CN105087503A - Human enter ovirus 71 3CD chimeric virus as well as rescue method and application thereof - Google Patents
Human enter ovirus 71 3CD chimeric virus as well as rescue method and application thereof Download PDFInfo
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Abstract
The invention relates to human enter ovirus 71 3CD chimeric virus (Latin word name) as well as a rescue method and an application thereof. The human enter ovirus 71 3CD chimeric virus is preserved in the China Center for Type Culture Collection (CCTCC) which is located in Wuhan University in Wuhan, Hubei province, the preservation date is June, 24th, 2015, and the preservation serial number is CCTCC NO.V201523. A study platform is provided for the rescue for the human enter ovirus 71 3CD chimeric virus. 3CD in EV71 plays an important role in duplication and pathopoiesis of viruses as a precursor protein, and therefore, the construction of the chimeric virus strain SDLY107 (1-3CD) provides study platforms for studying the action of the 3CD coding region in the virus pathogenic mechanism, studying the antiviral drugs and the development for vaccines.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of enterovirns type 71 3CD embedded virus and rescue methods and applications thereof.
Background technology
Hand foot mouth disease is global infectious disease, and world's most area all has this sick popular report.Nineteen fifty-seven, in New Zealand's reported first, isolates Coxsackie virus in 1958 first, and proposes HFMD name first in nineteen fifty-nine.The pathogenic agent of the hand foot mouth disease of early discovery is mainly CoxA16 type, and hand foot mouth disease and EV71 infect relevant report and start from early 1970s, and within 1972, EV71 confirms in the U.S. first.After this EV71 infection is infected with CoxA16 and is alternately occurred, becomes the main pathogens of hand foot mouth disease.It is multiple is born in less than 5 years old children, and can cause the bleb at the position such as hand, foot, oral cavity, minority infant can cause the complication such as pulmonary edema, AME.Indivedual children with serious disease disease progression is very fast, causes death.Severe and death cause primarily of EV71 virus infection.
Enterovirns type 71 (EV71) belongs to the member of Picornaviridae (Picornaradae) enterovirus genus (Enterovirus), belongs to Human enterovirus virus A.The positive rate of EV71 antibody in HAS reaches more than 65%, describes the wide-scale distribution of this virus, and all can enter when temperature raises annual spring and summer and infect peak period, fashion trend is tending towards having small-sized aggregation to break out gradually from distributing.
The related scientific research work of hand foot mouth disease is started late, developed country has good Scientific Research Platform, and the Asian-Pacific area is Major Epidemic region, but drops into not to the fundamental research of EV71, so far also there is no effective vaccine and medicine clinically, effective prevention and corntrol is carried out to EV71.Because enterovirns type 71 belongs to Picornaviridae, so its genomic manual operation is more difficult, this adds difficulty to the preparation of vaccine.
Therefore the current vaccine research to EV71 still mainly focuses on traditional inactivated vaccine, and the immune effect of this type of vaccine is general lower; Although the immune response producing and comprise neutralizing antibody can be induced, can not react by inducing cytotoxic T lymphocyte; The immune response time length that induction produces is shorter, needs repeatedly to inoculate; Need to use adjuvant, there is inactivator in preparation, inactivator has impact to virus antigen; Because the immune response level of induction is lower, and the vaccine response imbalance between each antigenic component, may induce an illness; General not by natural way inoculation, not easily produce local immunity reaction.Due to the existence of above problem, need research and development new generation vaccine badly, complete immunogenicity can be retained, effective stimulus immunne response, again can avoiding infection property.The development of this type of vaccine, needs further investigate the function of gene coding region each in viral genome and understand, and this target spot also contributing to screening antiviral of understanding in depth is studied.
Reverse-genetics approach research RNA viruses operates in plasmid level, studies RNA viruses, and the Revive virus phenotype of acquisition is stablized, and easy to operate, the further investigation for EV71 provides a good basic platform.But also there is no suitable virus so far as the instrument of vaccine research.
Investigator is not had to do EV71 Revive virus in prior art, the reverse genetics research that mainly there is technology prejudice EV71 cannot realize, in addition because EV71 Revive virus is a kind of virus newly, so just there is very large not predictability when studying rescue method, how overcoming this not predictability is cannot accomplish in prior art.
Summary of the invention
Object of the present invention is exactly to provide a kind of enterovirns type 71 3CD embedded virus and rescue methods and applications thereof.
To achieve these goals, the present invention adopts following technical scheme:
A kind of enterovirns type 71 3CD embedded virus, Latin name: (Humanenterovirus713CDchimericvirus), depositary institution's title: China typical culture collection center, depositary institution address: Wuhan City, Hubei Province Wuhan University in the school, preservation date: on June 24th, 2015, deposit number: CCTCCNO.V201523.
The morphological specificity of virus of the present invention describes:
The particle of this virus is the globosity of icosahedron cubic symmetry, and without coating with outstanding, diameter is 24 ~ 30nm about, and nucleic acid is single-stranded positive RNA.The four kinds of Structural protein VP1 ~ VP4 of EV71 C-type virus C genome encoding forms protomer, and the latter is assembled into the subunit with pentamer spline structure again, and 60 subunits are interconnected by respective structural domain, the final shell forming virus.After this virus infection sensitive cells RD cell, the CPE of formation is characterized as cell rounding, pyknosis, refractivity enhancing, then comes off.
A rescue method for enterovirns type 71 3CD embedded virus, step is as follows:
(1) with SDLY1 (GenBank, HM188481.1) full-length cDNA is pcr template, and primer 1 (sequence in table 2, SEQIDNo.1) is upstream primer, primer 5 (sequence in table 2, SEQIDNo.5) is downstream primer amplification 3CD protein-coding region;
(2) with SDLY107 (GenBank, JX244186.1) full-length cDNA is pcr template, and primer 6 (sequence in table 2, SEQIDNo.6) is upstream primer, primer 4 (sequence in table 2, SEQIDNo.4) is downstream primer amplification 3 ' UTR coding region;
(3) adopt fusion DNA vaccine method, with primer 1, SEQIDNo.1 for upstream primer, primer 4, SEQIDNo.4 is that two fragments couple together and obtain merging fragment 3CD (1)-3 ' UTR (107) by downstream primer;
(4) fusion DNA vaccine product 3CD (1)-3 ' UTR (107) clone in (3) is connected into pMD19-T carrier, form pMD19-T-3CD (1)-3 ' UTR (107);
(5) pMD19-T-3CD (1)-3 ' UTR (107) plasmid that pMD19-T-SDLY107 plasmid and step (4) obtain is carried out HindIII and XbaI double digestion respectively, after reclaiming endonuclease bamhi, utilize T4 ligase enzyme to carry out connection and obtain recombinant virus plasmid pMD19-T-107 (1-3CD);
(6) pMD19-T-107 (1-3CD) that step (5) obtains is carried out plasmid amplification, and use HindIII and XbaI double digestion, reclaim 107 (1-3CD), be transfected into RD cell after in-vitro transcription, 37 DEG C, 5%CO
2cultivate in cell culture incubator, go down to posterity more than 3 times until cytopathy is stable be enterovirns type 71 3CD embedded virus.
The concrete steps that in described step (5), (6), enzyme is cut are as follows: each 2.5 μ L of HindIII and XbaIFastDigestenzyme, and plasmid (is pMD19-T-SDLY107 plasmid and pMD19-T-3CD (1)-3 ' UTR (107) plasmid in 5; Be pMD19-T-107 (1-3CD) plasmid in 6) <2.5 μ g, 10 × FastDigestGreenBuffer5 μ L, water polishing to 50 μ l, 37 DEG C of enzymes cut 3-4h.
Described step is specially in (5): carry out RNA transfection after double digestion obtains 107 (1-3CD), in-vitro transcription after pMD19-T-107 (1-3CD) is carried out plasmid amplification, after RD cell transfecting RNA, the rescue to virus is cultivated in 37 DEG C of CO2gas incubator, the appearance of Revive virus particle is found by observation of cell pathology every day, when CPE reaches 90% pathology, freeze thawing once (is avoided thawing) and gathers in the crops supernatant being first-generation Revive virus; Get in first-generation Revive virus inoculating cell culture plate and (preferably get 500 μ l to inoculate in 24 porocyte culture plates), put into 37 DEG C of CO2gas incubator to continue to cultivate, cultivate and do not occur obvious CPE after 5 days, freeze thawing once (avoids thawing), 10, the centrifugal 10min of 000g gathers in the crops supernatant, called after two generation Revive virus; It is viral that continuation above-mentioned steps inoculated for two generations, and occur pathology after about 36h, after 72h, pathology reaches 90%, freeze thawing three results viruses.Repeatable operation goes down to posterity more than 3 times until cytopathy is stable be rescue successfully virus.
Above-mentioned enterovirns type 71 3CD embedded virus is preparing the application in vaccine.
Beneficial effect of the present invention:
The 3CD of EV71 is as the copying and all playing a significant role in causing a disease in virus of a precursor protein, and be therefore configured to the research effect of 3CD coding region in viral pathogenesis mechanism, antiviral research and the vaccine development of embedded virus strain SDLY107 (1-3CD) all provide research platform.
Accompanying drawing explanation
A kind of enterovirns type 71 3CD embedded virus, Latin name: (Humanenterovirus713CDchimericvirus), depositary institution's title: China typical culture collection center, depositary institution address: Luo Jia Shan, wuchang, wuhan Wuhan University, preservation date: June 24 in 2015, deposit number: CCTCCNO.V201523.
Fig. 1 is pMD19-T-107 total length plasmid figure;
Fig. 2 EV71 full-length clone pcDNA3.1 (+)-SDLY107 forming types schematic diagram.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
1 materials and methods
1.1 experiment material
1.1.1 cell and virus
Cell behaviour human rhabdomyosarcoma cells (rhabdomyosarcomacells, RD) used.RD Growth of Cells is to virus inoculation during logarithmic phase, and time until about cytopathy to 80%, between-40 DEG C and room temperature, 4 DEG C of 10,000g centrifugal 10 minutes results after multigelation three times, are stored in-80 DEG C of refrigerators.
1.1.2 experiment reagent
Main agents used is that viral RNA extracts test kit (OMEGA, China), DNA glue reclaims test kit and plasmid extraction kit (OMEGA, China); Reverse Transcriptase kit (TOYOBO, Japan); In-vitro transcription test kit (Promega, the U.S.); Efficient fidelity enzyme PrimeSTARGXLDNAPolymerase, pMD19-T (TaKaRa, China); Restriction enzyme, transfection reagent (Thermo, the U.S.); Cell culture medium, serum (HyClone, the U.S.); Primer is synthesized by Shanghai Sheng Gong biotechnology limited-liability company; Order-checking is completed by Shanghai Bo Shang biotech company.
1.2 experimental technique
1.2.1 the cDNA of amplification EV71SDLY107 and SDLY1 strain virus
Use the ViralRNAkit test kit of OMEGA company to extract the RNA of SDLY107 and SDLY1 strain virus, use the ReverTraAceqPCRKit reverse transcription of TOYOBO company to generate cDNA.
1.2.2 design of primers and RCR increase
Virus strain SDLY107 and SDLY1 are the hand foot mouth disease infant that this seminar is separated from Shandong, SDLY107 is dead infant strain isolated, through being accredited as virulent strain, (cytopathy accelerates and strong, animal pathological characters is obvious), SDLY1 is separated from mild infant, through being accredited as low virulent strain, strain all carries out complete sequence analysis.
The basis of the pMD19-T-107 total length plasmid preserved in this room is carried out the rescue of recombinant virus SDLY107 (1-3CD).As Fig. 1 marked the pMD19-T-107 total length plasmid figure choosing restriction enzyme site.Restriction enzyme site Apa I self contains jointly for SDLY107 strain and SDLY1 strain virus; Xba I is for introducing site; The site that Hind III contains for pMD19-T.
The step that pMD19-T builds:
1. utilize restriction enzyme site (BsiWI, Bsp1407I) intrinsic in SDLY107 strain whole genome sequence, point three sections of amplification EV71 full-length gene groups.Fig. 2 is shown in by structure
(1) according to SDLY107 strain full-length cDNA as amplification template, be that (sequence is in table 1 for upstream primer with ORF107F, SEQIDNo.2), take ORF107R as large portion open reading frame (BsiWI-Bsp1407I) of downstream primer (sequence in table 1, the SEQIDNo.3) EV71 that increases.Amplified fragments ORF (BsiWI-Bsp1407I) is carried out HindIII and XbaI double digestion with pcDNA3.1 (+) plasmid.After endonuclease bamhi reclaims, carry out T4 and connect acquisition pcDNA3.1 (+)-ORF (BsiWI-Bsp1407I).
(2) with SDLY107 strain full-length cDNA for amplification template, (sequence is in table 1 for upstream primer for 5 ＇ UTR107F (containing T7 promotor), SEQIDNo.7), VP4107R is the 5 ' end (5 ' UTR-BsiWI) of downstream primer (sequence in table 1, the SEQIDNo.8) EV71 that increases.Amplified fragments 5 ' UTR-BsiWI and carrier pcDNA3.1 (+)-ORF (BsiWI-Bsp1407I) is carried out HindIII and BsiWI enzyme cut.After endonuclease bamhi reclaims, carry out T4 and connect acquisition pcDNA3.1 (+)-5 ' UTR-ORF (BsiWI-Bsp1407I).
(3) with 3 ＇ UTRBsp1407IF, for upstream primer, (sequence is in table 1, SEQIDNo.9), 3 ＇ UTR52TR are the 3 ' end (Bsp1407I-3 ' UTR) of downstream primer (sequence in table 1, the SEQIDNo.10) EV71 that increases.Amplified fragments Bsp1407I-3 ' UTR and carrier pcDNA3.1 (+)-5 ' UTR-ORF (BsiWI-Bsp1407I) is carried out Bsp1407I and XbaI enzyme cutting.After endonuclease bamhi reclaims, carry out T4 and connect acquisition total length EV71 clone pcDNA3.1 (+)-SDLY107.
2. use HindIII and XbaI to carry out double digestion to pcDNA3.1 (+)-SDLY107, reclaim 107 fragments; T-A clones 107 fragments and enters pMD19-T plasmid, obtains pMD19-T-107 total length.
Table 1 primer sequence
1.2.2.1PCR increase 3CD (1)-3 ' UTR (107)
Primer is in table 2.Use primer 1 (SEQIDNo.1), primer 5 (SEQIDNo.5), with SDLY1 strain cDNA for template, amplification 3CD protein-coding region; Use primer 6 (SEQIDNo.6), primer 4 (SEQIDNo.4), with SDLY107 strain cDNA for template, amplification 3 ' UTR coding region; Use primer 1 (SEQIDNo.1), primer 4 (SEQIDNo.4), the SDLY1 strain 3CD obtained to increase and SDLY107 strain 3 ' UTR is that template carries out over-lap PCR, obtains recombinant PCR product 3CD (1)-3 ' UTR (107).The base CAG of 3 3CD albumen coded sequence upstreams is introduced in 5 ' UTR upstream of primer 1 (SEQIDNo.1); Complementary fragment is contained in 5 ' UTR upstream of primer 5 (SEQIDNo.5) and primer 6 (SEQIDNo.6).
Table 2 primer sequence
Note: primer 1: italic is the restriction enzyme site primer 4 that the EV71 self chosen exists: italic is the restriction enzyme site primer 2 and primer 3 that insert, primer 5 and primer 6: underscore is complementary fragment.
1.2.3pMD19-T-107 the structure of (1-3CD)
PCR primer 3CD (1)-3 ' UTR (107) clone is connected into pMD19-T carrier, forms pMD19-T-3CD (1)-3 ' UTR (107).Use restriction enzyme A pa I and Xba I pair of pMD19-T-3CD (1)-3D-3 ' UTR (107) and pMD19-T-107 to carry out double digestion, reclaim 3CD (1)-3 ' UTR (107) and pMD19-T-107
-(107
-for removing the rest segment of 3CD-3 ' UTR coding region).T4 ligase enzyme is used to connect pMD19-T-107
-with 3CD (1)-3 ' UTR (107), obtain pMD19-T-107 (1-3CD).
The acquisition of 107 (1-3CD) total length of 1.2.4 recombinating and in-vitro transcription
Use Xba I and Hind III pair of pMD19-T-107 (1-3CD) to carry out double digestion, make its linearizing, and reclaim 107 (1-3CD).The RiboMAX III LargeScaleRNAProductionSystems-T7 test kit using Promega company to produce carries out in-vitro transcription, and uses the RNA of OMEGA company extraction test kit to extract RNA.
1.2.5 transfection and Revive virus
According to transfection reagent ThermoScientificTurboFect using method, 107 (1-3CD) full-length RNA of 1 μ g is transfected into the RD cell of firm confluent monolayers, harvested cell after about 72h hour, put (avoiding multigelation) after-40 DEG C and room temperature freeze thawing once, be seeded to the RD cell of firm confluent monolayers, every hole 200 μ l.Cultivate harvested cell after 72h and freeze thawing once, be seeded to the RD cell of firm confluent monolayers.Omnidistance observation of cell pathology situation, and feminine gender and SDLY107, SDLY1 strain are set in contrast.
1.2.6 the qualification of recombinant virus
Extract the RNA of Revive virus SDLY107 (1-3CD), reverse transcription generates cDNA, use primer 1 (SEQIDNo.1), primer 4 (SEQIDNo.4) are reclaimed after increasing to 3CD-3 ' UTR coding region, serve the order-checking of Hai Boshang biotech company and identify.
The specific embodiment of the present invention is described although above-mentioned in conjunction with the embodiments; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (5)
1. an enterovirns type 71 3CD embedded virus, is characterized in that, is preserved in China typical culture collection center, depositary institution address: Wuhan City, Hubei Province Wuhan University in the school, preservation date: on June 24th, 2015, deposit number: CCTCCNO.V201523.
2. the rescue method of enterovirns type 71 3CD embedded virus as claimed in claim 1, it is characterized in that, step is as follows:
(1) with SDLY1 full-length cDNA for pcr template, primer 1 is upstream primer, and primer 5 is the 3CD of downstream primer amplification SDLY1 strain;
(2) with SDLY107 full-length cDNA for pcr template, primer 6 is upstream primer, and primer 4 is 3 ' UTR fragment of downstream primer amplification SDLY107 strain;
(3) adopt fusion DNA vaccine method, with primer 1 for upstream primer, primer 4 obtains merging fragment 3CD (1)-3 ' UTR (107) for two fragments couple together by downstream primer;
(4) fusion DNA vaccine product 3CD (1)-3 ' UTR (107) clone in (3) is connected into pMD19-T carrier, form pMD19-T-3CD (1)-3 ' UTR (107);
(5) pMD19-T-3CD (1)-3 ' UTR (107) that pMD19-T-SDLY107 plasmid and step (4) obtain is carried out HindIII and XbaI double digestion respectively, after reclaiming endonuclease bamhi, utilize T4 ligase enzyme to carry out connection and obtain recombinant virus plasmid pMD19-T-107 (1-3CD);
(6) pMD19-T-107 (1-3CD) that step (5) obtains is increased, and use HindIII and XbaI double digestion, reclaim 107 (1-3CD), be transfected into RD cell after in-vitro transcription, 37 DEG C, 5%CO
2cultivate in cell culture incubator, Secondary Culture is enterovirns type 71 3CD embedded virus until cytopathy is stable.
3. save method as claimed in claim 2, it is characterized in that, the concrete steps that in described step (5), (6), enzyme is cut are as follows: each 2.5 μ L of HindIII and XbaIFastDigestenzyme, plasmid <2.5 μ g, 10 × FastDigestGreenBuffer5 μ L, water polishing to 50 μ l, 37 DEG C of enzymes cut 3-4h.
4. enterovirns type 71 3CD embedded virus as claimed in claim 1 is preparing the application in vaccine.
5. the application of enterovirns type 71 3CD embedded virus as claimed in claim 1 in preparation antiviral.
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Application publication date: 20151125 |