CN102533671B - Coxsackie virus A16-type virus strain and applications thereof - Google Patents
Coxsackie virus A16-type virus strain and applications thereof Download PDFInfo
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Abstract
The invention provides a coxsackie virus A16-type virus strain. The collection number of the coxsackie virus A16-type virus strain is CGMCC No.5372, wherein CGMCC refers to China General Microbiological Culture Collection Center. The virus is a 20-face stereoscopic symmetrical sphere under observation through an electron microscope, and the diameter of the virus is 23-30nm. VP1 conserved region sequence analysis and mass spectrum analysis are respectively performed on the virus strain, and a result shows a CA16 virus. The CA16 virus can be efficiently proliferated in Vero cells (African green monkey kidney cells), and the virus titer can reach 6.61g CCID50/ml. Moreover, the virus strain has no external pollution, better immunogenicity and a good effect.
Description
Technical field
The present invention relates to virusology, field of molecular biotechnology, specifically, relate to a kind of new coxsackie virus A 16-type virus strain and application thereof.
Background technology
Hand foot mouth disease (Hand foot mouth disease, HFMD) be the transmissible disease being caused by enterovirus, multiplely be born in 5 years old following children, can cause the bleb at the positions such as hand, foot, oral cavity, minority infant can cause the complication such as pulmonary edema, AME.If indivedual children with serious disease PD are fast, cause death.The enterovirus that causes hand foot mouth disease has kind more than 20 (type), 16,4,5,9,10 types of CA group, and 2,5 types of B group, and enterovirns type 71, be the more common pathogenic agent of hand foot mouth disease.Mainly through excrement-mouth, respiratory tract and close contact, propagate, can pass to fetus by placenta and cause intrauterine infection.Wherein Coxsackie virus (Coxasckievirus) A16 type (Cox A16, CA16) is one of hand foot mouth disease main pathogens, and it is the single strand plus RNA virus of Picornaviridae, is 20 three-dimensional symmetries spherical.Although severe and death are mainly caused by EV71 virus infection, it is worth noting that especially Cox A16 is relevant to comprehensive diseases such as myocarditis, pericarditis, Refractory Shocks.
All there is this sick popular report most areas, the world, and China saw this disease from 1981 in the Shanghai beginning, and all there is report in tens provinces and cities such as later Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, Hubei, Xining, Guangdong.The outburst of hand foot mouth disease and the popular daily life that had a strong impact on, cause huge financial loss and burden on society, utilizing virus vaccines fundamentally to cut off viral propagation is comparatively effective one of prevention approach at present, therefore there is no at present the report of CA16 virus vaccines, isolate the CAl6 virus strain that is suitable for production of vaccine and there is huge economic and social benefit.
Summary of the invention
The object of this invention is to provide a kind of new coxsackie virus A 16-type virus strain and application thereof.
In order to realize the object of the invention, a kind of coxsackie virus A 16-type virus strain of the present invention, its preserving number is CGMCC No.5372, now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on October 18th, 2011.At this strain of electric Microscopic observation, it is spherical that virion is 20 three-dimensional symmetries, diameter 23-30nm.
Aforesaid application, be by described coxsackie virus A 16-type virus strain after cells infected (as Vero cell, human diploid cell or other sensitive cells), cultivation, results virus liquid, deactivation and purifying, obtain vaccinogen liquid, obtain coxsackie virus A 16-type vaccine after adding immunological adjuvant (as aluminium hydroxide etc.).
Aforesaid application, after virus strain cells infected, in 37 ℃ of cultivations, reaches 75% when above until cytopathy, results virus liquid.
Aforesaid application, beta-propiolactone or formaldehyde solution are used in deactivation.Preferably use 1:100-1:10000(v/v) beta-propiolactone or 1:1000-1:10000(v/v) formaldehyde solution deactivation.Inactivation of virus also can carry out after viral purification.
The present invention also provides, and take coxsackie virus A 16-type virus strain of the present invention (CGMCC No.5372) as immunogen Dispersal risk or hybridoma or antiserum(antisera).Wherein, sero-fast preparation method is: virus strain, after cells infected, cultivation, results virus liquid, deactivation and purifying, obtains vaccinogen liquid, uses vaccinogen liquid immune animal, obtains antiserum(antisera).Described animal is preferably monkey, sheep, rabbit etc.
The VP1 albumen producing by further research virus strain of the present invention, the sequential analysis of VP1 conserved regions and mass spectrometry results show that this strain is CA16 virus, and pollute without xenobiotics, have good immunogenicity, are the respond well virus strain of a strain.
By technique scheme, the present invention at least has following advantages and beneficial effect:
(1) the mono-clonal CA16 virus strain of the present invention obtaining by the separation of plaque method, its progeny virus shows as inheritance stability, can be used for people and uses CA16 production of vaccine.
(2) utilize CA16 virus strain of the present invention or can be used for the disease that prevention causes by CA16 virus (hand foot mouth disease for example by the vaccine of its production, children's hand foot mouth disease), and have the advantages that titre is stable, immunogenicity is better, immunizing dose is little especially.
(3) CA16 virus strain of the present invention can be in Vero cell high efficiently multiplying, virus titer can reach 6.6lg CCID50/ml.
Accompanying drawing explanation
Fig. 1 is CA16 virus strain electron microscopic observation result of the present invention (magnification ratio: 100,000 times).
Fig. 2 is CA16 virus strain protein spectrum analytical results of the present invention.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Separation, the culture & identification of embodiment 1CA16 virus strain
(1) virus separation, cultivation
From brothers' mouth epidemic-stricken area, Zhejiang Province in 2010, (brothers' mouth accumulative total number of the infected reaches 111783) collects the Feces of Patients sample of the CA16 virus PCR result positive, be seeded to after treatment on African green monkey kidney passage cell (Vero), cultivate three generations and carry out virus separation, obtain after CA16 virus, by plaque method, obtain CA16 virus strain.
(2) virus is identified
1, VP1 conserved regions the sequencing results
This strain VP1 conserved regions sequence is analyzed, and itself and Reference Strains sequence (human coxsackievirus A16 C-type virus C strain shzh00-1, complete genome group sequence GenBank:AY790926.1) are compared, and nucleotide homology is more than 93.8%.
2, electron microscopic examination result
In this strain of electric Microscopic observation CA16 virus, it is spherical that virion is 20 three-dimensional symmetries, diameter 23-30nm.(Fig. 1)
3, mass spectrometry results
This strain CA16 viral protein is carried out to mass spectroscopy, and result shows that this strain albumen is really CA16 viral protein (Fig. 2).
4, aseptic, mycoplasma check result
This strain CA16 virus is carried out to aseptic, mycoplasma inspection, and result shows without mycoplasma and other microbial contamination.(according to < < Chinese Pharmacopoeia > > method, detecting)
5, virus titer detected result
Adopt microtitrimetry to measure the virus titer of this strain virus harvest liquid, virus titer can reach 6.6lg CCID50/ml.Method is as follows: adopt Vero cell to carry out the mensuration of virus titer.By 96 porocyte culture plate Cultivation of Vero, after cell is in blocks, use cell maintenance medium from 10 virus
-1doubling dilution to 10
-8, discard Vero cell conditioned medium, add respectively each dilution virus liquid, inoculation 50 μ l/ holes, each extent of dilution is inoculated 8 holes, establishes cell contrast simultaneously.Put 37 ℃, 5%CO
2incubator is cultivated, and 7d observation of cell infects viral situation, calculates virus titer.
6, antigenic content detects
Adopt euzymelinked immunosorbent assay (ELISA) to detect this strain CA16 virus antigen content, antigenic content is not less than 200U/ml.Adopt CA16 antigenic content in double antibody sandwich method quantitative assay sample.First specific C A16 polyclonal antibody is coated in to enzyme plate and forms insolubilized antibody; Then add CA16 sample, form solid phase antigen antibody complex with insolubilized antibody; Finally add enzymic-labelled antibody, the CA16 antigen in sample can with enzyme labelled antibody generation specific binding, when adding substrate, there is dye-forming reaction, by microplate reader, at suitable wavelength, measure OD value, by EXCEL statistic analysis result.
The preparation of embodiment 2CA16 vaccine
CA16 virus strain, after vero cells infection, cultivation, results virus liquid, deactivation and purifying, obtains vaccinogen liquid, for further preparing CA16 vaccine.
(1) set up the main seed of cell and work seed bank (Vero cell)
To be derived from U.S. ATCC, 120 generation African green monkey kidney cell (Vero cell) seed recoveries, concrete operations are: in liquid nitrogen, take out cell cryopreservation tube, be placed in 39-40 ℃ of sterilized water, cell thawed within one minute, aseptic sucking-off suspension, the centrifugal 3min of 1000rpm, abandons supernatant, adds the MEM cell growth medium containing 10% calf serum, gently piping and druming it is mixed, by the cell suspension inoculation mixing in 25cm
2tissue Culture Flask in, put 37 ℃, 5%CO
2incubator is cultivated, and changes liquid, then put 37 ℃, 5%CO after it is adherent
2incubator is cultivated, and when within 5-7 days, cell degree of converging reaches 100%, by 1:4, goes down to posterity, and the cell generation that often goes down to posterity increases a generation.Prepare as stated above 129 generation Vero cell work seed banks.According to aforesaid method, researcher in this field can prepare the work seed bank that meets requirement of the present invention equally.
The main seed bank of cell and work seed bank are all kept in liquid nitrogen (196 ℃).
(2) set up main generation virus seed bank and work seed bank
The hand foot mouth disease patient's of the CA16 virus PCR result positive clinical samples, is seeded to African green monkey kidney passage cell (Vero) upper after treatment, cultivates three generations and carries out virus separation, obtains after CA16 virus, by plaque method, obtains CA16 virus strain.According to < < Chinese Pharmacopoeia > >, about the requirement of seed bank establishment method, set up viral main seed bank and work seed bank, all freezing preservations of seed bank (60 ℃ following).
Banking process is as follows: by CA16 virus strain by 1:1000(volume ratio) be seeded to degree of converging (substratum is the MEM cell virus growth media containing 2% calf serum) in 100% Vero cell (from cell work seed bank) bottle, put 37 ℃, 5%CO
2incubator is cultivated, observation of cell pathology every day (CPE).When CPE reaches +++ extremely ++++time results virus, put-20 ℃ freezing, after room temperature is melted, freeze thawing thing is continued to go down to posterity as stated above and sets up viral main generation seed bank and work seed bank.Bing Song China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation.Its preserving number is CGMCC No.5372.
Every a collection of harvest liquid is designated as a generation.
(3) virus harvest liquid preparation
Recovery Vero work seed bank cell, after amplification, is inoculated in CA16 virus strain of the present invention in the cell bottle that grows to thin individual layer in proportion, 37 ℃ of cultivations, every day observation of cell pathology, when cytopathy reaches 50% when above, results virus liquid, mixes postposition-20 ℃ preservation, standby.
(4) inactivation of virus and purifying
By the CA16 virus harvest liquid 1:100-1:10000(v/v of above-mentioned preparation) beta-propiolactone or 1:1000-1:10000(v/v) formaldehyde solution deactivation.Inactivation of virus also can carry out after viral purification.
After inactivation of virus, above-mentioned deactivation liquid is carried out, after centrifugal clarification, carrying out ultrafiltration dialysis, volume is concentrated into the 1/10-50 of original volume.The viral ultrafiltrated obtaining adds magnetic stir bar, is placed on magnetic stirring apparatus and stirs, and simultaneously slowly adding PEG(molecular weight is 6000) to make PEG final concentration be 5-15%, adjust pH, between 5.0-7.0, continues to stir 10-60min.Centrifugal 20-60min after 2-8 ℃ of precipitation 8-24h, precipitation is heavy molten with PBS damping fluid, the viral precipitated liquid of centrifugal rear acquisition.Virus precipitated liquid, again through sucrose density gradient centrifugation, is collected after the band of viral location, through ultrafiltration desugar, obtains viral desugar liquid.Virus desugar liquid obtains viral chromatographic solution after sieve chromatography, and chromatographic solution obtains vaccinogen liquid after degerming.
(5) vaccine preparation
Above-mentioned vaccinogen liquid and aluminum hydroxide adjuvant obtained to CA16 vaccine work in-process after by suitable proportion absorption.CA16 work in-process, through packing, are finished product vaccine after calibrating.
Embodiment 3CA16 virus strain Study On Immunogenicity
The CA16 strain vaccinogen liquid of preparation in embodiment 2 is carried out to purifying, and after deactivation, immune mouse, new zealand rabbit, sheep all obtain better protecting effect.
Former liquid and preparation method thereof is with described in embodiment 2.
Wherein, Study On Immunogenicity sheep being carried out is as follows:
By after vaccinogen liquid and the absorption of aluminum hydroxide adjuvant equal proportion, in the 0th day, 7 days, 14 days, 21 days, sheep is carried out to immunity, 2ml//time, within whole trial period, the health condition of animal, behavior variation etc. is done to detailed record.Immunity animal on the same day, should observe half an hour.Observe animal for twice every day and have or not death condition.Respectively at the 0th day, 7 days, 14 days, 21 days, 28 days, take a blood sample.Vein is adopted a small amount of blood, the centrifugal 10min of 3000rpm, separation of serum.Carrying out anti-CA16 NAT measures, method is as follows: in 96 porocyte culture plates, add the separated sheep blood serum obtaining, by maintenance medium, carry out doubling dilution to finite concentration, add the CA16 calibrating strain virus liquid that is diluted in advance 100CCID50, in and 1.5h-2h after add calibrating cell suspension, cell concn is 1.5-2.0 * 10
5individual/ml, is placed in 37 ℃, CO
2incubator is cultivated.Observation of cell pathology situation after 5-7 days, wherein take and occurs that the high dilution of cytopathic serum tires as serum neutralization, positively judges index: neutralization is tired and is greater than 1:8, and negative control sera is tired and is less than 1:8.The results are shown in Table 1.
Table 1 pair sheep carries out the neutralizing antibody detected result of Study On Immunogenicity
| Blood sampling time | Serum title | (1 :) is tired in neutralization |
| 0d | Negative serum | <8 |
| 7d | CA16 vaccine immunity sheep blood serum | 16 |
| 14d | CA16 vaccine immunity sheep blood serum | 48 |
| 21d | CA16 vaccine immunity sheep blood serum | 64 |
| 28d | CA16 vaccine immunity sheep blood serum | 96 |
As can be seen from Table 1, use CA16 vaccine prepared by strain of the present invention from immune animal the 7th day, just can bring out sheep body and produce anti-CA16 antiviral antibody, and in rising trend with the increase of immune time.This result shows, uses vaccine prepared by this strain to have better protecting effect to sheep.
Embodiment 4 be take CA16 virus strain and is prepared polyvalent antibody as immunogen
The vaccinogen liquid that uses CA16 strain to prepare in embodiment 2 is purified, and immune new zealand rabbit after deactivation, has obtained the higher anti-CA16 polyvalent antibody of rabbit of NAT.
Former liquid and preparation method thereof is with described in embodiment 2.
By above-mentioned stoste and Freund's complete adjuvant fully emulsified after, take nape portion subcutaneous with muscle multi-point injection, 3ml/ only, 5/batches.Within after first immunisation two weeks, strengthen, after booster immunization vaccinogen liquid and Freund's incomplete adjuvant are fully emulsified, take equally nape portion subcutaneous with muscle multi-point injection, 2ml/ only/time, later weekly booster immunization once, 2ml/ only/time, booster immunization is 4 times altogether, blood sampling before each booster immunization, carries out NAT mensuration to gained serum, judges last blood sampling time.
At NAT, reach after higher level, blood sampling in a week after immunity, under 4 ℃ of conditions, the centrifugal 4min of 3500r/min, collects serum, carry out NAT mensuration, method is as follows: in 96 porocyte culture plates, add the separated rabbit anteserum obtaining, by maintenance medium, carry out doubling dilution to finite concentration, add the CA16 calibrating strain virus liquid that is diluted in advance 100CCID50, in and 1.5h-2h after add calibrating cell suspension, cell concn is 1.5-2.0 * 10
5individual/ml, is placed in 37 ℃, CO
2incubator is cultivated.Observation of cell pathology situation after 5-7 days, wherein take and occurs that the high dilution of cytopathic serum tires as serum neutralization, positively judges index: neutralization is tired and is greater than 1:8, and negative control sera is tired and is less than 1:8.Result under testing into and premising, five the anti-CA16 serum of new zealand rabbit mixed solutions, serum titer can reach 1:6144.This result shows to use CA16 virus strain immunity new zealand rabbit of the present invention, can obtain the anti-CA16 polyvalent antibody of rabbit that serum titer is higher.
For further verifying the immanoprotection action of CA16 vaccine of the present invention; contriver has carried out experimentation on animals; be about to after CA16 vaccine inoculation laboratory animal of the present invention; laboratory animal is carried out to poison attacks; compare with not vaccinated laboratory animal; result shows, CA16 vaccine of the present invention has good vaccine provide protection.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. a coxsackie virus A 16-type virus strain, its preserving number is CGMCC No.5372.
2. described in claim 1, virus strain is prevented or treats by the vaccine of transmissible disease due to coxsackie virus A 16-type and the application in diagnostic reagent in preparation.
3. application according to claim 2, is characterized in that, virus strain, after cells infected, cultivation, results virus liquid, deactivation and purifying, obtains vaccinogen liquid described in claim 1, obtains coxsackie virus A 16-type vaccine after adding immunological adjuvant.
4. application according to claim 3, is characterized in that, the cell that virus strain infects is African green monkey kidney cell or human diploid cell.
5. application according to claim 4, is characterized in that, after cells infected, in 37 ℃ of cultivations, until cytopathy, reaches 75% when above, results virus liquid.
6. according to the application described in claim 3-5 any one, it is characterized in that, beta-propiolactone or formaldehyde solution are used in deactivation.
7. according to the application described in claim 3-5 any one, it is characterized in that, immunological adjuvant is aluminium hydroxide.
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| CN102839159B (en) * | 2012-09-07 | 2014-03-19 | 江苏康淮生物科技有限公司 | CoxA16 virus strain and human CoxA16 inactivated vaccine |
| CN103555672B (en) * | 2013-10-10 | 2015-05-13 | 北京科兴生物制品有限公司 | Coxsackie virus A16 type mouse-adapted strain and application thereof |
| CN107744530B (en) * | 2017-10-12 | 2020-03-27 | 泰山医学院 | Establishment and evaluation of animal model infected with coxsackie virus A10 domesticated strain TA151R-1 |
| CN107746832B (en) * | 2017-10-12 | 2020-06-09 | 山东第一医科大学(山东省医学科学院) | High-titer Coxsackie virus A10 domesticated strain and application thereof |
| CN113564130B (en) * | 2021-09-23 | 2022-01-07 | 北京民海生物科技有限公司 | Coxsackie virus A10 type strain and application thereof |
| CN113564133B (en) * | 2021-09-23 | 2022-01-07 | 北京民海生物科技有限公司 | Coxsackie virus A16 type strain and immunogenic composition and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
| CN101897963A (en) * | 2010-03-18 | 2010-12-01 | 北京绿竹生物技术有限责任公司 | Vaccine for hand-foot-and-mouth disease viruses |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
| CN101897963A (en) * | 2010-03-18 | 2010-12-01 | 北京绿竹生物技术有限责任公司 | Vaccine for hand-foot-and-mouth disease viruses |
Non-Patent Citations (2)
| Title |
|---|
| 扈晓晴.柯萨奇病毒A16型的监测报告.《医学理论与实践》.2010,第23卷(第12期),1501-1502. |
| 柯萨奇病毒A16型的监测报告;扈晓晴;《医学理论与实践》;20101231;第23卷(第12期);1501-1502 * |
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