CN106085932A

CN106085932A - A kind of structure of the genetic engineering bacterium of VEGF Yu GnRH fusion protein of recombinating

Info

Publication number: CN106085932A
Application number: CN201610343170.9A
Authority: CN
Inventors: 曹荣月; 叶佳; 井亮亮; 李岩; 李曼曼; 苗梓韬; 刘淑君
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2016-05-19
Filing date: 2016-05-19
Publication date: 2016-11-09

Abstract

The invention belongs to genetic engineering field, present disclosure provides the coli strain about a kind of VEGF Yu GnRH that recombinate and preparation method.Coli strain BL21 (DE3)/VGD, CCTCC NO:M2016132 that the present invention provides.This engineering bacteria contains recombiant plasmid pET32a VEGF M₂‑GnRH₃Hinge MVP NRLLLTG, plasmid has recombination mutation VEGF gene, and this gene has the gene order shown in SEQ ID NO.1.BL21 (DE3)/VGD bacterial strain, can highly effective expressing recombinant protein VEGF I M through lactose-induced₂‑GnRH₃‑hinge‑MVP‑NRLLLTG.The expection of this recombiant protein can improve the body immunne response to tumor cell, and break immune tolerates, and reaches to identify and kill the purpose of tumor cell, has good potential applicability in clinical practice for antineoplaston.

Description

A kind of structure of the genetic engineering bacterium of VEGF Yu GnRH fusion protein of recombinating

Technical field

In genetic engineering field, the invention discloses a kind of new fusion protein recombination engineering and construction method thereof, with And the purification of this fusion protein.

Background technology

Recombiant plasmid refers to, in engineered field, genes of interest and carrier are combined into one and has self replication energy The DNA molecular of power, converts or is transfected into host strain or cell then, filters out the recon containing genes of interest.

Genetic engineering (Genetic engineering): be with molecular genetics as theoretical basis, with molecular biology and Microbiological modernism is means, by the gene of separate sources by the blueprint being pre-designed, builds hybrid DNA in vitro and divides Son, is then introduced into living cells, to change biological original inherited character, to obtain new varieties, production new product, passes through genetic engineering Technology can build and express the novel destination protein with several functions.

Tumor vaccine by the type of antigenic component can be divided into tumor cell-based vaccines, tumor-antigen peptide and protein vaccine, DNA vaccination and dendritic cell vaccine etc..The most what type of tumor vaccine, its key developed all is to select suitably Target antigen and raising immunogenicity for tumor.

Tumor cell can expressing tumor related antigen or tumor specific antigen, the tumor grown up for these antigens Specific active immunotherapy, is the important method of tumor biotherapy.

Tumor associated antigen (Tumor-associated antigen, TAA): refer to that not a certain tumor institute is peculiar, The antigen molecule that there is also on other tumor cells or normal cell.Normal cell and the tumor cell only difference of the amount of having in propagation Different, because of referred to herein as related antigen.

VEGF (Vascular endothelial growth factor), i.e. VEGF, is that endothelium is special The mitogen of the opposite sex, can strengthen the survival ability of endotheliocyte, mitosis promoting, induce angiogenesis, increase Microvascular permeability makes tumor continued propagation, promotes the several functions such as cell migration and suppression apoptosis.Most of tumor cells are equal High level expression VEGF, and in normal structure, the only minority such as kidney, ovary internal organs have the expression of higher level, therefore VEGF is anti-swollen The preferable target site of tumor vascular treatment.

The methods such as monoclonal antibody, little molecules in inhibiting thing, soluble recepter are applied to lead to by blocking its signal transduction at present Road or exhaustion tumor cell produce VEGF, suppress tumor-blood-vessel growth, reach to suppress tumor growth and the effect of transfer.With tumor Angiogenesis is the targeted therapy of target spot, is concentrated mainly in anti-vegf treatment.Currently for the gene therapy of VEGF pathway, In the treatment of tumor, prospect is more optimistic.VEGF₁₂₁Mutant I is that two strong T of this use for laboratory tetanus toxin assist epi-position 618-627 and 831-838 replaces hVEGF₁₂₁Two peptide fragments of 1-8 and 115-121.Result explanation is keeping cysteine original On the premise of dimeric structure, after two strong T auxiliary epi-position hVEGF critical residues displacements of tetanus toxin, can be effectively improved The immunogenicity of VEGF.

Gonadotropin releasing hormone (Gonadotropin-releasing hormone, GnRH) is by the specific god of hypothalamus Through unit's cell synthesis, and it is secreted into antepituitary by hypophyseal portal circulation in a pulsed fashion, high affine with on pituicyte film The GnRH receptor (GnRHR) of power combines specifically, causes synthesis and the release of FSH and LH, thus promotes that in gonad, steroid swashs The synthesis of element.Neutralize the GnRH-I in body by the anti-GnRH-I antibody of induced high titers, itself and I type on hypophysis can be suppressed The combination of GnRHR, causes the gonadotropin releasing hormone levels such as LH and FSH to be decreased obviously, and lowers the synthesis of steroids, fall The low supply to tumor cell, has good therapeutic effect to hormone-dependent tumor.GnRH can be by suppression V EGF etc. simultaneously Role of Vasculogenesis and the regulation and control of down-regulated expression tumor cell induction or act on somatomedin, as at prostate gland cancer cell Middle GnRH-A has anti-EGF/TGFa systemic effect, reduces cell division；It is likely to relate to apoptotic induction or suppression The signalling channels such as intracellular such as P13/PKB.Therefore GnRH and receptor thereof are the promising target of oncotherapy.

GnRH-I is a kind of small haptens, and immunogenicity is the most weak, individually is difficult to cause immunity to answer as vaccine Answering, therefore this laboratory utilizes auxiliary T epi-position to improve the immunogenicity of GnRH-I.Research shows, Fusion protein of measles virus 288-302 amino acid fragment in sequence is a typical T epi-position (SEIKGVIVRLEGVAK is the most all called for short MVP), can For vaccine for man and significantly antibody for this T epi-position will not be produced.There are some researches show, containing multicopy linearly connected The immunogenicity of self-peptide sequences albumen can be greatly improved, thus, coding three sections is joined end to end by this laboratory The nucleotide fragments of GnRH-I be cloned into efficient expression vector.Also there are some researches show, haptenic dimerization physical ability produces the strongest Immunogenicity.Human IgG1 hinge region (hinge region) oligopeptide fragments 226-232/226 '-232 ' (CPPCPAP/ CPPCPAP) it is the pivot of natural immunity albumen.This experiment will have the polypeptide of high affinity with DnaK polypeptide binding structural domain Fragment NRLLLTG merges the C-end of previously constructed GnRH-I polypeptide vaccine fusion protein.Therefore, the design is at coding The corresponding nucleotide sequence of this oligopeptide fragments is introduced, it is desirable to GnRH-I between GnRH-I repetitive sequence and the nucleotide sequence of MVP Can thereby form dimer, strengthen immunogenicity.

Owing to independent VEGF or GnRH immunogenicity is relatively low, and jointly act on tumor from multiple sites and become For trend.The present invention is by VEGF₁₂₁I-M2 gene couples together with GnRH gene, sets up pET32a-VEGF I-M₂-GnRH3- Hinge-MVP-Dnak binding site recombiant plasmid, abduction delivering fusion protein, produce as boosting vaccine body Active immunity, break immune tolerates, and produces the antibody for VEGF and GnRH specificity peptide fragment, thus suppresses the growth of tumor.

Summary of the invention

The present invention discloses a kind of genetic engineering bacterium expressing restructuring VEGF fusion protein.

It is an object of the present invention to provide the preparation method of this recombinant bacterium, method is easy, it is easy to operation.

Another object of the present invention is to disclose the purification process of this recombiant protein.

In the first aspect of the invention, this coli strain named Escherichia coli BL21 (DE3)/ VGD:pET32a-VEGF-M₂-GnRH₃-hinge-MVP-NRLLLTG, bacterial strain has been filed on China typical culture collection center, protects Address, Tibetan: China, Wuhan, Wuhan University.Preservation date: on March 21st, 2015, deposit number is CCTCC NO:M2016132, Classification And Nomenclature is Escherichia coli BL21/pET32a-VEGF-M₂-GnRH₃-hinge-MVP-NRLLLTG, its cell In containing recombiant plasmid pET32a-VEGF-M₂-GnRH₃-hinge-MVP-NRLLLTG, containing shown in SEQ ID NO.1 in plasmid Recombination mutation VEGF gene order.

In the second aspect of the invention, it is provided that the preparation method of this recombiant plasmid, details are as follows for its technology path:

1. recombiant plasmid pET32a-VEGF I-M₂-GnRH₃-hinge-MVP-NRLLLTG and the structure of corresponding gene engineering bacteria Build

Existing about recombiant plasmid pET28a-(ansB-C)-GnRH according to this laboratory₃-hinge-MVP-NR LLLTG and pET28a-VEGF I-M₂Sequence information, utilize primer-design software Primer 5 and Oligo7 to design primer V1、V2.Forward primer V1 introduces Nco I, downstream primer V2 introduces BamH I restriction enzyme site and GSGSG connection peptides sequence Row.Use round pcr from pET28a-VEGF I-M₂In plasmid, clone obtains gene VEGF I-M₂-GSGSG.By Nco I and BamH I double digestion, T4 ligase connect and transformation technology is by the product VEGF I-M of PCR₂-GSGSG inserts pEDGD plasmid and carries In the corresponding restriction enzyme site of body, obtain middle transformant pET28a-VEGF-M₂-GnRH₃-hinge-MVP-NRLLLTG, then pass through N co I and Hind III double digestion, T4 ligase connect, and genes of interest inserts pET32a and forms recombiant plasmid pE T32a- VEGF-M₂-GnRH₃-hinge-MVP-NRLLLTG, obtains the weight needed by recombinant plasmid transformed E. coli BL21 The engineering bacteria of group gene.

2. the abduction delivering of fusion protein

Recombination engineering is activated, is seeded in the LB culture medium containing ammonia benzyl mycin shake overnight incubation, by 1:100 Transfer in fresh LB culture fluid, when 37 DEG C of cultivation to A600 values are 0.5-0.8, add lactose (final concentration 7mM) and induce Express, be centrifuged after 7h and collect bacterial sediment, carry out 15%SDS-PAGE analysis, observe destination protein pillar location and expression.

3. fusion protein is isolated and purified

Collecting thalline, every gram of wet thallus adds 10mL cellular lysate buffer solution, after thalline ultrasonic degradation, and centrifugal collection Supernatant.Through ammonium sulfate precipitation, filter out ammonium sulfate saturated concentration when destination protein mainly settles and collect now heavy Form sediment.Precipitation being melted into again in ion exchange liquid, 4 DEG C of stirrings, to fully dissolving, are centrifuged and collect supernatant.Supernatant is loaded on 8000- In the bag filter of 14000KDa, to ion exchange liquid in 4 DEG C of abundant dialysis, being centrifuged and abandon precipitation, supernatant crosses anion-exchange column DEAE-cellulose is further purified, and is 0-1M NaCl chromatography buffer gradient elution with Concentraton gradient, collects eluting Peak, combining data detection destination protein peak component, after distilled water is fully dialysed, lyophilizing preserves.

Accompanying drawing explanation

Fig. 1 construction of recombinant plasmid principle schematic.

Fig. 2 0.8% agarose gel electrophoresis detection PCR primer VEGF₁₂₁I-M₂The result of-GSGSG gene.

Lane1:Protein Marker, Lane2:VEGF₁₂₁I-M₂-GSGSG gene

Fig. 3 plasmid pET28a-VEGF₁₂₁I-M₂The forward sequencer map of-GSGSG gene

Fig. 4 recombiant plasmid pET28a-(ansB-C)-GnRH₃The forward of-hinge-MVP-NRLLLTG (pEDGD) gene Sequencer map.

Fig. 5 0.8% agarose gel electrophoresis detection VEGF-M₂-GnRH₃The knot of-hinge-MVP-NRLLLTG genes of interest Really.

Lane1:Protein Marker, Lane2: recombiant plasmid, the checking of Lane3: single endonuclease digestion, Lane4: double digestion obtains VGD gene

Fig. 6 recombiant plasmid pET28a-VEGF-M₂-GnRH₃The gene forward of-hinge-MVP-NRLLLTG (pEDVG2) is surveyed Sequence figure.

Fig. 7 0.8% agarose gel electrophoresis detection PCR primer pET32a-VEGF-M₂-GnRH₃-hinge-MVP- The result of NRLLLTG gene.

Fig. 8 recombiant plasmid pET32a-VEGF-M₂-GnRH₃The gene of-hinge-MVP-NRLLLTG (pET32a-VG) is just To sequencer map.

Fig. 9 15%SDS-PAGE electrophoresis detection recombinant bacterium is through the induction curves of lactose-induced expressed fusion protein.

Lane1:Protein Marker, Lane2: the whole bacterial protein sample before induction, Lane3-10: after induction, 1-8h is every The whole bacterial protein sample of individual hour

Scheme after Figure 10 15%SDS-PAGE electrophoresis detection fusion protein is broken.

Lane1:Protein Marker, Lane2: whole bacterial protein before induction, Lane3: 6h whole bacterial protein after induction, Lane4: centrifugal supernatant, Lane5: ultrasonication postprecipitation after ultrasonication

The selection of Figure 11 15%SDS-PAGE electrophoresis detection ammonium sulfate saturated concentration

Lane1:Protein Marker, Lane2: whole bacterial protein before induction, Lane3:0～5% ammonium sulfate saturated concentration Thoroughly, Lane4:5%～10% ammonium sulfate saturated concentration, Lane5:10%～20% ammonium sulfate saturated concentration, Lane6:20%～ 30% ammonium sulfate saturated concentration, Lane7:30%～40% ammonium sulfate saturated concentration, Lane8:40%～50% ammonium sulfate are saturated Concentration, Lane9:50%～60% ammonium sulfate saturated concentration

Detailed description of the invention

Material

(1) bacterial strain

Carrier pET28a-(ansB-C)-GnRH₃-hinge-MVP-NRLLLTG, Escherichia coli B L21 (DE3), bacterial strain pET28a-VEGF₁₂₁I-M₂It is this laboratory to preserve.

(2) toolenzyme and reagent

Molecular cloning toolenzyme is Fermentas Products；Plasmid extraction test kit is that Shanghai JaRa biotechnology has Limit company；Product Purification Kit and PCR glue reclaim the product that test kit is Sheng Gong bio tech ltd, Shanghai.

(3) culture medium

LB culture medium, formula sees reference document Sambrook J, FristshE F, Maniatis T.Molecular Cloning；A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989。

Plasmid extraction, PCR reaction, endonuclease digestion, the recovery of PCR primer, connection in method mentioned by this specification Enzyme connects and converts escherichia coli, and these are all the conventional practices of genetic engineering research field, referring specifically to Sambrook J, FristshE F, Maniatis T.Molecular Cloning；A Laboratory M anual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, pp.16-340.

Embodiment 1 pET28a-VEGF-M₂-GnRH₃The clone of-hinge-MVP-NRLLLTG gene

According to this laboratory built about pET28a-VEGF₁₂₁I-M₂Sequence information, utilize primer-design software Primer, Oligo separately design two couples of primers V1, V2:

V1:5 '-CATGCCATGGACATCATCGACGACTTCACTA-3 '

V2:5 '-CGGGATCCACCGCTGCCGCTACCCTTGTTGT-3 '

Forward primer V1 introduces restriction enzyme site Nco I, downstream V2 introduces restriction enzyme site BamH I and flexible peptide GSGSG.Plasmid pET28a-VEGF is extracted with plasmid extraction kit₁₂₁I-M₂With plasmid pET28a-(ansB-C)-GnRH₃- hinge-MVP-NRLLLTG.Primer V1, V2 is utilized to clone VEGF₁₂₁I-M₂-GSGSG gene, PCR reaction system is shown in Table 1, reaction Parameter is shown in Table 2.

Table 1 VEGF I-M2-GSGSG PCR reaction system

Table 2 VEGF I-M2-GSGSG PCR response parameter

0.8% agarose gel electrophoresis identifies PCR primer (seeing Figure of description 2), pillar location and intended 534bp Unanimously.

Acquisition purpose fragment VEGF is reclaimed by cutting glue₁₂₁I-M₂-GSGSG, by purpose fragment and pET28a-(ansB-C)- GnRH₃-hinge-MVP-NRLLLTG vector plasmid respectively through Nco I, BamH I double digestion, double digestion reaction system is shown in Table 3, Table 4.

Table 3 VEGF I-M₂-GSGSG Gene Double endonuclease reaction system

Table 4 pET28a-(ansB-C)-GnRH3-hinge-MVP-NRLLLTG double digestion reaction system

Digestion products passes through PCR primer Purification Kit, and agarose gel electrophoresis is separately recovered genes of interest fragment It is connected with E.coli DNA ligase 4 DEG C with after vector gene fragment, uses cold CaCl₂Method prepares the competence of e. coli bl21 Cell, is applied on the LB solid medium containing kanamycin after enzyme connects product transformed competence colibacillus cell, 37 DEG C of incubated overnight Rear picking list bacterium colony, by list colony inoculation to LB fluid medium, overnight incubation, extraction plasmid, respectively by PCR, single enzyme Cutting and the method primary dcreening operation positive colony such as double digestion, and deliver to raw work order-checking company and check order, sequencing result is after software comparison The most correct (seeing Figure of description 4).

Acquisition purpose fragment is reclaimed, by purpose fragment VEGF-M by cutting glue₂-GnRH₃-hinge-MVP-NR LLLTG and PET32a vector plasmid is respectively through Nco I, Hind III double digestion, and double digestion reaction system is shown in Table 3, table 4.

Table 3 VEGF-M2-GnRH3-hinge-MVP-NRLLLTG Gene Double endonuclease reaction system

Table 4 empty carrier pET32a double digestion reaction system

Digestion products passes through PCR primer Purification Kit, and agarose gel electrophoresis is separately recovered genes of interest fragment It is connected with E.coli DNA ligase 4 DEG C with after vector gene fragment, uses cold CaCl₂Method prepares the competence of e. coli bl21 Cell, is applied to after enzyme connects product transformed competence colibacillus cell on the LB solid medium containing ammonia benzyl mycin, 37 DEG C of incubated overnight Rear picking list bacterium colony, by list colony inoculation to LB fluid medium, overnight incubation, extract plasmid, by PCR, single endonuclease digestion and Method primary dcreening operation positive colonies such as double digestion, and deliver to raw work order-checking company and check order, sequencing result after software comparison completely Correctly (see Figure of description 8).

Embodiment 2 VEGF-M₂-GnRH₃The expression in the escherichia coli of-hinge-MVP-NRLLLTG gene and cultivation

Recombinant bacterium with 1% inoculum concentration in LB liquid medium test tube 37 DEG C of shaken cultivation overnight, then the inoculum concentration with 1% Switching shaking flask bulk cultures, cultivates and adds the lactose-induced expression of final concentration of 5mM after 4h, collects thalline after induction 6h, keep sample into Row SDS-PAGE analyzes.Fusion protein 6h after induction reaches stable maximum expression, is melted by Bandscan software analysis The expression of hop protein is up to 65% (seeing Figure of description 9) of bacterial protein amount.

The initial gross separation purification of embodiment 3 fusion protein

Select high expressed amount bacterial strain, be inoculated in LB culture medium, 37 DEG C of incubated overnight；Incubated overnight liquid is transferred and is cultivated into LB Base, 37 DEG C of amplification culture, choose optimum fermentation condition and obtain fermentation thalli.5000r/min is centrifuged 15min, supernatant and precipitation all Keep sample.The precipitation cellular lysate liquid (10mL/g thalline) prepared is dissolved, ultrasonic degradation thalline.12000r/min is centrifuged 20min, collects supernatant and precipitates the labelling that all keeps sample.SDS-PAG E electrophoresis result display destination protein is primarily present on thalline In Qing, therefore can determine whether that fusion protein is solubility expression (seeing Figure of description 10).

The ammonium sulfate precipitation of embodiment 4 solubility destination protein

Take the centrifugal rear supernatant of recombinant bacterium lysate and in ice bath, add finely ground ammonium sulfate powder while stirring to corresponding full With degree (saturation is respectively 5%, 10%, 20%, 30%, 40%, 50%, 60%), add speed should be slow, with in liquid not Occur that ammonium sulfate solids is advisable.After reaching corresponding saturation, 4 DEG C stand 1h, fully precipitate for protein, and 12000r/min is centrifuged 20min, every one-level saturation takes cleer and peaceful precipitation and prepares sample, carries out SDS-PAGE detection, analyzes mesh according to protein band Albumen concentrate precipitation ammonium sulfate saturation.Destination protein is selected to collect precipitation when concentrating the ammonium sulfate saturated concentration precipitated multiple It is melted in ion exchange liquid, in (25mM Tris HCl, pH8.0), 1% (v/v) beta-mercaptoethanol during redissolution, must be added.Will be multiple Molten protein solution is in 4 DEG C to ion exchange buffering liquid dialysed overnight, and then at 4 DEG C, 12000r/min is centrifuged 20min, abandons precipitation, Supernatant DEAE-52 anion exchange resin carries out the most isolated and purified.

Embodiment 5 DEAE-52 anion-exchange chromatography

The DEAE52 that this experiment uses is weak-type anionite, by DEAE-52 with NaOH, HCl and NaOH respectively Being washed till neutrality with distilled water after process, then loaded by DEAE-52 in chromatographic column, chromatographic column upper end inlet connects constant current Pump, lower outlet connects nucleic acid-protein detector, utilizes ion exchange buffering liquid (pH8.0,20mM Tris HCl) to chromatograph The balance of post, carries out the loading operation of protein solution, repeats balancing run after loading with the speed of 1mL/min after balance, Carry out gradient elution with NaCl (0-1M) again, collect eluting peak and keep sample, carry out SDS-PAGE electrophoretic analysis.

Embodiment 6 desalination lyophilizing

Collect the higher eluent of purity of protein, 4 DEG C to RO water dialysed overnight, by the eluent after dialysis in freeze dryer Lyophilizing, scrapes protein dry powder freezen protective.

In addition to the above-mentioned fact, the present invention can also have other embodiments, all employing equivalents or equivalent transformation Technical scheme, all falls within the protection domain of application claims.

Claims

1. one kind produces recombiant protein VEGF-M₂-GnRH₃The coli strain of-hinge-MVP-NRLLLTG, it is characterised in that Coli strain Escherichia coli.BL21 (DE3)/VGD, deposit number: CCTCC NO:M2016132.

2. according to this coli strain BL21 (the DE3)/VGD described in claims 1, it is characterised in that: escherichia coli are thin Containing recombiant plasmid pET32a-VEGF-M in born of the same parents₂-GnRH₃-hinge-MVP-NRLLLTG。

3. according to recombiant plasmid pET32a-VEGF-in this coli strain BL21 (the DE3)/VGD described in claims 2 M₂-GnRH₃-hinge-MVP-NRLLLTG, it is characterised in that: containing the VEGF-M of restructuring₂-GnRH₃-hinge-MVP- NRLLLTG, can expressed fusion protein, it has the aminoacid sequence shown in SEQ ID NO:2.

4. the method preparing this coli strain described in claims 1, it comprises the following steps:

(I) plasmid pET28a-(ansB-C)-GnRH is extracted with plasmid extraction kit₃-hinge-MVP-NRLLLTG and pET28a-VEGF I-M₂, amplify purpose fragment VEGF I-M by PCR method₂-GSGSG。

(II) VEGF I-M is cut with restricted enzyme Nco I, BamH I₂-GSGSG and pET28a-(ansB-C)-GnRH₃- Hinge-MVP-NRLLLTG plasmid, connects overnight with E.coli DNA ligase 4 DEG C, is converted coli strain, it is thus achieved that Containing plasmid pET28a-VEGF-M₂-GnRH₃The bacterial strain of-hinge-MVP-NRLLLTG.

(III) plasmid pET28a-VEGF-M is extracted with plasmid extraction kit₂-GnRH₃-hinge-MVP-NRLLLTG and plasmid PET32a, cuts plasmid pET28a-VEGF-M with restricted enzyme Nco I, Hind III₂-GnRH₃-hinge-MVP- NRLLLTG and plasmid pET32a, connects overnight with E.coli DNA ligase 4 DEG C, is converted coli strain, it is thus achieved that BL21 (DE3)/VGD bacterial strain.

(IV) recombiant plasmid is identified: use PCR method and single endonuclease digestion, double digestion method to verify, checked order by recombinant bacterial strain Checking.

A kind of new fusion protein VEGF-M the most according to claim 3₂-GnRH₃-hinge-MVP-NRLLLTG, it is special Levying and be: the fusion protein actual molecular weight of this gene expression is about 27kDa, its N end is VEGF I and M₂(Hsp70(407- 426) two sections of tandem repetitive sequences), C end is the GnRH sequence of three sections of tandem sequence repeats and the hinge region of IgG and Measles virus is melted 288-302 amino acid fragment MVP in hop protein sequence, utilizes GSGSG flexibility peptide to connect between N end and C end.

VEGF-M the most according to claim 5₂-GnRH₃The recombination method of-hinge-MVP-NRLLLTG peptide, its feature exists In: comprise epitope VEGF, GnRH of anti-tumor vaccine, and Inner adjuvant M₂, there is higher anti-tumor function.

7. a VEGF-M₂-GnRH₃The preparation method of-hinge-MVP-NRLLLTG peptide, it is characterised in that the method comprises step Rapid:

(I) step builds Host Strains according to claim 4

(II) by lactose-induced, ultrasonication, ammonium sulfate precipitation tentatively obtains recombiant protein

(III) it is separated by decantation to VEGF-M by DEAE-52 ion exchange layer₂-GnRH₃-hinge-MVP-NRLLLTG, this fusion egg Bai Chundu is pure up to SDS-PAGE electrophoresis.