CN1224467A - Process for producing maturation bone morphogenetic protein - Google Patents

Abstract

A process for producing a maturation bone morphogenetic protein by the action of a processing enzyme on a bone morphogenetic protein precursor, which comprises either introducing an expression vector of a bone morphogenetic protein precursor and an expression vector of a processing enzyme into an aminol cell strain, culturing the resultant strain to yield a maturation bone morphogenetic protein, and separating the same from the culture, or alternatively adding a solution of a processing enzyme to a solution of a bone morphogenetic protein precursor and incubating the obtained solution mixture. A suitable process comprises introducing an expression vector of a human MP52 precursor and an expression vector of a secretory furin variant into an established animal cell strain, culturing the resultant strain to yield a maturation bone morphogenetic protein, and separating the same from the culture.

Description

Produce the method for maturation bone morphogenetic protein

Technical field

The present invention relates to produce a kind of method of maturation bone morphogenetic protein.More specifically, the present invention relates to produce the method for ripe Delicious peptide, comprise making processive enzyme act on the Delicious peptide precursor.

Background of invention

Exist the Delicious peptide factor to find (Science, 150, pp.893-899,1965) and be named as " Delicious peptide " (" BMP " after this slightly) in the ground substance of bone by Urist et al..In recent years, the gene that many BMP are relevant is cloned and known transforming growth factor-beta (" TGF-β " after this slightly) superfamily that all belongs to.Produce recombinant protein by some of them.They are used for confirming that the bone form takes place active and expectation is applied to the treatment of bone disease.

Think that from gene structure the albumen of the above-mentioned TGF-of belonging to beta superfamily synthesizes precursor in vivo,, form adult form (active form) the peptide dimer that isozygotys again through various processing.Known person adult form TGF-β 1 holds the dimer (Nature, 316,701-705,1985) of 112 residue peptide for COOH.In fact, in the known mammalian cell, introduce the coding precursor cDNA and produce BMP-2 or BMP-6/Vgr-1, except that with the effect of mature peptide dimer, also with culture supernatant in many all kinds of peptide dimers (by the macromolecule monomer form isozygoty dimer and by a macromolecule monomer and the heterozygosis dimer that the adult form monomer is formed) act on, its molecular weight is greater than adult form (Growth Factors 7, pp.139-150,1992; J.Biol.Chem.126, pp.1595-1609,1994).These molecular weight are considered to corresponding to the molecule from precursor to the adult form course of processing greater than the peptide dimer of adult form.After this molecular weight is called " precursor dimer " greater than the peptide dimer of adult form.Technical being difficult to selected the peptide dimer of production certain molecular weight, on a large scale as the adult form dimer from above-mentioned all kinds of dimers; Or effectively separate the adult form dimer.Therefore this technical barrier is captured in strong request.

Disclosure of the Invention

The objective of the invention is to provide a kind of effective means, in the precursor dimer mixture of mammalian cell Delicious peptide different molecular weight, optionally produces the adult form dimer of same molecular weight on a large scale.

In recent years, one group like Kex-2 proteolytic enzyme, furin, and PC-2, PC-3, PACE4, PC6 and analogue are accredited as the processive enzyme (Biochem.J.299, PP.1-18,1994) of higher animal amyloid protein precursor.Wherein, furin COOH end tool hydrophobic transmembrane territory and part are positioned at golgi's membrane.The cDNA sequence of coding people furin is by van den Ouweland et al. report (Nucleic Acids Res.18, pp.664,1990).Disclosed furin identification Arg-X ₁-X ₂-Arg (X wherein ₁And X ₂Represent any amino acid, but mainly be Lys or Arg) as 4 amino acid whose sequences in its cracking site upstream (J.Biol.Chem.266, pp.12127-12130,1991).Further, reported and only expressed NH in the mammalian cell ₂When the end territory did not have the not woods albumen of membrane-spanning domain, it was secreted the extracellular, and single-minded lytic activity in aminoacid sequence both just still kept (J.Biol.Chem.269, pp.25830-25837,1994) at the secretion position.

The inventor uses above-mentioned furin for the first time successfully based on recombinant DNA technology, and in mammalian cell, effectively production has only the adult form dimer and do not have the precursor dimer on a large scale.

According to the present invention, can produce maturation bone morphogenetic protein by making processive enzyme act on the Delicious peptide precursor.

According to the present invention, in the mammalian cell strain, introduce Delicious peptide precursor expression carrier and processive enzyme expression vector, make processive enzyme act on the Delicious peptide precursor.Culturing cell system separates obtaining maturation bone morphogenetic protein then from culture supernatant.

According to the present invention, also can in the culture supernatant that contains the Delicious peptide precursor, add the culture supernatant that contains processive enzyme, make processive enzyme act on the Delicious peptide precursor.The insulation product mixtures is produced maturation bone morphogenetic protein.

Preferably, introduce people MP52 precursor expression carrier and secretion furin mutant expression vector in mammal cell line, culturing cell system separates the maturation bone morphogenetic protein of so producing then from culture supernatant.

Term used herein " maturation bone morphogenetic protein " means basically by coming from one section Delicious peptide that aminoacid sequence is formed that active form people TGF-β 1 COOH holds 112 amino-acid residues together.Think that the Delicious peptide activity is positioned at homology in the aminoacid sequence district that active form people TGF-β 1 COOH holds 112 amino-acid residues, expects that Delicious peptide does not contain other aminoacid sequence districts.

The embodiment of available processive enzyme comprises one group like Kex2 proteolytic enzyme, furin, PC-2, PC-3, PACE4 and PC6 among the present invention.Wherein, furin is particularly preferred.Because the cDNA of coding furin, the DNA base sequence of the whole aminoacid sequences of available code furin, but the DNA base sequence of a part of aminoacid sequence of preferably encoding (after this being called " secretor type furin mutant ") does not wherein comprise membrane-spanning domain and keeps single-minded lytic activity in aminoacid sequence in addition.

In the preferred embodiment of the invention, use the synthetic dna primer, by the RT-PCR method, from total RNA of human cell line HepG2 (ATCC HB8065), cloned the dna fragmentation that contains secretion furin mutants cDNA from extracting.Secretion furin mutants cDNA as its DNA base sequence, has the 163rd to the 2014th Nucleotide, is disclosed in SEQ IDNo:1 in the sequence table.This dna fragmentation inserts in the expression vector, makes up the expression vector of secretion furin mutant thus.The expression vector of usefulness contains the eukaryotic promoter downstream herein, the poly of interpolation (A) signal, and this is expressing gene clone's a restriction enzyme sites.This expression vector can further contain anti-medicine mark and be beneficial to screen transformant.The embodiment of this expression vector comprises human cytomegalic inclusion disease virus promotor enhanser, and many [site] joint, Polisac poly (A) add signal and contain the pRc/CMV (from INVITROGEN company) of neomycin resistance mark.

The present invention can be used for producing a kind of Delicious peptide of people TGF-beta superfamily, more specifically, the maturation bone morphogenetic protein of forming by the monomer of same molecular weight, as MP52, BMP-2, BMP-4, BMP-6 or BMP-7).As Delicious peptide, be disclosed in PCT application WO93/16099 or WO95/04819 and have the bone form active people MP52 to take place especially by preferably.In preferred embodiments, the cDNA of coding people MP52 precursor is inserted the expression vector that expression vector makes up people MP52 precursor, introduce mammalian cell subsequently, precursor and the dimeric mammal cell line of ripe MP52 are produced in preparation thus.Be suitable for comprising Chinese hamster ovary (CHO) cell as the mammalian cell embodiment of host cell, bhk cell, 293 cells and mouse L cell.Wherein, Chinese hamster ovary celI is preferred.At the product people MP52 mammal cell line that so obtains (MC-2: preserving number FERM BP-5142), introduce the expression vector of secretion furin mutant, come two kinds of albumen of coexpression, in culture supernatant, only produce ripe MP52 dimer thus.

The solution and the solution that contains precursor that also can contain processive enzyme by mixing, and with in 32 to 40 ℃ in mixture, preferred 37 ℃, incubated overnight makes amyloid protein precursor change adult form into.

The inventive method gained maturation bone morphogenetic protein can be by adding required pharmaceutically acceptable carrier, and additive, thinner and/or vehicle are used for the treatment of or prevent bone, and cartilage or injury of teeth or artificial tooth are taken root in.

For the osteopathia that the treatment dysostosis causes, can pass through traditional way systemic administration maturation bone morphogenetic protein, as injection, intravenous injection, intramuscularly, or peritoneal injection, Orally administered or parenteral administration such as suppository.

Be the treatment fracture, can be by injection, Orally administered or parenteral administration whole body or topical application adult form morphogenetic proteins.Also can near fracture region, implant the matrix that contains the adult form morphogenetic proteins.Suitable matrix embodiment comprises the multipolymer of natural polymer such as collagen protein or fibrin sealant and synthetic polymer such as poly(lactic acid) and oxyacetic acid.

Rebuild in strong type, bone is transplanted or when planting artificial tooth, maturation bone morphogenetic protein can be used for bone to be implanted or tooth surface, coated with collagen protein cream, and fibrin sealant or other tackiness agents.When bone was transplanted, it can be used for natural and artificial bone.

Determine the dosage of maturation bone morphogenetic protein according to purpose and application process.Usually, systemic administration dosage is per kilogram 1 to 100 μ g, the every 30 μ g to 30mg of place of topical application.

The accompanying drawing summary

Fig. 1 behaves and secretes furin mutant expression vector pDfurpRC/CMV (7.2kb) plasmid figure.

Fig. 2 MP52 expression vector pMSS99 (5.0kb) plasmid figure that behaves.

Fig. 3 is the Western engram analysis photo under the reductive condition, shows that coexpression adult form people MP52 dimer and people secrete the serum-free culture supernatant of furin mutant cells system.

Fig. 4 is the Western engram analysis photo under the reductive condition, shows that secreting the furin mutant by the people acts on dimer and make various precursor people MP52 dimers be converted into adult form MP52.

The preferred embodiment explanation

Advantage of the present invention will be in after this being described more specifically by embodiment.Embodiment 1

Chinese hamster ovary celI system by coexpression human secretion furin mutant and people MP52 produces adult form people's MP52 dimer (1) human cloning secretor type furin mutants cDNA and makes up its expression vector

People's furin protein structure is from NH ₂The end structure territory is followed successively by signal peptide, like proteolytic enzyme structural domain, membrane spaning domain and the tenuigenin side structure territory of subtilisin.Among the present invention, by RT-PCR method clones coding NH ₂End protein enzyme territory and the people that do not have a membrane spaning domain secretes the furin mutants cDNA, and for expressing.

From human HepG2 cell's extracted total RNA, and as template by the rTth RNA polymerase, upstream antisense primer 1 (No. the 931st to the 914th, people's furin cDNA sequence is disclosed in SEQ ID No:1 in the sequence table) and downstream antisense primer 2 (the 2095th to No. 2071) are used for reverse transcription.These products are united respectively adopted primer 3 (sequence table SEQ ID No:2) and antisense primer 4 (SEQ ID No:3 in the sequence table), there are adopted primer 5 (SEQ ID No:4 in the sequence table) and antisense primer 6 (SEQ ID No:5 in the sequence table) to be used for the PCR reaction, obtain two cDNA fragments of upstream and downstream thus.Hind III-Sal I the site that connects these fragments and insert plasmid pUC119 (from TakaraShuzo company limited), 595 the amino acid whose people that obtain thus encoding secrete the furin mutants cDNA.Product cDNA measures checking by restriction enzyme digestion and DNA base sequence.The finder secretes the furin mutant and has DNA base sequence as sequence table SEQ ID No:1 the 163rd to the 2014th Nucleotide.Different being of the dna sequence dna that the people secretes the furin mutants cDNA and van den Ouweland et al. report, deleted may be to translating unfavorable and nonessential atg start codon thereby No. 165 base is VITAMIN B4; No. 2004 base is thereby that VITAMIN B4 forms terminator codon.Then, downcut the people with Hind III-Xba I digestion and secrete the furin mutants cDNA, insert Hind III-Xba I site subsequently, prepare the expression vector that the people secretes the furin mutants cDNA thus available from the pRc/CMV carrier of INVITROGEN company, pDfurpRC/CMV is shown in Fig. 1.(2) make up people MP52 expression vector

From the pSK52s plasmid that contains people MP52 gene that doctor Hoetten of Biopharm GmbH provides, separate the dna fragmentation that contains people MP52 gene with the digestion of Hind III, insert the Hind III site of the pABstop carrier that doctor Zettlmeissl of Behringwerke AG provides subsequently, prepare people MP52 expression vector pMSS99 thus, be shown in Fig. 2.Its structure is measured by the DNA base sequence and restriction enzyme digestion checking.The people MP52 DNA base sequence that found that pMSS99 is the 576th to the 2279th Nucleotide of SEQ ID No:6 in the sequence table.(3) set up MC2, promptly produce the various dimeric Chinese hamster ovaries of precursor people MP52 (CHO) clone

At the CHO-DUKX-Bll cell that doctor Zettlmeissl of Behringwerke AG provides, promptly in the Chinese hamster ovary celI mutantion line, introduce pMSS99 and the pSVOAdhfr that doctor Zettlmeissl provides with calcium phosphate DNA cosedimentation method.Then, set up people MP52 high yield clone with methotrexate (MTX) gene amplification method.

PMSS99 (10 μ g) and pSVOAdhfr (2 μ g) are dissolved in 1ml 25mM HEPES, 140mM NaCl and 0.75mM Na ₂HPO ₄(pH7.05), back and 50 μ l 2.5M CaCl ₂Mix.Throw out places on the interior CHO-DUKX-Bll cell of 10cm ware, room temperature 30 minutes.On cellular layer, add MEM-ALPHA (the MEM-α of 8ml qiagen rnase and thymus nucleic acid and 10% foetal calf serum ⁺) substratum, the gained mixture is in CO ₂Cultivated 4 to 6 hours in the insulation can.After 3 minutes, cell cultures is in the MEM-α that contains 10%FBS with 10% glycerin treatment for room temperature ⁺Substratum.And then culturing cell is inoculated in no Yeast Nucleic Acid and thymus nucleic acid, contain MEM-ALPHA (the MEM-α of 10% dialysis FBS ^-) substratum and screen transformant.People MP52 produces by the Western engram analysis and detects, and is set forth in down (5).

In the substratum that produces people MP52 clone, add MTX.By progressively increasing MTX concentration, the clone of screening amplification MP52 gene.When MTX concentration is 400nM, obtain producing the precursor people MP52 dimer and the dimeric clone MC-2 of adult form people MP52 of different molecular weight.Gained clone MC-2 is preserved in industry and science and technology June 21 nineteen ninety-five and economizes life science and human body Institute for Research and Technology (Ibaraki, Japan build east, ripple city 1 fourth order a kind No. 3), preserving number FERM BP-5142.(4) by being that MC-2 introduces the people and secretes furin mutant expression vector and set up the coexpression clone that people MP52 and people secrete the furin mutant to the Chinese hamster ovary celI that produces people MP52

Producing various precursor people MP-52 dimers and the dimeric Chinese hamster ovary celI of adult form MP52 by calcium phosphate DNA cosedimentation normal direction is to introduce the people among the MC-2 to secrete furin mutant expression vector, under 333 μ g/ml G418 and 400nM MTX condition, screen transformant then, set up the coexpression clone that people MP52 and people secrete the furin mutant.

To be similar to the mode of embodiment 1 (2), form calcium phosphate DNA cosedimentation with pDfurpRC/CMV (4.8 μ g).Cover the MC-2 cell in the 10cm ware, room temperature was put 30 minutes.8ml does not have MEM-ALPHA (the MEM-α-) substratum that Yeast Nucleic Acid and thymus nucleic acid contain 10%FBS and is added to cellular layer, and the gained mixture is in CO ₂Insulation can was cultivated 4 to 6 hours.With 10% glycerine room temperature treatment after 3 minutes, cell was in the MEM-α-culture medium culturing that contains 10%FBS 2 days.Culturing cell and then be inoculated in and contain 10%FBS, MEM-α-substratum of 400nM MTX and 333 μ g/ml G418, and screening transforms strain.People MP52 produces by the Western engram analysis and detects, following being set forth in (5).The people secretes the furin mutant and produces by the detection of enzymic activity amount method, following being set forth in (6).Income earner MP52 furin and people secrete one of the serum-free culture supernatant of furin mutant coexpression clone every day and change substratum, and the culture supernatant of collection is used for polyacrylamide gel electrophoresis and continues with the Western engram analysis under reductive condition.The results are shown in Fig. 3 photo.Sample 0.5 μ l culture supernatant on each swimming lane.Swimming lane 1,2 and 3 is respectively 1,2 and 3 day culture supernatant.Adult form MP52 monomer band shows with arrow.Found to detect in the supernatant precursor MP52 less than macromolecule, all MP52 are the peptides of the about 15K of molecular weight, conform to adult form MP52 monomer.Analytical results under the non-reduced condition is that these peptides respectively form the adult form MP52 dimer of the about 28K of molecular weight.The dimerization scale of construction of producing with coexpression clone is about 8 μ g/ml/24 hours, be about with the MP52 express cell be MC-2 three to octuple.Like this, secrete the furin mutant by end user MP52 and people, present inventors successfully produce adult form MP52 dimer on a large scale than traditional method for the first time.(5) with the people MP52 in the Western engram analysis detection culture supernatant

With the albumen in sds polyacrylamide gel electrophoresis (15-25% polyacrylamide gradient gel, Daiichi KagaKu company limited) the separation and Culture supernatant, then transfer protein to pvdf membrane (Clear Blot Membrane-P, ATTO).Film envelope, is spent the night with 10 μ g/ml chicken anti-human MP 52 antibody treatment with Tris salt buffer (TBS) rinsing with Block Ace (DainipponPharmaceutical company limited) a hour again.TBS (TTBS) rinsing of film to contain 0.1% polysorbas20 handled with the anti-chicken IgG of alkaline phosphatase rabbit mixture (Sigma A9171) then.Film manifests the band corresponding to MP52 with the TTBS rinsing and with alkaline phosphatase substrate test kit (BIO-RAD).(6) people secretes furin mutant activity in the detection culture supernatant

20 μ l culture supernatant are with 30 μ l pure water dilution and mixed with 200 μ l fluorogenic substrate solution; To contain 100 μ M Boc-Arg-Arg-Val-Arg-MCA (available from Protein Technology Institute) and 1.25mM CaCl ₂37 ℃ of 125 mM MES/NaOH (pH7.0) insulation 10 minutes.Measure AMC fluorescence in 380nm excitation wavelength and 450nm transmitted wave strong point.Furin activity unit is that the activity that per minute is discharged 1pmol AMC is defined as 1 unit (U).(7) purifying and people secrete the adult form MP52 of furin mutant coexpression production and analyze NH ₂Terminal amino acid sequence

The serum-free culture supernatant of people MP52 and people being secreted furin mutant coexpression clone mixes with the 0.2M sodium phosphate buffer (pH6.0) of 0.1 volume.The gained mixture imposes on the HiTrap SF that crosses with 20mM sodium phosphate buffer (pH6.0) balance that contains 50mM NaCl, and (1ml, Pharmacia) post is then with same damping fluid drip washing.Albumen is to contain 0.1M sodium phosphate buffer (pH6.0) wash-out of 6M Guanidinium hydrochloride.Eluate imposes on reversed-phase HPLC post ResourceRPC, and (3ml, Pharmacia), albumen is with 25-55% acetonitrile wash-out.The isolate that contains adult form MP52 is analyzed NH with pulse liquid gas phase sequenator ₂Terminal amino acid sequence (AppliedBiosystems model 476).The results are shown in table 1.

Table 1 loop amino acid sequence 1 (pmol) amino acid sequence 2 (pmol) 1 Arg 11.95 Ala 25.51 2 Ala 28.75 Pro 14.75 3 Pro 17.55 Leu 18.07 4 Leu 16.47 Ala 14.46 5 Ala 16.99 Thr 5.02 6 Thr 7.15 Arg 7.38 7 Arg 9.21 Gln 9.08 8 Gln 9 54 Gly 13.23 9 Gly 11.29 Lys 5.29 10 Lys 8.04 Arg 6.52

By table 1, speculating acid sequence 1 and aminoacid sequence 2 are respectively from the aminoacid sequence that starts from Arg 354 and Ala 355 of SEQ IDNo:6 in the sequence table, and its mol ratio is about 1: 1.Embodiment 2

Secreting the furin mutant with the people makes people MP52 precursor dimer be converted into the Chinese hamster ovary celI system that adult form dimer (1) foundation production people secretes the furin mutant

In the CHO-DUKX-Bll cell that provides with doctor Zettlmeissl of calcium phosphate DNA cosedimentation normal direction Behringwerke AG, introduce the people who is set forth in embodiment 1 (1) and secrete furin mutant expression vector pDfurpRC/CMV.In the presence of G418, screen transformant, set up the high expression level strain that the people secretes the furin mutant thus.

To be similar to embodiment 1 (3) described method, the co-precipitation of preparation pDfurpRC/CMV and calcium phosphate is overlying in the 10cm ware and placed 30 minutes on the CHO-DUKX-Bll cell and in room temperature.Add MEM-ALPHA (the MEM-α that 8ml contains 10%FBS and Yeast Nucleic Acid and thymus nucleic acid toward cellular layer ⁺) substratum, then in CO ₂Insulation can was cultivated 4 to 6 hours.Cell after 3 minutes, is inoculated in the MEM-α that contains 10%FBS and 400 μ g/ml G418 with 10% glycerine room temperature treatment ⁺Substratum screens transformant thus.The enzymic activity amount method that more than is set forth in embodiment 1 (6) detect to be produced the people and is secreted the clone of furin mutant, and to obtain the furin activity be 500 to 1000 U/ml/24 hours clone.(2) secreting furin mutant conversion people precursor MP52 dimer with the people is adult form

Be that serum-free culture supernatant (containing precursor and adult form MP52 dimer) and the people of MC-2 secretes the serum-free culture supernatant (1000U/ml) that furin mutant express cell is and mix in varing proportions with the Chinese hamster ovary celI that produces people MP52,37 ℃ of incubated overnight then.After the reaction, be set forth in the Western engram analysis of embodiment 1 (5) under reductive condition, carrying out and detect precursor MP52 and be converted into adult form.The results are shown in Fig. 4 photo.At swimming lane 1 to 5, it is culture supernatant that the people of last sample 1 μ l MC-2 culture supernatant and different volumes secretes the furin mutant cells.The final concentration that the people secretes the furin mutant is respectively, 1 swimming lane 0U/ml, 2 swimming lane 50U/ml, 3 swimming lane 100U/ml, 4 swimming lane 200U/ml and 5 swimming lane 400U/ml.Adult form MP52 monomer band shows with arrow A and B, and adult form MP52 monomer band shows with arrow C.Show as Fig. 4, add the culture supernatant of expressing human secretion furin mutant cells system so that the final active concentration that MP52 is converted into adult form fully is 200U/ml at least.As a result, adult form MP52 dimer increment is about 3 times.

Industrial applicability

Osseous maturation type morphogenetic proteins is by the tool different molecular of producing from mammalian cell traditionally Separate obtaining in the bone morphogenetic protein dimer mixture of amount, produce but be difficult to extensive the selection Maturation bone morphogenetic protein or effectively obtain maturation bone morphogenetic protein is because not yet send out Putting on display a kind of technology is effectively separated it from said mixture. Yet, method of the present invention Become possibility so that prepare maturation bone morphogenetic protein by the bone morphogenetic protein precursor, thereby Promote production on a large scale. Further, the maturation bone morphogenetic protein that provides of the inventive method be by High-purity thing that the peptide of substantially the same molecular weight forms is so that it is suitable for use as medicine especially.

Sequence table SEQ ID NO:1 length: 4180 types: amino acid chain number: double-stranded topology: linear molecule type: fragments of peptides type: source: organism: people types of organization: people's liver cancer characteristic: location: other information: processive enzyme furin sequence description: SEQ ID NO:1:GCGGGGAAGC AGCAGCGGCC AGGATGAATC CCAGGTGCTC TGGAGCTGGA TGGTGAAGGT 60 CGGCACTCTT CACCCTCCCG AGCCCTGCCC GTCTCGGCCC CATGCCCCCA CCAGTCAGCC 120 CCGGGCCACA GGCAGTGAGC AGGCACCTGG GAGCCGAGGC CCTAAGACCA GGCCAAGGAG 180 ACGGGCGCTC CAGGGTCCCA GCCACCTGTC CCCCCC ATG GAG CTG AGG CCC TGG 234

                                    Met Glu Leu Arg Pro Trp

                                     1                5  TTG CTA TGG GTG GTA GCA GCA ACA GGA ACC TTG GTC CTG CTA GCA GCT     282 Leu Leu Trp Val Val Ala Ala Thr Gly Thr Leu Val Leu Leu Ala Ala

         10                  15                  20 GAT GCT CAG GGC CAG AAG GTC TTC ACC AAC ACG TGG GCT GTG CGC ATC     330 Asp Ala Gln Gly Gln Lys Val Phe Thr Asn Thr Trp Ala Val Arg Ile

     25                  30                  35 CCT GGA GGC CCA GCG GTG GCC AAC AGT GTG GCA CGG AAG CAT GGG TTC     378 Pro Gly Gly Pro Ala Val Ala Asn Ser Val Ala Arg Lys His Gly Phe

 40                  45                  50 CTC AAC CTG GGC CAG ATC TTC GGG GAC TAT TAC CAC TTC TGG CAT CGA     426 Leu Asn Leu Gly Gln Ile Phe Gly Asp Tyr Tyr His Phe Trp His Arg  55                 60                  65                  70 GGA GTG ACG AAG CGG TCC CTG TCG CCT CAC CGC CCG CGG CAC AGC CGG     474 Gly Val Thr Lys Arg Ser Leu Ser Pro His Arg Pro Arg His Ser Arg

             75                  80                  85 CTG CAG AGG GAG CCT CAA GTA CAG TGG CTG GAA CAG CAG GTG GCA AAG     522 Leu Gln Arg Glu Pro Gln Val Gln Trp Leu Glu Gln Gln Val Ala Lys

         90                  95                 100 CGA CGG ACT AAA CGG GAC GTG TAC CAG GAG CCC ACA GAC CCC AAG TTT     570 Arg Arg Thr Lys Arg Asp Val Tyr Gln Glu Pro Thr Asp Pro Lys Phe

    105                 110                 115 CCT CAG CAG TGG TAC CTG TCT GGT GTC ACT CAG CGG GAC CTG AAT GTG     618 Pro Gln Gln Trp Tyr Leu Ser Gly Val Thr Gln Arg Asp Leu Asn Val

120                 125                 130 AAG GCG GCC TGG GCG CAG GGC TAC ACA GGG CAC GGC ATT GTG GTC TCC     666 Lys Ala Ala Trp Ala Gln Gly Tyr Thr Gly His Gly Ile Val Val Ser 135                 140                 145                 150 ATT CTG GAC GAT GGC ATC GAG AAG AAC CAC CCG GAC TTG GCA GGC AAT     714 Ile Leu Asp Asp Gly Ile Glu Lys Asn His Pro Asp Leu Ala Gly Asn

            155                 160                 165 TAT GAT CCT GGG GCC AGT TTT GAT GTC AAT GAC CAG GAC CCT GAC CCC     762 Tyr Asp Pro Gly Ala Ser Phe Asp Val Asn Asp Gln Asp Pro Asp Pro

        170                 175                 180 CAG CCT CGG TAC ACA CAG ATG AAT GAC AAC AGG CAC GGC ACA CGG TGT     810 Gln Pro Arg Tyr Thr Gln Met Asn Asp Asn Arg His Gly Thr Arg Cys

    185                 190                 195 GCG GGG GAA GTG GCT GCG GTG GCC AAC AAC GGT GTC TGT GGT GTA GGT     858 Ala Gly Glu Val Ala Ala Val Ala Asn Asn Gly Val Cys Gly Val Gly

200                 205                 210 GTG GCC TAC AAC GCC CGC ATT GGA GGG GTG CGC ATG CTG GAT GGC GAG     906 Val Ala Tyr Asn Ala Arg Ile Gly Gly Val Arg Met Leu Asp Gly Glu 215                 220                 225                 230 GTG ACA GAT GCA GTG GAG GCA CGC TCG CTG GGC CTG AAC CCC AAC CAC     954 Val Thr Asp Ala Val Glu Ala Arg Ser Leu Gly Leu Asn Pro Asn His

            235                 240                 245 ATC CAC ATC TAC AGT GCC AGC TGG GGC CCC GAG GAT GAC GGC AAG ACA    1002 Ile His Ile Tyr Ser Ala Ser Trp Gly Pro Glu Asp Asp Gly Lys Thr

        250                 255                 260 GTG GAT GGG CCA GCC CGC CTC GCC GAG GAG GCC TTC TTC CGT GGG GTT    1050 Val Asp Gly Pro Ala Arg Leu Ala Glu Glu Ala Phe Phe Arg Gly Val

265 270 275AGC?CAG?GGC?CGA?GGG?GGG?CTG?GGC?TCC?ATC?TTT?GTC?TGG?GCC?TCG?GGG 1098Ser?Gln?Gly?Arg?Gly?Gly?Leu?Gly?Ser?Ile?Phe?Val?Trp?Ala?Ser?Gly

280 285 290AAC?GGG?GGC?CGG?GAA?CAT?GAC?AGC?TGC?AAC?TGC?GAC?GGC?TAC?ACC?AAC 1146Asn?Gly?Gly?Arg?Glu?His?Asp?Ser?Cys?Asn?Cys?Asp?Gly?Tyr?Thr?Asn295 300 305 310AGT?ATC?TAC?ACG?CTG?TCC?ATC?AGC?AGC?GCC?ACG?CAG?TTT?GGC?AAC?GTG 1194Ser?Ile?Tyr?Thr?Leu?Ser?Ile?Ser?Ser?Ala?Thr?Gln?Phe?Gly?Asn?Val

315 320 325CCG?TGG?TAC?AGC?GAG?GCC?TGC?TCG?TCC?ACA?CTG?GCC?ACG?ACC?TAC?AGC 1242Pro?Trp?Tyr?Ser?Glu?Ala?Cys?Ser?Ser?Thr?Leu?ALa?Thr?Thr?Tyr?Ser

330 335 340AGT?GGC?AAC?CAG?AAT?GAG?AAG?CAG?ATC?GTG?ACG?ACT?GAC?TTG?CGG?CAG 1290Ser?Gly?Asn?Gln?Asn?Glu?Lys?Gln?Ile?Val?Thr?Thr?Asp?Leu?Arg?Gln

345 350 355AAG?TGC?ACG?GAG?TCT?CAC?ACG?GGC?ACC?TCA?GCC?TCT?GCC?CCC?TTA?GCA 1338Lys?Cys?Thr?Glu?Ser?His?Thr?Gly?Thr?Ser?Ala?Ser?Ala?Pro?Leu?Ala

360 365 370GCC?GGC?ATC?ATT?GCT?CTC?ACC?CTG?GAG?GCC?AAT?AAG?AAC?CTC?ACA?TGG 1386Ala?Gly?Ile?Ile?Ala?Leu?Thr?Leu?Glu?Ala?Asn?Lys?Asn?Leu?Thr?Trp375 380 385 390CGG?GAC?ATG?CAA?CAC?CTG?GTG?GTA?CAG?ACC?TCG?AAG?CCA?GCC?CAC?CTC 1434Arg?Asp?Met?Gln?His?Leu?Val?Val?Gln?Thr?Ser?Lys?Pro?Ala?His?Leu

395 400 405AAT?GCC?AAC?GAC?TGG?GCC?ACC?AAT?GGT?GTG?GGC?CGG?AAA?GTG?AGC?CAC 1482Asn?Ala?Asn?Asp?Trp?Ala?Thr?Asn?Gly?Val?Gly?Arg?Lys?Val?Ser?His

410 415 420TCA?TAT?GGC?TAC?GGG?CTT?TTG?GAC?GCA?GGC?GCC?ATG?GTG?GCC?CTG?GCC 1530Ser?Tyr?Gly?Tyr?Gly?Leu?Leu?Asp?Ala?Gly?Ala?Met?Val?Ala?Leu?Ala

425 430 435CAG?AAT?TGG?ACC?ACA?GTG?GCC?CCC?CAG?CGG?AAG?TGC?ATC?ATC?GAC?ATC 1578Gln?Asn?Trp?Thr?Thr?Val?Ala?Pro?Gln?Arg?Lys?Cys?Ile?Ile?Asp?Ile

440 445 450CTC?ACC?GAG?CCC?AAA?GAC?ATC?GGG?AAA?CGG?CTC?GAG?GTG?CGG?AAG?ACC 1626Leu?Thr?Glu?Pro?Lys?Asp?Ile?Gly?Lys?Arg?Leu?Glu?Val?Arg?Lys?Thr455 460 465 470GTG?ACC?GCG?TGC?CTG?GGC?GAG?CCC?AAC?CAC?ATC?ACT?CGG?CTG?GAG?CAC 1674Val?Thr?Ala?Cys?Leu?Gly?Glu?Pro?Asn?His?Ile?Thr?Arg?Leu?Glu?His

475 480 485GCT?CAG?GCG?CGG?CTC?ACC?CTG?TCC?TAT?AAT?CGC?CGT?GGC?GAC?CTG?GCC 1722Ala?Gln?Ala?Arg?Leu?Thr?Leu?Ser?Tyr?Asn?Arg?Arg?Gly?Asp?Leu?Ala

490 495 500ATC?CAC?CTG?GTC?AGC?CCC?ATG?GGC?ACC?CGC?TCC?ACC?CTG?CTG?GCA?GCC 1770Ile?His?Leu?Val?Ser?Pro?Met?Gly?Thr?Arg?Ser?Thr?Leu?Leu?Ala?Ala

505 510 515AGG?CCA?CAT?GAC?TAC?TCC?GCA?GAT?GGG?TTT?AAT?GAC?TGG?GCC?TTC?ATG 1818Arg?Pro?His?Asp?Tyr?Ser?Ala?Asp?Gly?Phe?Asn?Asp?Trp?Ala?Phe?Met

520 525 530ACA?ACT?CAT?TCC?TGG?GAT?GAG?GAT?CCC?TCT?GGC?GAG?TGG?GTC?CTA?GAG 1866Thr?Thr?His?Ser?Trp?Asp?Glu?Asp?Pro?Ser?Gly?Glu?Trp?Val?Leu?Glu535 540 545 550ATT?GAA?AAC?ACC?AGC?GAA?GCC?AAC?AAC?TAT?GGG?ACG?CTG?ACC?AAG?TTC 1914Ile?Glu?Asn?Thr?Ser?Glu?Ala?Asn?Asn?Tyr?Gly?Thr?Leu?Thr?Lys?Phe

555 560 565ACC?CTC?GTA?CTC?TAT?GGC?ACC?GCC?CCT?GAG?GGG?CTG?CCC?GTA?CCT?CCA 1962Thr?Leu?Val?Leu?Tyr?Gly?Thr?Ala?Pro?Glu?Gly?Geu?Pro?Val?Pro?Pro

570 575 580GAA?AGC?AGT?GGC?TGC?AAG?ACC?CTC?ACG?TCC?AGT?CAG?GCC?TGA 2004Glu?Ser?Ser?Gly?Cys?Lys?Thr?Leu?Thr?Ser?Ser?Gln?Ala?***

585 590 595GTGGTGTGCG AGGAAGGCTT CTCCCTGCAC CAGAAGAGCT GTGTCCAGCA CTGCCCTCCA 2064GGCTTCGCCC CCCAAGTCCT CGATACGCAC TATAGCACCG AGAATGACGT GGAGACCATC 2124CGGGCCAGCG TCTGCGCCCC CTGCCACGCC TCATGTGCCA CATGCCAGGG GCCGGCCCTG 2184ACAGACTGCC TCAGCTGCCC CAGCCACGCC TCCTTGGACC CTGTGGAGCA GACTTGCTCC 2244CGGCAAAGCC AGAGCAGCCG AGAGTCCCCG CCACAGCAGC AGCCACCTCG GCTGCCCCCG 2304GAGGTGGAGG CGGGGCAACG GCTGCGGGCA GGGCTGCTGC CCTCACACCT GCCTGAGGTG 2364GTGGCCGGCC TCAGCTGCGC CTTCATCGTG CTGGTCTTCG TCACTGTCTT CCTGGTCCTG 2424CAGCTGCGCT CTGGCTTTAG TTTTCGGGGG GTGAAGGTGT ACACCATGGA CCGTGGCCTC 2484ATCTCCTACA AGGGGCTGCC CCCTGAAGCC TGGCAGGAGG AGTGCCCGTC TGACTCAGAA 2544GAGGACGAGG GCCGGGGCGA GAGGACCGCC TTTATCAAAG ACCAGAGCGC CCTCTGATGA 2604GCCCACTGCC CACCCCCTCA AGCCAATCCC CTCCTTGGGC ACTTTTTAAT TCACCAAAGT 2664ATTTTTTTAT CTTGGGACTG GGTTTGGACC CCAGCTGGGA GGCAAGAGGG GTGGAGACTG 2724TTTCCCATCC TACCCTCGGG CCCACCTGGC CACCTGAGGT GGGCCCAGGA CCAGCTGGGG 2784CGTGGGGAGG GCCGTACCCC ACCCTCAGCA CCCCTTCCAT GTGGAGAAAG GAGTGAAACC 2844TTTAGGGCAG CTTGCCCCGG CCCCGGCCCC AGCCAGAGTT CCTGCGGAGT GAAGAGGGGC 2904AGCCCTTGCT TGTTGGGATT CCTGACCCAG GCCGCAGCTC TTGCCCTTCC CTGTCCCTCT 2964AAAGCAATAA TGGTCCCATC CAGGCAGTCG GGGGCTGGCC TAGGAGATAT CTGAGGGAGG 3024AGGCCACCTC TCCAAGGGCT TCTGCACCCT CCACCCTGTC CCCCAGCTCT GGTGAGTCTT 3084GGCGGCAGCA GCCATCATAG GAAGGGACCA AGGCAAGGCA GGTGCCTCCA GGTGTGCACG 3144 TGGCATGTGG CCTGTGGCCT GTGTCCCATG ACCCACCCCT GTGCTCCGTG CCTCCACCAC 3204 CACTGGCCAC CAGGCTGGCG CAGCCAAGGC CGAAGCTCTG GCTGAACCCT GTGCTGGTGT 3264 CCTGACCACC CTCCCCTCTC TTGCACCCGC CTCTCCCGTC AGGGCCCAAG TCCCTGTTTT 3324 CTGAGCCCGG GCTGCCTGGG CTGTTGGCAC TCACAGACCT GGAGCCCCTG GGTGGGTGGT 3384 GGGGAGGGGC GCTGGCCCAG CCGGCCTCTC TGGCCTCCCA CCCGATGCTG CTTTCCCCTG 3444 TGGGGATCTC AGGGGCTGTT TGAGGATATA TTTTCACTTT GTGATTATTT CACTTTAGAT 3504 GCTGATGATT TGTTTTTGTA TTTTTAATGG GGGTAGCAGC TGGACTACCC ACGTTCTCAC 3564 ACCCACCGTC CGCCCTGCTC CTCCCTGGCT GCCCTGGCCC TGAGGTGTGG GGGCTGCAGC 3624 ATGTTGCTGA GGAGTGAGGA ATAGTTGAGC CCCAAGTCCT GAAGAGGCGG GCCAGCCAGG 3684 CGGGCTCAAG GAAAGGGGGT CCCAGTGGGA GGGGCAGGCT GACATCTGTG TTTCAAGTGG 3744 GGCTCGCCAT GCCGGGGGTT CATAGGTCAC TGGCTCTCCA AGTGCCAGAG GTGGGCAGGT 3804 GGTGGCACTG AGCCCCCCCA ACACTGTGCC CTGGTGGAGA AAGCACTGAC CTGTCATGCC 3864 CCCCTCAAAC CTCCTCTTCT GACGTGCCTT TTGCACCCCT CCCATTAGGA CAATCAGTCC 3924 CCTCCCATCT GGGAGTCCCC TTTTCTTTTC TACCCTAGCC ATTCCTGGTA CCCAGCCATC 3984 TGCCCAGGGG TGCCCCCTCC TCTCCCATCC CCCTGCCCTC GTGGCCAGCC CGGCTGGTTT 4044 TGTAAGATAC TGGGTTGGTG CACAGTGATT TTTTTCTTGT AATTTAAACA GGCCCAGCAT 4104 TGCTGGTTCT ATTTAATGGA CATGAGATAA TGTTAGAGGT TTTAAAGTGA TTAAACGTGC 4164 AGACTATGCA AACCAG 4180SEQ IN NO:2：29：：：：： ： ：：3：SEQ ID NO:2:GCGAAGCTTA AGACCAGGCC AAGGAGACG 29SEQ IN NO:3：26：：：：： ： ：：4：SEQ ID NO:3:CCTCGCCATC CAGCATGCGC ACCCCT 26SEQ IN NO:4：26：：：：： ： ：：5：SEQ ID NO:4:TGGAGGGGTG CGCATGCTGG ATGGCG 26SEQ IN NO:5：30：：：：： ： ：：6：SEQ ID NO:5:GGCGTCGACG CACACCACTC AGGCCTGACT 30SEQ IN NO:6：2703：：：：：： ： ：： ： ：MP52：SEQ ID NO:6:CCATGGCCTC GAAAGGGCAG CGGTGATTTT TTTCACATAA ATATATCGCA CTTAAATGAG 60TTTAGACAGC ATGACATCAG AGAGTAATTA AATTGGTTTG GGTTGGAATT CCGTTTCCAA 120TTCCTGAGTT CAGGTTTGTA AAAGATTTTT CTGAGCACCT GCAGGCCTGT GAGTGTGTGT 180GTGTGTGTGT GTGTGTGTGT GTGTGTGTGA AGTATTTTCA CTGGAAAGGA TTCAAAACTA 240GGGGGAAAAA AAAACTGGAG CACACAGGCA GCATTACGCC ATTCTTCCTT CTTGGAAAAA 300TCCCTCAGCC TTATACAAGC CTCCTTCAAG CCCTCAGTCA GTTGTGCAGG AGAAAGGGGG 360CGGTTGGCTT TCTCCTTTCA AGAACGAGTT ATTTTCAGCT GCTGACTGGA GACGGTGCAC 420GTCTGGATAC GAGAGCATTT CCACTATGGG ACTGGATACA AACACACACC CGGCAGACTT 480CAAGAGTCTC AGACTGAGGA GAAAGCCTTT CCTTCTGCTG CTACTGCTGC TGCCGCTGCT 540TTTGAAAGTC CACTCCTTTC ATGGTTTTTC CTGCCAAACC AGAGGCACCT TTGCTGCTGC 600CGCTGTTCTC TTTGGTGTCA TTCAGCGGCT GGCCAGAGG ATG AGA CTC CCC AAA 654

Met?Arg?Leu?Pro?Lys

-25CTC?CTC?ACT?TTC?TTG?CTT?TGG?TAC?CTG?GCT?TGG?CTG?GAC?CTG?GAA?TTC 702Leu?Leu?Thr?Phe?Leu?Leu?Trp?Tyr?Leu?Ala?Trp?Leu?Asp?Leu?Glu?Phe

-20 -15 -10ATC?TGC?ACT?GTG?TTG?GGT?GCC?CCT?GAC?TTG?GGC?CAG?AGA?CCC?CAG?GGG 750Ile?Cys?Thr?Val?Leu?Gly?Ala?Pro?Asp?Leu?Gly?Gln?Arg?Pro?Gln?Gly

-5 1 5 10ACC?AGG?CCA?GGA?TTG?GCC?AAA?GCA?GAG?GCC?AAG?GAG?AGG?CCC?CCC?CTG 798Thr?Arg?Pro?Gly?Leu?Ala?Lys?Ala?Glu?Ala?Lys?Glu?Arg?Pro?Pro?Leu

15 20 25GCC?CGG?AAC?GTC?TTC?AGG?CCA?GGG?GGT?CAC?AGC?TAT?GGT?GGG?GGG?GCC 846Ala?Arg?Asn?Val?Phe?Arg?Pro?Gly?Gly?His?Ser?Tyr?Gly?Gly?Gly?Ala

30 35 40ACC?AAT?GCC?AAT?GCC?AGG?GCA?AAG?GGA?GGC?ACC?GGG?CAG?ACA?GGA?GGC 894Thr?Asn?Ala?Asn?Ala?Arg?Ala?Lys?Gly?Gly?Thr?Gly?Gln?Thr?Gly?Gly

45 50 55CTG?ACA?CAG?CCC?AAG?AAG?GAT?GAA?CCC?AAA?AAG?CTG?CCC?CCC?AGA?CCG 942Leu?Thr?Gln?Pro?Lys?Lys?Asp?Glu?Pro?Lys?Lys?Leu?Pro?Pro?Arg?Pro

60 65 70GGC?GGC?CCT?GAA?CCC?AAG?CCA?GGA?CAC?CCT?CCC?CAA?ACA?AGG?CAG?GCT 990Gly?Gly?Pro?Glu?Pro?Lys?Pro?Gly?His?Pro?Pro?Gln?Thr?Arg?Gln?Ala?75 80 85 90ACA?GCC?CGG?ACT?GTG?ACC?CCA?AAA?GGA?CAG?CTT?CCC?GGA?GGC?AAG?GCA 1038Thr?Ala?Arg?Thr?Val?Thr?Pro?Lys?Gly?Gln?Leu?Pro?Gly?Gly?Lys?Ala

95 100 105CCC?CCA?AAA?GCA?GGA?TCT?GTC?CCC?AGC?TCC?TTC?CTG?CTG?AAG?AAG?GCC 1086Pro?Pro?Lys?Ala?Gly?Ser?Val?Pro?Ser?Ser?Phe?Leu?Leu?Lys?Lys?Ala

110 115 120AGG?GAG?CCC?GGG?CCC?CCA?CGA?GAG?CCC?AAG?GAG?CCG?TTT?CGC?CCA?CCC 1134Arg?Glu?Pro?Gly?Pro?Pro?Arg?Glu?Pro?Lys?Glu?Pro?Phe?Arg?Pro?Pro

125 130 135CCC?ATC?ACA?CCC?CAC?GAG?TAC?ATG?CTC?TCG?CTG?TAC?AGG?ACG?CTG?TCC 1182Pro?Ile?Thr?Pro?His?Glu?Tyr?Met?Leu?Ser?Leu?Tyr?Arg?Thr?Leu?Ser

140 145 150GAT?GCT?GAC?AGA?AAG?GGA?GGC?AAC?AGC?AGC?GTG?AAG?TTG?GAG?GCT?GGC 1230Asp?Ala?Asp?Arg?Lys?Gly?Gly?Asn?Ser?Ser?Val?Lys?Leu?Glu?Ala?Gly155 160 165 170CTG?GCC?AAC?ACC?ATC?ACC?AGC?TTT?ATT?GAC?AAA?GGG?CAA?GAT?GAC?CGA 1278Leu?Ala?Asn?Thr?Ile?Thr?Ser?Phe?Ile?Asp?Lys?Gly?Gln?Asp?Asp?Arg

175 180 185GGT?CCC?GTG?GTC?AGG?AAG?GAG?AGG?TAC?GTG?TTT?GAC?ATT?AGT?GCC?CTG 1326Gly?Pro?Val?Val?Arg?Lys?Gln?Arg?Tyr?Val?Phe?Asp?Ile?Ser?Ala?Leu

190 195 200GAG?AAG?GAT?GGG?CTG?CTG?GGG?GCC?GAG?CTG?CGG?ATC?TTG?CGG?AAG?AAG 1374Glu?Lys?Asp?Gly?Leu?Leu?Gly?Ala?Glu?Leu?Arg?Ile?Leu?Arg?Lys?Lys

205 210 215CCC?TCG?GAC?ACG?GCC?AAG?CCA?GCG?GCC?CCC?GGA?GGC?GGG?CGG?GCT?GCC 1422Pro?Ser?Asp?Thr?Ala?Lys?Pro?Ala?Ala?Pro?Gly?Gly?Gly?Arg?Ala?Ala

220 225 230 CAG?CTG?AAG?CTG?TCC?AGC?TGC?CCC?AGC?GGC?CGG?CAG?CCG?GCC?TCC?TTG 1470Gln?Leu?Lys?Leu?Ser?Ser?Cys?Pro?Ser?Gly?Arg?Gln?Pro?Ala?Ser?Leu235 240 245 250CTG?GAT?GTG?CGC?TCC?GTG?CCA?GGC?CTG?GAC?GGA?TCT?GGC?TGG?GAG?GTG 1518Leu?Asp?Val?Arg?Ser?Val?Pro?Gly?Leu?Asp?Gly?Ser?Gly?Trp?Glu?Val

255 260 265TTC?GAC?ATC?TGG?AAG?CTC?TTC?CGA?AAC?TTT?AAG?AAC?TCG?GCC?CAG?CTG 1566Phe?Asp?Ile?Trp?Lys?Leu?Phe?Arg?Asn?Phe?Lys?Asn?Ser?Ala?Gln?Leu

270 275 280TGC?CTG?GAG?CTG?GAG?GCC?TGG?GAA?CGG?GGC?AGG?GCC?GTG?GAC?CTC?CGT 1614Cys?Leu?Glu?Leu?Glu?Ala?Trp?Glu?Arg?Gly?Arg?Ala?Val?Asp?Leu?Arg

285 290 295GGC?CTG?GGC?TTC?GAC?CGC?GCC?GCC?CGG?CAG?GTC?CAC?GAG?AAG?GCC?CTG 1662Gly?Leu?Gly?Phe?Asp?Arg?Ala?Ala?Arg?Gln?Val?His?Glu?Lys?Ala?Leu

300 305 310TTC?CTG?GTG?TTT?GGC?CGC?ACC?AAG?AAA?CGG?GAC?CTG?TTC?TTT?AAT?GAG 1710Phe?Leu?Val?Phe?Gly?Arg?Thr?Lys?Lys?Arg?Asp?Leu?Phe?Phe?Asn?Glu315 320 325 330ATT?AAG?GCC?CGC?TCT?GGC?CAG?GAC?GAT?AAG?ACC?GTG?TAT?GAG?TAC?CTG 1758Ile?Lys?Ala?Arg?Ser?Gly?Gln?Asp?Asp?Lys?Thr?Val?Tyr?Glu?Tyr?Leu

335 340 345TTC?AGC?CAG?CGG?CGA?AAA?CGG?CGG?GCC?CCA?CTG?GCC?ACT?CGC?CAG?GGC 1806Phe?Ser?Gln?Arg?Arg?Lys?Arg?Arg?Ala?Pro?Leu?AlaThr?Arg?Gln?Gly

350 355 360AAG?CGA?CCC?AGC?AAG?AAC?CTT?AAG?GCT?CGC?TGC?AGT?CGG?AAG?GCA?CTG 1854Lys?Arg?Pro?Ser?Lys?Asn?Leu?Lys?Ala?Arg?Cys?Ser?Arg?Lys?Ala?Leu

365 370 375CAT?GTC?AAC?TTC?AAG?GAC?ATG?GGC?TGG?GAC?GAC?TGG?ATC?ATC?GCA?CCC 1902His?Val?Asn?Phe?Lys?Asp?Met?Gly?Trp?Asp?Asp?Trp?Ile?Ile?Ala?Pro

380 385 390CTT?GAG?TAC?GAG?GCT?TTC?CAC?TGC?GAG?GGG?CTG?TGC?GAG?TTC?CCA?TTG 1950Leu?Glu?Tyr?Glu?Ala?Phe?His?Cys?Glu?Gly?Leu?Cys?Glu?Phe?Pro?Leu395 400 405 410CGC?TCC?CAC?CTG?GAG?CCC?ACG?AAT?CAT?GCA?GTC?ATC?CAG?ACC?CTG?ATG 1998Arg?Ser?His?Leu?Glu?Pro?Thr?Asn?His?Ala?Val?Ile?Gln?Thr?Leu?Met

415 420 425AAC?TCC?ATG?GAC?CCC?GAG?TCC?ACA?CCA?CCC?ACC?TGC?TGT?GTG?CCC?ACG 2046Asn?Ser?Met?Asp?Pro?Glu?Ser?Thr?Pro?Pro?Thr?Cys?Cys?Val?Pro?Thr

430 435 440CGG?CTG?AGT?CCC?ATC?AGC?ATC?CTC?TTC?ATT?GAC?TCT?GCC?AAC?AAC?GTG 2094Arg?Leu?Ser?Pro?Ile?Ser?Ile?Leu?Phe?Ile?Asp?Ser?Ala?Asn?Asn?Val

445 450 455GTG?TAT?AAG?CAG?TAT?GAG?GAC?ATG?GTC?GTG?GAG?TCG?TGT?GGC?TGC?AGG 2142Val?Tyr?Lys?Gln?Tyr?Glu?Asp?Met?Val?Val?Glu?Ser?Cys?Gly?Cys?Arg

460 465 470TAG?CAGCACTGGC?CCTCTGTCTT?CCTGGGTGGC?ACATCCCAAG?AGCCCCTTCC 2195***475TGCACTCCTG?GAATCACAGA?GGGGTCAGGA?AGCTGTGGCA?GGAGCATCTA?CACAGCTTGG 2255GTGAAAGGGG?ATTCCAATAA?GCTTGCTCGC?TCTCTGAGTG?TGACTTGGGC?TAAAGGCCCC 2315CTTTTATCCA?CAAGTTCCCC?TGGCTGAGGA?TTGCTGCCCG?TCTGCTGATG?TGACCAGTGG 2375CAGGCACAGG?TCCAGGGAGA?CAGACTCTGA?ATGGGACTGA?GTCCCAGGAA?ACAGTGCTTT 2435CCGATGAGAC?TCAGCCCACC?ATTTCTCCTC?ACCTGGGCCT?TCTCAGCCTC?TGGACTCTCC 2495TAAGCACCTC?TCAGGAGAGC?CACAGGTGCC?ACTGCCTCCT?CAAATCACAT?TTGTGCCTGG 2555TGACTTCCTG?TCCCTGGGAC?AGTTGAGAAG?CTGACTGGGC?AAGAGTGGGA?GAGAAGAGGA 2615GAGGGCTTGG ATAGAGTTGA GGAGTGTGAG GCTGTTAGAC TGTTAGATTT AAATGTATAT 2675TGATGAGATA AAAAGCAAAA CTGTGCCT 2703

Claims (8)

1. a method of producing maturation bone morphogenetic protein is characterized in that handling the Delicious peptide precursor with processive enzyme.

2. method of producing maturation bone morphogenetic protein is characterized in that may further comprise the steps:

In mammalian cell, introduce the expression vector of Delicious peptide precursor and be transformed into processive enzyme;

Cultivate this mammal cell line, produce maturation bone morphogenetic protein thus; The maturation bone morphogenetic protein that separates purifying from culture supernatant then.

3. the method for the production maturation bone morphogenetic protein of claim 1, wherein maturation bone morphogenetic protein is selected from: adult form MP52, BMP-2, BMP-4, BMP-6 and BMP-7.

4. the method for claim 1 to 3 production maturation bone morphogenetic protein one of at least, wherein processive enzyme is a furin.

5. the method for claim 1 to 4 production maturation bone morphogenetic protein one of at least, wherein the processive enzyme furin that whole aminoacid sequences are formed of serving as reasons.

6. the method for claim 1 to 4 production maturation bone morphogenetic protein one of at least, wherein processive enzyme is a secretion furin mutant.

7. the method for claim 1 to 5 production maturation bone morphogenetic protein one of at least is characterized in that comprising step:

In mammalian cell, introduce MP52 precursor expression carrier and be transformed into secretion furin mutant;

Cultivate this mammal cell line, produce maturation bone morphogenetic protein thus; Then

The maturation bone morphogenetic protein of separation purifying from culture supernatant.

8. method of producing maturation bone morphogenetic protein is characterized in that:

The solution that will contain processive enzyme adds the solution that contains the Delicious peptide precursor, is incubated this mixture then.

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