CN102533671A - 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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- CN102533671A CN102533671A CN2011104479365A CN201110447936A CN102533671A CN 102533671 A CN102533671 A CN 102533671A CN 2011104479365 A CN2011104479365 A CN 2011104479365A CN 201110447936 A CN201110447936 A CN 201110447936A CN 102533671 A CN102533671 A CN 102533671A
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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 that causes by enterovirus; Pilosity is born in children below 5 years old, can cause the bleb at positions such as hand, foot, oral cavity, and the minority infant can cause complication such as wet lung, AME.If indivedual severe infant 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 are the more common pathogenic agent of hand foot mouth disease.Mainly through excrement-mouth, respiratory tract and contact transmission closely, can pass to fetus through placenta and cause in the official and infect.Wherein (Cox A16 CA16) is one of hand foot mouth disease main pathogens to Coxsackie virus (Coxasckievirus) A16 type, and it is the sub-thread positive chain RNA virus of Picornaviridae, and it is spherical to be 20 three-dimensional symmetries.Though severe and death are mainly caused by the EV71 virus infection, it is worth noting that especially Cox A16 is relevant with comprehensive diseases such as myocarditis, pericarditis, intractable shocks.
World's most of areas all has this sick popular report, and China began to see this disease in Shanghai that all there was report in tens provinces and cities such as later Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, Hubei, Xining, Guangdong from 1981.The outburst of hand foot mouth disease and the popular daily life that had a strong impact on; Cause enormous economic loss and burden on society; The propagation that utilizes virus vaccines fundamentally to cut off virus is comparatively effective one of prevention approach at present; Still the report that does not have at present the CA16 virus vaccines, therefore isolating the CA16 virus strain that is suitable for production of vaccine has huge economic and social benefit.
Summary of the invention
The purpose 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; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City at present, preservation date on October 18th, 2011.Under Electronic Speculum, observe this strain, it is spherical that virion is 20 three-dimensional symmetries, the about 23-30nm of diameter.
Aforesaid application; Be with said coxsackie virus A 16-type virus strain through cells infected (like Vero cell, human diploid cell or other sensitive cells), cultivate, gather in the crops viral liquid, deactivation and purifying after; Obtain vaccinogen liquid, promptly get the coxsackie virus A 16-type vaccine after adding immunological adjuvant (like white lake etc.).
Aforesaid application behind the virus strain cells infected, in 37 ℃ of cultivations, treats that cytopathy reaches 75% when above, gathers in the crops viral liquid.
Aforesaid application, beta-propiolactone or formaldehyde solution are used in deactivation.Preferably use 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 later at viral purification.
The present invention also provides, and is immunogen preparing antibody or hybridoma or antiserum(antisera) with coxsackie virus A 16-type virus strain of the present invention (CGMCC No.5372).Wherein, sero-fast preparation method is: behind virus strain process cells infected, cultivation, results virus liquid, deactivation and the purifying, obtain vaccinogen liquid, use the vaccinogen liquid immune animal, obtain antiserum(antisera).Said animal is preferably monkey, sheep, rabbit etc.
Through the VP1 albumen that further research virus strain of the present invention produces, sequential analysis of VP1 conserved regions and mass spectrometry results show that this strain is a CA16 virus, and do not have xenobiotics and pollute, and better immunogenicity is arranged, and are the respond well virus strain of a strain.
By technique scheme, the present invention has advantage and beneficial effect at least:
(1) separate the mono-clonal CA16 virus strain of the present invention that obtains through the plaque method, its progeny virus shows as inheritance stability, can be used for human CA16 production of vaccine.
(2) utilize CA16 virus strain of the present invention or can be used for preventing the disease (for example hand foot mouth disease, especially children's hand foot mouth disease) that causes by CA16 virus, and have the advantages that titre is stable, immunogenicity is better, immunizing dose is little by the vaccine of its production.
(3) CA16 virus strain of the present invention can be in the Vero cell high efficiently multiplying, virus titer can reach 6.61g CCID50/ml.
Description of drawings
Fig. 1 is CA16 virus strain electron microscopic observation result of the present invention (magnification ratio: 100,000 times).
Fig. 2 is a CA16 virus strain protein spectrum analytical results of the present invention.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
Separation, cultivation and the evaluation of embodiment 1 CA16 virus strain
(1) virus is separated, is cultivated
(brothers' mouth accumulative total number of the infected reaches 111783) collected CA16 virus PCR male patient stool sample as a result from brothers' mouth epidemic-stricken area, Zhejiang Province in 2010; Be seeded to after treatment on the African green monkey kidney passage cell (Vero); Cultivate the three generations and carry out the virus separation; After obtaining CA16 virus, obtain the CA16 virus strain through the plaque method.
(2) virus is identified
1, VP1 conserved regions The sequencing results
This strain VP1 conserved regions sequence is analyzed, its with compare with reference to strain sequence (human coxsackievirus A16 C-type virus C strain shzh00-1, complete genome group sequence GenBank:AY790926.1), nucleotide homology is more than 93.8%.
2, electron microscopic examination result
Under Electronic Speculum, observe this strain CA16 virus, it is spherical that virion is 20 three-dimensional symmetries, the about 23-30nm of diameter.(Fig. 1)
3, mass spectrometry results
This strain CA16 viral protein is carried out mass spectroscopy, and the result shows that this strain albumen really is CA16 viral protein (Fig. 2).
4, aseptic, mycoplasma check result
This strain CA16 virus is carried out aseptic, mycoplasma inspection, and the result shows no mycoplasma and other microbial contamination.(according to " the Chinese pharmacopoeia method detects)
5, virus titer detected result
Adopt microtitrimetry to measure the virus titer of this strain virus results liquid, virus titer can reach 6.61g CCID50/ml.Method is following: adopt the Vero cell to carry out the mensuration of virus titer.With 96 porocyte culture plate Cultivation of Vero, cell in flakes after with virus with cell maintenance medium from 10
-1Doubling dilution to 10
-8, discard the Vero cell conditioned medium, add each dilution viral liquid respectively, inoculate 50 μ l/ holes, each extent of dilution is inoculated 8 holes, establishes the cell contrast simultaneously.Put 37 ℃, 5%CO
2Incubator is cultivated, and 7d observation of cell infective virus situation is calculated virus titer.
6, antigenic content detects
Adopt ELISA that this strain CA16 virus antigen content is detected, antigenic content is not less than 200U/ml.Adopt CA16 antigenic content in the double antibody sandwich method quantitatively determined sample.At first specific C A16 polyclonal antibody is encapsulated in enzyme plate and form insolubilized antibody; Add the CA16 sample then, form the solid phase antigen antibody complex with insolubilized antibody; Add enzymic-labelled antibody at last, the CA16 antigen in the sample can combine with enzyme labelled antibody generation specificity, dye-forming reaction when adding substrate, occurs, measures the OD value with ELIASA at suitable wavelength, through the EXCEL statistic analysis result.
The preparation of embodiment 2 CA16 vaccines
Behind CA16 virus strain process vero cells infection, cultivation, results virus liquid, deactivation and the purifying, obtain vaccinogen liquid, be used for further preparing the CA16 vaccine.
(1) sets up cell master seed and work seed bank (Vero cell)
To be derived from U.S. ATCC, the recovery of 120 generation African green monkey kidney cell (Vero cell) seeds, concrete operations are: in liquid nitrogen, take out the cell cryopreservation pipe; Place 39-40 ℃ of sterilized water, the cell that thaws within a minute, aseptic sucking-off suspension; The centrifugal 3min of 1000rpm abandons supernatant, adds the MEM cell growth medium that contains 10% calf serum; Piping and druming makes its mixing gently, with the cell suspension inoculation of mixing in 25cm
2Tissue Culture Flask in, put 37 ℃, 5%CO
2Incubator is cultivated, and treats that it changes liquid after adherent, puts 37 ℃ again, 5%CO
2Incubator is cultivated, and 5-7 days cell degree of converging reach at 100% o'clock and went down to posterity by 1: 4, and the cell generation that whenever goes down to posterity increases a generation.Prepare 129 generation Vero cell work seed banks as stated above.According to aforesaid method, the researchist of this area can prepare equally and satisfy the work seed bank that the present invention requires.
Cell master seed bank all is kept in the liquid nitrogen (196 ℃) with the work seed bank.
(2) set up the master for viral seed bank and work seed bank
The CA16 virus PCR is male hand foot mouth disease patient's clinical samples as a result, is seeded to after treatment on the African green monkey kidney passage cell (Vero), cultivates the three generations and carries out virus and separate, obtain CA16 virus after, through plaque method acquisition CA16 virus strain.According to " Chinese pharmacopoeia is set up viral main seed bank and work seed bank, all freezing preservation of seed bank (below 60 ℃) about the requirement of seed bank establishment method.
Banking process is following: the CA16 virus strain is seeded to degree of converging in 100% Vero cell (from the cell work seed bank) bottle (substratum is the MEM cell virus growth media that contains 2% calf serum) by 1: 1000 (volume ratio), puts 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 was melted, the freeze thawing thing continued to go down to posterity as stated above, and to set up virus main for seed bank and work seed bank.And send China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation.Its preserving number is CGMCC No.5372.
Each batch results liquid is designated as a generation.
(3) virus results 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 the observation of cell pathology, when cytopathy reaches 50% when above; Gather in the crops viral liquid, rearmounted-20 ℃ of preservations of mixing, subsequent use.
(4) inactivation of virus and purifying
The CA16 virus of above-mentioned preparation is gathered in the crops liquid 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 later at viral purification.
After behind the inactivation of virus above-mentioned deactivation liquid being carried out centrifugal clarification, carry out ultrafiltration dialysis, volume is concentrated into the 1/10-50 of original volume.The viral ultrafiltrated that obtains adds magnetic agitation, places on the magnetic stirring apparatus and stirs, and it is 5-15% that while slow adding PEG (molecular weight is 6000) makes the PEG final concentration, and adjust pH continues to stir 10-60min between the 5.0-7.0.Centrifugal 20-60min behind 2-8 ℃ of deposition 8-24h, precipitate heavily molten with the PBS damping fluid, the viral precipitated liquid of centrifugal acquisition afterwards.Virus precipitated liquid again through SDGC, collect viral location band after, obtain viral desugar liquid through the ultrafiltration desugar.Virus desugar liquid obtains viral chromatographic solution behind sieve chromatography, chromatographic solution obtains vaccinogen liquid after degerming.
(5) vaccine production
Above-mentioned vaccinogen liquid and aluminum hydroxide adjuvant are promptly obtained CA16 vaccine work in-process after by suitable proportion absorption.The CA16 work in-process are the finished product vaccine through packing after the calibrating.
The test of embodiment 3 CA16 virus strain immunogenicities
The CA16 strain vaccinogen liquid of preparation among the embodiment 2 is carried out purifying, and immune mouse, new zealand rabbit, sheep all obtain the better protecting effect after the deactivation.
Former liquid and preparation method thereof is said with embodiment 2.
The immunogenicity of wherein, sheep being carried out is tested as follows:
After the absorption of vaccinogen liquid and aluminum hydroxide adjuvant equal proportion, in the 0th day, 7 days, 14 days, 21 days sheep is carried out immunity, 2ml/ only/inferior, in whole test in the phase, detail record is done in the health condition of animal, behavior variation etc.Should observe half a hour immunity animal on the same day.Twice observation every day animal has 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 following: in 96 porocyte culture plates, add and separate the sheep blood serum that obtains; Carry out doubling dilution to finite concentration with keeping liquid; Add the CA16 calibrating strain virus liquid be diluted to 100CCID50 in advance, in 1.5h-2h after add calibrating to use cell suspension, cell concn be 1.5-2.0 * 10
5Individual/ml, place 37 ℃, CO
2Incubator is cultivated.Observation of cell pathology situation after 5-7 days is that the serum neutralization is tired the high dilution of cytopathic serum to occur wherein, positively judges index: neutralization is tired greater than 1: 8, and negative control sera is tired less than 1: 8.The result sees table 1.
Table 1 pair sheep carries out the neutralizing antibody detected result of immunogenicity test
| Blood sampling time | The 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 |
Can find out that from table 1 the CA16 vaccine that uses strain preparation of the present invention just can bring out the sheep body and produce anti-CA16 antiviral antibody from immune animal beginning in the 7th day, and in rising trend with the increase of immune time.This result shows, uses the vaccine of this strain preparation that sheep is had the better protecting effect.
Embodiment 4 is the immunogen preparing polyvalent antibody with the CA16 virus strain
The vaccinogen liquid that uses the preparation of CA16 strain among the embodiment 2 is purified, and immune new zealand rabbit after the deactivation has obtained the higher anti-CA16 polyvalent antibody of rabbit of NAT.
Former liquid and preparation method thereof is said with embodiment 2.
With above-mentioned stoste and Freund's complete adjuvant fully emulsified after, take nape portion subcutaneous with the muscle multi-point injection, 3ml/ only, 5/batches.Two weeks were strengthened after the first immunisation, after booster immunization vaccinogen liquid and Freund's incomplete adjuvant are fully emulsified, taked the subcutaneous and muscle multi-point injection of nape portion equally; 2ml/ only/inferior, later on weekly booster immunization once, 2ml/ only/inferior; Booster immunization is 4 times altogether; Blood sampling before each booster immunization is carried out NAT to gained serum and is measured, and judges last blood sampling time.
After NAT reached higher level, then week blood sampling after immunity was under 4 ℃ of conditions; The centrifugal 4min of 3500r/min collects serum, carries out NAT and measures; Method is following: in 96 porocyte culture plates, add and separate the rabbit anteserum that obtains, carry out doubling dilution to finite concentration with keeping liquid, add the CA16 calibrating strain virus liquid that is diluted to 100CCID50 in advance; In with 1.5h-2h after add calibrating to use cell suspension, cell concn be 1.5-2.0 * 10
5Individual/ml, place 37 ℃, CO
2Incubator is cultivated.Observation of cell pathology situation after 5-7 days is that the serum neutralization is tired the high dilution of cytopathic serum to occur wherein, positively judges index: neutralization is tired greater than 1: 8, and negative control sera is tired less than 1: 8.The result is testing to become to premise down, five the anti-CA16 serum of new zealand rabbit mixed solutions, and serum titer can reach 1: 6144.This result shows use CA16 virus strain immunity of the present invention new zealand rabbit, can obtain the anti-CA16 polyvalent antibody of the higher rabbit of serum titer.
For further verifying the immanoprotection action of CA16 vaccine of the present invention; The contriver has carried out experimentation on animals; After being about to CA16 vaccine inoculation laboratory animal of the present invention, laboratory animal being carried out poison attack, compare with not vaccinated laboratory animal; The result shows that CA16 vaccine of the present invention has vaccine provide protection preferably.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. coxsackie virus A 16-type virus strain, its preserving number is CGMCC No.5372.
2. the said virus strain of claim 1 is preparing prevention or treatment by the medicine of transmissible disease due to the Coxsackie virus or the application in vaccine and the diagnostic reagent.
3. application according to claim 2 is characterized in that, behind the said virus strain process of claim 1 cells infected, cultivation, results virus liquid, deactivation and the purifying, obtains vaccinogen liquid, promptly gets the coxsackie virus A 16-type vaccine after the 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, behind the cells infected, in 37 ℃ of cultivations, treats that cytopathy reaches 75% when above, gathers in the crops viral liquid.
6. according to each described application of claim 3-5, it is characterized in that beta-propiolactone or formaldehyde solution are used in deactivation.
7. according to each described application of claim 3-5, it is characterized in that immunological adjuvant is a white lake.
8. be antibody or hybridoma or the antiserum(antisera) that immunogen makes with the described virus strain of claim 1.
9. be the sero-fast method of immunogen preparing with the described virus strain of claim 1; It is characterized in that, behind the said virus strain process of claim 1 cells infected, cultivation, results virus liquid, deactivation and the purifying, obtain vaccinogen liquid; Use the vaccinogen liquid immune animal, obtain antiserum(antisera).
10. method according to claim 9 is characterized in that said animal is monkey, sheep, exempts from.
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| CN102839159A (en) * | 2012-09-07 | 2012-12-26 | 江苏康淮生物科技有限公司 | CoxA16 virus strain and human CoxA16 inactivated vaccine |
| CN103555672A (en) * | 2013-10-10 | 2014-02-05 | 北京科兴生物制品有限公司 | Coxsackie virus A16 type mouse-adapted strain and application thereof |
| CN107746832A (en) * | 2017-10-12 | 2018-03-02 | 泰山医学院 | The Coxsackie virus A 10 of one plant height titre tames strain and its application |
| CN107744530A (en) * | 2017-10-12 | 2018-03-02 | 泰山医学院 | Coxsackie virus A 10 tames the foundation and evaluation of the infected animal models of strain TA151R 1 |
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| CN102839159A (en) * | 2012-09-07 | 2012-12-26 | 江苏康淮生物科技有限公司 | CoxA16 virus strain and human CoxA16 inactivated vaccine |
| CN103555672A (en) * | 2013-10-10 | 2014-02-05 | 北京科兴生物制品有限公司 | Coxsackie virus A16 type mouse-adapted strain and application thereof |
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| CN107746832A (en) * | 2017-10-12 | 2018-03-02 | 泰山医学院 | The Coxsackie virus A 10 of one plant height titre tames strain and its application |
| CN107744530A (en) * | 2017-10-12 | 2018-03-02 | 泰山医学院 | Coxsackie virus A 10 tames the foundation and evaluation of the infected animal models of strain TA151R 1 |
| 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 |
| CN113564133A (en) * | 2021-09-23 | 2021-10-29 | 北京民海生物科技有限公司 | Coxsackie virus A16 type strain and immunogenic composition and application thereof |
| CN113564130A (en) * | 2021-09-23 | 2021-10-29 | 北京民海生物科技有限公司 | Coxsackie virus A10 type 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 |
| WO2023045426A1 (en) * | 2021-09-23 | 2023-03-30 | 北京民海生物科技有限公司 | Coxsackie virus a16 strain and immunogenic composition and application thereof |
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