CN103360497A - Novel antitumor fusion protein vaccine, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of gene engineering preparation of bioactive peptides, and discloses an antitumor fusion protein vaccine VEGF121-M2-X10-beta hCG-CTP37 peptide, and a preparation method and an application thereof. With a fusion expression preparation technology platform of a laboratory, engineering bacteria for fusion expression of a VEGF121-M2-X10-hCG-CTP37 peptide in an escherichia coli cell are constructed. An objective protein is obtained after separation through an ion exchange chromatography. Correct expression of the fusion protein vaccine and immunogenicity of two epitopes contained in the fusion protein vaccine are identified and detected by Western blotting in vitro; with an animal experiment in vivo, the vaccine is expected to significantly inhibit tumor angiogenesis and tumor cell proliferation, induce a specific antitumor immune response, and have a good clinical application prospect for the antitumor treatment.

Description

A kind of new antitumoral amalgamation protein vaccine and its preparation method and application

Technical field

The invention belongs to preparation bioactive peptide field, the structure, the DEAE52 ion exchange chromatography that disclose the amalgamation and expression engineering bacteria of a kind of antineoplastic amalgamation protein vaccine VEGF121-M2-X10-β hCG-CTP37 peptide and peptide prepare method and the correction of external Western blotting evaluation amalgamation protein vaccine and the immunogenicity of contained two kinds of epitopes of VEGF121-M2-X10-β hCG-CTP37 peptide.

Background technology

Biotherapy is a widely concept, relates to the method that all applying biological macromole are treated, if kind is very various. come minute acellular treatment and a cell therapy from operator scheme.Biotherapy is the 4th class cancer treatment method that develops behind operation, radiation and chemotherapy, is that the immune response of utilization and excitating organism resists, suppresses and kill cancer cells.The major advantage of biotherapy has: use the normal people to depend on for existence and tumour patient is expressed the lower biomass cells factor and transferred the immune strength of body self and reach antitumor action, compare with radiation and chemotherapy, side effect is very little; Can excite the anti-tumour effect of general by active immunity, sphere of action is more extensive, is specially adapted to multiple focus or the malignant tumour of extensive transfer is arranged; Adopt molecular targeted agents to treat, with clearly defined objective, without impact, to the middle and advanced stage tumour patient that should not perform the operation, can obviously contain the progress of tumour to the normal cell beyond the tumour cell, prolong patient's life.

Tumour polypeptide vaccine namely belongs to a kind of important non cellular organism treatment pattern, the novel method simple because of production process, that the advantages such as expense is cheap, stable chemical nature, non-carcinogenesis become immunotherapy of tumors.Desirable polypeptide vaccine immunogenicity is strong, can the activation antigen specific CTL and htl response, and effective killing tumor cell and to normal cell toxicological harmless effect.But common polypeptide antigen is single because of its epi-position, molecular weight Xiao Yi degraded etc. is former thereby cause a little less than the immunogenicity, can only excite low-level ctl response, can not obtain desirable antitumous effect, improve immunogenic problem therefore in the polypeptide vaccine design process, need emphasis to solve.

Molecular targeted therapy has highly selective, hypotoxicity and high therapeutic index for the unusual signal path of tumour, but long-term prescription, thus might make malignant tumour be converted into " chronic disease ".At present, existing more than ten plant molecular targeted agents is approved for treatment of solid tumors, and other has tens of kinds to be in the clinical study.Molecular targeted agents has become the main direction of anti-cancer agent research and development.But, the small molecules class pharmacological agent narrow range of single target spot, and easily produce immunologic escape and resistance.Most of noumenal tumours all are the regulation processes of many target spots, too many levels, block an acceptor or target position and differ and block surely all cells signal transduction.Many target drugs have been simplified treatment procedure, have represented the new developing direction of neoplasm targeted therapy medicine.

Vascular endothelial growth factor (VEGF) is a kind of important angiogenesis factor, is to promote one of key factor that the angiogenic growth effect is the strongest, is the main adjusting factor that induction of vascular generates.And the generation of blood vessel is tumour Fast Growth and the necessary condition of transfer that cancerates.Finding altogether at present has five kinds of Mammals VEGF parts and three kinds of VEGF tyrosine kinase receptors, and some co-receptors also can indirectly be regulated the biological effect of VEGF.There were significant differences in the expression of VEGF in healthy tissues and tumor tissues, has vital role in the genesis of tumour, for the treatment of tumour provides new action target spot.

Human chorionic gonadotrophin (hCG) is one of characteristic sign of tumour cell at the ectopic expression of tumor cell surface, the histiocytic embryonic gene of being grown up under normal circumstances is in quiescent condition, do not express or only express the extremely hCG of trace, when cell generation vicious transformation, the embryonic gene of tranquillization is activated and expresses.But benign tumor cells is not generally expressed dystopy hCG, and the tumour that differentiation degree is lower is expressed higher, and grade malignancy is also higher; And tumor growth and escape from immune system attack played an important role.HCG is the generation of induced tumor not, but tumour is simulated the fetal development pattern and promoted self growth in case formation will be expressed dystopy hCG.37 amino acid of hCG β subunit C-terminal (β hCG-CTP37) position has been concentrated in contained 6 sugar chains of ehCG β 4 (on C-terminal the 121st, 127, be connected with four O-sugar chains on 132,138 Serines), may be closer with the relation of the characteristics such as the transfer of tumour, immunological tolerance; And have the specificity epitope of β hCG, but the immunne response of inducing specific is avoided the generation of cross reaction.So the immunne response (comprising antibody and effector cell) for the crucial antigen of β hCG-CTP37 may have more anti-knurl effect to the ehCG+ tumour.

Because VEGF and hCG are autologous protein, very easily produce immunological tolerance, immunogenicity is lower, therefore can strengthen by the method for series connection repetition epi-position the immunogenicity of the two.M is the peptide section between the upper 407-426 of heat shock protein(HSP) Hsp70, and M2 then is that the series connection of two sections M repeats to connect, and M2 can be used as adjuvant in the molecule, strengthens the immunogenicity of polypeptide vaccine, and does not rely on Hsp70 and play a role.X is the peptide section between the 109-118 on the hCG β subunit, and the gene fragment repeat techniques by based on the isocaudarner technology constructs the X10 of ten sections repetitions, is used for strengthening the immunogenicity of β hCG-CTP37.

This fusion rotein is many target spots tumor protein p53 vaccine, for different epitopes, bring out specific immune response, and depend on the help of M2 and X10, produce VEGF antibody and the anti-β hCG antibody of high titre, two kinds of antibody act on respectively different antitumor target spots, reach the effect that suppresses tumor-blood-vessel growth and specificity inhibition tumor cell propagation.

Summary of the invention

A kind of antitumor bifunctional fusion proteins vaccine VEGF121-M2-X10-β hCG-CTP37 peptide preparation method and application are disclosed.

An object of the present invention is to provide corresponding encoding sequence, carrier and Host Strains.

Another object of the present invention is as the boosting vaccine body by VEGF121-M2-X10-β hCG-CTP37 peptide, break immunological tolerance, produce simultaneously the antibody for VEGF and β hCG specific peptide section, the antineoplastic immune by antigen-specific and antineoplastic vascular generate the growth that two aspects suppress tumour.

In another aspect of this invention, provide a kind of VEGF121-M2-X10-β hCG-CTP37 peptide, it is to utilize the flexible peptide of DPTGG that the epitope on two kinds of anti-tumor vaccines is connected preparation New-type bifunctional amalgamation protein vaccine.

In a second aspect of the present invention, a kind of preparation method of VEGF121-M2-X10-β hCG-CTP37 peptide is provided, it is characterized in that, the method comprises step:

(a) under conditions suitable for the expression, cultivate claim 4,5 described plasmid vector and engineering bacterias thereof.

(b) from culture, isolate the VEGF121-M2-X10-β hCG-CTP37 peptide of aminoacid sequence shown in the SEQ NO:1.

(c) be purified into VEGF121-M2-X10-β hCG-CTP37 peptide.

In a preference, in the method for the present invention, described step (b) comprising:

By bacteriolyze, ammonium sulfate precipitation, the methods such as ion exchange chromatography are isolated the fusion rotein of VEGF121-M2-DPTGG and X10-β hCG-CTP37 composition.

Description of drawings

Fig. 1 construction of recombinant plasmid principle schematic.Linker represents connection peptides DPTGG, and D represents Asp, and P represents Pro, and T represents Thr, and G represents Gly.

Fig. 2 PCR obtains the agarose gel electrophoresis detected result of X10-β hCG-CTP37 gene.

M is DNA Marker, the Lane1:PCR product.

Fig. 3 PCR obtains the agarose gel electrophoresis detected result of VEGF-M2-DPTGG gene.

M is DNA Marker, the Lane1:PCR product.

The gene forward sequencer map of Fig. 4 fusion rotein.

The gene backward sequencing figure of Fig. 5 fusion rotein.

Figure 61 2%SDS-PAGE electrophoresis detection recombinant bacterium is through lactose-induced expressed fusion protein result, and namely recombinant bacterium induces curve.

M is Protein Marker, Lane1: the whole bacterial protein sample before inducing, Lane2-9: induce the rear 1-8 whole bacterial protein sample of hour each hour

Figure 71 2%SDS-PAGE electrophoresis detection ultrasonic degradation thalline result.

M is Protein Marker, Lane1: induce front whole bacterial protein, Lane2: induce rear 6h whole bacterial protein, Lane3: supernatant behind the ultrasonic degradation, Lane4: ultrasonic degradation postprecipitation

Figure 81 2%SDS-PAGE electrophoresis detection ammonium sulfate precipitation target protein result.

M is Protein Marker, Lane1: cellular lysate supernatant, albumen precipitation during the Lane2:10%-15% saturation ratio, albumen precipitation during the Lane3:15%-20% saturation ratio, albumen precipitation during the Lane4:20%-25% saturation ratio, albumen precipitation during the Lane5:25%-30% saturation ratio, albumen precipitation during albumen precipitation Lane7:35%-40% saturation ratio during the Lane6:30%-35% saturation ratio.

Figure 91 2%SDS-PAGE electrophoresis detection DEAE52 anion-exchange chromatography is to the target protein purification result.

M is Protein Marker, Lane1: protein solution sample before the loading, Lane2-9: the every 8mL sampling of albumen elution peak sample.

Figure 101 2%SDS-PAGE electrophoresis detection fusion rotein lyophilized powder redissolution result.

M is Protein Marker, Lane1: induce front whole bacterial protein, Lane2: induce rear 6h whole bacterial protein, Lane3: lyophilized powder redissolution sample.

Figure 11 Western bloting detects β hCG antigen result in the fusion rotein.

M is that albumen dyes Marker, Lane1 in advance: the fusion rotein behind the purifying.

Figure 12 Western bloting detects VEGF antigen result in the fusion rotein.

M is that albumen dyes Marker, Lane1 in advance: the fusion rotein behind the purifying.

Embodiment

Below the invention will be further described by drawings and Examples.

The material of mentioning in this specification sheets:

(1) Host Strains and plasmid

Host Strains Escherichia coli BL21 is genetically engineered engineering strain commonly used, and generally there is preservation in the laboratory that genetically engineered research is relevant.The pET-28a carrier makes up and preserves for this laboratory.

(2) enzyme and reagent

The molecular cloning toolenzyme is Fermentas company product; It is that worker's bio tech ltd product is given birth in Shanghai that plasmid extraction test kit, PCR glue reclaim test kit; DAB colouring reagents box is the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state product.

(3) substratum

The LB substratum, the document Sambrook J that sees reference that fills a prescription, FristshE F, Maniatis T.Molecular Cl oning; A Laboratory Manual2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989.

Recovery, connection and the conversion intestinal bacteria of plasmid extraction, PCR reaction, endonuclease digestion, dna fragmentation in the mentioned method of this specification sheets, these all are the routine operation methods of genetically engineered research field, specifically referring to Sambrook J, FristshE F, Maniatis T.Molecular Cloning; A Laboratory Manual2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, pp.16-340.

(4) antibody

The anti-human β hCG of rabbit monoclonal antibody is Beijing Bo Aosen Bioisystech Co., Ltd product, and the anti-human VEGF monoclonal antibody of rabbit is that worker's biotechnology limited-liability company product is given birth in Shanghai, and it is Wuhan Boster Biological Technology Co., Ltd.'s product that goat anti-rabbit igg two resists.

Embodiment 1VEGF121-M2-X10-β hCG-CTP37 gene cloning

This experiment utilizes round pcr, utilizes synthetic 4 primer V1, V2, C1, C2, obtains respectively the fragment of goal gene from template plasmid, two purpose fragments is inserted in the pET-28a expression vector one by one again, thereby obtains the fusion rotein sequence.Article four, primer sequence is as follows:

V1:5 '-CATGCCATGGACATCATCGACGACT-3 ' contains protection base and Nco I restriction enzyme site

V2:5 '-GGAATTCGCCACCAGTCGGATCCTTGTTGTGAGCGGCAATCT-3 ' contains protection base and EcoR I restriction enzyme site

C1:5 '-CGGCGACATGGGTGGCATGGAATTC-3 ' contains protection base and EcoR I restriction enzyme site

C2:5 '-GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTA-3 ' contains protection base and Hind III restriction enzyme site

Primer is synthetic by JaRa order-checking company.The PCR reaction is carried out at eppendorf pcr amplification instrument.C1 and C2 mix in 1: 1 ratio, in 94 ℃, and 30s; 60 ℃, 45s; 72 ℃, 1min; Totally 35 circulations.1.5% agarose gel electrophoresis is identified PCR product (referring to Fig. 3), and pillar location is consistent with the 522bp of expection.

The PCR product through cutting glue and reclaim obtaining the goal gene fragment and the pET-28a vector plasmid respectively through EcoR I and Hind III double digestion, agarose gel electrophoresis is used T after reclaiming respectively goal gene fragment and vector gene fragment ₄4 ℃ of connections of dna ligase, be applied on the LB solid medium that contains the 50mg/mL kantlex after being converted into E.coli BL21 competent cell, 37 ℃ of overnight incubation, picking list bacterium colony, enzyme blanking method primary dcreening operation positive colony, positive colony is delivered to living worker's order-checking company check order, sequencing result is entirely true after the software comparison.

Again V1 and V2 are mixed in 1: 1 ratio, in 94 ℃, 30s, 58 ℃, 45s; 72 ℃, 1min; Totally 35 circulations.1.5% agarose gel electrophoresis is identified PCR product (referring to Fig. 3), and pillar location is consistent with the 538bp of expection.

The PCR product through cutting glue and reclaim obtaining the goal gene fragment and the vector plasmid that makes up of the first step respectively through EcoR I and Nco I double digestion, agarose gel electrophoresis is used T after reclaiming respectively goal gene fragment and vector gene fragment ₄4 ℃ of connections of dna ligase, other steps are cloned with the first step, positive colony is delivered to living worker's order-checking company check order, and sequencing result is after the software comparison entirely true (referring to Fig. 4), and so far antigen-4 fusion protein gene makes up and finishes.

Embodiment 2VEGF121-M2-X10-β hCG-CTP37 peptide gene Expression in Escherichia coli and cultivate recombinant expressed bacterium with 1% inoculum size, 37 ℃ of shaken overnight in liquid LB substratum, 1% switching shaking flask bulk culture again, adding final concentration behind the cultivation 3h is the lactose-induced expression of 7mM, collect thalline after inducing 6h, keep sample and carry out SDS-PAGE analysis (referring to accompanying drawing 4).Fusion rotein 6h after inducing reaches stable maximum expression amount, and the expression amount by Bandscan software analysis fusion rotein can reach 35% of bacterial protein amount, and expresses with soluble form in born of the same parents.

The initial gross separation purifying of embodiment 3 fusion roteins

Select high expression level amount bacterial strain, be inoculated in the LB substratum, 37 ℃ of incubated overnight; Incubated overnight liquid is transferred into the LB substratum, and 37 ℃ of enlarged culturing are chosen optimum fermentation condition and obtained the fermentation thalline.The centrifugal 15min of 8000r/min, supernatant and precipitation all keep sample.PH8.0,2 bacterial sediments of the TrisHCl rinsing of 50mM; In the thalline of collecting, add the cellular lysate liquid (10mL/g thalline) for preparing, be positioned over 37 ℃ of shaking table constant temperature thermal agitations and carry out the ultrasonic degradation operation after 2 hours, with abundant cracking thalline; Cracking is not taken out lysate during viscous to solution, and the centrifugal 15min of 12000r/min collects supernatant and the precipitation mark that all keeps sample.The SDS-PAGE electrophoresis result shows that target protein mainly is present in the cellular lysate supernatant, is solubility expression in the born of the same parents therefore can judge fusion rotein.

Get the cracking supernatant and in ice bath, add while stirring the ammonium sulfate powder of porphyrize to corresponding saturation ratio, 4 ℃ leave standstill the centrifugal 20min of 12000r/min after 1 hour, every one-level saturation ratio is got cleer and peaceful precipitation sample, carry out the SDS-PAGE electrophoresis detection, the result shows that target protein mainly concentrates precipitation during to 25% saturation ratio at ammoniumsulphate soln 15%, although also have target protein to separate out when high saturation more, content is less, can ignore.Get above-mentioned precipitation and redissolve in ion-exchange damping fluid (25mM TrisHCl, pH8.0), in 4 ℃ of dialysed overnight, again in 4 ℃, 12000r/min, centrifugal 20min abandons precipitation, and supernatant carries out further separation and purification with the DEAE-52 anion-exchange column.

Embodiment 4DEAE-52 anion-exchange chromatography

What the present embodiment adopted is that DEAE-Mierocrystalline cellulose DE52 is the weak-type anionite, DEAE-52 is washed till neutrality with distilled water with it with HCl and NaOH after processing respectively, again DEAE-52 is packed in the chromatography column, chromatography column upper end fluid inlet connects constant flow pump, lower outlet connects the nucleic acid-protein detector, utilize ion-exchange damping fluid (pH8.0,25mM TrisHCl) carries out the balance of chromatography column, carry out the loading operation of protein solution after balance is complete with the speed of 1mL/min, repeat balancing run after the loading, use again NaCl (0-500mM) to carry out gradient elution, collect elution peak, and every 8ml keeps sample, and carries out the SDS-PAGE electrophoretic analysis.

Embodiment 5 desalination freeze-drying

Collect the higher elutriant of purity of protein, 4 ℃ are spent the night with distill water dialysis to it, and the elutriant after the dialysis is tiled in the glass dish, keep flat in-20 ℃ of refrigerators and are refrigerated in advance solid, be transferred to rapidly again in the Freeze Drying Equipment subzero 40 ℃ of freeze-drying 12 hours, the freezing preservation of scraping albumen dry powder.

The Western blotting of embodiment 6 amalgamation protein vaccines identifies

The external Western blotting authentication method of β hCG antigen fragment is as follows in the amalgamation protein vaccine: the preparation sample carried out the SDS-PAGE electrophoresis after albumen dry powder was redissolved, just run out of the offset plate stop electrophoresis to tetrabromophenol sulfonphthalein, stripping glue, cut glue to suitable size, according to big or small cutting nitrocellulose (NC) film of glue, for the albumen about 40kDa, current stabilization 110mA transferring film 3h, after protein transduction moves on the NC film, the skimmed milk confining liquid with 5% or 5%BSA37 ℃ of sealing 1h.The taking-up of NC film is placed in the hybridization bag, in the positive primary antibodie (the anti-human β hCG of rabbit monoclonal antibody) that adds after diluting of film, the exhaust bubble, sealing is hatched 2h for 37 ℃, film is taken out, TBST (pH7.6,100mM TrisHCl, 0.9%NaCl, 0.1%tween-20) wash three times, each 10min.With two anti-(goat anti-rabbit igg of horseradish peroxidase-labeled) the same 1h of hatching after the dilution, TBST washes three times again, and each 10min behind washing 10min, takes out the NC film and carries out DAB colour developing observations again.

The external Western blotting authentication method of VEGF antigen fragment is the same in the amalgamation protein vaccine, and difference is that primary antibodie is the anti-human VEGF monoclonal antibody of rabbit.Other reagent and the operation item all with aforesaid method in consistent.

Except the above-mentioned fact, the present invention can also have other embodiments, and all employings are equal to the technical scheme of replacement or equivalent transformation, all falls within the protection domain of requirement of the present invention.

Claims (9)

1. the amino acid sequence of an antineoplastic amalgamation protein vaccine VEGF121-M2-X10-β hCG-CTP37 is: Met, Asp, Ile, Ile, Asp, Asp, Phe, Thr, Asn, Glu, Ser, Gln, Asn, His, His, Glu, Val, Val, Lys, Phe, Met, Asp, Val, Tyr, Gln, Arg, Ser, Tyr, Cys, His, Pro, Ile, Glu, Thr, Leu, Val, Asp, Ile, Phe, Gln, Glu, Tyr, Pro, Asp, Glu, Ile, Glu, Tyr, Ile, Phe, Lys, Pro, Ser, Cys, Val, Pro, Leu, Met, Arg, Cys, Gly, Gly, Cys, Cys, Asn, Asp, Glu, Gly, Leu, Glu, Cys, Val, Pro, Thr, Glu, Glu, Ser, Asn, Ile, Thr, Met, Gln, Ile, Met, Arg, Ile, Lys, Pro, His, Gln, Gly, Gln, His, Ile, Gly, Glu, Met, Ser, Phe, Leu, Gln, His, Asn, Lys, Cys, Glu, Cys, Arg, Pro, Lys, Lys, Asp, Arg, Ala, Arg, Gln, Glu, Lys, Tyr, Ile, Lys, Ala, Asn, Ser, Lys, Phe, Ser, Ser, Gln, Pro, Ser, Val, Gln, Ile, Gln, Val, Tyr, Gln, Gly, Glu, Arg, Glu, Ile, Ala, Ala, His, Asn, Lys, Ser, Ser, Gln, Pro, Ser, Val, Gln, Ile, Gln, Val, Tyr, Gln, Gly, Glu, Arg, Glu, Ile, Ala, Ala, His, Asn, Lys, Asp, Pro, Thr, Gly, Gly, Glu, Phe, Ala, Arg, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ala, Arg, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ala, Arg, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ala, Arg, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ala, Arg, Thr, Cys, Asp, Asp, Pro, Arg, Phe, GIn, Asp, Ser, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ala, Ser, Ala, Asp, Pro, Thr, Gly, Gly, Thr, Cys, Asp, Asp, Pro, Arg, Phe, Gln, Asp, Ser, Ser, Ser, Ser, Lys, Ala, Pro, Pro, Pro, Ser, Leu, Pro, Ser, Pro, Ser, ArgLeu, Pro, Gly, Pro, SerAsp, Thr, Pro, Ile, Leu, Pro, Gln (SEQ, ID, NO:1).

2. VEGF121-M2-X10-β hCG-CTP37 according to claim 1 is characterized in that, it comprises the nucleotide sequence shown in the SEQ ID NO:2.

3. a kind of antitumor bifunctional fusion proteins vaccine VEGF121-M2-X10-β hCG-CTP37 according to claim 1, it is characterized in that: the theoretical molecular of this polypeptide is 37657.05Da, actual molecular weight is about 42kDa, its N end is VEGF mutant 1 (VEGF121 head and the tail 1-8 and two sections aminoacid sequences of 115-121 are replaced with tetanus toxin Th epi-position) and M2 (two sections tandem repetitive sequences of HSP70 (407-426) peptide section), the C end is 37 amino acid (β hCG-CTP37) of the C-terminal of X10 (ten sections tandem repetitive sequences of β hCG (109-118)) and β hCG, utilizes the flexible peptide of DPTGG to connect between N end and the C end.

4. carrier, it is characterized in that: it contains nucleotide sequence claimed in claim 2 and recombination method claimed in claim 3: first X10-β hCGCTP37 gene is inserted on the pET-28a carrier of preserving in this laboratory, screening and cloning, again VEGF mutant 1 gene is inserted on this clone, consist of new antigen-4 fusion protein gene.This antigen-4 fusion protein gene is positioned at the downstream of pET-28a carrier T7 promotor.The fusion expression plasmid of this restructuring claims pET-28a-VEGF121-M2-X10-β hCG-CTP37.

5. a genetic engineering bacterium is characterized in that: contain carrier claimed in claim 4.

6. comprise the recombination method of VEGF121-M2-X10-β hCG-CTP37 peptide claimed in claim 4, it is characterized in that: comprise epitope and the interior adjuvant M2 of molecule and the X10 of two kinds of anti-tumor vaccines, have simultaneously two kinds of different antitumor mechanism and function.

7. the preparation method of a VEGF121-M2-X10-β hCG-CTP37 peptide is characterized in that, the method comprises step:

(I) under the expression condition that is fit to, cultivate Host Strains claimed in claim 5.

(II) by bacteriolyze, ultrasonication, ammonium sulfate precipitation, the fusion rotein after the initial gross separation restructuring; This fusion rotein has the aminoacid sequence of the VEGF121-M2-X10-β hCG-CTP37 shown in the claim 1.

(III) be separated by decantation to VEGF121-M2-X10-β hCG-CTP37 by the DEAE-52 ion exchange layer, this fusion rotein purity SDS-PAGE electrophoresis is pure.

8. the activity of recombinant polypeptide according to claim 1, it is characterized in that: described VEGF121-M2-X10-β hCG-CTP37 tests (Western blotting) at the external protein immunoblotting that carries out, can produce positive reaction with VEGF monoclonal antibody and β hCG monoclonal antibody respectively, show that this fusion rotein has VEGF and two kinds of epitopes of β hCG simultaneously, and do not interfere with each other between epi-position, possess correct space conformation.

9. method as claimed in claim 7, it is characterized in that: after the VEGF121-M2-X10-β hCG-CTP37 peptide in the described step (III) enters in the body, stimulate the body generation for two strain specific antibodies of VEGF and β hCG-CTP37 epitope, reach the purpose of prevention and treatment tumour by the growth that suppresses tumor-blood-vessel growth and specific inhibition tumor cell.

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