CN103555672B - Coxsackie virus A16 type mouse-adapted strain and application thereof - Google Patents
Coxsackie virus A16 type mouse-adapted strain and application thereof Download PDFInfo
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Abstract
The invention provides a Coxsackie virus A16 type mouse-adapted strain and an application thereof. The Coxsackie virus A16 type mouse-adapted strain is mCA16V-1243 with a preservation number of CGMCCNo.7860. The mouse-adapted strain provides powerful tool for research of Coxsackie A16 type viral vaccines and screen of efficient antibacterial drugs and for the study of etiology and virus infection mechanisms.
Description
Technical field
The present invention relates to virusology and biology field, specifically, relate to a kind of coxsackie virus A 16-type mouse adapted strain and application 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 is reported first in New Zealand, within 1958, isolates Coxsackie virus first, and proposes HFMD name first in nineteen fifty-nine.The pathogenic agent of the hand foot mouth disease of early discovery is mainly Cox A16 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 Cox A16 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.If indivedual children with serious disease PD is fast, cause death.Wherein Coxsackie virus (Coxasckievirus) A16 type (Cox A16, CA16) is one of main pathogens of hand foot mouth disease, and it is the single strand plus RNA virus of Picornaviridae, spherical in 20 cubic symmetry.Although severe and death cause primarily of EV71 virus infection, it is worth noting that Cox A16 is relevant to comprehensive diseases such as myocarditis, pericarditis, Refractory Shocks especially.
China began to see this disease in Shanghai from 1981, and all there are the relevant report of this disease 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 popularly had a strong impact on daily life, causes huge financial loss and burden on society.Development CA16 virus vaccines and medicine, effectively controlling brothers' mouth epidemic situation is the key subjects that current global brothers' mouth epidemic-stricken area faces.Simultaneously because CA16 virus has kind restriction, only responsive to host, insensitive to other animal, the strict security causing CA16 virus vaccines and medicine to carry out needed for before listing and efficiency evaluation method also become international a difficult problem.Therefore stable CA16 infected animal model is obtained and CA16 mouse adapted strain is particularly important.
Summary of the invention
The object of this invention is to provide a kind of coxsackie virus A 16-type mouse adapted strain and application thereof.
In order to realize the object of the invention, a kind of coxsackie virus A 16-type mouse adapted strain mCA16V-1243 of the present invention, now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC No.7860, preservation date on July 10th, 2013.
The preparation method of above-mentioned coxsackie virus A 16-type mouse adapted strain is: by the coxsackie virus A 16-type virus strain of separation after centrifugal acquisition supernatant, be 6.12lg CCID50/mL by every only 30 μ L(virus titers) carry out going down to posterity 3-6 time in Neonatal Mouse brain, the virus liquid of centrifugal treating collection is mouse adapted strain; Last goes down to posterity inoculation 6 4-6 age in days BALB/c suckling mouses, after 5-9 days, the cerebral tissue of collection, skeletal muscle is carried out homogenate, is coxsackie virus A 16-type mouse adapted strain mCA16V-1243 after centrifugal treating.
In aforementioned preparation process, coxsackie virus A 16-type virus strain is separated from CA16 clinical samples, through being separated, after Plaque Clone differentiates, infects RD cell, in 37 ± 1 DEG C of cultivation, goes down to posterity after 1-3 time, gathers in the crops virus liquid.
The authentication method of described coxsackie virus A 16-type mouse adapted strain mCA16V-1243: CA16 mouse adapted strain seed culture of viruses is inoculated 6 4-6 age in days BALB/c suckling mouses, all virus inoculation suckling mouses all present 1-3 bar limb paralysis symptom after 7 days, are and successfully prepare CA16 mouse adapted strain.
The present invention also provides the application of described coxsackie virus A 16-type mouse adapted strain mCA16V-1243 in research COxsackie A16 C-type virus C pathogenesis.
The present invention also provides a kind of coxsackie virus A 16-type infected animal model, 4-6 age in days BALB/c suckling mouse brain is inoculated with described coxsackie virus A 16-type mouse adapted strain mCA16V-1243, inoculum size is every suckling mouse 50 μ L(virus titer is 3.0-7.0LD50/mL), to obtain final product.
The present invention also provides above-mentioned animal model evaluating the application for preventing or treat in the vaccine safety of transmissible disease caused by COxsackie A16 C-type virus C and validity.
The present invention also provides above-mentioned animal model evaluating the application in the antiviral security and validity for the treatment of transmissible disease caused by COxsackie A16 C-type virus C.
The present invention has the following advantages:
(1) by virus culture and animal experiment technology, obtain coxsackie virus A 16-type BALB/c suckling mouse adapted strain mCA16V-1243, this mouse adapted strain is the research and development of COxsackie A16 C-type virus C vaccine and the screening of high-efficient antiviral medicament, for the research of etiology and virus infection mechanism provides strong instrument.
(2) the present invention establishes stable coxsackie virus A 16-type infected animal model.
(3) be the research and development of COxsackie A16 C-type virus C vaccine and the screening of high-efficient antiviral medicament, for the research of etiology and virus infection mechanism provides technical support.
Accompanying drawing explanation
Fig. 1 is the RT-PCR detected result of clinical isolated viral strain in the embodiment of the present invention 1; Wherein, 1 is CA16 virus strain, and 2 is negative cells contrast, and 3 is EV71 positive control, and 4 is blank reagent contrast, and 5 is DNA molecular amount standard.
Fig. 2 is the qualification result of CA16 mouse adapted strain in the embodiment of the present invention 2, is illustrated as injecting virus group suckling mouse tetraplegia schematic diagram.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's specification sheets advising.
The separation of embodiment 1CA16 virus strain, culture & identification
(1) virus purification, cultivation
The Feces of Patients sample of the CA16 virus PCR result positive is collected from brothers' mouth epidemic-stricken area, Zhejiang Province in 2010 (brothers' mouth adds up number of the infected and reaches 111783), be seeded on African green monkey kidney passage cell (Vero) after treatment, cultivate three generations and carry out virus purification, obtain CA16 virus strain.
(2) viruses indentification
1, RT-PCR detects
The extraction of CA16 geneome RNA is carried out to this strain, reverse transcription becomes cDNA, adopt VP1 conserved regions primer (upstream primer: 5 '-TTGCAGACATGATTGACCAG-3 ', downstream primer: 5 '-GAGTGATGGTTCAACACACA-3 ') it is identified, result shows that isolated strain has single band to occur (Fig. 1) at 212bp place, and the provable strain be separated through clinical samples is CA16 virus.
2, virus titer detected result
Adopt microtitrimetry to measure the virus titer of this strain virus harvest liquid, virus titer can reach 6.12lg CCID50/mL.Method is as follows: adopt Vero cell to carry out virus titer mensuration.By 96 porocyte culture plate Cultivation of Vero, cell in flakes after by virus cell maintenance medium from 10-1 doubling dilution to 10-8, discard Vero cell conditioned medium, add each dilution virus liquid respectively, inoculate 50 μ L/ holes, each extent of dilution inoculates 8 holes, establishes cell controls simultaneously.Put 37 ± 1 DEG C, 5%CO2 incubator is cultivated, and 6-8d observation of cell infects viral situation, calculates virus titer.
3, 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 (purchased from Beijing Kexing Biotech Products Co., Ltd) is coated in 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, occur dye-forming reaction when adding substrate, measure OD value by microplate reader at suitable wavelength, by EXCEL statistic analysis result, antigenic content is 400U/mL.
The preparation of embodiment 2CA16 mouse adapted strain
Virus liquid: by above-mentioned CA16 virus RD cell, 7 days results virus liquids, after freeze thawing three times, centrifuging and taking viral supernatant liquid is for subsequent use.
Animal: buy the pregnant mouse of cleaning grade BALB/c, provides sufficient food and drinking-water, and bedding and padding changed by clean cage house weekly.
The preparation of CA16 mouse adapted strain with build storehouse: inoculate in suckling mouse brain by ready CA16 virus liquid after suckling mouse be born, inoculum size is 30 μ L/ (virus titer is 6.12lg CCID50/mL), establishes normal suckling mouse to contrast simultaneously.After virus inoculation, every day observes suckling mouse state.The cerebral tissue collecting ill mouse carries out homogenate, and after centrifugal treating, continuing to go down to posterity according to upper method obtains mouse adapted strain 4 times.
The qualification of CA16 mouse adapted strain and amplification:
1, CA16 mouse adapted strain seed culture of viruses is inoculated 65 age in days BALB/c suckling mouses, all virus inoculation suckling mouses all present 1-3 bar limb paralysis symptom (Fig. 2) after 7 days, the cerebral tissue of collection, skeletal muscle are carried out homogenized, collected by centrifugation supernatant continues to go down to posterity after 1-2 time and the cerebral tissue of collection, skeletal muscle is carried out homogenized, CA16 virus mouse adapted strain Virus seed library is after the packing of collected by centrifugation supernatant, called after mCA16V-1243, its preserving number is CGMCC No.7860.
2, virus titer detected result
Adopt microtitrimetry to measure the virus titer of this mouse adapted strain virus harvest liquid, virus titer can reach 5.29lg CCID50/mL(Vero cell detection).
The application of embodiment 3CA16 mouse adapted strain
Calibrated mouse adapted strain is diluted to 20LD50/0.1mL; CA16 virus convalescent phase serum balanced mix is infected, 37 DEG C, after neutralization with CA16 virus immunity sheep blood serum and people; inject 5 age in days suckling mouses, the protected effect of human serum after serum and natural infection after preliminary assessment CA16 virus immunity.Result is as shown in table 1:
Poison protection result attacked by table 1
Result shows, and the mouse adapted strain of preparation can the protectiveness of effectively evaluating CA16 virus immunity serum and natural infection CA16 human serum.May further be research COxsackie A16 type etiology, pathogenesis and the screening of high potency drugs and the development of vaccine lays the foundation.
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 without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (2)
1. a coxsackie virus A 16-type mouse adapted strain mCA16V-1243, its preserving number is CGMCC No.7860.
2. the application of coxsackie virus A 16-type mouse adapted strain mCA16V-1243 according to claim 1 in the vaccine of preparation transmissible disease caused by COxsackie A16 C-type virus C.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102533671A (en) * | 2011-12-28 | 2012-07-04 | 北京科兴生物制品有限公司 | Coxsackie virus A16-type virus strain and applications thereof |
| CN103087994A (en) * | 2011-11-03 | 2013-05-08 | 北京科兴生物制品有限公司 | Coxsackievirus A16-type virus strain and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103087994A (en) * | 2011-11-03 | 2013-05-08 | 北京科兴生物制品有限公司 | Coxsackievirus A16-type virus strain and use thereof |
| CN102533671A (en) * | 2011-12-28 | 2012-07-04 | 北京科兴生物制品有限公司 | Coxsackie virus A16-type virus strain and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation;Qunying Mao,et al;《Journal of Virology》;20121130;第86卷(第22期);11967–11976 * |
| Qingwei Liu,et al.A virus-like particle vaccine for coxsackievirus A16 potently elicits neutralizing antibodies that protect mice against lethal challenge.《Vaccine》.2012,第30卷6642-6648. * |
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