CN106636001B - Construction method and application of recombinant enterovirus phenotype mixing system - Google Patents
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Abstract
The invention provides a construction method of a recombinant enterovirus phenotype mixing system, which is characterized in that a VP1 recombinant cell line is constructed, and the attenuated enterovirus strain is infected and amplified in a VP1 recombinant cell line to obtain the recombinant enterovirus phenotype mixing system; the VP1 recombinant cell line is a cell line which singly or simultaneously expresses one or two of VP1 of enterovirus 71 type and Coxsackie virus A group 16 type; the attenuated enterovirus strain is selected from coxsackievirus group B3, enterovirus 71 or poliovirus I; the attenuated enterovirus strain infects VP1 recombinant cell line, and obtains progeny virus after cytopathic effect to obtain mixed phenotype recombinant attenuated strains containing various enterovirus VP1 capsids with different sources and genome core of the attenuated enterovirus strain; the progeny virus formed by the system can activate host immunity and generate immune protection against various enteroviruses, and the phenotype hybrid system has practical and wide application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a construction method and application of a recombinant enterovirus phenotype mixing system.
Background
Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus infection, is mostly seen in infants under 5 years old, and is clinically manifested as fever, anorexia, crying and screaming, and herpes or ulcer appears in the hands, feet, oral mucosa, perianal area, etc. The hand-foot-and-mouth disease is usually self-limiting, most children can heal the disease in one week or so, but a few children can have complications of central nervous system, cardiovascular system and respiratory system such as aseptic meningitis, encephalitis, myocarditis, pulmonary edema and the like, rapidly progress to severe hand-foot-and-mouth disease, and can cause death in severe cases. The hand-foot-and-mouth disease is highly developed in China, brings great threat to public health of China, and is brought into class C infectious diseases by the health department in 2008 for management.
The enteroviruses causing HFMD include coxsackievirus A groups 2-10, 12, 16 and 24, enterovirus B groups 1-5, enterovirus 71 (EV 71) and the like, wherein the most important pathogenies are EV71 and coxsackievirus A group 16 (CA 16). 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 nervous system complications, and the separation rate of CA6 is increasingly higher in the prevalence of 2012. At present, effective treatment medicines for treating hand-foot-and-mouth diseases are mainly used for symptomatic and supportive treatment.
CA16, EV71 and CA6 all belong to enterovirus genus of picornaviridae, are forward single-stranded RNA viruses without envelope, the capsid is icosahedral symmetrical structure, the capsid is composed of VP1, VP2, VP3 and VP4, wherein VP1 is the main capsid protein, and is also the virus adsorption protein, and plays a determining role in the virus adsorption and penetration process, therefore VP1 is the virus neutralizing antigen. The hand-foot-and-mouth disease is serious in epidemic situation, only inactivated vaccines aiming at EV71 virus are on the market at present in the aspect of prevention means, and no vaccine is applied to CA 16.
Coxsackie group B3 viruses, poliovirus type I, II and III also belong to the genus Enterovirus of the family picornaviridae, the viruses of the genus Enterovirus being similar in biological properties. In the replication cycle, viruses of the enterovirus genus all use genomes as templates, are read and transcribed into a mRNA, are translated into precursor proteins, and are self-cleaved under the action of viral proteases to form a plurality of viral proteins. During the packaging stage, viruses VP1, VP2, VP3 and VP4 in the infected cells will form progeny virus capsids, wrap the progeny genome and eventually form complete progeny viruses for release. During packaging, viruses VP1 of Enterovirus have similar functions, and thus, if a cell is infected by more than two Enterovirus, a phenomenon of mixed phenotypes occurs, i.e., the genome of one virus is enveloped by the capsid of another virus, and the capsid and genome of the viruses are derived from different Enterovirus.
The phenomenon of phenotype mixing of enteroviruses has potential value in vaccine research, but problems exist in the actual operation process of natural phenotype mixing. Two or more than two enteroviruses can cause interference phenomenon by cells at the same time, so that the proliferation efficiency of the viruses is reduced, the replication and proliferation efficiency of different viruses after mixed infection is different, and effective quality control cannot be carried out, while high replication efficiency is required for strains with application value of the vaccines, which means that single-type viruses are respectively proliferated in the current vaccine research field, and then the separately prepared vaccines are mixed according to the requirement without simultaneously infecting the cells with multiple viruses to proliferate the viruses, and a phenotype mixing mechanism is not utilized.
For the virus with mixed 'natural' phenotype, except the bottleneck of interference phenomenon and different proliferation efficiency, if the virus is a virulent strain source, inactivation treatment is needed to prepare the inactivated vaccine, on one hand, the inactivated vaccine utilizes the inactivated and non-infectious virus capsid, after intramuscular injection, the inactivated vaccine is processed by depending on the in vivo professional antigen presenting cells, the organism is activated to generate immune response, the exogenous antigen presentation depending on MHC-II molecules is realized, and the virus infection under natural conditions is more to activate the organism immunity by depending on the endogenous antigen presentation mode of MHC-I molecules, so the protective force is not as good as that of the attenuated live vaccine based on attenuated strains. On the other hand, if attenuated enterovirus strains are used for phenotype mixing, the bottleneck of interference phenomenon and inconsistent propagation efficiency exists, and meanwhile, if the mixed phenotype application is more limited, a plurality of attenuated enterovirus strains are mixed to infect cells, because the strains can generate functional complementation and unexpected gene recombination, the risk of returning the attenuated strains to virulence can be greatly increased.
Therefore, if the enterovirus phenotype mixing phenomenon is applied to the research and development of an enterovirus vaccine, a safe and efficient phenotype mixing system needs to be established, so that the interference phenomenon, the inconsistent propagation efficiency, the unexpected gene recombination and the virus strain reversion virulence caused by the simultaneous infection of a plurality of viruses can be avoided, and the immune protection effect is better than that of an inactivated vaccine.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a construction method of a recombinant enterovirus phenotype mixing system.
The second purpose of the invention is to provide a recombinant enterovirus phenotype mixing system obtained by the method.
A third object of the invention is to provide an application of the hybrid system.
The purpose of the invention is realized by the following technical scheme:
a construction method of a recombinant enterovirus phenotype mixing system comprises the steps of constructing a VP1 recombinant cell line, and carrying out infection amplification on an attenuated enterovirus strain in a VP1 recombinant cell line to obtain the recombinant enterovirus phenotype mixing system; the VP1 recombinant cell line is a cell line which singly or simultaneously expresses one or two of VP1 of enterovirus 71 type and Coxsackie virus A group 16 type; the attenuated enterovirus strain is selected from coxsackievirus group B type 3, enterovirus type 71 or poliovirus type I.
Stably transfecting cells for expressing various enterovirus VP1 proteins by exogenous recombination, amplifying attenuated enteroviruses, and obtaining the attenuated enteroviruses containing various different enterovirus VP1 virus capsids. The system comprises two parts, a VP1 recombinant cell line and an attenuated enterovirus strain. In this system, after infection of the enterovirus VP1 recombinant expression cell line by the attenuated enterovirus strain, progeny viruses will mix VP1 from other enteroviruses into the progeny virus capsid, for example, during the packaging phase of virus propagation. The proliferated progeny virus has Coxsackie virus B group 3 attenuated strain genome as the virus core and surface virus capsid with VP1 mixed and containing VP1 from enterovirus 71 and Coxsackie virus A group 16. The progeny virus obtained can infect a host, but its replication and pathogenicity in the host is limited because the viral core is the genome of an attenuated enterovirus strain; and the progeny virus contains VP1 proteins of various enteroviruses, and VP1 is a neutralizing antigen with type specificity of the enteroviruses, so that the progeny virus formed by the system can activate host immunity and generate immune protection against various enteroviruses.
Preferably, the method for preparing the recombinant enterovirus phenotype mixing system comprises the following steps:
s1, amplifying VP1 genes of enterovirus 71 type and/or coxsackievirus A group 16 type, connecting to a eukaryotic expression vector, and obtaining a VP1 recombinant cell line after transfection and screening;
s2, infecting the recombinant attenuated enterovirus strain with the VP1 recombinant cell line obtained from S1.
The VP1 recombinant cell line and the preparation method of the recombinant attenuated enterovirus strain loaded with cytokine genes are all conventional techniques in the field of molecular biology.
Preferably, the VP1 recombinant cell line is prepared by extracting RNA of a corresponding virus, performing reverse transcription by utilizing polyA or random primers to obtain cDNA, utilizing specific primers of the corresponding virus, performing PCR amplification to obtain VP1 gene of the corresponding virus, inserting VP1 gene fragment into a eukaryotic expression vector by utilizing enzyme digestion connection, wherein the eukaryotic expression vector can use Neo or Puyo resistance gene, the constructed eukaryotic expression vector is transfected into eukaryotic Vero cells (or 293, Hep2, He L a and other cells) by utilizing liposome, then screening by utilizing G418 or puromycin, and obtaining one part of the phenotype hybrid system, namely the VP1 recombinant cell line after immunoblot identification and immunofluorescence identification.
The invention also provides a recombinant enterovirus phenotype mixing system obtained by the method.
Thus, the attenuated enterovirus strain infects VP1 recombinant cell line, and obtains progeny virus after cytopathic effect to obtain mixed phenotype recombinant attenuated strains containing various enterovirus VP1 capsids with different sources and genome core of the attenuated enterovirus strain; the progeny virus formed by the system can activate host immunity and generate immune protection against various enteroviruses.
The invention also provides application of the recombinant enterovirus phenotype mixing system in a method for preparing a vaccine for treating enterovirus.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a construction method of a recombinant enterovirus phenotype mixing system, which is characterized in that a VP1 recombinant cell line is constructed, and the attenuated enterovirus strain is infected and amplified in a VP1 recombinant cell line to obtain the recombinant enterovirus phenotype mixing system; the VP1 recombinant cell line is a cell line which singly or simultaneously expresses one or two of VP1 of enterovirus 71 type and Coxsackie virus A group 16 type; the attenuated enterovirus strain is selected from coxsackievirus group B3, enterovirus 71 or poliovirus I; the attenuated enterovirus strain infects VP1 recombinant cell line, and obtains progeny virus after cytopathic effect to obtain mixed phenotype recombinant attenuated strains containing various enterovirus VP1 capsids with different sources and genome core of the attenuated enterovirus strain; the progeny virus formed by the system can activate host immunity and generate immune protection against various enteroviruses, and the phenotype hybrid system has practical and wide application value.
Drawings
FIG. 1 is a PCR amplification electrophoretogram of VP1 fragment of CA16 and EV71 strains; m: NEB 1kb DNA ladder; 1: the PCR product of VP1 fragment of CA16 strain; 2: EV71 strain VP1 fragment PCR product.
FIG. 2 is the restriction enzyme recovery of the VP1 fragment of CA16 and EV71 strains and the eukaryotic expression vector pcFlag; m: NEB 1kbDNA ladder; 1: the enzyme digestion product of the VP1 fragment of CA16 strain; 2: the enzyme digestion product of the EV71 strain VP1 fragment; 3: the pcFlag enzyme digestion product.
FIG. 3 is a colony PCR identification of VP1 eukaryotic expression plasmid pcFlag-CA16-VP1 of CA16 and VP1 eukaryotic expression plasmid pcFlag-EV71-VP1 of EV 71; m: NEB 1kb DNA ladder; 1-8: PCR product of pcFlag-CA16-VP1 colony; 9-16: PCR product from colony of pcFlag-EV71-VP 1.
FIG. 4 shows pcFlag-CA16-VP1 and pcFlag-EV71-VP1HindIII andXhoi, double enzyme digestion identification; m: NEB 1kb DNA ladder; 1-2: pcFlag-CA16-VP1 plasmidHindIII andXhoi, double enzyme digestion identification; 9-16: pcFlag-EV71-VP1 plasmidHindIII andXhoi, double enzyme digestion identification.
FIG. 5 shows the immunofluorescence detection of the expression of CA16 and EV71 recombinant VP1 proteins in cells.
Detailed Description
The following examples are presented to further illustrate the present invention and should not be construed as limiting the invention. It is within the scope of the present invention to make simple modifications or alterations to the methods, procedures or conditions of the present invention without departing from the spirit and substance of the invention; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Construction of cells stably expressing VP1 of CA16 and EV71
Clinical and laboratory confirmed samples of coxsackie virus A group 16 (hereinafter referred to as CA 16) and enterovirus 71 (hereinafter referred to as EV 71) infected cases were prepared by extracting viral RNA from infected cases with a viral RNA extraction kit, and performing reverse transcription with SuperScript III reverse transcriptase and random primers to obtain viral cDNA of CA16 and EV 71.
The upstream primer of CA16-VP1 gene takes CA16 and EV71 virus cDNA as templates: 5 '-CCTAAGCTTTCTGGGTACTTTGACTATTACACC, and a downstream primer 5' -CAGCTCGAGTCATGTTGTTATCTTGTCTCTACTAC. Upstream primer of EV71-VP1 gene: 5 '-CCTAAGCTTTATGCCCGAGATGGAGTGTTTGAC, and a downstream primer 5' -CAGCTCGAGTCATTTCCCAAGAGTGGTGATTGCTG.
And (3) amplification reaction conditions: pre-denaturation at 94 ℃ for 3min and then circulation, denaturation at 94 ℃ for 30s, renaturation at 52 ℃ for 30s, extension at 72 ℃ for 2min, and extension at 72 ℃ for 5min after 30 cycles. The corresponding product fragments were recovered by electrophoresis on a 1.2% agarose gel. The size of the major band of the PCR product was about 1100bp, and the result was consistent with the prediction (FIG. 1).
The PCR products of the VP1 of the CA16 and EV71 strains were subjected to agarose gel electrophoresis to recover the main amplification band of the expected size, and the PCR products were usedHindIII andXhoi carries out double enzyme digestion on the VP1 fragment and the pCMV-C-Flag plasmid (pcFlag plasmid for short), the enzyme digestion product utilizes agarose gel electrophoresis again to recover the corresponding fragment, and the CA16 strain and the EV71 strain VP1 are respectively connected with the pCMV-C-Flag plasmid (figure 2). CA16 and EV71 strain VP1 are respectively connected with pCMV-C-Flag plasmid, after competent cells are transformed, 8 single colonies subjected to resistance screening are respectively selected for colony PCR identification, and as shown in figure 3, 16 colonies of VP1 eukaryotic expression plasmid pcFlag-CA16-VP1 of CA16 and VP1 eukaryotic expression plasmid pcFlag-EV71-VP1 of EV71 can amplify corresponding VP1 fragments.
2 clones of each colony positive by colony PCR identification were selected for bacterial culture and plasmid extraction, and enzyme digestion identification was performed as shown in FIG. 4HindIII andXhoafter double digestion, the VP1 exogenous fragment of about 1100bp and the pcFlag vector fragment of about 5kb both appear. The sequencing analysis of the plasmid identified by enzyme digestion is carried out, the result is completely correct, and the plasmids are named pcFlag-CA16-VP1 and pcFlag-EV71-VP1 respectively.
293T cells, RD cells, Vero cells were all DMEM (containing 10% FBS) at 37 ℃ with 5% CO2The cells growing to a fusion rate of more than 95% are inoculated on a 6-well cell culture plate before transfection, and transfection is carried out when the cells grow for 20-24 h until the cell density reaches 70-90%, and the transfection operation is carried out according to the operation manual of L ipofectamine 2000 transfection reagent.
293T and RD cells are transfected by pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids which are verified by enzyme digestion and sequencing, and immunoblotting detection is carried out by using F L AG antibody, a predicted size band appears at a position of about 32Kd, which indicates that the eukaryotic expression plasmid can express F L AG fused VP1 recombinant protein in 293T and RD cells.
The pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids transfect 293T cells, and the expression of recombinant VP1 is detected by immunofluorescence, as shown in FIG. 5, CA16 and EV71 recombinant VP1 proteins are mainly distributed in cell cytoplasm.
The pcFlag-CA16-VP1 and pcFlag-EV71-VP1 plasmids were ligated in a 1: 1, carrying out cotransfection on Vero cells at a ratio of 1, carrying out screening by using G418 72 hours after transfection, obtaining resistant cell clones after screening for about 3 weeks, and obtaining a Vero cell line of VP1 for stably expressing CA16 and EV71 through detection.
Preparation of recombinant attenuated enterovirus strains
Attenuated enterovirus strains attenuated coxsackievirus group B3, attenuated enterovirus 71, or attenuated poliovirus I (Sabin vaccine strain) can be used. The attenuated virus is infected in Vero cell line of VP1 of stable expression CA16 and EV71, cell supernatant is harvested after 48h, and recombinant enterovirus with mixed phenotypes is obtained by ultracentrifugation.
SEQUENCE LISTING
<110> Shantou university college of medicine
<120> construction method and application of recombinant enterovirus phenotype mixing system
<130>
<160>4
<170>PatentIn version 3.3
<210>1
<211>33
<212>DNA
<213> upstream primer of CA16-VP1 gene
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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 of 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 (3)
1. A construction method of a recombinant enterovirus phenotype mixing system is characterized in that a VP1 recombinant cell line is constructed, and the recombinant enterovirus phenotype mixing system is obtained by infecting and amplifying attenuated enterovirus strains in a VP1 recombinant cell line; the VP1 recombinant cell line is a cell line which singly or simultaneously expresses one or two of VP1 of enterovirus 71 type and Coxsackie virus A group 16 type; the attenuated enterovirus strain is selected from coxsackievirus group B3, enterovirus 71 or poliovirus I; the construction method comprises the following steps:
s1, amplifying VP1 genes of enterovirus 71 type and/or coxsackievirus A group 16 type, connecting the VP1 genes to a eukaryotic expression vector, and performing transfection and screening to obtain a VP1 recombinant cell line; and (3) amplification reaction conditions: carrying out pre-denaturation at 94 ℃ for 3min and then circulating, carrying out denaturation at 94 ℃ for 30s, renaturation at 52 ℃ for 30s, extending at 72 ℃ for 2min, and extending at 72 ℃ for 5min after 30 cycles; subjecting to 1.2% agarose gel electrophoresis, and recovering corresponding product fragments;
s2, infecting the recombinant attenuated enteric virus strain with a VP1 recombinant cell line obtained from S1 to obtain the recombinant enteric virus strain; attenuated enterovirus strains attenuated coxsackievirus group B3, attenuated enterovirus 71, or attenuated poliovirus type I; the attenuated virus is infected in Vero cell line of VP1 of stable expression CA16 and EV71, cell supernatant is harvested after 48h, and recombinant enterovirus with mixed phenotypes is obtained by ultracentrifugation.
2. A recombinant enterovirus phenotype mixing system obtained by the method of claim 1.
3. Use of the recombinant enterovirus phenotypic mixing system of claim 2 in a method of preparing a vaccine for the treatment of enteroviruses.
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| CN111235114B (en) * | 2020-01-22 | 2023-10-17 | 东莞市第八人民医院（东莞市儿童医院） | EV71 replication-defective virus, and preparation method and application thereof |
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| CN102284058A (en) * | 2011-01-17 | 2011-12-21 | 中国科学院武汉病毒研究所 | Preparation method of replication-defective bivalent vaccine for resisting EV71 and CA16 |
| WO2016073929A1 (en) * | 2014-11-07 | 2016-05-12 | Takeda Vaccines, Inc. | Hand, foot, and mouth vaccines and methods of manufacture and use thereof |
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| CN102284058A (en) * | 2011-01-17 | 2011-12-21 | 中国科学院武汉病毒研究所 | Preparation method of replication-defective bivalent vaccine for resisting EV71 and CA16 |
| WO2016073929A1 (en) * | 2014-11-07 | 2016-05-12 | Takeda Vaccines, Inc. | Hand, foot, and mouth vaccines and methods of manufacture and use thereof |
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| Title |
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| Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens;JuanXu,et al.;《Biochemical and Biophysical Research Communications》;20120317;第420卷(第4期);第755-761页 * |
| MATURATION OF POLIOVIRUS RNA WITH CAPSID PROTEIN CODED BY HETEROLOGOUS ENTEROVIRUSES;JOHN J. HOLLAND AND CARL E. CORDS;《Proc Natl Acad Sci USA》;19640630;第51卷(第6期);1082页第1段至第1085页最后1段 * |
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