CN105543181A - Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine - Google Patents
Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine Download PDFInfo
- Publication number
- CN105543181A CN105543181A CN201610063861.3A CN201610063861A CN105543181A CN 105543181 A CN105543181 A CN 105543181A CN 201610063861 A CN201610063861 A CN 201610063861A CN 105543181 A CN105543181 A CN 105543181A
- Authority
- CN
- China
- Prior art keywords
- virus
- cell
- novel
- vaccine
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a novel EV71 virus. The virus strain is collected from herpes fluid or throat swab samples of sick children diagnosed to suffer from the hand-foot-and-mouth disease in Guizhou province in 2012-2014, the complete genome length of the virus stain is 7405bp, a base sequence is as shown in SEQ ID NO: 1, 297 amino acids are encoded in a VP1 area of the code area of the virus strain, and an amino acid sequence is as shown in SEQ ID NO: 2. The invention also discloses applications of the EV71 virus in preparation of EV71 vaccine and diagnostic products, which belongs to the technical field of biological medicines.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a strain new E V71 virus and preparing the application in vaccine for hand-foot-mouth disease and diagnostic products.
Background technology
Guizhou Disease Control and Prevention Center is since monitoring hand foot mouth disease in 2008, and it is in first place in Class C transmissible disease rate always, and sickness rate is the highest in all kinds of infectious intestinal disease, is in national medium level.
Hand foot mouth disease (HFMD) is a kind of global infectious disease, and world's most area all has this sick popular report.Enterovirns type 71 (enterovirus71, and coxsackie virus A 16 (coxackievirusA16 EV71), CA16) be cause hand foot mouth disease to distribute and two large main pathogens of outbreak of epidemic, EV71 more easily causes serious neurological complication as aseptic meningitis, BBE, poliomyelitis sample paralysis and neurogenic pulmonary edema etc., has higher case fatality rate and disability rate.The pathogenesis of EV71 is that this virus is invaded by pharyngeal or enteron aisle, breeds and gets rid of, cause local symptom in local mucous membrane or Lymphoid tissue; Then virus invades regional nodes again, and enter blood circulation cause first time viremia; Virus invades place's amount reproductions such as deep lymph node, liver, spleen, marrow through blood circulation and enters circulation of blood thus, causes second time viremia; Virus enters systemic organs with circulation of blood: the places such as central nervous system, heart, lung, skin mucosa, breeds and causes pathology.There are some researches show, after susceptible person infects EV71, occur blood vessel transformation reactions and tissue inflammatory pathology.When virus adds up central nervous system, tissue inflammation comparatively neurotoxic effect is more strong, and central nervous system thin vessels endothelium is vulnerable to infringement most.
Enterovirns type 71 (EV71) belongs to the member of Picornaviridae (Picomaradae) enterovirus genus (Enterovirus), belongs to Human enterovirus virus A.Cause HFMD and central nervous system disease, for hazard rating is only second to the Neural invasion enterovirus of poliovirus.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.Polyprotein, while synthesis, is constantly produced P1, P2-P3 bis-precursor proteins by the 2A proteolytic enzyme of himself cutting.P1 precursor protein is again through a series of processing, final formation molecular weight is respectively VP1, VP2, VP3 and VP4 virus capsid protein of 34KD, 30KD, 26KD and 7KD, for forming the structural protein of virus particle, wherein VP1 albumen is considered to main epitope point concentrated area, is once repeatedly reported and is used for developing virus vaccines.And P2 and P3 precursor protein is cut into activated functional protein further, form Nonstructural Protein intermediate, and then form ripe Nonstructural Protein, be responsible for copying of viral RNA and assemble with albumen.Phase starts copying of promotor gene group RNA to virus upon translation, and the coat protein that processing is ripe and the RNA copied pack, and produce ripe virion, virus causes the cracking of host cell to cause the release of progeny virus.
Also do not have at present effective medicine and vaccine to be used for preventing, the infection of test-and-treat EV71, avoid being considered to preventive measures effectively with the infected's close contact.Due to EV71, to infect the harm that brings day by day serious, so research and develop effective vaccine protection Susceptible population, finds suitable diagnostic products, suitable pharmacological agent patient seems increasingly important.
Summary of the invention
The EV71 virus that the object of the present invention is to provide a strain novel, this virus picks up from infant bleb liquid or the throat swab sample that Guizhou province 2012-2014 is diagnosed as HFMD, and its full genome group leader 7405bp, its sequence is as shown in SEQIDNO:1.
Another object of the present invention is to provide above-mentioned EV71 virus and applies preparing in vaccine for hand-foot-mouth disease and diagnostic products.
Above-mentioned EV71 virus, it is characterized in that encoding 297 amino acid altogether in this VP1 district, virus strain coding region, aminoacid sequence is as shown in SEQIDNO:2.
Utilize new E V71 provided by the invention virus, can for set up various Evaluation model in the future, the Evaluation model as vaccine for hand-foot-mouth disease Evaluation of Immunogenicity animal model, EV71 curative drug and therapeutic vaccine provides reference.Utilize its animal model can also deepen the etiologic understanding of EV71, promote the research of the mechanism of causing a disease of enterovirus EV 71 C-type virus C.Meanwhile, the invention also discloses this EV71 virus to apply in preparation EV71 vaccine and diagnostic products.
Accompanying drawing explanation
Accompanying drawing 1 is EV71 virus infection RD cellular form change procedure figure;
Accompanying drawing 2 is albumen Western-Blot qualification result figure (1:Western-blotMarker; 2:EV71 virus infection RD cell; 3: normal RD cell).
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, the condition as described in " molecular cloning: laboratory manual " (NewYork:ColdSpringHarborLaboratoryPress, 1989) is carried out.
Embodiment 1EV71 full-length genome bioinformatics analysis
The cellar culture of 1.RD cell
Cell recovery: first water-bath is adjusted to 37 degree, from-80 DEG C of refrigerators or liquid nitrogen container, take out RD people's pernicious embryo's rhabdomyoma cell (being purchased from Shanghai Chinese Academy of Sciences cell bank) cryopreservation tube drops into rapidly in 37 DEG C of warm water, and shake makes it melt rapidly gently, take out cryopreservation tube, teetertotter cryopreservation tube slightly, open with after 75% alcohol disinfecting, cell suspension after dissolving with suction pipe sucking-off, inject centrifuge tube and add 5ml containing the cell culture fluid of 10% foetal calf serum (FBS).With the centrifugal 5min of 800 ~ 1000 turns/min, removing supernatant liquor, adds the piping and druming of 1ml nutrient solution, makes it form suspension.After suitably diluting with the cell culture fluid containing 20%FBS, be inoculated in culturing bottle, be placed in 37 DEG C, saturated humidity, 5%CO2 incubator cultivate, a nutrient solution is changed after 24h, visual cell's growing state and nutrient solution color (generally treating that nutrient solution turns yellow) change nutrient solution subsequently, continue to cultivate.
Cell changes liquid: absorb old substratum in culturing bottle, wash 2 ~ 3 times with PBS, continues to cultivate or experiment after adding new substratum.
Passage: examine under a microscope adherent of culturing bottle and merge to 70% ~ 80%, Bechtop operates, first first should absorb old substratum in culturing bottle, 2 ~ 3 times are washed with PBS, 50ml culturing bottle adds trysinization liquid 1-3ml, digest in this ratio, rock and make Digestive system spread evenly, put 37 DEG C of incubators about 5 minutes, after seeing under mirror that cellular contraction change circle or minority come off, vibrating gently at the bottom of bottle makes cell all come off, after adding 2-3ml perfect medium, blow and beat gently, collecting cell is to centrifuge tube, abandon supernatant, add nutrient solution, blow and beat into cell suspension gently, be passaged in sterile culture flask according to the ratio of 1:2 or 1:3, continue after adding substratum to cultivate or experiment.
2. virus infection RD cell
EV71 inoculates: virus picks up from infant bleb liquid or the throat swab sample that Guizhou province 2012-2014 is diagnosed as HFMD.Sample disposal liquid is seeded to healthy individual layer RD cell, under 36 DEG C of conditions, observe and have distinctive enterovirus cytopathic effect to occur, in negative patient blind passage two generation, two cultures still then sentence feminine gender for feminine gender; Frozen and passed for two generations after positive one week, again identify with two culture things.Operation steps is as follows:
1. monolayer cell is observed under inverted microscope, to guarantee that cell is healthy;
2. outwell growth media (GM), change the maintenance medium (MM) of 1 ~ 1.2ml;
3. every a sample Simultaneous vaccination 2 RD cells and 2 HEp-2;
4. the sample suspension of test tube inoculation 0.2ml is often propped up, culture temperature 36 DEG C;
5. inverted microscope observation of cell every day culturing bottle is used, to observe the appearance having distinctive enterovirus cytopathic effect (CPE);
6. change at least one week that inoculation bottle and control bottle cell occur is recorded;
If 7. there is distinctive enterovirus CPE to occur, the cell being recorded to 75% there occurs change, is then stored at-20 DEG C and goes down to posterity in order to secondary;
8. generation positive isolates passed for two generations again, if there is again obvious CPE to occur, and-20 DEG C of preservations, with use to be identified:
If 9. do not have CPE to occur after 7d, can continue in blind passage 1 generation to observe 7d;
10., after blind passage 2 generation, still do not have CPE to occur, be then judged to feminine gender.
Amplification positive-virus:
1. disinfection table top and Biohazard Safety Equipment;
2. from incubator, RD cell is taken out;
3. the liquid in bottle is outwelled;
4. the virus liquid of strain selected by 0.2ml is slowly blown into Growth of Cells face, put into 36 DEG C of incubators, every 15 minutes jogs once, make virus be attached to completely on cell;
5. the wash bottle at twice of 4mlMEM liquid is used;
6. add 4mlMM, put into 36 DEG C of incubators, after one week, receive poison.
7. by after cell toxicant multigelation 3 times, in 4 DEG C of centrifugal 10min of 2000r/min, put less than-20 DEG C after getting supernatant liquor packing and save backup.
Virus infection RD cell results (as Fig. 1) shows, and has virus increment, and cause apoptosis in positive group.
3.EV71 full genome amplification and order-checking
1. extract RNA: get the RD cell upper strata substratum that 200 μ l infect virus, extract test kit with TAKARA viral RNA and extract viral RNA (concrete steps are see TAKARA test kit specification sheets).
②RT-PCR：
Reverse transcription pre-mixing:
65 DEG C, 5min; Following reagent is added after 4 DEG C of precoolings:
42 DEG C, 45min; 72 DEG C, 15min; 4 DEG C of coolings.
PCR reaction system:
Primer sequence:
95 DEG C of sex change 5min, 95 DEG C of 30sec, 52 ~ 58 DEG C of 45sec, 72 DEG C extend 30sec, and 30 circulations, 72 DEG C of 10min, are cooled to 4 DEG C.
3. recombinant expression vector is built: reclaimed by above-mentioned PCR primer glue, reclaim product with BamHI and EcoRI double digestion PMD18-T carrier and PCR glue, EV71 gene is inserted enzyme and cut back to close (concrete steps are with reference to Takara company DNALigationKitVer.2.1 test kit specification sheets) in PMD18-T carrier.Linked system is as follows:
Hatch 30min for 16 DEG C.Get connection product 10ul and add the middle piping and druming of 100ulDH5a competent cell (purchased from Takara company) evenly, leave standstill 20min on ice, put into 42 DEG C of water-bath 90s again, be placed in ice rapidly, leave standstill 3min, add 500ulLB liquid nutrient medium, 180rpm, 37 DEG C of shaking culture 1h, get bacterium liquid 100ul and be spread evenly across LB solid medium (chlorampenicol resistant), 37 DEG C of overnight incubation.Picking mono-clonal bacterium colony, in 5mlLB substratum (chlorampenicol resistant), extracts plasmid after 180rpm, 37 DEG C of cultivation 12h and does PCR and double digestion qualification, positive colony called after PMD18-T-EV71.The order-checking of Shanghai biotechnology company limited is delivered in sampling, and its base sequence is as shown in SEQIDNO:1.
4.EV71 full-length genome bioinformatics analysis
EV71 genome sequence after assembling is carried out Blastn comparison with the genome sequence of other EV71 virus reported respectively.By the ORFFinder of NCBI, the genome sequence assembled is inquired about, obtain CDs sequence, and be translated into aminoacid sequence with DNAMAN8.0, then carry out Blastp comparison with the protein sequence of other EV71 virus reported respectively.EV71 virus sequence on the EV71 coding region VP1 sequence of this experiment acquisition and NCBI is carried out Blastn comparison, with software MEGA5.2 constructing system evolutionary tree.
Experimental result shows: the present invention identifies pnca gene group total length 7405bp, 297 amino acid of encoding altogether, this strain isolated and other strain homology of EV71 higher, and lower with the homology of Cox.A16.The display of VP1 district, coding region amino acid (as shown in SEQIDNO:2) homologous sequence comparison result, virus strain of the present invention and EV71 other strain VP1 district amino acid identity very high.Virus strain of the present invention and other EV71 type strain VP1 region nucleotide sequence evolutionary analysis show, according to EV71 type strain VP1 region nucleotide sequence, A, B, C1, C2, C3, C4a, C4b and C5 type can be divided into, qualification strain VP1 region sequence gathers the C4a hypotype in C4 type, and gather in same branch with SDLY107 strain, show that its sibship is very near.
Embodiment 2Western-Blot identifies the antigenicity of recombinant protein
1. preparation of samples
The inoculation that embodiment 1 step 2 is collected there is the RD cell (1 × 10 of EV71 virus
7) add 200ul cell pyrolysis liquid, at cracking 30min, centrifuging and taking supernatant 40ul on ice, add 2 × loading Buffer of equivalent, boil 10min, make the abundant sex change of albumen.
2. albumen sample SDS-PAGE electrophoresis
1. use distilled water flushing glass plank and comb and adhesive tape, dry.Sheet glass and adhesive tape are put into folder after aliging and are clamped, and then vertical card prepares encapsulating on the top of the shelf.
2. join 10% separation gel, shake up rear encapsulating and be about 5cm height, glue adds a water seal, about after half an hour, glue fully solidifies the water on upper strata of just can removing photoresist and blots with thieving paper.
3. join 5% concentrated glue, shake up rear encapsulating, remaining space is filled concentrated glue, then comb is inserted in concentrated glue.After gelling to be concentrated is solid, the both sides that two hands pinch comb are respectively extracted straight up.
4. removing comb is placed in electrophoresis chamber, pours into electrophoretic buffer and drives the bubble in loading hole away, each hole loading 20ul, and leftmost albumen hole adds Marker (Biomiga company).
5. electrophoresis.
3. the transfer of protein
Prepare the pvdf membrane of 6 6cm × 8cm filter paper and 1 5cm × 7.5cm.The pvdf membrane cut first is soaked 1-2min in methyl alcohol.The clip of transferring film is put into, the pvdf membrane that sponge pad, glass rod, filter paper and methyl alcohol are dipped in the pallet being added with transferring film liquid.Clip is opened and makes black one side maintenance level.Pad a sponge pad above, roll with glass rod several all over the bubble to roll away the inside back and forth.Mat pads three metafiltration paper, rolls the bubble that degass.
Gel is taken out from electrophoresis chamber, carefully peels separation gel and be placed on filter paper, by membrane cover on glue, then cover 3 filter paper and remove bubble, finally covering another sponge pad, close clip.Clip is put into transfer groove, electrophoresis apparatus ice bath, 400mA, transferring film 90min.
4. target protein is combined with primary antibodie and two immunity resisted
1., after transferring film terminates, after pvdf membrane PBS is soaked, put into confining liquid (5% skim-milk that TBST dissolves) and close, put shaking table 2h.
2. under room temperature, film is washed 3 times, each 10min with TBST damping fluid (20mmol/LTris-HCl, 150mmol/LNaCl, 0.05%Tween-20) in allograft reaction box.
3. rabbitanti-EV71 (Humanenterovirus71) VP1 (primary antibodie, purchased from Beijing Bo Aosen) of 1: 500 confining liquid dilution is added, room temperature effect 2h.
4. TBST washes film 3 times, each 10min.
5. film is placed in two anti-(sheep anti-mouse igg of HRP mark, purchased from Beijing Bo Aosen) of 1: 2000 confining liquid dilution, in room temperature, reacts 1h.
6. TBST washes film 3 times, each 10min.
5. develop
By film compressing tablet, exposure, colour developing (Bio-Rad gel imaging system carries out IMAQ) after lucifuge immersion 3min in ECL solution (1mlSolutionA+1mlSolutionB).
The capsid protein of EV71 virus comprises VP1, VP2, VP3 and VP4, size is respectively 34,30,26,7KD.
Western-blot adopts the specific monoclonal antibody of VP1 to detect, Western-blot result display (accompanying drawing 2): expression product can be combined with monoclonal antibody specificity, and have at about 34KD place and comparatively significantly react band, illustrate that this expression product has immunoreactivity well.
Claims (3)
1. a strain new E V71 virus, its full genome group leader 7405bp, base sequence is as shown in SEQIDNO:1.
2. EV71 according to claim 1 virus, it is characterized in that encoding 297 amino acid altogether in described EV71 virus VP 1 coding region, aminoacid sequence is as shown in SEQIDNO:2.
3. EV71 virus according to claim 1 and 2, is characterized in that the application of described EV71 virus in preparation EV71 vaccine and diagnostic products.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610063861.3A CN105543181A (en) | 2016-01-25 | 2016-01-25 | Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610063861.3A CN105543181A (en) | 2016-01-25 | 2016-01-25 | Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105543181A true CN105543181A (en) | 2016-05-04 |
Family
ID=55822739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610063861.3A Pending CN105543181A (en) | 2016-01-25 | 2016-01-25 | Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105543181A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604969A (en) * | 2012-02-09 | 2012-07-25 | 中国医学科学院医药生物技术研究所 | Enterovirus 71 cDNA (complementary deoxyribose nucleic acid) infectious clone as well as construction method and application of enterovirus 71 cDNA infectious clone |
| CN103194475A (en) * | 2013-04-17 | 2013-07-10 | 深圳市疾病预防控制中心 | Enterovirus 71 infectious clone, as well as construction method and application thereof |
| CN103233030A (en) * | 2013-04-17 | 2013-08-07 | 深圳市疾病预防控制中心 | Enterovirus type 71 infection fluorescent reporter system and construction method thereof |
| CN103320451A (en) * | 2013-03-01 | 2013-09-25 | 江汉大学 | Genetic engineering protein vaccine for preventing EV71, and preparation method thereof |
| CN103374580A (en) * | 2012-04-27 | 2013-10-30 | 中国医学科学院医药生物技术研究所 | Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain |
| CN103374549A (en) * | 2012-04-26 | 2013-10-30 | 广东华南联合疫苗开发院有限公司 | Novel EV71 virus strain and application thereof |
| WO2015092668A1 (en) * | 2013-12-16 | 2015-06-25 | Mab Explorations Sdn Bhd | Antibodies specific for enteroviruses that infect humans |
-
2016
- 2016-01-25 CN CN201610063861.3A patent/CN105543181A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604969A (en) * | 2012-02-09 | 2012-07-25 | 中国医学科学院医药生物技术研究所 | Enterovirus 71 cDNA (complementary deoxyribose nucleic acid) infectious clone as well as construction method and application of enterovirus 71 cDNA infectious clone |
| CN103374549A (en) * | 2012-04-26 | 2013-10-30 | 广东华南联合疫苗开发院有限公司 | Novel EV71 virus strain and application thereof |
| CN103374580A (en) * | 2012-04-27 | 2013-10-30 | 中国医学科学院医药生物技术研究所 | Enterovirus 71 (EV 71) Fuyang strain and cDNA (deoxyribonucleic acid) infectious clone of attenuated strain of enterovirus 71 (EV 71) Fuyang strain as well as application of enterovirus 71 (EV 71) Fuyang strain |
| CN103320451A (en) * | 2013-03-01 | 2013-09-25 | 江汉大学 | Genetic engineering protein vaccine for preventing EV71, and preparation method thereof |
| CN103194475A (en) * | 2013-04-17 | 2013-07-10 | 深圳市疾病预防控制中心 | Enterovirus 71 infectious clone, as well as construction method and application thereof |
| CN103233030A (en) * | 2013-04-17 | 2013-08-07 | 深圳市疾病预防控制中心 | Enterovirus type 71 infection fluorescent reporter system and construction method thereof |
| WO2015092668A1 (en) * | 2013-12-16 | 2015-06-25 | Mab Explorations Sdn Bhd | Antibodies specific for enteroviruses that infect humans |
| CN105992594A (en) * | 2013-12-16 | 2016-10-05 | Mab探索私人有限公司 | Antibodies specific for enteroviruses that infect humans |
Non-Patent Citations (6)
| Title |
|---|
| WEN,H.-L.: "《Human enterovirus 71 strain SDLY107, complete genome》", 《GENBANK: JX244186.1》 * |
| YAN,Y. AND JUN,G.: "《capsid protein, partial [Enterovirus A71]》", 《GENBANK: AFQ94252.1》 * |
| 刘静等: "《１株ＥＶ７１临床分离株的全基因组序列测定和结构分析及对乳鼠致病性研究》", 《中国病毒病杂志》 * |
| 李静等: "《湖北肠道病毒ＥＶ７１型流行株ＶＰ１基因以及蛋白结构特征分析》", 《中国人兽共患病学报》 * |
| 潘家华主编: "《实用小儿手足口病诊疗指南》", 30 June 2010 * |
| 阎岩等: "《００８～２０１１年贵州省肠道病毒７１型分子流行特征分析 阎岩，郭军，周敬祝，唐光鹏，王定明》", 《病毒学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Halstead | Dengue and dengue hemorrhagic fever | |
| Bank-Wolf et al. | Zoonotic aspects of infections with noroviruses and sapoviruses | |
| Koci et al. | Avian astroviruses | |
| Melnick | Enteroviruses | |
| CN107513524A (en) | One plant of pig Sai Neijia paddy virus stain and its application | |
| BRPI0510396B1 (en) | ATTENUED PESTIVIRUS AND ITS USE, COMPOSITION, NUCLEIC ACID MOLECULE AND METHOD FOR ATTENUING PESTIVIRUS | |
| CN108384762B (en) | Porcine alpha-enterocoronavirus and culture method and application thereof | |
| Lennette et al. | Picornaviridae: the enteroviruses (polioviruses, coxsackieviruses, echoviruses) | |
| CN106591242A (en) | Canine parvovirus strain CPV-YH and applications thereof | |
| CN105866408B (en) | A kind of four kinds of enterovirus joint-detection immune chromatography reagent kits and preparation method thereof | |
| CN102167735A (en) | Recombinant antigen protein of S.japonicum SjTollip and preparation method and application thereof | |
| CN103539839A (en) | Neutralizing epitope peptide of enterovirus 71-type VP2 antigen and application thereof | |
| CN111073862B (en) | Bovine viral diarrhea type2 attenuated strain and application thereof | |
| CN106854647A (en) | Duck virus hepatitis divalence yolk antibody and its preparation method and application | |
| CN109536464B (en) | Chikungunya virus infectious clone with deletion of capsid protein gene, construction method and application in preparation of attenuated vaccine | |
| CN105543181A (en) | Novel EV71 virus and application of novel EV71 virus in preparation of EV71 vaccine | |
| CN103374549B (en) | One strain new E V71 virus and application thereof | |
| Wang et al. | Hepatitis E virus | |
| Greene et al. | Venezuelan equine encephalitis virus in the guinea pig model: evidence for epizootic virulence determinants outside the E2 envelope glycoprotein gene | |
| Peralta et al. | The Tacaribe complex: the close relationship between a pathogenic (Junin) and a nonpathogenic (Tacaribe) arenavirus | |
| STRAUSS et al. | Plus-strand RNA viruses | |
| CN102120986B (en) | Cell culture method of rotavirus | |
| Richards | Foodborne and waterborne enteric viruses | |
| CN106520789B (en) | A kind of DNA molecular and recombinant virus and their preparation method and purposes | |
| CN104651378B (en) | A kind of hog cholera vaccine infectious CDNA and its construction method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination |