CN103087994B - Coxsackievirus A16-type virus strain and use thereof - Google Patents
Coxsackievirus A16-type virus strain and use thereof Download PDFInfo
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- CN103087994B CN103087994B CN201110344375.6A CN201110344375A CN103087994B CN 103087994 B CN103087994 B CN 103087994B CN 201110344375 A CN201110344375 A CN 201110344375A CN 103087994 B CN103087994 B CN 103087994B
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Abstract
The invention provides a coxsackievirus A16-type virus strain and a use thereof. The coxsackievirus A16-type virus strain has the preservation number of CGMCC No. 5371. The full-length sequence analysis and the mass spectrometry analysis on a VP1 protein produced by the coxsackievirus A16-type virus strain prove that the oxsackievirus A16-type virus strain is a good CA16 virus strain which is not polluted by allothigenes and has good immunogenicity. The CA16 virus strain can efficiently proliferate in Vero cells and has virus titer of 7.41g CCID 50/ml. The CA16 virus strain or a vaccine prepared from the CA16 virus strain can be used for preventing diseases caused by CA16 viruses, and has the characteristics of stable titer, good immunogenicity and less immunizing dose.
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) is global infectious disease, and world's most area all has this sick popular report, is common in infant, is caused by enterovirus.Occur that with positions such as heating and hand, foot, oral cavities fash, ulcer etc. show as master clinically, relevant to comprehensive diseases such as myocarditis, pericarditis, Refractory Shocks.
Coxsackie virus (Coxasckievirus) A16 type (Cox A16, CA16) is one of hand foot mouth disease main pathogens, is the single strand plus RNA virus of Picornaviridae, is 20 three-dimensional symmetries spherical, diameter 23-30nm.Mainly propagate through excrement-mouth, respiratory tract and close contact, can pass to fetus by placenta and cause intrauterine infection.The pathogenic agent of the hand foot mouth disease of early discovery is mainly Cox A16 type, after 20 century 70s, infects alternately and occurs with EV71, becomes the main pathogens of hand foot mouth disease.All there is report in multiple provinces such as China Beijing, Fujian, Tianjin, Jilin, Shandong, Guangdong, Shenzhen, Zhejiang, Sichuan, Anhui.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, there is no at present the report of CA16 virus vaccines, therefore isolate the CA16 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.5371, 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 17th, 2011.At this strain of electric Microscopic observation, it is spherical that virion is 20 three-dimensional symmetries, diameter 30nm.
Polymerase, glutelin, glycoprotein, nucleoprotein or Nonstructural Protein that the present invention also provides above-mentioned virus strain to produce.
The present invention also provides the gene of the above-mentioned polymerase of coding, glutelin, glycoprotein, nucleoprotein or Nonstructural Protein.
The host cell that the present invention also provides the carrier that contains said gene and contains this carrier.
The present invention also provides described virus strain, described polymerase, glutelin, glycoprotein, nucleoprotein or Nonstructural Protein, the encode gene of described polymerase, glutelin, glycoprotein, nucleoprotein or Nonstructural Protein, the carrier that contains described gene or the host cell that contains described carrier in preparation prevention or treatment by the application in medicine or vaccine and the diagnostic reagent of transmissible disease due to Coxsackie virus.
Aforesaid application, its 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, for further preparing CA16 vaccine.
The present invention also provides with coxsackie virus A 16-type virus strain, and preserving number CGMCCNo.5371 is antibody or hybridoma or the antiserum(antisera) that immunogen makes.
The present invention is also provided for detecting the primer of described coxsackie virus A 16-type virus strain, comprises CA16 upstream primer: 5 '-TTGCAGACATGATTGACCAG-3 ' and CA16 downstream primer: 5 '-GAGTGATGGTTCAACACACA-3 '.
The present invention also provides the test kit for detection of coxsackie virus A 16-type that contains above-mentioned primer.
The present invention further provides the VP1 albumen that described coxsackie virus A 16-type virus strain produces, its aminoacid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of this VP1 albumen of encoding is as shown in SEQ ID NO.1.The analysis of VP1 full length sequence and mass spectrometry results being shown to this strain is CA16 virus, and pollute without xenobiotics, have good immunogenicity, is the respond well virus strain of a strain.
The present invention also provides a kind of Dispersal risk or hybridoma or sero-fast method, and it is take coxsackie virus A 16-type virus strain of the present invention (CGMCC No.5371) as immunogen.
By technique scheme, the present invention at least has following advantages and beneficial effect:
(1) separate by plaque method the mono-clonal CA16 virus strain of the present invention obtaining, 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 by the vaccine of its production disease (for example hand foot mouth disease that prevention is caused by CA16 virus, especially children's hand foot mouth disease), and have the advantages that titre is stable, immunogenicity is better, immunizing dose is little.
(3) CA16 virus strain of the present invention can be in Vero cell high efficiently multiplying, virus titer can reach 7.41g 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 separates, cultivates
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, obtain CA16 virus strain by plaque method.
(2) virus is identified
1, VP1 full length sequence analytical results
The VP1 albumen that this strain is produced carries out the analysis of VP1 full length sequence, and its aminoacid sequence is as shown in SEQ ID NO.2, and the gene order of this VP1 albumen of encoding is as shown in SEQ ID NO.1.
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 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.(detecting according to " Chinese Pharmacopoeia " method)
5, virus titer detected result
Adopt microtitrimetry to measure the virus titer of this strain virus harvest liquid, virus titer can reach 7.41g 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, in the time adding substrate, there is dye-forming reaction, measure OD value at suitable wavelength by microplate reader, by EXCEL statistic analysis result.
Embodiment 2 detects primer and the test kit of CA16 virus strain
1, for detection of the primer of CA16 virus strain, comprising:
CA16 upstream primer: 5 '-TTGCAGACATGATTGACCAG-3 ' and
CA16 downstream primer: 5 '-GAGTGATGGTTCAACACACA-3 '.
2, the detection kit for detection of CA16 virus strain that contains above-mentioned primer.
The preparation of embodiment 3CA16 vaccine
CA16 virus strain, through infecting after vero cell, 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. ATCC120 recovers for African green monkey kidney cell (Vero cell) seed, concrete operations are: in liquid nitrogen, take out cell cryopreservation tube, be placed in 39-40 ℃ of sterilized water, the cell that thaws within one minute, aseptic sucking-off suspension, the centrifugal 3min of 1000rpm, abandon supernatant, add the MEM cell growth medium containing 10% calf serum, piping and druming mixes it gently, 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%, goes down to posterity by 1: 4, 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, obtains CA16 virus strain by plaque method.Set up viral main seed bank and work seed bank according to " Chinese Pharmacopoeia " about the requirement of seed bank establishment method, all freezing preservations of seed bank (60 ℃ following).
Banking process is as follows: CA16 virus strain is seeded to degree of converging (substratum is the MEM cell growth medium containing 2% calf serum) in 100% Vero cell (from cell work seed bank) bottle by 1: 1000 (volume ratio), 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.And send China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation.Its preserving number is CGMCC No.5371.
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, for subsequent use.
(4) inactivation of virus and purifying
By the CA16 virus harvest liquid of above-mentioned preparation with 1: 100-1: the beta-propiolactone of 10000 (v/v) or 1: 1000-1: the formaldehyde solution deactivation of 10000 (v/v).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 slowly adds PEG simultaneously, and making PEG final concentration is 5-15%, adjusts pH value 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, obtains viral desugar liquid through ultrafiltration desugar.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 4CA16 virus strain Study On Immunogenicity
The vaccinogen liquid CA16 strain of preparation in embodiment 3 is carried out to enlarged culturing on Vero cell, purifying, after deactivation, immune mouse, new zealand rabbit, sheep all obtain better protecting effect.
Former liquid and preparation method thereof is with described in embodiment 3.
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, detailed record is done in health condition, behavior variation etc. to animal.Should observe half an hour immunity animal on the same day.Observe animal for twice every day and have or not death condition.Took a blood sample respectively at the 0th day, 7 days, 14 days, 21 days, 28 days.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 and separate the sheep blood serum obtaining, carry out doubling dilution to finite concentration by maintenance medium, add the CA16 calibrating strain solution that is diluted in advance 100CCID50, in and 1.5h-2h after add inspection 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 to occur 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 carries out the neutralizing antibody detected result of Study On Immunogenicity to sheep
| 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 | 32 |
| 21d | CA16 vaccine immunity sheep blood serum | 48 |
| 28d | CA16 vaccine immunity sheep blood serum | 64 |
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 5 prepares polyvalent antibody take CA16 virus strain as immunogen
The vaccinogen liquid CA16 strain of preparation in embodiment 3 is carried out to enlarged culturing on Vero cell, purifying, 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 3.
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 4 times altogether, blood sampling before each booster immunization, carries out NAT mensuration to gained serum, judges last blood sampling time.
Reach after higher level at NAT, 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 and separate the rabbit anteserum obtaining, carry out doubling dilution to finite concentration by maintenance medium, 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 to occur 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.
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 (4)
1. a coxsackie virus A 16-type virus strain, its preserving number is CGMCC No.5371.
2. described in claim 1, virus strain is prevented or treats by the application in the vaccine of transmissible disease due to coxsackie virus A 16-type 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, for further preparing coxsackie virus A 16-type vaccine described in claim 1.
4. the polyvalent antibody making take virus strain claimed in claim 1 as immunogen.
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| CN104513310B (en) * | 2013-09-27 | 2017-12-08 | 中国科学院上海巴斯德研究所 | A kind of preparation and its application of the monoclonal antibody of anti-coxsackie virus A 16-type |
| 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 |
| CN105483289B (en) * | 2015-12-29 | 2019-10-08 | 深圳澳东检验检测科技有限公司 | A kind of detection kit and reaction system of the triple direct fluorescence RT-PCRs of enterovirus |
| CN113564132B (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 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 |
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