin, alteplase, tpa, reteplase, tpa, disappearance is 3 in tpa-5 the structural domain, Regular Insulin, Insulin lispro, aspartame's Regular Insulin, Lantus, Recent Development of Long-acting Insulin Analogs, glucagon, tsh, follitropin-β, fsh, pdgh, inf-β 1b, ifn-α 1, ifn-α, ifn-α 1a, ifn-α 1b, ifn-α 2, ifn-α 2a, ifn-α 2b, ifn-β 1a, ifn-γ 1b, il-2, il-11, hbsag, ospa, dornase alfa-DNA enzyme, the β glucocerebrosidase, tnf-α, il-2-diphtheria toxin fusion rotein, tnfr-lgg fragment fusion rotein, the La Luoni enzyme, the DNA enzyme, Ah method's Saite, tositumomab, the mouse monoclonal antibody, Ah coming organizes monoclonal antibody, rasburicase, Ah add'sing carbohydrase β, teriparatide, parathyroid hormone derivative, adalimumab (lgg1), Ah that is plain from stagnating, the bio-modification agent, Nesiritide, people β-type natriuretic peptide (hbnp), G CFS, pegvisomant, the growth hormone receptor antagonist, the albumen c of recombination activation, the Ao Mazuo monoclonal antibody, ige (lge) blocking-up thing, ibritumomab tiuxetan, ACTH, glucagon, Somatostatin, tethelin, thymosin, Rat parathyroid hormone 1-34, pigment hormone, somatomedin, lutropin, chorionic gona dotropin, hypothalamic releasing factor, etanercept, antidiuretic hormone, prolactin antagonist and thyrotropic hormone, immunoglobulin polypeptides, immunoglobulin polypeptides D district, immunoglobulin polypeptides J district, immunoglobulin polypeptides C district, light chain immunoglobulin, heavy chain immunoglobulin, immunoglobulin heavy chain variable region, immunoglobulin light chain variable region and joint peptide.

100., it is characterized in that described composition is in pharmaceutical preparation as the described composition of claim 99.

101., it is characterized in that described protein is that N-is glycosylated as the described composition of claim 99.

102., it is characterized in that described protein is that O-is glycosylated as the described composition of claim 99.

Produce the genetically modified transgenic avian contain the erythropoietin of encoding 103. a method for preparing glycosylated erythropoietin, described method comprise, wherein said erythropoietin is packaged in the hard shelled egg that described bird produces.

104. the egg that bird produced, it contains EPO or G-CSF.

105. the egg that bird produced, it contains the described protein of claim 99.

106. an egg white, every milliliter of exogenous protein that contains greater than 0.5 microgram.