CN101624581A - Pseudovirus based on Vasohibin gene and preparation method and application thereof - Google Patents

Abstract

The invention discloses pseudovirus based on Vasohibin gene and a preparation method and application thereof. The pseudovirus is a substance simulating a natural virus structure, which is obtained in such a way that viral capsid chimeric protein HPV16L1-RGD wraps a Vasohibin gene recombination eukaryotic expression vector, wherein the HPV16L1-RGD is formed by inserting RGC peptide between the 266th amino acid and the 267th amino acid of the HPV16L1. The preparation method of the pseudovirus comprises three steps: constructing the HPV16L1-RGD, constructing the Vasohibin gene recombination eukaryotic expression vector and wrapping the Vasohibin gene recombination eukaryotic expression vector with the HPV16L1-RGD. The pseudovirus can escape from organic cleaning mechanism at the maximum, be exclusively absorbed by new vascular endothelial cells of a tumor and tumor cells, play the roles of inhibiting tumor neovascularization and inducing the apoptosis of the tumor cells, and can be used for preparing antineoplastic.

Description

Based on pseudo-virus of Vasohibin gene and its production and application

Technical field

The present invention relates to the genetically engineered field, particularly based on pseudo-virus of Vasohibin gene and its production and application.

Background technology

Tumour is the high incidence and the high mortality disease of serious threat human health.After operation, radiotherapy and chemotherapy, biotherapy has become the important means in the combined therapy of tumour.Current, the tumor biotherapy strategy has the targeted therapy of tumor-related gene or tumor suppressor gene, the immunotherapy of tumor associated antigen etc., but all, be very limited in actual applications owing to problems such as technology maturation, validity, security and economy.

Discover that the human tumor more than 90% is a solid tumor, and the growth of solid tumor and transfer need new vessel to form, suppress new vessel formation and can effectively suppress growth of tumor and transfer to a certain extent.Therefore, suppress tumor-blood-vessel growth and become important replenishment strategy in the oncotherapy.In past 10 years, the investigator is devoted to the research of various angiogenesis inhibitors both at home and abroad, but does not obtain gratifying effect up to now as yet.In addition, research is also found, suppresses tumor-blood-vessel growth merely and still have certain limitation in oncotherapy, if coupling simultaneously will produce the anti-knurl effect of ideal more at other treatment means of tumour cell itself.Therefore, be that the therapeutic strategy of dual-target becomes a new direction in the tumor research with endothelial cells in tumor neogenetic blood vessels and tumour cell.

Vasohibin is first vasculogenesis reverse feedback regulatory factor of finding recently, the selective expression is in vascular endothelial cell, can be promoted the factor such as vascular endothelial growth factor (VEGF), fibroblast grwoth factor-2 also bFG (FGF-2) etc. to induce generation by vasculogenesis, and regulate the inhibition vasculogenesis by reverse feedback.Studies show that the Vasohibin gene transfection can make Vasohibin release increase in the blood plasma to murine liver tissue, and shows the angiogenesis inhibitor effect at remote part; In mouse retinal neovascularization model, vascular endothelial cell VEGF high expression level and Vasohibin mRNA rising companion lines, cause retinal neovascularization to increase after knocking out the Vasohibin gene, on the contrary, intraocular injection reorganization Vasohibin gene or the adenovirus carrier that has a Vasohibin gene can form by the strongly inhibited retinal neovascularization; Can also suppress propagation, migration and the microtubule formation etc. of Human umbilical vein endothelial cells by adenovirus carrier transfection Vasohibin gene.Therefore, Vasohibin may be the desirable target spot that suppresses tumor-blood-vessel growth.

The RGD peptide is the little peptide that a class contains Arg-Gly-Asp (RGD) sequence, extensively is present in the various tissues that reach in the multiple organism with a kind of organism.As a kind of important cell recognition site and signal enabling molecule, in many vital movements, bringing into play important regulatory role.Thereby what research was more at present is to compete by its contained RGD sequence about the RGD peptide to produce aspects such as anti-platelet aggregation, antitumor migration and antineoplastic vascular nucleus formation on the integrin receptor that is attached to cell surface.Studies confirm that: α in the integrin family _υβ ₃The closest with the relation that cell adhesion and new vessel form, its part RGD peptide has obvious restraining effect to the adhesion and the migration of endothelial cells in tumor neogenetic blood vessels, and but caspase-3 and caspase-7 express and increase in the inducing tumor cell, thereby promote the apoptosis of tumour cell.

Human papillomavirus 16 types (Human Papillomavirus type-16, HPV16) main capsid protein (L1) has and self is assembled into virus-like particle (Virus-like Particle, VLP) characteristic, and VLP can be packaged into the pseudo-virus (Pseudo Virus) with natural viral spline structure with foreign gene or small-molecule substance under optimum conditions, therefore, HPV16L1 can be used as gene or pharmaceutical carrier.But HPV16L1VLP has stronger immunogenicity, can induce body to produce the neutralizing antibody of high titre during immune animal, is unfavorable for that the efficient transfection of goal gene is to target cell.Discover that HPV16L1 VLP and neutralizing antibody produce and be positioned at the high variable loop district of HPV16L1 in conjunction with relevant conformation dependent epitope, this zone when being assembled into VLP towards the particulate outside surface; Insert an exogenous peptide between the 266th of HPV16L1 and the 267th amino acids, can not influence the ability that HPV16L1 self is assembled into VLP, the NAT that body is produced obviously descends.

Summary of the invention

In view of this, one of purpose of the present invention is to provide a kind of pseudo-virus based on the Vasohibin gene, can escape the body purge mechanism to greatest extent, specificity ground is by endothelial cells in tumor neogenetic blood vessels and tumour cell picked-up, performance suppresses the effect of tumor-blood-vessel growth and inducing apoptosis of tumour cell simultaneously, improves the validity and the security of oncotherapy greatly.

For reaching this purpose, in a first aspect of the present invention, a kind of pseudo-virus based on the Vasohibin gene is provided, and this puppet virus is the material of the simulation natural viral spline structure that obtained by viral capsid chimeric protein HPV16L1-RGD parcel Vasohibin gene recombination carrier for expression of eukaryon; Described viral capsid chimeric protein HPV16L1-RGD inserts the RGD peptide between the 266th of HPV16L1 and the 267th amino acids.

Further, described viral capsid chimeric protein HPV16L1-RGD has the aminoacid sequence shown in SEQ ID No.2;

Further, the encoding gene of described viral capsid chimeric protein HPV16L1-RGD has the nucleotide sequence shown in SEQ IDNo.1;

Further, described Vasohibin gene recombination carrier for expression of eukaryon contains the Vasohibin gene of nucleotide sequence shown in SEQ IDNo.9;

Further, described Vasohibin gene recombination carrier for expression of eukaryon adopts the p3xFLAG-CMV-14 carrier for expression of eukaryon.

Two of purpose of the present invention is to provide described pseudo-viral preparation method based on the Vasohibin gene, and is easy to operation.

For reaching this purpose, in a second aspect of the present invention, provide described pseudo-viral preparation method based on the Vasohibin gene, may further comprise the steps:

The structure of a, viral capsid chimeric protein HPV16L1-RGD

The structure of HPV16L1-RGD gene recombined vector: extract the total RNA of human cervical carcinoma cell strain Caski; Synthetic following primer: primers F 1 has sequence shown in the SEQ ID No.3, and primer R1 has sequence shown in the SEQ ID No.4, and mutagenic primer Fm has sequence shown in the SEQ ID No.5, and mutagenic primer Rm has sequence shown in the SEQ IDNo.6; With the Caski cell total rna is reverse transcription and the overlapping extension PCR method site-directed mutagenesis that template is carried out the HPV16L1 gene, makes up the HPV16L1-RGD gene with sequence shown in the SEQ ID No.1; Overlapping extension PCR method site-directed mutagenesis comprises two-wheeled PCR: first round PCR comprises 2 reactions, with reverse transcription gained HPV16L1 cDNA is template, 1 reaction is the upstream and downstream primer with primers F 1 and mutagenic primer Rm, makes the sheet segment DNA FRm that contains mutational site and upstream sequence thereof; 1 reaction is the upstream and downstream primer with mutagenic primer Fm and primer R1 in addition, makes the sheet segment DNA FmR that contains mutational site and downstream sequence thereof; First round PCR product carries out second and takes turns PCR after glue recovery purifying is identified, cut to agarose gel electrophoresis, FRm and FmR pass through terminal complementary pairing primer each other, it is the HPV16L1-RGD gene that extension makes the full length DNA FR that contains mutational site and upstream and downstream sequence thereof, is that template, primers F 1 and primer R1 increase for the upstream and downstream primer again with FR; Second takes turns the PCR product promptly gets the HPV16L1-RGD gene after glue recovery purifying is identified, cut to agarose gel electrophoresis; The reaction conditions of above-mentioned two-wheeled PCR is identical: 94 ℃ of pre-sex change of temperature 5 minutes, and 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, last 72 ℃ were extended 4 minutes; The pCR-TOPO carrier is gone in gained HPV16L1-RGD gene clone, be transformed into bacillus coli DH 5 alpha again, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt single endonuclease digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows insertion fragment sequence and the consistent person of sequence shown in the SEQ ID No.1, is the HPV16L1-RGD gene recombined vector pCRII-TOPO/HPV16L1-RGD that successfully constructs;

The structure of HPV16L1-RGD gene recombination baculovirus shuttle vectors: synthetic following primer: primers F 2 has sequence shown in the SEQ ID No.7, and primer R2 has sequence shown in the SEQ ID No.8; With pCRII-TOPO/HPV16L1-RGD is that template, primers F 2 and primer R2 carry out PCR for the upstream and downstream primer, the PCR reaction conditions is: 94 ℃ of pre-sex change of temperature 5 minutes, 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, and last 72 ℃ were extended 4 minutes; The PCR product is identified through agarose gel electrophoresis, after cutting glue recovery purifying, carry out double digestion with restriction enzyme EheI and XhoI, be connected through the baculovirus shuttle vectors pFastBacDual of EheI and XhoI double digestion again with equally, connect product and be transformed into bacillus coli DH 5 alpha, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt double digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows insertion fragment sequence and the consistent person of sequence shown in the SEQ ID No.1, is the HPV16L1-RGD gene recombination baculovirus shuttle vectors pFastBacDual/HPV16L1-RGD that successfully constructs;

The structure of HPV16L1-RGD gene recombination baculovirus: pFastBacDual/HPV16L1-RGD and linearized baculovirus dna cotransfection are carried out homologous recombination in the born of the same parents to the insect cell Sf-9 of logarithmic phase, make up the HPV16L1-RGD gene recombination baculovirus, after the transfection 3 days, collect culture supernatant as virus stock solution used;

The cell inner expression of viral capsid chimeric protein HPV16L1-RGD: the Sf-9 cell that the gained virus stock solution used is infected logarithmic phase, temperature is cultivated after 3 days for 27 ℃, collecting cell, resuspended with PBS, ultrasonic broken wall, low-speed centrifugal, supernatant liquor is transferred to fills in the centrifuge tube of sucrose solution that concentration is 400g/L, ultracentrifugation, abandon supernatant, to precipitate and use the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/mL in the collection centrifuge tube, promptly gets the viral capsid chimeric protein HPV16L1-RGD with sequence shown in the SEQ ID No.2;

B, Vasohibin gene recombination Construction of eukaryotic

Extract the total RNA of people's spleen tissue vascular endothelial cell; Synthetic following primer: primers F 3 has sequence shown in the SEQ IDNo.10, and primer R3 has sequence shown in the SEQ ID No.11; With the total RNA of people's spleen tissue vascular endothelial cell is that template, primers F 3 and primer R3 are reverse transcription and the pcr amplification that the upstream and downstream primer carries out the Vasohibin gene, the PCR reaction conditions is: 95 ℃ of pre-sex change of temperature 10 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of sex change of temperature were extended 4 minutes for 1 minute, 72 ℃ then, totally 30 circulations, final temp extended 10 minutes for 72 ℃; The PCR product is cut glue and is reclaimed purifying purpose fragment after agarose gel electrophoresis is identified, promptly gets the Vasohibin gene; Gained Vasohibin gene is carried out double digestion with restriction enzyme NotI and XbaI, be connected through the carrier for expression of eukaryon p3xFLAG-CMV-14 of NotI and XbaI double digestion again with equally, connect product and be transformed into bacillus coli DH 5 alpha, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt double digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows nucleotide sequence person in full accord shown in insertion fragment sequence and the SEQ ID No.9, is the Vasohibin gene recombination carrier for expression of eukaryon p3xFLAG-CMV-14/Vasohibin that successfully constructs; Get the bacillus coli DH 5 alpha that contains p3xFLAG-CMV-14/Vasohibin, amplification cultivation, a large amount of extracting recombinant plasmids promptly get p3xFLAG-CMV-14/Vasohibin;

C, based on the preparation of the pseudo-virus of Vasohibin gene

Get step a gained viral capsid chimeric protein HPV16L1-RGD, add denaturing agent and make protein denaturation, add step b gained Vasohibin gene recombination carrier for expression of eukaryon p3xFLAG-CMV-14/Vasohibin again, dialysed overnight in PBS, make metaprotein renaturation gradually, HPV16L1-RGD parcel p3xFLAG-CMV-14/Vasohibin in renaturation process, self-assembly forms the pseudo-virus based on the Vasohibin gene.

Three of purpose of the present invention is to provide described pseudo-viral application based on the Vasohibin gene.

For reaching this purpose,, provide the described application of pseudo-virus in the preparation antitumor drug based on the Vasohibin gene in a third aspect of the present invention.

The invention provides a kind of pseudo-virus based on the Vasohibin gene and its production and application, have following beneficial effect: (1) is inserted the RGD peptide and is made up viral capsid chimeric protein HPV16L1-RGD between the 266th of HPV16L1 and the 267th amino acids, can not influence the ability that HPV16L1 self is assembled into VLP, and the NAT of body generation is obviously descended, thereby make the pseudo-virus of the present invention can escape the body purge mechanism to greatest extent; (2) the RGD peptide is exposed to the outside surface of VLP after the HPV16L1-RGD self-chambering is made into VLP, thereby the pseudo-virus of the present invention can be absorbed on specificity ground by the mediation of the integrin receptor of endothelial cells in tumor neogenetic blood vessels and tumor cell surface, reduce the side effect that the irrelevant non-special picked-up of cell is brought, improve the security of oncotherapy greatly; (3) the RGD peptide has obvious restraining effect to the adhesion and the migration of endothelial cells in tumor neogenetic blood vessels, but and in the inducing tumor cell caspase-3 and caspase-7 express and increase, thereby make the pseudo-virus of the present invention have the effect of inducing apoptosis of tumour cell; (4) Vasohibin gene recombination carrier for expression of eukaryon can be expressed justacrine Vasohibin at the endothelial cells in tumor neogenetic blood vessels internal specific, thereby makes the pseudo-virus of the present invention have the effect that suppresses tumor-blood-vessel growth; (5) preparation method of the pseudo-virus of the present invention is simple and easy to do; In sum, the pseudo-virus of the present invention can be escaped the body purge mechanism to greatest extent, specificity ground is by endothelial cells in tumor neogenetic blood vessels and tumour cell picked-up, performance suppresses the effect of tumor-blood-vessel growth and inducing apoptosis of tumour cell simultaneously, improve the validity and the security of oncotherapy greatly, can be used for preparing antitumor drug, have a good application prospect in the oncotherapy field.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:

Fig. 1 detects HPV16L1-RGD gene transcribing in insect cell for RT-PCR;

Fig. 2 detects the expression of HPV16L1-RGD gene in insect cell for SDS-PAGE;

Fig. 3 detects the self-assembly ability of HPV16L1-RGD for transmission electron microscope;

Fig. 4 detects the expression of Vasohibin gene recombination carrier for expression of eukaryon for Western blot;

Fig. 5 detects based on the viral form of the puppet of Vasohibin gene for transmission electron microscope.

Embodiment

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

In a preferred embodiment, at first 3 placed in-line RGD peptides are inserted between the 266th of HPV16L1 and the 267th amino acids and make up viral capsid chimeric protein HPV16L1-RGD (certainly, also can only insert 1 RGD peptide, or insert 2,4 or more a plurality of placed in-line RGD peptide, can reach the object of the invention); Secondly the Vasohibin gene clone is gone into to make up in the carrier for expression of eukaryon Vasohibin gene recombination carrier for expression of eukaryon; With HPV16L1-RGD parcel Vasohibin gene recombination carrier for expression of eukaryon, self-assembly makes the pseudo-virus based on the Vasohibin gene at last; And animal lotus knurl level is investigated the effect that the pseudo-virus of gained suppresses tumor-blood-vessel growth and inducing apoptosis of tumour cell in cell in vitro level and body.

(1) based on the viral preparation of the puppet of Vasohibin gene

One, the structure of viral capsid chimeric protein HPV16L1-RGD and detection

1, the structure of viral capsid chimeric protein HPV16L1-RGD

(1) structure of HPV16L1-RGD gene recombined vector

Get human cervical carcinoma cell strain Caski (Sciencell company), adopt RNA to extract test kit (TaKaRa company) and extract cell total rna.

According to the GenBank accession number be the HPV16L1 gene order of EU430688 and overlapping extension PCR (Overlap-extension PCR, OE-PCR) legal some mutagenesis principle design following 4 primers and entrust Shanghai to give birth to worker company and synthesize: primers F 1:5 '- AgatctgCcaccatgtctctttggctgcct-3 ' (SEQ IDNo.3), underscore partly are Bgl II restriction enzyme site; Primer R1:5 '-atgtcgtactaccatcaccatcaccatcac-3 ' (SEQ ID No.4); Mutagenic primer Fm:5 '-aagcttttta Tagatcacgtagttcgct-3 ' (SEQID No.5), underscore partly are the mutational site; Mutagenic primer Rm:5 '-aactgctaaagcta Gcaattgtcagtta-3 ' (SEQ ID No.6), underscore partly is the mutational site.

Adopt RT-PCR reverse transcription amplification test kit (TaKaRa company) to carry out the reverse transcription and the OE-PCR method site-directed mutagenesis of HPV16L1 gene, between the 798th of the HPV16L1 gene and the 799th bit base, insert the encoding gene of placed in-line 3 RGD peptides, make up HPV16L1-RGD gene (SEQ ID No.1); Wherein, reverse transcription is to be template, Oligo (dT) with the Caski cell total rna that extracts ₁₆Be primer, make HPV16L1 cDNA; OE-PCR method site-directed mutagenesis comprises two-wheeled PCR totally 3 reactions: first round PCR comprises 2 reactions, 1 reaction is that template, primers F 1 and mutagenic primer Rm are the upstream and downstream primer with HPV16L1 cDNA, makes the sheet segment DNA FRm that contains mutational site and upstream sequence thereof; 1 reaction is that template, mutagenic primer Fm and primer R1 are the upstream and downstream primer with HPV16L1 cDNA in addition, makes the fragment DNAFmR that contains mutational site and downstream sequence thereof, and FmR and FRm are terminal complementary; First round PCR product carries out second and takes turns PCR after agarose gel electrophoresis evaluation, glue recovery test kit (TIANGEN company) are cut glue recovery purifying, FRm and FmR pass through terminal complementary pairing primer each other, it is the HPV16L1-RGD gene that extension makes the full length DNA FR that contains mutational site and upstream and downstream sequence thereof, is that template, primers F 1 and primer R1 increase for the upstream and downstream primer again with FR; Second takes turns the PCR product promptly gets the HPV16L1-RGD gene through agarose gel electrophoresis evaluation, glue recovery test kit after cutting glue recovery purifying; The reaction conditions of above-mentioned two-wheeled PCR is identical: 94 ℃ of pre-sex change of temperature 5 minutes, and 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, last 72 ℃ were extended 4 minutes.

The HPV16L1-RGD gene is carried out PCR with dATP under Taq archaeal dna polymerase (TaKaRa company) catalysis, 72 ℃ of reactions of temperature 30 minutes, make 3 ' end of HPV16L1-RGD gene add the A base, after the PCR product is cut glue recovery purifying with glue recovery test kit, carrying out T-A with pCR II-TOPO carrier (sigma company) that 3 ' end has a T base under T4DNA ligase enzyme (TaKaRa company) effect is connected, connect product and be transformed into bacillus coli DH 5 alpha (TIANGEN company) competent cell, with the LB plate screening positive colony that contains penbritin (AMP), single positive colony is inoculated in the LB liquid nutrient medium that contains AMP, after 37 ℃ of jolting overnight incubation of temperature, extract test kit (TIANGEN company) in a small amount with plasmid and extract recombinant plasmid in a small amount, the gained recombinant plasmid is identified with Bgl II single endonuclease digestion method and PCR method, single endonuclease digestion method and PCR method all are accredited as the male recombinant plasmid and entrust Shanghai living worker company to check order to inserting fragment, sequencing result shows that the insertion fragment sequence is consistent with sequence shown in the SEQ ID No.1, show the gene constructed success of HPV16L1-RGD, HPV16L1-RGD gene recombined vector pCR II-TOPO/HPV16L1-RGD successfully constructs.

(2) structure of HPV16L1-RGD gene recombination baculovirus shuttle vectors

According to the restriction enzyme site of HPV16L1-RGD gene order and baculovirus shuttle vectors pFastBacDual, design following 1 pair of primer and entrust Shanghai living worker company to synthesize: primers F 2:5 '-gg GgcgccTctctttggctgcctagtgag-3 ' (SEQ ID No.7), underscore partly is the EheI restriction enzyme site; Primer R2:5 '-gg CtcgagTtacagcttacgttttttgcg-3 ' (SEQ ID No.8), underscore partly is the XhoI restriction enzyme site.

With pCRII-TOPO/HPV16L1-RGD is that template, primers F 2 and primer R2 carry out PCR for the upstream and downstream primer, the PCR reaction conditions is: 94 ℃ of pre-sex change of temperature 5 minutes, 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, and last 72 ℃ were extended 4 minutes; The PCR product is identified through agarose gel electrophoresis, after glue recovery test kit is cut glue recovery purifying, carry out double digestion with EheI and XhoI, be connected through the pFastBacDual of EheI and XhoI double digestion (sigma company) again with equally, connect product and be transformed into the bacillus coli DH 5 alpha competent cell, with the LB plate screening positive colony that contains AMP, single positive colony is inoculated in the LB liquid nutrient medium that contains AMP, after 37 ℃ of jolting overnight incubation of temperature, extract test kit in a small amount with plasmid and extract recombinant plasmid in a small amount, the gained recombinant plasmid is identified with EheI and XhoI double digestion method and PCR method, double digestion method and PCR method all are accredited as the male recombinant plasmid and entrust Shanghai living worker company to check order to inserting fragment, sequencing result shows that the insertion fragment sequence is consistent with sequence shown in the SEQ ID No.1, shows that HPV16L1-RGD gene recombination baculovirus shuttle vectors pFastBacDual/HPV16L1-RGD successfully constructs.

(3) structure of HPV16L1-RGD gene recombination baculovirus

Is that the TMN-FH of the foetal calf serum of 100mL/L cultivates based on 27 ℃ of cultivations of temperature with insect cell Sf-9 (Beijing North great achievement company) with containing concentration, when treating that cell grows to 50～70% individual layers in culture dish, adopt BaculoGold transfection reagent box (sigma company) that pFastBacDual/HPV16L1-RGD and BaculoGold DNA (linearized baculovirus dna) cotransfection are carried out in the born of the same parents homologous recombination to the Sf-9 cell with structure HPV16L1-RGD gene recombination baculovirus, after the transfection 3 days, the cytopathy variability reaches more than 90%, visible cell nuclear increases, the kytoplasm muddiness, recombinate shape virus infection features such as cell rounding are collected culture supernatant as virus stock solution used.

(4) cell inner expression of viral capsid chimeric protein HPV16L1-RGD

With the gained virus stock solution used is the Sf-9 cell of 5 infection logarithmic phases with infection multiplicity, temperature is cultivated after 3 days for 27 ℃, obvious pathology appears in cell, collecting cell, concentration with precooling is 0.1mol/L, the pH value is that 7.4 PBS is resuspended, ultrasonic broken wall, low-speed centrifugal, supernatant liquor is transferred to fills in the centrifuge tube of sucrose solution that concentration is 400g/L, ultracentrifugation, abandon supernatant, to precipitate and use the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/mL in the collection centrifuge tube, promptly gets viral capsid chimeric protein HPV16L1-RGD, aminoacid sequence has inserted placed in-line 3 RGD peptides between the 266th of HPV16L1 and the 267th amino acids shown in SEQ ID No.2.

2, the detection of viral capsid chimeric protein HPV16L1-RGD

(1) RT-PCR detects HPV16L1-RGD gene transcribing in insect cell

Get the Sf-9 cell that infects the HPV16L1-RGD gene recombination baculovirus, extract cell total rna and carry out RT-PCR, the PCR product carries out the agarose gel electrophoresis detection that concentration is 10g/L, the result as shown in Figure 1, wherein the M swimming lane is a dna molecular amount standard, 1 swimming lane is the PCR product, and visible 1 swimming lane has a tangible DNA band at about 1600bp place, and is consistent with the expection size of HPV16L1-RGD gene; After the PCR product cut glue and reclaim purifying, entrust Shanghai to give birth to worker company and check order, sequencing result shows that its sequence is consistent with sequence shown in the SEQ IDNo.1, shows that the HPV16L1-RGD gene transcribes at Sf-9 cell internal specific.

(2) SDS-PAGE detects the expression of HPV16L1-RGD gene in insect cell

Get the Sf-9 cell that infects the HPV16L1-RGD gene recombination baculovirus, after an amount of sample-loading buffer mixes, boiled 30 minutes, with mass percentage concentration 5% concentrated glue, mass percentage concentration is that 12.5% separation gel carries out SDS-PAGE and coomassie brilliant blue staining analysis, the result as shown in Figure 2, wherein the M swimming lane is a protein molecular weight standard, 1 swimming lane is for infecting the Sf-9 cell of HPV16L1-RGD gene recombination baculovirus, 2 swimming lanes are normal Sf-9 cell, as seen 1 swimming lane has a tangible protein band at the about 63kD of molecular weight place, consistent with the expection molecular weight size of HPV16L1-RGD, and 2 swimming lanes are not seen respective strap, show that the HPV16L1-RGD gene expresses at Sf-9 cell internal specific.

(3) transmission electron microscope detects the self-assembly ability of HPV16L1-RGD

With the HPV16L1-RGD drips of solution on 200 order copper mesh, be after 1% the acetic acid uranium solution negative staining with mass percentage concentration, put the form of observing HPV16L1-RGD under the transmission electron microscope, the result as shown in Figure 3, as seen the rounded granular structure of HPV16L1-RGD, the about 50nm of diameter shows that HPV16L1-RGD can self-chambering be made into VLP under the situation that lacks other viral protein.

Two, Vasohibin gene recombination Construction of eukaryotic and detection

1, Vasohibin gene recombination Construction of eukaryotic

Get people's spleen tissue of excision, add liquid nitrogen and fully grind, take by weighing 100mg again and put in the centrifuge tube of no RNA enzyme, adopt RNA to extract test kit and extract the total RNA of vascular endothelial cell.

According to the GenBank accession number is the Vasohibin gene order of NM_014909, designs following 1 pair of primer and entrusts Shanghai living worker company to synthesize: primers F 3:5 '- GcggccgcAgatccccataccgagtgtg-3 ' (SEQ ID No.10), underscore partly is the NotI restriction enzyme site; Primer R3:5 '- TctagaGggcctctttggtcatttcc-3 ' (SEQ ID No.11), underscore partly is the XbaI enzyme cutting site.

With the total RNA of vascular endothelial cell is that template, primers F 3 and primer R3 are the upstream and downstream primer, adopt RT-PCR reverse transcription amplification test kit to carry out the reverse transcription and the pcr amplification of Vasohibin gene, the PCR reaction conditions is: 95 ℃ of pre-sex change of temperature 10 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of sex change of temperature were extended 4 minutes for 1 minute, 72 ℃ then, totally 30 circulations, final temp extended 10 minutes for 72 ℃; The PCR product reclaims test kit through agarose gel electrophoresis evaluation, glue and cuts glue recovery purifying, promptly gets the Vasohibin gene.

The Vasohibin gene is carried out double digestion with NotI and XbaI (TaKaRa company), be connected under the effect of T4 dna ligase through the carrier for expression of eukaryon p3xFLAG-CMV-14 of NotI and XbaI double digestion (Sigma-Aldrich company) again with equally, connect product and be transformed into the bacillus coli DH 5 alpha competent cell, with the LB plate screening positive colony that contains AMP, single positive colony is inoculated in the LB liquid nutrient medium that contains AMP, after 37 ℃ of joltings of temperature are cultivated 12 hours, extract test kit in a small amount with plasmid and extract recombinant plasmid in a small amount, the gained recombinant plasmid adopts double digestion method and PCR method to identify, double digestion method and PCR method all are accredited as the male recombinant plasmid and entrust Shanghai living worker company to check order to inserting fragment, sequencing result is presented between the NotI of p3xFLAG-CMV-14 and the XbaI site and is inserted with and the on all four dna fragmentation of nucleotide sequence shown in the SEQ ID No.9, shows that Vasohibin gene recombination carrier for expression of eukaryon p3xFLAG-CMV-14/Vasohibin successfully constructs.

Get the bacillus coli DH 5 alpha that contains p3xFLAG-CV-14/Vasohibin, be inoculated in the LB liquid nutrient medium that contains AMP, in 37 ℃ of jolting overnight incubation of temperature, the OD490nm value for the treatment of nutrient solution reaches at 1～1.5 o'clock, adopt a large amount of extracting recombinant plasmids of ultrapure plasmid extraction test kit (TIANGEN company), the gained recombinant plasmid is made p3xFLAG-CMV-14/Vasohibin solution with the ultrapure water dissolving, measures purity and concentration with ultraviolet spectrophotometer, put temperature-20 ℃ preservation, standby.

2, Western blot detects the expression of Vasohibin gene recombination carrier for expression of eukaryon

Is 37 ℃, CO with the PRIM RPMI-1640 in temperature with the cos-7 cell ₂Gas concentration is to cultivate 24 hours under the condition of 50mL/L, treat that cell confluency reaches at about 80% o'clock, adopt lipofectamine Lipofectamine 2000 that p3xFLAG-CMV-14/Vasohibin or p3xFLAG-CMV-14 are transfected into the cos-7 cell, after the transfection 4 hours, nutrient solution is replaced by the PRIM RPMI-1640, continue to cultivate 48 hours, collecting cell, with PBS washing and resuspended, the ultrasonic degradation cell, centrifugal, collect supernatant liquor, with His Bind protein purification test kit (Novage company) purifying Vasohibin; Get purifying protein solution, adopting mass percentage concentration is that 5% concentrated glue, mass percentage concentration are that 12.5% separation gel carries out SDS-PAGE, electric transfer printing pvdf membrane after electrophoresis finishes, add the anti-mouse Vasohibin of rabbit monoclonal antibody after washing membrane closure, temperature was hatched 1 hour for 37 ℃, added goat anti-rabbit igg again after washing film, and temperature was hatched 1 hour for 37 ℃, develop the color after washing film, observe.The result as shown in Figure 4, wherein 1 to 3 swimming lane is the protein solution that extracts purifying from the cos-7 cell of p3xFLAG-CMV-14/Vasohibin transfection, 4 swimming lanes are the protein solution that extracts purifying from the cos-7 cell of p3xFLAG-CMV-14 transfection, as seen 1 to 3 swimming lane all has a tangible protein band at the about 40kD of molecular weight place, consistent with Vasohibin expection molecular weight size, and 4 swimming lanes are not seen respective strap, show that the p3xFLAG-CMV-14/Vasohibin of structure can correctly express Vasohibin in eukaryotic cell.

Three, based on the puppet of Vasohibin gene viral preparation and detection

1, based on the viral preparation of the puppet of Vasohibin gene

With HPV16L1-RGD concentration is 0.1mol/L, the pH value is that the solution that concentration is 1mg/mL is made in 7.4 PBS dissolving, adding 2 mercapto ethanol to final volume percentage concentration is 5%, 4 ℃ of reactions of temperature made protein denaturation in 16 hours, add p3xFLAG-CMV-14/Vasohibin 2mg again, be transferred in the dialysis tubing behind the mixing, in concentration is 0.1mol/L, the pH value is in 4 ℃ of dialysed overnight of temperature among 7.4 the PBS, make metaprotein renaturation gradually, HPV16L1-RGD parcel p3xFLAG-CMV-14/Vasohibin in renaturation process, self-assembly forms the pseudo-virus based on the Vasohibin gene.

2, transmission electron microscope detects the form based on the pseudo-virus of Vasohibin gene

To drip based on the pseudo-viral suspension of Vasohibin gene on 200 order copper mesh, be after 1% the acetic acid uranium solution negative staining with mass percentage concentration, put and observe its form under the transmission electron microscope, the result as shown in Figure 5, wherein A is HPV16L1-RGD, B is the HPV16L1-RGD after the sex change, C is the pseudo-virus based on the Vasohibin gene, as seen HPV16L1-RGD rounded granular structure before sex change, be dissociated into tiny molecular structure after the sex change, but can be in PBS renaturation gradually, wrap up p3xFLAG-CMV-14/Vasohibin simultaneously, revert to the circular granular spline structure again.

(2) based on the viral experimental study that suppresses tumor cell proliferation, adhesion, invasive ability and inducing apoptosis of tumour cell of the puppet of Vasohibin gene

One, experiment in vitro

1, MTT (thiazalylblue, tetrazolium bromide) colorimetry detects the ability that suppresses tumor cell proliferation based on the pseudo-virus of Vasohibin gene

To be in the normal cell DC2.4 of exponential phase of growth or cervical cancer cell Hela is that to make cell density be 1 * 10 for the perfect medium of the new-born calf serum of 100g/L with containing concentration ⁶The suspension of/mL is seeded in 96 orifice plates, and every hole 100 μ L are divided into two groups: control group and experimental group, establish 3 multiple holes for every group, and the every hole of control group adds complete culture solution 100 μ L; It is the pseudo-viral suspension 100 μ L based on the Vasohibin gene of 1000 μ g/mL that the every hole of experimental group adds concentration; In temperature is 37 ℃, CO ₂Gas concentration is to cultivate under the condition of 50mL/L after 72 hours, every hole adds the MTT 20 μ L that concentration is 5g/L again, continue to cultivate after 4 hours, supernatant is removed in suction, every hole adds dimethyl sulfoxide (DMSO) 150 μ L again, jog 10 minutes is measured each hole OD value with microplate reader at wavelength 490nm place, calculate cell proliferation inhibition rate.

The result: is 8% based on the pseudo-virus of Vasohibin gene to the proliferation inhibition rate of DC2.4 cell, is 64% to the proliferation inhibition rate of Hela cell, shows that the pseudo-virus of the present invention can effectively suppress the propagation of tumour cell.

2, the MTT colorimetry detects the ability that suppresses tumor cell adhesion based on the pseudo-virus of Vasohibin gene

Basilar membrane Matrigel (BD company) is made the solution that concentration is 1 μ g/ μ L with the serum-free medium dilution, add in 96 orifice plates, every hole 8 μ L, temperature was hatched 1 hour for 37 ℃, be divided into three groups: control group, DC2.4 groups of cells and Hela groups of cells, establish 3 multiple holes for every group, the every hole of control group adds serum-free medium 100 μ L; The every hole adding of DC2.4 groups of cells concentration is that pseudo-viral suspension 20 μ L and the cell density based on the Vasohibin gene of 1000 μ g/mL is 1 * 10 ⁶The DC2.4 cell suspension 200 μ L of/mL; The every hole adding of Hela groups of cells concentration is that pseudo-viral solution 20 μ L and the cell density based on the Vasohibin gene of 1000 μ g/mL is 1 * 10 ⁶The Hela cell suspension 200 μ L of/mL; 37 ℃ of joltings of temperature are after 30 minutes, every hole adds serum-free DMEM substratum 50 μ L again and concentration is the MTT 50 μ L of 1mg/mL, after 37 ℃ of temperature are hatched 4 hours, every hole adds dimethyl sulfoxide (DMSO) 100 μ L again, jog 10 minutes, detect each hole OD value at wavelength 490nm place with microplate reader, calculate the cell adhesion rate.

The result: the adhesion rate of DC2.4 cell is 87%, and the adhesion rate of Hela cell is 32%, shows that the pseudo-virus of the present invention can effectively suppress the adhesion of tumour cell.

3, Boyden cell method detects the ability that suppresses tumor cell invasion based on the pseudo-virus of Vasohibin gene

Get Boyden cell (kylin medical apparatus company), at cell the millipore filtration that the aperture is 8 μ m is set between the chamber up and down, the basilar membrane Matrigel of shop, film surface dilution in even 1: 2, following chamber adds previously prepared NIH3T3 cell culture supernatant as chemokine, and pseudo-viral suspension 20 μ L and cell density that last chamber adds based on the Vasohibin gene are 1 * 10 ⁶The DC2.4 of/mL or Hela cell suspension 200 μ L are 37 ℃, CO in temperature ₂Gas concentration is to hatch 6 hours under the condition of 50mL/L, discard chamber liquid, take out millipore filtration, Matrigel reaches the cell that does not pass, formaldehyde fixed, haematoxylin dyeing on the most film of wiping, microscopically is observed, get 5 high power fields at random, count in each visual field and attack to the cell count at the filter membrane back side, the result represents with mean value.

The result: attack to the DC2.4 cell count at the filter membrane back side be 6.4, the Hela cell count is 2.3, shows that the pseudo-virus of the present invention can effectively suppress invasion by tumor cells.

4, the two stainings of flow cytometer-Annexin V/PI detect the ability based on the pseudo-viral inducing apoptosis of tumour cell of Vasohibin gene

With DC2.4 or Hela cell with cultivate 48 hours altogether based on the pseudo-viral suspension of Vasohibin gene after, the PBS washing, add concentration again and be the Annexin V 5 μ L of FITC mark of 250 μ g/mL and the PI 5 μ L that concentration is 250 μ g/mL, the ice bath lucifuge was hatched 10 minutes, the PBS washing detects the apoptosis situation with FACS Calibur flow cytometer.

The result: the DC2.4 apoptosis rate is 8.5%, and the Hela apoptosis rate is 42.7%, shows the effectively apoptosis of inducing tumor cell of the pseudo-virus of the present invention.

Two, experiment in the body

Tumor bearing nude mice is divided into three groups at random: control group I, control Group II and experimental group, control group I tail vein injection saline; The control Group II tail vein injection does not contain the pseudo-virus of RGD peptide and Vasohibin gene recombination carrier for expression of eukaryon; The experimental group tail vein injection is based on the pseudo-virus of Vasohibin gene; Observe survival rate and the gross tumor volume of respectively organizing mouse in 60 days, draw tumor growth curve; Get the mouse interior tumor tissue, the row check pathological section, and detect tumor neogenetic blood vessels density (is one to resist with anti-CD31 monoclonal antibody) with the immunohistochemical methods method.

Result: compare experimental mice survival rate height, the slow and tumor neogenetic blood vessels density reduction of tumor growth with control group I and control Group II.

Based on above-mentioned experimental result, can draw as drawing a conclusion: the pseudo-virus based on the Vasohibin gene of the present invention can be escaped the body purge mechanism to greatest extent, specificity ground is by endothelial cells in tumor neogenetic blood vessels and tumour cell picked-up, performance suppresses the effect of tumor-blood-vessel growth and inducing apoptosis of tumour cell simultaneously, improve the validity and the security of oncotherapy greatly, can be used as active substance, use separately or use with other compound and/or extract composition compound with pharmacologically active, make the antitumor drug of various formulations again according to the conventional formulation method of pharmaceutical field with acceptable accessories, and, reach efficient by suitable administration, treat the purpose of tumour safely.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Sequence table

＜110〉Military Medical Univ No.3, P.L.A

＜120〉based on pseudo-virus of Vasohibin gene and its production and application

<160>11

<210>1

<211>1599

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(1599)

<220>

＜223〉description of artificial sequence: HPV16L1-RGD gene

<400>1

tgt?cgt?act?acc?atc?acc?atc?acc?atc?acg?att?acg?ata?tcc?caa?cga 48

Met?Ser?Tyr?Tyr?His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr

1 5 10 15

ccg?aaa?acc?tgt?att?ttc?agg?gcg?cct?ctc?ttt?ggc?tgc?cta?gtg?agg 96

Thr?Glu?Asn?Leu?Tyr?Phe?Gln?Gly?Ala?Ser?Leu?Trp?Leu?Pro?Ser?Glu

20 25 30

cca?ctg?tct?act?tgc?ctc?ctg?tcc?cag?tat?cta?agg?ttg?taa?gca?cgg 144

Ala?Thr?Val?Tyr?Leu?Pro?Pro?Val?Pro?Val?Ser?Lys?Val?Val?Ser?Thr

35 40 45

atg?aat?atg?ttg?cac?gca?caa?aca?tat?att?atc?atg?cag?gaa?cat?cca 192

Asp?Glu?Tyr?Val?Ala?Arg?Thr?Asn?Ile?Tyr?Tyr?His?Ala?Gly?Thr?Ser

50 55 60

gac?tac?ttg?cag?ttg?gac?atc?cct?att?ttc?cta?tta?aaa?aac?cta?aca 240

Arg?Leu?Leu?Ala?Val?Gly?His?Pro?Tyr?Phe?Pro?Ile?Lys?Lys?Pro?Asn

65 70 75 80

ata?aca?aag?tat?tag?ttc?cta?aag?tat?cag?gat?tac?aat?aca?ggg?tat 288

Asn?Asn?Lys?Val?Leu?Val?Pro?Lys?Val?Ser?Gly?Leu?Gln?Tyr?Arg?Val

85 90 95

tta?gaa?tac?att?tac?ctg?acc?cca?ata?agt?ttg?gtt?ttc?ctg?aca?cct 336

Phe?Arg?Ile?His?Leu?Pro?Asp?Pro?Asn?Lys?Phe?Gly?Phe?Pro?Asp?Thr

100 105 110

cat?ttt?ata?atc?cag?ata?cac?agc?ggc?tgg?ttt?ggg?cct?gtg?tag?gtg 384

Ser?Phe?Tyr?Asn?Pro?Asp?Thr?Gln?Arg?Leu?Val?Trp?Ala?Cys?Val?Gly

115 120 125

ttg?agg?tag?gtc?gtg?gtc?agc?cat?tag?gtg?tgg?gca?tta?gtg?gcc?atc 432

Val?Glu?Val?Gly?Arg?Gly?Gln?Pro?Leu?Gly?Val?Gly?Ile?Ser?Gly?His

130 135 140

ctt?tat?taa?ata?aat?tgg?atg?aca?cag?aaa?atg?cta?gtg?ctt?atg?cag 480

Pro?Leu?Leu?Asn?Lys?Leu?Asp?Asp?Thr?Glu?Asn?Ala?Ser?Ala?Tyr?Ala

145 150 155 160

caa?atg?cag?gtg?tgg?ata?ata?gag?aat?gta?tat?cta?tgg?att?aca?aac 528

Ala?Asn?Ala?Gly?Val?Asp?Asn?Arg?Glu?Cys?Ile?Ser?Met?Asp?Tyr?Lys

165 170 175

aaa?cac?aat?tgt?gtt?taa?ttg?gtt?gca?aac?cac?cta?tag?ggg?aac?act 576

Gln?Thr?Gln?Leu?Cys?Leu?Ile?Gly?Cys?Lys?Pro?Pro?Ile?Gly?Glu?His

180 185 190

ggg?gca?aag?gat?ccc?cat?gta?cca?atg?ttg?cag?taa?atc?cag?gtg?att 624

Trp?Gly?Lys?Gly?Ser?Pro?Cys?Thr?Asn?Val?Ala?Val?Asn?Pro?Gly?Asp

195 200 205

gtc?cac?cat?tag?agt?taa?taa?aca?cag?tta?ttc?agg?atg?gtg?ata?tgg 672

Cys?Pro?Pro?Leu?Glu?Leu?Ile?Asn?Thr?Val?Ile?Gln?Asp?Gly?Asp?Met

210 215 220

ttg?ata?ctg?gct?ttg?gtg?cta?tgg?act?tta?cta?cat?tac?agg?cta?aca 720

Val?Asp?Thr?Gly?Phe?Gly?Ala?Met?Asp?Phe?Thr?Thr?Leu?Gln?Ala?Asn

225 230 235 240

aaa?gtg?aag?ttc?cac?tgg?ata?ttt?gta?cat?cta?ttt?gca?aat?atc?cag 768

Lys?Ser?Glu?Val?Pro?Leu?Asp?Ile?Cys?Thr?Ser?Ile?Cys?Lys?Tyr?Pro

245 250 255

att?ata?tta?aaa?tgg?tgt?cag?aac?cat?atg?cgc?ggc?gac?cgc?ggc?gac 816

Asp?Tyr?Ile?Lys?Met?Val?Ser?Glu?Pro?Tyr?Arg?Gly?Asp?Arg?Gly?Asp

260 265 270

cgc?ggc?gac?aac?aaa?tgt?ttg?tta?gac?att?tat?tta?ata?ggg?ctg?gtg 864

Arg?Gly?Asp?Glu?Gln?Met?Phe?Val?Arg?His?Leu?Phe?Asn?Arg?Ala?Gly

275 280 285

ctg?act?ggg?agt?act?cgg?ttg?gtg?aaa?atg?tac?cag?acg?att?tat?aca 912

Ala?Asp?Trp?Glu?Tyr?Ser?Val?Gly?Glu?Asn?Val?Pro?Asp?Asp?Leu?Tyr

290 295 300

tta?aag?gct?ctg?ggt?cta?ctg?caa?att?tag?cca?gtt?cga?att?att?ttc 960

Ile?Lys?Gly?Ser?Gly?Ser?Thr?Ala?Asn?Leu?Ala?Ser?Ser?Asn?Tyr?Phe

305 310 315 320

cta?cac?cta?gtg?gtt?cta?tgg?tta?cct?ctg?atg?ccc?aaa?tat?tca?ata 1008

Pro?Thr?Pro?Ser?Gly?Ser?Met?Val?Thr?Ser?Asp?Ala?Gln?Ile?Phe?Asn

325 330 335

aac?ctt?att?ggt?tac?aac?gag?cac?agg?gcc?aca?ata?atg?gca?ttt?gtt 1056

Lys?Pro?Tyr?Trp?Leu?Gln?Arg?Ala?Gln?Gly?His?Asn?Asn?Gly?Ile?Cys

340 345 350

ggg?gta?acc?aac?tat?ttg?tta?ctg?ttg?ttg?ata?cta?cac?gca?gta?caa 1104

Trp?Gly?Asn?Gln?Leu?Phe?Val?Thr?Val?Val?Asp?Thr?Thr?Arg?Ser?Thr

355 360 365

ata?tgt?cat?tat?gtg?ctg?cca?tat?cta?ctt?cag?aaa?cta?cat?ata?aaa 1152

Asn?Met?Ser?Leu?Cys?Ala?Ala?Ile?Ser?Thr?Ser?Glu?Thr?Thr?Tyr?Lys

370 375 380

ata?cta?act?tta?agg?agt?acc?tac?gac?atg?ggg?agg?aat?atg?att?tac 1200

Asn?Thr?Asn?Phe?Lys?Glu?Tyr?Leu?Arg?His?Gly?Glu?Glu?Tyr?Asp?Leu

385 390 395 400

agt?tta?ttt?ttc?aac?tgt?gca?aaa?taa?cct?taa?ctg?cag?acg?tta?tga 1248

Gln?Phe?Ile?Phe?Gln?Leu?Cys?Lys?Ile?Thr?Leu?Thr?Ala?Asp?Val?Met

405 410 415

cat?aca?tac?att?cta?tga?att?cca?cta?ttt?tgg?agg?act?gga?att?ttg 1296

Thr?Tyr?Ile?His?Ser?Met?Asn?Ser?Thr?Ile?Leu?Glu?Asp?Trp?Asn?Phe

420 425 430

gtc?tac?aac?ccc?ccc?cag?gag?gca?cac?tag?aag?ata?ctt?ata?ggt?ttg 1344

Gly?Leu?Gln?Pro?Pro?Pro?Gly?Gly?Thr?Leu?Glu?Asp?Thr?Tyr?Arg?Phe

435 440 445

taa?cat?ccc?agg?caa?ttg?ctt?gtc?aaa?gac?ata?cac?ctc?cag?cac?cta 1392

Val?Thr?Ser?Gln?Ala?Ile?Ala?Cys?Gln?Arg?His?Thr?Pro?Pro?Ala?Pro

450 455 460

aag?aag?atc?ccc?tta?aaa?aat?aca?ctt?ttt?ggg?aag?taa?att?taa?agg 1440

Lys?Glu?Asp?Pro?Leu?Lys?Lys?Tyr?Thr?Phe?Trp?Glu?Val?Asn?Leu?Lys

465 470 475 480

aaa?agt?ttt?ctg?cag?acc?tag?atc?agt?ttc?ctt?tag?gac?gca?aat?ttt 1488

Glu?Lys?Phe?Ser?Ala?Asp?Leu?Asp?Gln?Phe?Pro?Leu?Gly?Arg?Lys?Phe

485 490 495

tac?tac?aag?cag?gat?tga?agg?cca?aac?caa?aat?tta?cat?tag?gaa?aac 1536

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

500 505 510

gaa?aag?cta?cac?cca?cca?cct?cat?cta?cct?cta?caa?ctg?cta?aac?gca 1584

Arg?Lys?Ala?Thr?Pro?Thr?Thr?Ser?Ser?Thr?Ser?Thr?Thr?Ala?Lys?Arg

515 520 525

aaa?aac?gta?agc tgt 1599

Lys?Lys?Arg?Lys?Leu

530

<210>2

<211>533

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: viral capsid chimeric protein HPV16L1-RGD

<400>2

Met?Ser?Tyr?Tyr?His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr

1 5 10 15

Thr?Glu?Asn?Leu?Tyr?Phe?Gln?Gly?Ala?Ser?Leu?Trp?Leu?Pro?Ser?Glu

20 25 30

Ala?Thr?Val?Tyr?Leu?Pro?Pro?Val?Pro?Val?Ser?Lys?Val?Val?Ser?Thr

35 40 45

Asp?Glu?Tyr?Val?Ala?Arg?Thr?Asn?Ile?Tyr?Tyr?His?Ala?Gly?Thr?Ser

50 55 60

Arg?Leu?Leu?Ala?Val?Gly?His?Pro?Tyr?Phe?Pro?Ile?Lys?Lys?Pro?Asn

65 70 75 80

Asn?Asn?Lys?Val?Leu?Val?Pro?Lys?Val?Ser?Gly?Leu?Gln?Tyr?Arg?Val

85 90 95

Phe?Arg?Ile?His?Leu?Pro?Asp?Pro?Asn?Lys?Phe?Gly?Phe?Pro?Asp?Thr

100 105 110

Ser?Phe?Tyr?Asn?Pro?Asp?Thr?Gln?Arg?Leu?Val?Trp?Ala?Cys?Val?Gly

115 120 125

Val?Glu?Val?Gly?Arg?Gly?Gln?Pro?Leu?Gly?Val?Gly?Ile?Ser?Gly?His

130 135 140

Pro?Leu?Leu?Asn?Lys?Leu?Asp?Asp?Thr?Glu?Asn?Ala?Ser?Ala?Tyr?Ala

145 150 155 160

Ala?Asn?Ala?Gly?Val?Asp?Asn?Arg?Glu?Cys?Ile?Ser?Met?Asp?Tyr?Lys

165 170 175

Gln?Thr?Gln?Leu?Cys?Leu?Ile?Gly?Cys?Lys?Pro?Pro?Ile?Gly?Glu?His

180 185 190

Trp?Gly?Lys?Gly?Ser?Pro?Cys?Thr?Asn?Val?Ala?Val?Asn?Pro?Gly?Asp

195 200 205

Cys?Pro?Pro?Leu?Glu?Leu?Ile?Asn?Thr?Val?Ile?Gln?Asp?Gly?Asp?Met

210 215 220

Val?Asp?Thr?Gly?Phe?Gly?Ala?Met?Asp?Phe?Thr?Thr?Leu?Gln?Ala?Asn

225 230 235 240

Lys?Ser?Glu?Val?Pro?Leu?Asp?Ile?Cys?Thr?Ser?Ile?Cys?Lys?Tyr?Pro

245 250 255

Asp?Tyr?Ile?Lys?Met?Val?Ser?Glu?Pro?Tyr?Arg?Gly?Asp?Arg?Gly?Asp

260 265 270

Arg?Gly?Asp?Glu?Gln?Met?Phe?Val?Arg?His?Leu?Phe?Asn?Arg?Ala?Gly

275 280 285

Ala?Asp?Trp?Glu?Tyr?Ser?Val?Gly?Glu?Asn?Val?Pro?Asp?Asp?Leu?Tyr

290 295 300

Ile?Lys?Gly?Ser?Gly?Ser?Thr?Ala?Asn?Leu?Ala?Ser?Ser?Asn?Tyr?Phe

305 310 315 320

Pro?Thr?Pro?Ser?Gly?Ser?Met?Val?Thr?Ser?Asp?Ala?Gln?Ile?Phe?Asn

325 330 335

Lys?Pro?Tyr?Trp?Leu?Gln?Arg?Ala?Gln?Gly?His?Asn?Asn?Gly?Ile?Cys

340 345 350

Trp?Gly?Asn?Gln?Leu?Phe?Val?Thr?Val?Val?Asp?Thr?Thr?Arg?Ser?Thr

355 360 365

Asn?Met?Ser?Leu?Cys?Ala?Ala?Ile?Ser?Thr?Ser?Glu?Thr?Thr?Tyr?Lys

370 375 380

Asn?Thr?Asn?Phe?Lys?Glu?Tyr?Leu?Arg?His?Gly?Glu?Glu?Tyr?Asp?Leu

385 390 395 400

Gln?Phe?Ile?Phe?Gln?Leu?Cys?Lys?Ile?Thr?Leu?Thr?Ala?Asp?Val?Met

405 410 415

Thr?Tyr?Ile?His?Ser?Met?Asn?Ser?Thr?Ile?Leu?Glu?Asp?Trp?Asn?Phe

420 425 430

Gly?Leu?Gln?Pro?Pro?Pro?Gly?Gly?Thr?Leu?Glu?Asp?Thr?Tyr?Arg?Phe

435 440 445

Val?Thr?Ser?Gln?Ala?Ile?Ala?Cys?Gln?Arg?His?Thr?Pro?Pro?Ala?Pro

450 455 460

Lys?Glu?Asp?Pro?Leu?Lys?Lys?Tyr?Thr?Phe?Trp?Glu?Val?Asn?Leu?Lys

465 470 475 480

Glu?Lys?Phe?Ser?Ala?Asp?Leu?Asp?Gln?Phe?Pro?Leu?Gly?Arg?Lys?Phe

485 490 495

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

500 505 510

Arg?Lys?Ala?Thr?Pro?Thr?Thr?Ser?Ser?Thr?Ser?Thr?Thr?Ala?Lys?Arg

515 520 525

Lys?Lys?Arg?Lys?Leu

530

<210>3

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primers F 1

<400>3

agatctgcca?ccatgtctct?ttggctgcct 30

<210>4

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer R1

<400>4

atgtcgtact?accatcacca?tcaccatcac 30

<210>5

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: mutagenic primer Fm

<400>5

aagcttttta?tagatcacgt?agttcgct 28

<210>6

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: mutagenic primer Rm

<400>6

aactgctaaa?gctagcaatt?gtcagtta 28

<210>7

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primers F 2

<400>7

ggggcgcctc?tctttggctg?cctagtgag 29

<210>8

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer R2

<400>8

ggctcgagtt?acagcttacg?ttttttgcg 29

<210>9

<211>1098

<212>DNA

＜213〉homo sapiens (homo sapiens)

<220>

<221>CDS

<222>(1)...(1098)

<400>9

atgccagggg?ggaagaaggt?ggctgggggt?ggcagcagcg?gtgccactcc?aacgtccgct?60

gcggccaccg?ccccctctgg?ggtcaggcgt?ttggagacca?gcgaaggaac?ctcagcccag?120

agagatgagg?agccagaaga?ggaaggggaa?gaggacctgc?gagacggagg?cgtccccttc?180

tttgtcaacc?ggggtgggct?acctgtggat?gaggccacct?gggaaaggat?gtggaaacac?240

gtggccaaga?tccaccccga?tggagagaag?gtggcgcaac?ggatccgtgg?ggccacagac?300

ctgcccaaga?tccccatacc?gagtgtgcct?acgttccagc?cgtctacacc?tgtccctgag?360

cgcctggaag?ctgtgcagcg?ctacatcaga?gagctgcagt?acaatcacac?agggacacag?420

ttctttgaaa?ttaagaagag?cagacctctg?acagggctga?tggacctggc?caaggaaatg?480

accaaagagg?ccctgccaat?caaatgcctg?gaagccgtga?tcctgggaat?ttacctcacc?540

aacagcatgc?ccaccctgga?gcgcttcccc?atcagcttca?agacctactt?ctcagggaac?600

tacttccgcc?acatcgtgct?gggggtgaac?ttcgcgggcc?gctacggtgc?gctgggcatg?660

agtcggcgcg?aggacctgat?gtacaagccg?cccgccttcc?gcacgctcag?cgagctcgtg?720

ctggacttcg?aggccgccta?cggccgctgc?tggcacgtgc?tcaagaaggt?gaagctgggc?780

cagagcgtgt?cacacgaccc?gcacagcgtg?gagcagatcg?agtggaagca?ctcggtgctg?840

gacgtggagc?gcctgggccg?cgatgacttc?cgcaaggagc?tggagcgcca?cgcccgcgac?900

atgcggctca?agattggcaa?agggacgggc?cctccctctc?ccaccaagga?ccggaagaag?960

gatgtttctt?ccccgcagcg?ggcccagtcc?agcccccacc?gcaggaacag?ccgcagtgaa?1020

agacggccct?cgggtgacaa?gaagacttcc?gagcccaaag?ccatgccaga?ccttaacggg?1080

taccagatcc?gggtctga 1098

<210>10

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primers F 3

<400>10

gcggccgcag?atccccatac?cgagtgtg 28

<210>11

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: primer R3

<400>11

tctagagggc?ctctttggtc?atttcc 26

Claims (7)

1, based on the pseudo-virus of Vasohibin gene, it is characterized in that: this puppet virus is the material of the simulation natural viral spline structure that obtained by viral capsid chimeric protein HPV16L1-RGD parcel Vasohibin gene recombination carrier for expression of eukaryon; Described viral capsid chimeric protein HPV16L1-RGD inserts the RGD peptide between the 266th of HPV16L1 and the 267th amino acids.

2, the pseudo-virus based on the Vasohibin gene according to claim 1, it is characterized in that: described viral capsid chimeric protein HPV16L1-RGD has the aminoacid sequence shown in SEQ ID No.2.

3, the pseudo-virus based on the Vasohibin gene according to claim 2, it is characterized in that: the encoding gene of described viral capsid chimeric protein HPV16L1-RGD has the nucleotide sequence shown in SEQ ID No.1.

4, the pseudo-virus based on the Vasohibin gene according to claim 1, it is characterized in that: described Vasohibin gene recombination carrier for expression of eukaryon contains the Vasohibin gene of nucleotide sequence shown in SEQ ID No.9.

5, the pseudo-virus based on the Vasohibin gene according to claim 4, it is characterized in that: described Vasohibin gene recombination carrier for expression of eukaryon adopts the p3xFLAG-CMV-14 carrier for expression of eukaryon.

6, the described pseudo-viral preparation method based on the Vasohibin gene of claim 1 is characterized in that: may further comprise the steps:

The structure of a, viral capsid chimeric protein HPV16L1-RGD

The structure of HPV16L1-RGD gene recombined vector: extract the total RNA of human cervical carcinoma cell strain Caski; Synthetic following primer: primers F 1 has sequence shown in the SEQ ID No.3, and primer R1 has sequence shown in the SEQ ID No.4, and mutagenic primer Fm has sequence shown in the SEQ ID No.5, and mutagenic primer Rm has sequence shown in the SEQ IDNo.6; With the Caski cell total rna is reverse transcription and the overlapping extension PCR method site-directed mutagenesis that template is carried out the HPV16L1 gene, makes up the HPV16L1-RGD gene with sequence shown in the SEQ ID No.1; Overlapping extension PCR method site-directed mutagenesis comprises two-wheeled PCR: first round PCR comprises 2 reactions, with reverse transcription gained HPV16L1 cDNA is template, 1 reaction is the upstream and downstream primer with primers F 1 and mutagenic primer Rm, makes the sheet segment DNA FRm that contains mutational site and upstream sequence thereof; 1 reaction is the upstream and downstream primer with mutagenic primer Fm and primer R1 in addition, makes the fragment DNAFmR that contains mutational site and downstream sequence thereof; First round PCR product carries out second and takes turns PCR after glue recovery purifying is identified, cut to agarose gel electrophoresis, FRm and FmR pass through terminal complementary pairing primer each other, it is the HPV16L1-RGD gene that extension makes the full length DNA FR that contains mutational site and upstream and downstream sequence thereof, is that template, primers F 1 and primer R1 increase for the upstream and downstream primer again with FR; Second takes turns the PCR product promptly gets the HPV16L1-RGD gene after glue recovery purifying is identified, cut to agarose gel electrophoresis; The reaction conditions of above-mentioned two-wheeled PCR is identical: 94 ℃ of pre-sex change of temperature 5 minutes, and 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, last 72 ℃ were extended 4 minutes; The pCR-TOPO carrier is gone in gained HPV16L1-RGD gene clone, be transformed into bacillus coli DH 5 alpha again, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt single endonuclease digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows insertion fragment sequence and the consistent person of sequence shown in the SEQ ID No.1, is the HPV16L1-RGD gene recombined vector pCR II-TOPO/HPV16L1-RGD that successfully constructs;

The structure of HPV16L1-RGD gene recombination baculovirus shuttle vectors: synthetic following primer: primers F 2 has sequence shown in the SEQ ID No.7, and primer R2 has sequence shown in the SEQ ID No.8; With pCRII-TOPO/HPV16L1-RGD is that template, primers F 2 and primer R2 carry out PCR for the upstream and downstream primer, the PCR reaction conditions is: 94 ℃ of pre-sex change of temperature 5 minutes, 1 minute, 45 ℃ annealing of 94 ℃ of sex change were extended 2 minutes for 30 seconds, 72 ℃ then, totally 30 circulations, and last 72 ℃ were extended 4 minutes; The PCR product is identified through agarose gel electrophoresis, after cutting glue recovery purifying, carry out double digestion with restriction enzyme EheI and XhoI, be connected through the baculovirus shuttle vectors pFastBacDual of EheI and XhoI double digestion again with equally, connect product and be transformed into bacillus coli DH 5 alpha, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt double digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows insertion fragment sequence and the consistent person of sequence shown in the SEQ ID No.1, is the HPV16L1-RGD gene recombination baculovirus shuttle vectors pFastBacDual/HPV16L1-RGD that successfully constructs;

The structure of HPV16L1-RGD gene recombination baculovirus: pFastBacDual/HPV16L1-RGD and linearized baculovirus dna cotransfection are carried out homologous recombination in the born of the same parents to the insect cell Sf-9 of logarithmic phase, make up the HPV16L1-RGD gene recombination baculovirus, after the transfection 3 days, collect culture supernatant as virus stock solution used;

The cell inner expression of viral capsid chimeric protein HPV16L1-RGD: the Sf-9 cell that the gained virus stock solution used is infected logarithmic phase, temperature is cultivated after 3 days for 27 ℃, collecting cell, resuspended with PBS, ultrasonic broken wall, low-speed centrifugal, supernatant liquor is transferred to fills in the centrifuge tube of sucrose solution that concentration is 400g/L, ultracentrifugation, abandon supernatant, to precipitate and use the CsCl density gradient ultracentrifugation, buoyant density is the white protein band of 1.34g/mL in the collection centrifuge tube, promptly gets the viral capsid chimeric protein HPV16L1-RGD with sequence shown in the SEQ ID No.2;

B, Vasohibin gene recombination Construction of eukaryotic

Extract the total RNA of people's spleen tissue vascular endothelial cell; Synthetic following primer: primers F 3 has sequence shown in the SEQ IDNo.10, and primer R3 has sequence shown in the SEQ ID No.11; With the total RNA of people's spleen tissue vascular endothelial cell is that template, primers F 3 and primer R3 are reverse transcription and the pcr amplification that the upstream and downstream primer carries out the Vasohibin gene, the PCR reaction conditions is: 95 ℃ of pre-sex change of temperature 10 minutes, 1 minute, 58 ℃ annealing of 94 ℃ of sex change of temperature were extended 4 minutes for 1 minute, 72 ℃ then, totally 30 circulations, final temp extended 10 minutes for 72 ℃; The PCR product is cut glue and is reclaimed purifying purpose fragment after agarose gel electrophoresis is identified, promptly gets the Vasohibin gene; Gained Vasohibin gene is carried out double digestion with restriction enzyme NotI and XbaI, be connected through the carrier for expression of eukaryon p3xFLAG-CMV-14 of NotI and XbaI double digestion again with equally, connect product and be transformed into bacillus coli DH 5 alpha, with the LB plate screening positive colony that contains penbritin, extract recombinant plasmid, adopt double digestion method and PCR method to identify positive colony plasmid and order-checking, sequencing result shows nucleotide sequence person in full accord shown in insertion fragment sequence and the SEQ ID No.9, is the Vasohibin gene recombination carrier for expression of eukaryon p3xFLAG-CMV-14/Vasohibin that successfully constructs; Get the bacillus coli DH 5 alpha that contains p3xFLAG-CMV-14/Vasohibin, amplification cultivation, a large amount of extracting recombinant plasmids promptly get p3xFLAG-CMV-14/Vasohibin;

C, based on the preparation of the pseudo-virus of Vasohibin gene

Get step a gained viral capsid chimeric protein HPV16L1-RGD, add denaturing agent and make protein denaturation, add step b gained Vasohibin gene recombination carrier for expression of eukaryon p3xFLAG-CMV-14/Vasohibin again, dialysed overnight in PBS, make metaprotein renaturation gradually, HPV16L1-RGD parcel p3xFLAG-CMV-14/Vasohibin in renaturation process, self-assembly forms the pseudo-virus based on the Vasohibin gene.

7, the described application of pseudo-virus in the preparation antitumor drug of claim 1 based on the Vasohibin gene.

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