CN106754758A - A kind of construction method of the restructuring enterovirus phenotype hybrid system of factor-containing adjuvant and its application - Google Patents

Abstract

The invention provides a method for constructing a recombinant enterovirus phenotype mixing system containing a cytokine adjuvant. First, construct a VP1 recombinant cell line and a recombinant attenuated enterovirus strain loaded with cytokine genes; The enterovirus strain is obtained by infecting and amplifying the VP1 recombinant cell line; the VP1 recombinant cell line is a cell line expressing one or two types of VP1 of Enterovirus 71 and Coxsackievirus A group 16; Recombinant attenuated enterovirus strains with cytokine genes are selected from coxsackievirus group B type 3, enterovirus 71 or poliovirus type I; cytokines are selected from cholera toxin or interferon gamma; cytokine genes are loaded The recombinant attenuated enterovirus infects the VP1 recombinant cell line, and the progeny virus is harvested after cell lesions. The progeny virus can activate host immunity and produce immune protection against a variety of enteroviruses, which has application value.

Description

A construction method of recombinant enterovirus phenotype mixing system containing cytokine adjuvant law and its application

technical field

The invention relates to the field of biotechnology, more specifically, to a construction method and application of a recombinant enterovirus phenotype mixing system containing a cytokine adjuvant.

Background technique

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus infection. It mainly occurs in infants and young children under 5 years old. Herpes or ulcers appear in the oral mucosa, perianal and other parts. Hand, foot and mouth disease is usually self-limiting, and most children recover by themselves in about a week, but a small number of children will develop complications of the central nervous system, cardiovascular system, and respiratory system such as aseptic meningitis, encephalitis, myocarditis, and pulmonary edema. Rapidly progresses to severe hand, foot and mouth disease, which can cause death in severe cases. Hand, foot and mouth disease is highly prevalent in my country and poses a huge threat to public health in my country. In 2008, the Ministry of Health included it as a Class C infectious disease for management.

Enteroviruses that cause HFMD include Coxsackievirus A group 2-10, 12, 16, and 24, B group 1-5, and enterovirus 71 (EV71), among which the most important pathogens are EV71 and Coxsackievirus group A type 16 (CA16). Usually, the symptoms caused by CA16 are relatively mild but the number of infected cases is large. EV71 is more likely to cause severe hand, foot and mouth disease such as neurological complications, and the isolation rate of CA6 in the epidemic since 2012 is also increasing. At present, there is a lack of effective drugs for the treatment of HFMD, and symptomatic and supportive treatments are mainly used.

CA16, EV71, and CA6 all belong to the genus Enterovirus of the family Picornaviridae, and are non-enveloped, positive-sense, single-stranded RNA viruses. , in which VP1 is its main capsid protein, and it is also a virus adsorption protein, which plays a decisive role in the process of virus adsorption and penetration, so VP1 is also the neutralizing antigen of the virus. Hand, foot and mouth disease is a serious epidemic situation, and in terms of preventive measures, only inactivated vaccines against EV71 virus are currently on the market, and there is no vaccine application for CA16.

Coxsackie group B type 3 virus, poliovirus types I, II and III also belong to the genus Enterovirus in the family Picornaviridae, and the viruses of the genus Enterovirus have similar biological characteristics. Among them, during the replication cycle, viruses of the Enterovirus genus use the genome as a template to read through and transcribe into an mRNA, which is translated into a precursor protein, and then self-cleaved by viral proteases to form a variety of viral proteins. In the packaging stage, the viruses VP1, VP2, VP3 and VP4 in the infected cells will form progeny virus capsids, wrap the progeny genome and finally form a complete progeny virus for release. During the packaging process, the functions of VP1 of Enteroviruses are similar, so if a cell is infected by two or more Enteroviruses, phenotypic mixing will occur, that is, the genome of one virus will be replaced by another. The capsids and genomes of this virus are derived from different enteroviruses.

The phenomenon of phenotypic mixing of Enteroviruses has potential value in vaccine research, but there are problems in the actual operation of natural phenotypic mixing. Two or more enteroviruses at the same time will cause interference, which will reduce the proliferation efficiency of the virus, and the replication and proliferation efficiency of different viruses after mixed infection is different, and effective quality control cannot be carried out. However, for the application value of the vaccine Virus strains require high replication efficiency, which is why the current vaccine research field uses single-type viruses to multiply separately, and then mixes the various types of vaccines prepared separately as needed, instead of using multiple viruses to infect cells at the same time to multiply viruses, and not to use Mechanisms of phenotypic mixing.

For viruses mixed with "natural" phenotypes, in addition to interference phenomena and bottlenecks of different proliferation efficiencies, if they are sourced from strong virus strains, they need to be inactivated and prepared as inactivated vaccines. On the one hand, the inactivated vaccines are Utilize the inactivated non-infectious virus capsid, after intramuscular injection, rely on professional antigen-presenting cells in the body for processing, activate the body to generate an immune response, and present exogenous antigens dependent on MHC-II molecules, while natural Under certain conditions, virus infection is more likely to activate the body's immunity in the form of MHC-I molecule-dependent endogenous antigen presentation, so the protection is not as good as that of attenuated live vaccines based on attenuated strains. On the other hand, if attenuated enterovirus strains are used for phenotypic mixing, there will also be bottlenecks of interference and uneven proliferation efficiency. Mixed virus strains infect cells, because functional complementarity between strains can occur, and unexpected gene recombination will occur, which will greatly increase the risk of attenuated strains recovering virulence.

Therefore, if the enterovirus phenotypic mixing phenomenon is applied to the research and development of enterovirus vaccines, it is necessary to establish a safe and efficient method of phenotypic mixing system, which can avoid the interference phenomenon and multiplication caused by simultaneous infection of multiple viruses. Efficiency varies, unexpected genetic recombination, and attenuated strains restore virulence, and at the same time, the immune protection effect is better than that of inactivated vaccines.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art, and provide a method for constructing a recombinant enterovirus phenotype mixing system containing cytokine adjuvant.

The second object of the present invention is to provide the recombinant enterovirus phenotype mixing system containing cytokine adjuvant obtained by the method.

A third object of the invention is to provide the application of said mixing system.

The purpose of the present invention is achieved through the following technical solutions:

A method for constructing a recombinant enterovirus phenotype mixed system containing a cytokine adjuvant, first constructing a VP1 recombinant cell line and a recombinant attenuated enterovirus strain loaded with cytokine genes; Virus enterovirus strains are infected and amplified in the VP1 recombinant cell line to obtain a recombinant enterovirus phenotype mixed system containing cytokine adjuvant; the VP1 recombinant cell line expresses enterovirus type 71, Ke One or two cell lines of VP1 of Saatchie virus group A type 16; the recombinant attenuated enterovirus strain loaded with cytokine genes is selected from coxsackie virus group B type 3, enterovirus type 71 or Poliovirus type I; said cytokine is selected from cholera toxin or interferon gamma.

The system includes two parts: a VP1 recombinant cell line and an attenuated enterovirus strain loaded with cytokine genes. Among them, the VP1 recombinant cell line is: construct the VP1 eukaryotic expression vector of enterovirus type 71 and Coxsackie virus group A type 16, and obtain the recombinant expression cell line stably expressing VP1 protein from various sources through transfection and screening. The background strains of attenuated enterovirus loaded with cytokine genes are: attenuated strain of enterovirus type 71, or attenuated strain of saatchie virus group B type 3, or attenuated strain of poliovirus type I strain. On the basis of the above-mentioned background strains, construct an infectious clone, insert the cytokine gene between the P1 and P2 regions of the viral genome, and add a viral protease cleavage site, transfect the infectious clone vector and rescue the virus to obtain An attenuated enterovirus capable of exogenously expressing cytokines. The cytokines can be cholera toxin or interferon gamma. In this system, after the recombinant attenuated enterovirus containing the cytokine gene is infected with other enterovirus VP1 recombinant expression cell lines, the progeny virus will mix the VP1 of other enteroviruses into the offspring during the packaging stage of virus proliferation. in the viral capsid. For example, the virus core of the progeny virus after propagation is the genome of an attenuated strain of Coxsackievirus group B type 3, while the VP1 in the viral capsid on its surface is mixed, containing genes derived from enterovirus type 71 , VP1 of Coxsackievirus group A type 16. The obtained progeny virus can infect the host, but because the core of the virus is the genome of the recombinant attenuated enterovirus strain containing the loaded cytokine gene, its replication and pathogenicity in the host are limited; at the same time, the recombinantly expressed cells Factors can enhance the immune response of the host, and the progeny viruses contain VP1 proteins of various enteroviruses, and VP1 is a type-specific neutralizing antigen of enteroviruses, so the progeny viruses formed by this system can activate the host Immunity, produces immune protection against a variety of enteroviruses.

Preferably, the preparation method of the recombinant enterovirus phenotype mixing system containing cytokine adjuvant comprises the following steps:

S1. Amplify the VP1 gene of enterovirus type 71 and/or coxsackievirus group A type 16, connect it to a eukaryotic expression vector, and obtain a VP1 recombinant cell line after transfection and screening;

S2. Cloning the entire genome of coxsackievirus group B type 3, enterovirus type 71 or poliovirus type I, and inserting cytokines between the P1 and P2 regions of the virus genome under the reverse genetics system A recombinant attenuated enterovirus strain loaded with cytokine genes was obtained;

S3. The VP1 recombinant cell line obtained from S1 is obtained by infecting the recombinant attenuated enterovirus strain loaded with cytokine genes obtained in S2.

The preparation methods of the VP1 recombinant cell line and the recombinant attenuated enterovirus strain loaded with cytokine genes in the present invention are conventional techniques in the field of molecular biology.

Preferably, the preparation method of the VP1 recombinant cell line is to extract the RNA of the corresponding virus, and perform reverse transcription using polyA or random primers to obtain cDNA. Using the specific primers of the corresponding virus, the VP1 gene of the corresponding virus is amplified by PCR, and the VP1 gene fragment is inserted into the eukaryotic expression vector by enzyme-cut ligation. Eukaryotic expression vectors can use Neo or Puyo resistance genes, and the constructed eukaryotic expression vectors are transfected into eukaryotic Vero cells (or 293, Hep2, HeLa, etc. cells) using liposomes, and then use G418 or Puromycin After screening by protein and identified by immunoblotting and immunofluorescence, a part of the phenotypic mixed system, that is, the VP1 recombinant cell line, was obtained.

Preferably, the preparation method of the recombinant attenuated enterovirus loaded with cytokine genes is as follows: a certain type of the above enterovirus is subjected to whole-genome cloning to construct viral infectious clones, and under the reverse genetics system, the A region coding for a protease cleavage site and a specific restriction endonuclease region are introduced between the P1 and P2 regions, and then cytokines are inserted using the restriction endonuclease cleavage sites. The cytokine is cholera toxin to promote the mucosal immune response of the recombinant attenuated strain in the intestinal mucosa; interferon gamma can also be used to promote the presentation of MHC-II antigens and improve the subsequent immunity of VP1 containing a variety of enterovirus sources response efficiency.

The present invention also provides the recombinant enterovirus phenotype mixing system containing cytokine adjuvant obtained by the above method.

In this way, the recombinant attenuated enterovirus loaded with cytokine genes infects the VP1 recombinant cell line, harvests the progeny virus after the cell pathology, and obtains the VP1 capsid containing a variety of different sources of enterovirus, and the genome core of the attenuated enterovirus strain. , A mixed phenotype recombinant attenuated strain that can exogenously express cytokines; the progeny virus formed by this system can activate host immunity and produce immune protection against a variety of enteroviruses.

Therefore, the present invention also provides the application of the recombinant enterovirus phenotype mixing system containing cytokine adjuvant in the method of preparing a vaccine for treating enterovirus.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a method for constructing a recombinant enterovirus phenotype mixing system containing a cytokine adjuvant. First, respectively construct a VP1 recombinant cell line and a recombinant attenuated enterovirus strain loaded with cytokine genes; Recombinant attenuated enterovirus strains of the gene are infected and amplified in the VP1 recombinant cell line. Remember the recombinant enterovirus phenotype mixed system containing cytokine adjuvant; the VP1 recombinant cell line expresses enterovirus 71 alone or simultaneously One or two cell lines of VP1 of Coxsackie virus group A type 16; the recombinant attenuated enterovirus strain loaded with cytokine genes is selected from Coxsackie virus group B type 3, enterovirus Type 71 or poliovirus type I; the cytokine is selected from cholera toxin or interferon gamma; the recombinant attenuated enterovirus loaded with cytokine gene infects the VP1 recombinant cell line, harvests the progeny virus after the cell pathology, and obtains the virus containing A variety of enterovirus VP1 capsids from different sources, a mixed phenotype recombinant attenuated strain containing the genome core of an attenuated enterovirus strain, and exogenously expressing cytokines; the progeny virus formed by this system can activate host immunity , to generate immune protection against a variety of enteroviruses, and this phenotypic hybrid system has practical and broad application value.

Description of drawings

Figure 1 is the PCR amplification electrophoresis of VP1 fragment of CA16 and EV71 strains; M: NEB 1kb DNA ladder; 1: PCR product of VP1 fragment of CA16 strain; 2: PCR product of VP1 fragment of EV71 strain.

Figure 2 shows the digestion and recovery of VP1 fragments of CA16 and EV71 strains and the eukaryotic expression vector pcFlag; M: NEB 1kbDNA ladder; 1: Digestion products of VP1 fragments of CA16 strains; 2: Digestion products of VP1 fragments of EV71 strains; 3 : pcFlag digestion product.

Figure 3 is the colony PCR identification of the VP1 eukaryotic expression plasmid pcFlag-CA16-VP1 of CA16 and the VP1 eukaryotic expression plasmid pcFlag-EV71-VP1 of EV71; M: NEB 1kb DNA ladder; 1-8: pcFlag-CA16-VP1 colony PCR products; 9-16: pcFlag-EV71-VP1 colony PCR products.

Figure 4 is the Hind III and Xho I double digestion identification of pcFlag-CA16-VP1 and pcFlag-EV71-VP1; M: NEB 1kb DNA ladder; 1-2: pcFlag-CA16-VP1 plasmid Hind III and Xho I double digestion Identification; 9-16: pcFlag-EV71-VP1 plasmid Hind III and Xho I double enzyme digestion identification.

Figure 5 is the immunofluorescence detection of CA16 and EV71 recombinant VP1 protein expression in cells.

Figure 6(a) The full-length Sabin1 gene was amplified by three-segment PCR. The size of the F1 fragment was 2510bp, the size of the F2 fragment was 3210bp, and the size of the F3 fragment was 1880bp. (b) The full length of Sabin1 was obtained by PCR bridging of three fragments, and the size of Sabin1 was 7441bp. (c) The Sabin1 gene obtained by PCR bridging and the pMD19-T vector were connected by EcoR1 and Sal1 cohesive ends to obtain two cloning vectors, pv-1 and pv-2, which were identified by digestion with BamH1, and four fragment bands were theoretically obtained. They are 5730bp, 2471bp, 1879bp, and 30bp, respectively. There are only three target bands in electrophoresis analysis, and 30bp is too small to be identified, so it can be determined as a positive clone. (d) The pv-1 and pv-2 Sabin1 vectors were cut with EcoR1 and Sal1 restriction enzymes to obtain a Sabin1 fragment with a size of 7441bp and a pMD19-T vector fragment with a size of about 2692bp.

Figure 7(a) Recover target fragments by enzyme digestion of Sabin1-f1, Sabin1-f2, Sabin1-f3, and Sabin1-f4 cloned plasmids. The size of the Sabin1-f1 fragment is 2481bp, the size of the Sabin1-f2 fragment is 2136bp, and the Sabin1-f3 fragment The size is 1017bp, and the size of the Sabin1-f4+pMD19-T fragment is 4487bp. (b) Sabin1-f1, Sabin1-f2, Sabin1-f3, Sabin1-f4+pMD19-T segmented connection to obtain the full length of Sabin1 gene, identified by BamH1 restriction endonuclease digestion, theoretically obtained four fragment bands, The sizes are 5730bp, 2471bp, 1879bp, and 30bp. There are only three target bands in electrophoresis analysis, and 30bp is too small to be identified. It can basically be determined that Sabin1-1, Sabin1-2, and Sabin1-3 are positive clones.

Figure 8(a) The Sabin1 vector constructed by PCR bridging was digested with EcoR1 and Sal1 restriction endonucleases to recover the Sabin1 gene, and then ligated with pcDNA3 that was digested with EcoR1 and Xho1 restriction endonucleases to obtain the cohesive ends of EcoR1 and Xho1, The cloned plasmid was identified by digestion with BamH1. In theory, there were 5 fragments of different sizes, which were 8236bp, 2471bp, 1879bp, 256bp, and 30bp. Among them, for agarose gel electrophoresis identification, fragments of 8236bp, 2471bp, and 1879bp can be seen, and 256bp and 30bp are too small to be effectively identified, and they are initially determined to be positive clones. (b) The pMD19-T_Sabin1 vector constructed by fragment ligation was digested with EcoR1 and Not1 restriction endonucleases to recover the Sabin1 fragment and the clone obtained by pcDNA3 ligation using EcoR1 and Not1 restriction endonucleases, using EcoR1 and Sal1 Restriction endonuclease digestion identification, there are 5 fragments of different sizes in theory: 7441bp, 2285bp, 2188bp, 903bp, 34bp. The actual agarose gel electrophoresis identification can only distinguish two bands of 7441bp and (2285bp, 2188bp), and the preliminary analysis is a positive clone.

Figure 9 is a plasmid map of pCV-CS.

detailed description

The content of the present invention will be further described below in conjunction with specific examples, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, simple modifications or replacements made to the methods, steps or conditions of the present invention all belong to the scope of the present invention; unless otherwise specified, the technical means used in the embodiments are those skilled in the art. well-known conventional means.

Example 1

1. Construction of cells stably expressing VP1 of CA16 and EV71

The specimens of coxsackievirus A group 16 (hereinafter referred to as CA16) and enterovirus 71 (hereinafter referred to as EV71) infected cases confirmed by clinical and laboratory were used to extract the viral RNA of the infected cases respectively by using the virus RNA extraction kit. SuperScript III reverse transcriptase and random primers were used to reverse transcribe the viral cDNA of CA16 and EV71.

Using CA16 and EV71 viral cDNA as templates, the upstream primer of CA16-VP1 gene: 5'-CCTAAGCTTTCTGGGTACTTTGACTATTACACC, and the downstream primer 5'-CAGCTCGAGTCATGTTGTTATCTTGTCTCTACTAC. The upstream primer of EV71-VP1 gene: 5'-CCTAAGCTTTATGCCCGAGATGGAGTGTTTGAC, the downstream primer 5'-CAGCTCGAGTCATTTCCCAAGAGTGGTGATTGCTG.

Amplification reaction conditions: pre-denatured at 94°C for 3 minutes, then cycled, denatured at 94°C for 30 seconds, annealed at 52°C for 30 seconds, extended at 72°C for 2 minutes, and extended at 72°C for 5 minutes after 30 cycles. The corresponding product fragments were recovered by 1.2% agarose gel electrophoresis. The size of the main band of the PCR product was about 1100bp, and the result was in line with the prediction (Figure 1).

The VP1 PCR products of CA16 and EV71 strains were electrophoresed on agarose gel, and the amplified main band of the expected size was recovered, and the VP1 fragment and the pCMV-C-Flag plasmid (referred to as pcFlag plasmid) were double digested with Hind III and Xho I , the digested products were recovered by agarose gel electrophoresis again, and the VP1 of CA16 and EV71 strains were respectively ligated with the pCMV-C-Flag plasmid (Figure 2). The VP1 of the CA16 and EV71 strains were respectively connected with the pCMV-C-Flag plasmid. After transforming the competent cells, 8 single colonies were selected for colony PCR identification after resistance screening. As shown in Figure 3, the VP1 eukaryotic of CA16 The 16 colonies of VP1 eukaryotic expression plasmid pcFlag-EV71-VP1 expressing plasmid pcFlag-CA16-VP1 and EV71 could amplify the corresponding VP1 fragment.

Pick 2 clones from each of the positive colonies identified by colony PCR for bacterial culture and plasmid extraction, and carry out enzyme digestion identification. As shown in Figure 4, after double digestion with Hind III and Xho I, about 1100bp of VP1 exogenous Fragment and about 5kb pcFlag vector fragment. The identified plasmids were sequenced and analyzed, and the results were completely correct. The plasmids were named pcFlag-CA16-VP1 and pcFlag-EV71-VP1, respectively.

293T cells, RD cells, and Vero cells were all cultured in DMEM (containing 10% FBS) at 37°C, 5% CO ₂ , and saturated with water. Before transfection, cells grown to a confluence rate of more than 95% were inoculated in a 6-well cell culture plate, and transfected when the cells grew for 20-24 hours until the cell density reached 70-90%. The transfection operation was carried out according to the operation manual of Lipofectamine 2000 transfection reagent.

The pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids verified by enzyme digestion and sequencing were transfected into 293T and RD cells, and the FLAG antibody was used for western blot detection. A band of expected size appeared at the position of about 32Kd, indicating eukaryotic expression The plasmid can express FLAG-fused VP1 recombinant protein in 293T and RD cells.

The pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids were transfected into 293T cells, and the expression of recombinant VP1 was detected by immunofluorescence. As shown in Figure 5, CA16 and EV71 recombinant VP1 proteins were mainly distributed in the cytoplasm of the cells.

The pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids were co-transfected into Vero cells at a ratio of 1:1, and were screened with G418 72 hours after transfection. After about 3 weeks of screening, resistant cell clones were obtained. A Vero cell line stably expressing VP1 of CA16 and EV71 was obtained.

2. Preparation of recombinant attenuated enterovirus strains loaded with cytokine genes

Attenuated enterovirus type 3, attenuated enterovirus 71, or attenuated poliovirus type 1 (Sabin vaccine strain) can be used as the attenuated enteric background virus strain. Cloning the full length of virus strains, constructing infectious cloning and reverse genetics systems.

The poliovirus Sabin strain type I was used as the background virus, and cloned by segmental cloning. The primer sequences are as follows:

The full-length genome was cloned by bridging PCR, and the type I reverse genetic plasmid system of the Sabin strain was identified (Figure 6-8). Then use PCR to introduce a restriction enzyme cutting site containing a protease cutting site into the P1 and P2 regions of the Sabin strain type I reverse genetic system plasmid, and then insert the cholera toxin avirulent B subunit gene fragment. Then, after transfection into Vero cells, the recombinant attenuated enterovirus strain Sabin strain I containing the loaded cytokine gene was rescued.

The pCV-CS plasmid, which contains the whole genome of attenuated Coxsackievirus group B type 3, and a restriction enzyme site containing a protease site (Figure 9), inserts the avirulent B subunit of cholera toxin Gene fragment. After transfection into Vero cells, the recombinant attenuated enterovirus strain Coxsackievirus B group 3 containing the loaded cytokine gene was rescued.

Recombinant attenuated enterovirus strain Sabin strain type 1 containing loaded cytokine genes, or recombinant attenuated enterovirus strain Coxsackievirus group B type 3 containing loaded cytokine genes, in VP1 stably expressing CA16 and EV71 The Vero cell line was infected, and the cell supernatant was harvested after 48 hours, and recombinant enteroviruses with mixed phenotypes were obtained by ultracentrifugation.

SEQUENCE LISTING

<110> Shantou University School of Medicine

<120> Construction method and application of a recombinant enterovirus phenotype mixing system containing cytokine adjuvant

<130>

<160> 4

<170> PatentIn version 3.3

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<211> 33

<212>DNA

<213> Upstream primer for CA16-VP1 gene

<400> 1

cctaagcttt ctgggtactt tgactattac acc 33

<210> 2

<211> 35

<212>DNA

<213> Downstream primer of CA16-VP1 gene

<400> 2

cagctcgagt catgttgtta tcttgtctct actac 35

<210> 3

<211> 33

<212>DNA

<213> Upstream primer for EV71-VP1 gene

<400> 3

cctaagcttt atgcccgaga tggagtgttt gac 33

<210> 4

<211> 35

<212>DNA

<213> Downstream primer of EV71-VP1 gene

<400> 4

cagctcgagt catttcccaa gagtggtgat tgctg 35

Claims (4)

1. a kind of factor-containing adjuvant restructuring enterovirus phenotype hybrid system construction method, it is characterised in that first The recombinant attenuated enterovirus strain of VP1 recombinant cell lines and load cells factor gene is built respectively；Then by load cells because Subbase because recombinant attenuated enterovirus strain infection amplification carried out in VP1 recombinant cell lines obtain final product the weight of factor-containing adjuvant Group enterovirus phenotype hybrid system；The VP1 recombinant cell lines are for individually or simultaneously expression enterovirns type 71, COxsackie are sick One or two the cell line of the VP1 of the malicious type of A groups 16；The recombinant attenuated enterovirus of the load cells factor gene is selected good strains in the field for seed From Coxsackievirus B3, enterovirns type 71 or poliovirus I types；The cell factor is selected from cholera toxin Or interferon gamma.

2. according to claim 1 factor-containing adjuvant restructuring enterovirus phenotype hybrid system preparation method, its Feature is having, and comprises the following steps：

S1. the VP1 genes of amplification enterovirns type 71 and/or coxsackievirus A16, are connected in carrier for expression of eukaryon, VP1 recombinant cell lines are obtained after transfection, screening；

S2. Coxsackievirus B3, enterovirns type 71 or poliovirus I types are carried out into genome cloning, Under reverse genetics system, insertion cell factor obtains load cells factor gene between P1 the and P2 areas of viral full-length genome Recombinant attenuated enterovirus strain；

The VP1 recombinant cell lines that the recombinant attenuated enterovirus strain infection S1 of the load cells factor gene that S3. S2 is obtained is obtained Obtain final product.

3. the methods described of claim 1 or 2 obtain factor-containing adjuvant restructuring enterovirus phenotype hybrid system.

4. the restructuring enterovirus phenotype hybrid system of factor-containing adjuvant described in claim 3 is preparing treatment enterovirus Vaccine approach application.