CN103160474B - Enterovirus 71 type virus strain, vaccine, animal model establishment method - Google Patents
Enterovirus 71 type virus strain, vaccine, animal model establishment method Download PDFInfo
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- CN103160474B CN103160474B CN201110418318.8A CN201110418318A CN103160474B CN 103160474 B CN103160474 B CN 103160474B CN 201110418318 A CN201110418318 A CN 201110418318A CN 103160474 B CN103160474 B CN 103160474B
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Abstract
The invention provides an Enterovirus 71 (EV71 for short) type virus strain, a vaccine, and an animal model establishment method. The preservation number of the virus strain is CGMCC No.5539. The EV71 type virus strain provided in the invention has the advantages that: it has strong toxicity, and can be used for evaluation of an EV71 vaccine or drug. The establishment method of the animal model for evaluation of the EV71 vaccine effect provided in the invention can provide a stable animal model, thus providing the basis for development of the EV71 vaccine and screening of antiviral drugs. The vaccine prepared from the EV71 virus strain is in accordance with the relevant requirements of ''Chinese Pharmacopoeia (three parts) 2010 edition'', and has effective immunoactivity and toxicity attack protection capability. The preparation method of the vaccine has the advantages of easily controllable production process, good repeatability, and suitability for large-scale production, and the prepared vaccine has stable quality.
Description
Technical field
The invention belongs to production of vaccine field, in particular to a kind of enterovirus type 71 viruses strain and uses thereof, the vaccine prepared by this enterovirus type 71 viruses strain and preparation method and the establishment method for the animal model of evaluating enterovirns type 71 vaccine effect.
Background technology
Hand foot mouth disease (Hand, foot and mouth disease, HFMD) be a kind of acute infectious disease caused by enterovirus infection, the multiple infant being born in less than 5 years old, with the fash of the area skin mucous membranes such as hand, foot, oral cavity, bleb, ulcer for typical performance, small number of patients can cause the complication such as myocarditis, pulmonary edema, sterility cerebrospinal meningitis, encephalitis.The enterovirus causing hand foot mouth disease has kind more than 20 (type), wherein with coxsackie virus A 16-type (CA16) and enterovirns type 71 (hereinafter referred to as EV71) the most common, wherein EV71 not only causes hand foot mouth disease, and severe central nervous system complication can be caused, as meningitis, encephalitis, acute flaccid paralysis etc.Research finds, in grave infection's case, EV71 infects and accounts for more than 90%.
China was in Shanghai reported first hand foot mouth disease in 1981, and after this, tens provinces such as Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, Hubei, Qinghai and Guangdong all have this disease to report.The hand foot mouth disease outburst that nineteen eighty-three Tianjin generation Cox A16 causes, breaks out for 1986 again.China's Mainland Late Cambrian EV71 type infection is a hand foot mouth disease epidemic period in Hubei Province's winter in 1987.2010, hand foot mouth disease case 1593437 example was reported in the whole nation altogether, dead more than 839 people.
There is no the specificity vaccine, medicine etc. for this disease of prevention at present.Wherein, in the triturating of the specificity vaccine of this disease of prevention, enterovirus type 71 viruses vaccine belongs to the new generation vaccine of prevention human infectious disease, the country such as China Taiwan and Singapore started the research carrying out related vaccines once, comprise inactivated vaccine, recombiant vaccine, polypeptide vaccine and attenuated live vaccine etc., but all rest on the preclinical study stage.At present, the research of EV71 vaccine worldwide makes a breakthrough not yet, but seroepidemiological survey data shows, and the EV71 neutralizing antibody produced after population infection effectively can prevent the subinfection again of EV71 virus, and certain amynologic basis has been established in the research and development for EV71 vaccine.Because between different genotype strain, between homologous genes type different genes hypotype strain, even, between homologous genes hypotype different strain, antigenicity and immunogenicity may there are differences, thus cause the cross protection level of different strain there are differences, stern challenge is proposed to the selection of vaccine strain; Although EV71 infects for many years popular, EV71 infects repeatedly outburst and causes more severe even death in recent years, and its mechanism of causing a disease is still unclear; These all make the development of EV71 vaccine there is certain difficulty.
In addition, the research and development of domestic and international enterovirns type 71 vaccine also lock into stable animal model, therefore set up mature and stable animal model and seem particularly important.
Summary of the invention
For solving above-mentioned problems of the prior art, the invention provides a kind of enterovirus type 71 viruses strain and uses thereof.
Specifically, the invention provides:
(1) enterovirns type 71 (Enterovirus 71) virus strain, the deposit number of described virus strain is CGMCC No.5539.
(2) virus strain Gen Ju (1), the complete genome sequence of wherein said virus strain is the sequence shown in SEQ ID No.:2.
(3) virus strain Gen Ju (1) is for the preparation of the purposes prevented and/or treated in the medicine of hand foot mouth disease.
(4) purposes Gen Ju (3), wherein, described medicine is vaccine.
(5) for preventing and/or treating a vaccine for hand foot mouth disease, wherein, described vaccine contains: 1) through the virus strain described in (1) or (2) of deactivation; And optional 2) adjuvant.
(6) vaccine Gen Ju (5), wherein, with viral protein content meter, the content of described virus strain is 1 ~ 4 μ g/ml; And/or described adjuvant is aluminium hydroxide, and the content of described aluminium hydroxide is 0.5 ~ 1.5 μ g/ml.
(7) preparation method of a kind of basis (5) or the vaccine described in (6), it comprises:
Virus strain described in (1) or (2) is cultivated, and carries out deactivation, purifying, thus prepare described vaccine.
(8) Dispersal risk or hybridoma or a sero-fast method, wherein, be that immunogen is prepared with the vaccine described in the virus strain Gen Ju (1) or (2) or (5) or (6).
(9) Dispersal risk obtained according to the preparation method of (8) or hybridoma or antiserum(antisera).
(10) setting up for evaluating the purposes in the animal model of enterovirns type 71 vaccine effect according to (1) or the virus strain described in (2).
(11) purposes Gen Ju (10), described virus strain is used as to attack strain.
(12) for evaluating an establishment method for the animal model of enterovirns type 71 vaccine effect, it comprises:
1) virus strain described in (1) is cultivated, obtain virus-culturing fluid; And
2) by step 1) virus-culturing fluid of gained bestows animal, thus prepares described animal model;
Wherein, described animal is mouse.
(13) preparation method Gen Ju (12), wherein, described mouse is the suckling mouse of 1-14 age in days.
(14) preparation method Gen Ju (13), wherein, described suckling mouse obtains by the following method: will often only grow up female mouse and 1 Female odor mating, before described mating, enterovirns type 71 vaccine to be evaluated is adopted to carry out initial immunity, adopt this enterovirns type 71 vaccine to carry out booster immunization in described post-coitum 2-3 week to described female mouse, and nest is divided by itself and described male mouse after described female mouse pregnancy, the suckling mouse given birth to by described female mouse cultivates 1-14 days.
The present invention compared with prior art has the following advantages and positively effect:
1. the invention provides two kinds of new enterovirus type 71 viruses strains (EV71 virus strain 1 and 2); and the vaccine 1 and 2 (being respectively EV71 virus strain 1 and 2 prepared) for preventing hand foot mouth disease using this virus strain to produce; the relevant requirements that the vaccine 1 and 2 produced all meets " Chinese Pharmacopoeia (three) version in 2010 ", and there is effective immunocompetence and attack malicious protective capability.Cell adaptation is good, output is high, cross immunogenicity is good for virus strain of the present invention (EV71 virus strain 1 and 2); After the EV71 vaccine 1 adopting method of the present invention to prepare or EV71 vaccine 2 immune animal; body can be stimulated to produce the specific serum neutralizing antibody of high-titer; after animal immune; pathogenic stronger strain is adopted to carry out virus attack; animal indices is normal; and there is not clinical symptom, show that this vaccine has better protecting effect.
2. the preparation method of described vaccine 1 or 2 provided by the present invention, has the following advantages: production process is easy to control, reproducible, applicable large-scale production, steady quality.
3. EV71 virus strain 2 provided by the present invention also can be used as attacking poison virus strain, and its tool has the following advantages: virulence is strong, suckling mouse can be made to occur typical EV71 infection symptoms such as significantly benumbing, paralysis is even dead, can be used for the evaluation of EV71 vaccine or medicine.
4. present invention also offers the establishment method of the animal model for evaluating enterovirns type 71 vaccine effect, the method can provide stable animal model, for the screening of enterovirns type 71 vaccine development and antiviral provides the foundation.
Preservation information:
Enterovirus type 71 viruses strain about deposit number is CGMCC No.5540: the Classification And Nomenclature of this virus is human enterovirus 71 (Human Enterovirus 71), Latin name is Enterovirus 71, in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 5th, 2011, Institute of Microorganism, Academia Sinica) preservation carried out, deposit number is CGMCC No.5540.
Enterovirus type 71 viruses strain about deposit number is CGMCC No.5539: the Classification And Nomenclature of this virus is human enterovirus 71 (Human Enterovirus 71), Latin name is Enterovirus 71, in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 5th, 2011, Institute of Microorganism, Academia Sinica) preservation carried out, deposit number is CGMCC No.5539.
Accompanying drawing explanation
Fig. 1 is EV71 virus strain 1 of the present invention continuous passage titration results figure on Vero cell;
Fig. 2 is that malicious survival rate graphic representation (A) is attacked in the suckling mouse abdominal cavity of suckling mouse model after vaccine 1 of the present invention immunity set up by one embodiment of the invention, suckling mouse encephalocoele attacks malicious survival rate graphic representation (B);
Fig. 3 is suckling mouse survival rate graphic representation after employing EV71 virus strain 2 intracranial challenge of the present invention;
Fig. 4 attacks rear suckling mouse survival rate graphic representation for adopting EV71 virus strain 2 abdominal cavity of the present invention;
Fig. 5 attacks rear suckling mouse morbidity displaying chart for adopting EV71 virus strain 2 of the present invention, and wherein A figure is the normal suckling mouse of Normal group, and B figure is virus group hindlimb paralysis, paralysis suckling mouse;
Fig. 6 is that malicious survival rate graphic representation (A) is attacked in the suckling mouse abdominal cavity of suckling mouse model after vaccine 1 of the present invention immunity set up by another embodiment of the invention, encephalic attacks malicious survival rate graphic representation (B);
Fig. 7 is neutralizing antibody level view after vaccine 1 of the present invention immunity;
Fig. 8 is vaccine 1 inoculation group of the present invention and the horizontal comparison diagram of EV71RNA in non-vaccination control group tissue, and wherein in X-coordinate, C1 ~ C10 represents 10 individualities in non-vaccination group, i.e. 10 not vaccinated suckling mouses; P1 ~ P10 represents 10 individualities in vaccine inoculation group, i.e. 10 vaccinated suckling mouses;
Fig. 9 is that malicious survival rate graphic representation (A) is attacked in the suckling mouse abdominal cavity of suckling mouse model after vaccine 2 of the present invention immunity set up by one embodiment of the invention, encephalic attacks malicious survival rate graphic representation (B).
Embodiment
Below by way of embodiment description and the invention will be further described with reference to accompanying drawing, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
In this article, described " attacking poison strain " refers to the attack strain that animal can be caused to fall ill.EV71 virus strain 2 both can be used as attacking strain in the present invention, also can for the preparation of vaccine after deactivation.
The present inventor has filtered out the enterovirus type 71 viruses strain 1 and 2 that can be used for preventing hand foot mouth disease, and on this basis, develops the vaccine for preventing hand foot mouth disease.The experiment proved that, vaccine 1 and 2 of the present invention has effective immunocompetence and attacks malicious protective capability.The present inventor, by seed selection and purified enterovirus 71 type vaccine strain 1 and 2, sets up and examines and determine seed culture of viruses three grades of seed lot storehouses.And with (such as) bio-reactor (fermentor tank), cell factory or WAVE bioreactor culture Vero cell or human diploid cell, fine and close individual layer or cell is grown up to when grown on microcarriers is to proper density at cell, virus inoculation, cultivate after 4-7 days for 36 ± 1 DEG C and gather in the crops viral suspension, after the steps such as concentrated, purifying deactivation, prepare vaccinogen liquid.Can be passed through conversion dilution to add such as Al (OH)
3and so on adjuvant absorption be mixed with containing adjuvant work in-process, also can not add adjuvant Cheng Buhan adjuvant work in-process.Packing 0.5ml/ props up and namely can be enterovirns type 71 inactivated vaccine 1 and 2.
Specifically, another aspect of the present invention provides a kind of enterovirus type 71 viruses strain (EV71 virus strain 1).The Classification And Nomenclature of this virus is human enterovirus 71 (Human Enterovirus 71), Latin name is Enterovirus 71, in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 5th, 2011, Institute of Microorganism, Academia Sinica) preservation carried out, deposit number is CGMCC No.5540.The complete genome sequence (cDNA sequence) of described virus strain can as shown in SEQ ID No.:1.
The present invention also provides a kind of enterovirus type 71 viruses strain (EV71 virus strain 2).The Classification And Nomenclature of this virus is human enterovirus 71 (Human Enterovirus 71), Latin name is Enterovirus 71, in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 5th, 2011, Institute of Microorganism, Academia Sinica) preservation carried out, deposit number is CGMCC No.5539.The complete genome sequence (cDNA sequence) of described virus strain can as shown in SEQ ID No.:2.
EV71 virus strain 1 and 2 all can be used for preparing the medicine preventing and/or treating hand foot mouth disease.Preferably, described medicine is vaccine.
Another aspect of the present invention provides a kind of vaccine for preventing and/or treating hand foot mouth disease, and it contains through the EV71 virus strain 1 (corresponding vaccine 1 of the present invention) of deactivation or containing the EV71 virus strain 2 (corresponding vaccine 2 of the present invention) through deactivation.Vaccine 1 or 2 of the present invention can contain adjuvant, also can not contain adjuvant.
Those skilled in the art can determine EV71 virus strain 1 or the content of EV71 virus strain 2 in vaccine 1 or 2 of the present invention as required.Preferably, in protein content, the content of EV71 virus strain 1 or EV71 virus strain 2 is 1 ~ 4 μ g/ml.Protein content can adopt (such as) BCA method (can see with method disclosed in Publication about Document: Li Hailing, Peng Shuming, Li Lin, Zhang Xuemei, the comparison of 4 kinds of conventional determination of protein concentration methods. Chinese biochemical drug magazine 2008,29 (4): 277-282.) detect.
Preferably, adjuvant is contained in vaccine of the present invention.Adjuvant can be conventional adjuvant well known in the art, such as: aluminum hydroxide adjuvant, CpG adjuvant etc.Those skilled in the art can determine the content of adjuvant in vaccine of the present invention as required.Preferably, adjuvant is aluminium hydroxide.And preferably, the content of described aluminium hydroxide is 0.5 ~ 1.5 μ g/ml.
Preferably, every vaccinating agent is 0.1 ~ 2ml; More preferably, every vaccinating agent is 0.5ml.
Another aspect of the present invention provides the preparation method of vaccine, and it comprises: EV71 virus strain 1 or EV71 virus strain 2 are cultivated, and carry out deactivation, purifying, thus prepare described vaccine.Preferably, described purifying carried out and/or carries out after described deactivation before described deactivation.
Preferably, preparation method of the present invention comprises:
1) Vero cell or human diploid cell are provided;
2) in step 1) in the Vero cell that obtains or human diploid cell, EV71 virus strain 1 or EV71 virus strain 2 are cultivated, thus obtains viral suspension;
3) by step 2) viral suspension that obtains carries out purifying and deactivation, thus obtains vaccinogen liquid; And
4) by step 3) vaccinogen liquid that obtains dilutes, thus obtains described vaccine.
In this article, Vero cell refers to African green monkey kidney passage cell, can derive from ATCC company; Human diploid cell derives from Normal human fetal's tissue, can derive from ATCC company.Before carrying out virus inoculation, Vero cell or human diploid cell can be cultivated in cell factory or bio-reactor.The fine and close individual layer of proper density or cell can be grown up to when grown on microcarriers is to proper density, virus inoculation at cell.Wherein, described microcarrier is the class nontoxicity used in cell cultures, the small-particle that non-rigid, density is homogeneous, normally transparent, the adherent cell of dependence can be made to be attached to particle surface monolayer growth when suspension culture, thus increase the area of cell attachment growth, be conducive to large scale culturing and the collection of cell.
In this article, cell factory refers to a kind of consumptive material of cell cultures, and it and Tissue Culture Dish, bottle are similar, and all for cell cultures, just cell factory is used for fairly large cell cultures, can derive from (such as) NUNC company; When bio-reactor refers to that animal cells in vitro is cultivated, the container of one that provides for cell suitable growing environment, make it fast breeding and reach higher density, be usually used in extensive, concentration cultivation, it can derive from (such as) SARTORIS company.
Preferably, in step 2) in, described cultivation is carried out in bio-reactor (fermentor tank or WAVE bio-reactor) or cell factory.Preferably, described cultivation is carried out in bio-reactor, and culture condition is temperature 36 ± 1 DEG C, pH value 7.0 ± 0.5, dissolved oxygen concentration 20 ~ 80%, rotating speed 20 ~ 80rpm.Cultivation can carry out 4-7 days.
In step 3) in, can first carry out purifying, carry out deactivation again, also can first carry out deactivation, carry out purifying again.
Way of purification can be the conventional method in this area, as ultrafiltration and concentration and chromatography, chromatography can comprise: gel permeation chromatography (such as adopting the chromatography of Sepharose 4FF, Sepharose 6FF or Sephacryl S400 medium) and/or ion exchange chromatography (as anion-exchange chromatography (as adopted the chromatography of DEAE-Sepharose FF medium)).
Deactivation mode can be the conventional method in this area, as adopted formaldehyde as inactivator, in 1: 1500-1: 4000 ratios in 34-38 DEG C of deactivation 2-7 days.Also beta-propiolactone deactivation can be used, in 1: 2000-1: 4000 ratios in 2-8 DEG C of deactivation 2-3 days, then 37 DEG C of hydrolysis 2-4 hour.
Preferably, in step 4) in, with aluminum hydroxide solution to step 3) vaccinogen liquid that obtains dilutes, make in obtained vaccine, with viral protein content meter, the content of described virus strain is 1 ~ 4 μ g/ml, and the content of described aluminium hydroxide is 0.5 ~ 1.5 μ g/ml.
Another aspect of the present invention provides the antibody (such as, polyclonal antibody or monoclonal antibody or its functional fragment) or hybridoma or antiserum(antisera) prepared for immunogen with EV71 virus strain 1,2 or vaccine of the present invention 1 or 2.Can according to the conventional method in this area, come Dispersal risk or hybridoma or antiserum(antisera).Such as, family's rabbit back multiple spot hypodermic injection prepares antiserum(antisera) and cytogamy legal system for hybridoma, caprylic acid precipition and affinity chromatography purified polyclonal or monoclonal antibody.
The method for building animal model (the open method disclosed in No.CN 101982181A or CN101978970A of such as Chinese patent application) for evaluating enterovirns type 71 vaccine effect known in the art can be adopted to set up animal model, and evaluate the effect of vaccine 1 or 2 of the present invention.The effect of vaccine 1 or 2 of the present invention can also adopt to be evaluated by the animal model of method establishment of the present invention.
Current inventor provides a kind of stable method for building animal model for evaluating enterovirns type 71 vaccine effect, for the screening of enterovirns type 71 vaccine development and antiviral provides the foundation.For this reason, the invention provides EV71 virus strain 2 as attacking poison strain, described poison strain of attacking has following features: virulence is strong, suckling mouse can be made to occur typical EV71 infection symptoms such as significantly benumbing, paralysis is even dead, can be used for the evaluation of EV71 vaccine or medicine.
EV71 virus strain 2 can be used for setting up the animal model for evaluating enterovirns type 71 vaccine effect.Wherein, EV71 virus strain 2 can be used as attacking strain.
Another aspect of the present invention provides a kind of establishment method of the animal model for evaluating enterovirns type 71 vaccine effect, and it comprises:
1) EV71 virus strain 2 is cultivated, obtain virus-culturing fluid; And
2) by step 1) virus-culturing fluid of gained bestows animal (being such as expelled in animal body), thus prepares described animal model.
Preferably, the middle viral level of described virus-culturing fluid is 10
4~ 10
7cCID
50/ ml.
The animal that described animal can adopt this area conventional, such as: mouse, rat, cavy, rabbit.Preferably described animal is mouse.More preferably, described mouse is the suckling mouse of 1-14 age in days.More preferably, the product of described mouse are ICR, BALB/c, NIH or Kunming strain.
Preferably, in step 2) in, described injection is intracranial injection.Preferably, the injection volume of described virus-culturing fluid is every mouse 1000-10000CCID
50.
Preferably, in step 2) in, described injection is abdominal injection.Preferably, the injection volume of described virus-culturing fluid is every mouse 1000-10000CCID
50.
The method that described suckling mouse can adopt this area conventional obtains.Preferably, described suckling mouse obtains by the following method: will often only grow up female mouse and 1 Female odor mating, before described mating, vaccine 1 or 2 of the present invention is adopted to carry out initial immunity (0.5 ~ 4 μ g/ml to described female mouse, preferably 2 μ g/ml), adopt this vaccine to carry out booster immunization (0.5 ~ 4 μ g/ml in described post-coitum 2-3 week, preferably 2 μ g/ml), and nest is divided by itself and described male mouse after described female mouse pregnancy, the suckling mouse given birth to by described female mouse cultivates 1-14 days, preferably cultivates 1-7 days.
Animal model of the present invention may be used for the effect evaluating enterovirns type 71 vaccine known in the art (the open vaccine disclosed in No.CN101575593A or CN101897963A of such as Chinese patent application), also may be used for the effect evaluating vaccine of the present invention.
Mode by the following examples further explains and describes content of the present invention, but these embodiments are not to be construed as limiting the scope of the invention.
DMEN substratum in following examples can purchased from GIBCO company; Vero cell or human diploid cell can purchased from ATCC companies; Bovine serum can purchased from GIBCO company; Anti-EV71 immune serum can purchased from ATCC company; 199 cell growth mediums can purchased from SIGMA company; Pancreatin can purchased from GIBCO company; Cell factory can purchased from NUNC company; Ultra-filtration membrane can purchased from MILLIPORE company; Sepharose 4FF, DEAE-Sepharose FF medium can purchased from GE companies; Beta-propiolactone can purchased from SIGMA company; RD cell can purchased from ATCC company; ICR, BALB/c, NIH, kunming mice can tie up experimental animal Science and Technology Ltd. of tonneau China purchased from Beijing.
The Isolation and ldentification method of embodiment 1:EV71 virus strain 1
(1) separation method of EV71 virus strain 1
Candidate's strain will have complete record, history, source.Measure the biological characteristics of candidate's strain; inactivated vaccine prepared by viral candidates strain should possess that viral yield is high, the ability of induction of immunity protection is strong, cross protection spectrum is wide, biological characteristics is stablized; and to select according to epidemiology and molecular epidemiology, there is the virus strain of the potentiality that are widely current at present and in the future.Viral candidates immune animal, separation of serum, measures serum NAT, and screening protection spectrum is wide, and the strong virus of induction of immunity protective capability is as inactivated vaccine strain.
Collect hand foot mouth disease patient (from the yellow ×× of the 4 years old hand foot mouth disease infant in one, Fuyang) oropharyngeal swab specimen, under 4 DEG C of conditions, the centrifugal 30min of 2000 ~ 4000rpm, gets supernatant degerming with 0.2 μm of frit, and the supernatant after filtering is inoculated Vero cell or human diploid cell.Before inoculation sample, outwell growth media, the sample suspension of every bottle of cell inoculation 0.2 ~ 1ml, culture temperature is 36 ± 1 DEG C; Adsorb after 1 ~ 2 hour, the DMEN substratum changed containing 2% bovine serum continues to cultivate.Use inverted microscope observation of cell pathology day by day, if the appearance of 7 days acellular pathology effects (CPE), in blind passage 1 generation, continues observation 7 days, until the cell of 75% ~ 90% changes, is then stored at-20 DEG C and goes down to posterity in order to secondary.The s-generation is cultivated when seeing suspicious cells pathology and is continued to go down to posterity, until cytopathy is stable occur after-70 DEG C frozen, feminine gender is then discarded.Thus obtain EV71 virus strain 1, send center (CGMCC) preservation of China Microbiological bacterium kind preservation management committee's common micro-organisms, preserving number is: CGMCC No.5540.
(2) authentication method of virus
Morphological observation: observe the pathology of EV71 virus on cell under an optical microscope, the contracting of cell circle, dispersion, endochylema endoparticle increase.After EV71 virus ultrafiltration and concentration, after 2% Salkowski's solution negative staining, under putting Electronic Speculum, can be observed spherical virus particle.
Telling test: by the anti-EV71 immune serum of virus-culturing fluid and equivalent in 36 ± 1 DEG C and after 1 ~ 2 hour, be inoculated in Tissue Culture Plate, continue cultivation 7 days, observe sick cell hole count, establish serum and cell controls, the titre of virus control should be not less than 500CCID simultaneously
50/ ml, experiment effectively.All there is cytopathy in result display virus control group hole, serum control group, cell controls group cell all do not occur pathology; Candidate's strain all can be neutralized by anti-EV71 immune serum, proves that they are really EV71.
Molecular biology identification: get virus liquid 0.2-0.5ml, extracts viral RNA, adopts EV71 Auele Specific Primer to carry out full genome amplification, sequencing and gene type, and above-mentioned steps can be matched genome research center by Beijing promise and carry out.Result shows, and its full-length genome 7406bp, belongs to C4 gene hypotype.Complete genome sequence is as shown in SEQ ID No.:1.
The purifying of embodiment 2:EV71 virus strain 1 and mitotic stability analysis
(1) plaque purification
The EV71 viral suspension serial dilution that embodiment 1 is obtained, after basis of microscopic observation cell grows up to individual layer, discard original nutrient solution in six porocyte culture plates, add viral suspension 0.1-0.5ml/ hole, 36 ± 1 DEG C of absorption 1-2 hour, after absorption terminates, outwell liquid in plate, wash once with aseptic PBS, enrobe, be placed in carbonic acid gas incubator 36 ± 1 DEG C cultivation.3-7 days after virus inoculation, Microscopic observation plaque formation, picking plaque, does further plaque purification, as this method repurity 2 times.
(2) the mitotic stability analysis of virus
For Vero cell, EV71 is selected virus strain continuous passage on Vero cell, gather in the crops when pathology is 75% ~ 90%, adopt Microdose cytopathic effect assay to measure titre, observe the titre stability of its continuous passage on Vero cell.Result shows, and the titre that this strain more than continuous 15 generations is gone down to posterity on Vero cell all maintains 7.0Lg CCID
50/ more than ml, has good mitotic stability.Shown in result Fig. 1.
The foundation of the seed lot of embodiment 3:EV71 virus strain 1 and calibrating
According to " Chinese Pharmacopoeia " requirement about seed bank establishment method, production of vaccine seed culture of viruses should carry out three-level management based on viral seed lot system, namely primordial seed criticize, main seed lot and working seed lots, seed lot cell all need in less than-60 DEG C freezen protective.Primordial seed is criticized and should be identified its record, history source and biological nature.Main seed lot, should examine and determine comprehensively, and calibration scope comprises the projects such as telling test, sterility test, mycoplasma inspection, titration of virus, viral exogenous factor inspection, immunogenicity inspection.Working seed lots carries out the project calibratings such as telling test, sterility test, mycoplasma inspection, titration of virus, can use after qualified.
For Vero cell, the establishment method (but the present invention is not limited thereto) of summary three grades of seed lots: by the virus inoculation criticized from primordial seed to the Vero cell (M.O.I=0.001 ~ 1) growing up to individual layer, use inverted microscope observation of cell pathology day by day, until when there is the cytopathy of 75% ~ 90%, culture is placed in-20 DEG C freezing, after room temperature melts secondary, be seeded to new Vero cell monolayer cell according to the method described above, until there is the cytopathy of 75% ~ 90%, so repeatedly carry out viral passages.Every a collection of cutting is designated as a generation.
The cultivation of embodiment 4:EV71 virus strain 1 and results
(1) cell: Cells for production is Vero cell or human diploid cell.
(2) virus: the EV71 working seed lots of preparation.
(3) cell and virus culture
Freeze-stored cell is taken out, transplanted cells suspension 1ml (concentration: 8 × 10 from liquid nitrogen container
6/ ml) in 199 cell growth medium 10ml, be placed in 36 ± 1 DEG C of cultivations, after 4 hours, change nutrient solution, continue to be placed in 36 ± 1 DEG C of cultivations, go down to posterity after growing into fine and close individual layer.Discard original growth media in bottle, add 0.25% pancreatin 20ml to digest, take off after wall until cell, supplementing 199 cell growth medium 100ml piping and druming is dispersible suspension shape to cell, obtained cell suspension 20ml is seeded in rolling bottle, carry out cell amplification, after same method carries out trysinization, obtained cell suspension 200ml is seeded in bio-reactor or cell factory, cultivate after 4 ~ 7 days, observation of cell growth conditions, determine the virus inoculation time, the ratio being M.O.I=0.001 ~ 1 in virus inoculation amount adds maintenance medium 20000ml, mixing, be seeded to and grow up in the cell of individual layer, continue to cultivate and observe pathology, according to lesion degree results virus liquid.Reactor cell culture condition: microcarrier content 2 ~ 15 grams per liter, temperature 36 ± 1 DEG C, pH value 7.0 ± 0.5, dissolved oxygen concentration 20 ~ 80%, rotating speed 20 ~ 80rpm; Virus culture condition: temperature 36 ± 1 DEG C, pH value 7.0 ± 0.5, DO concentration 20 ~ 80%, rotating speed 20 ~ 80rpm.
(4) virus harvest: after obvious pathology appears in 48-120h, cell after virus to be seeded, results virus-culturing fluid.
Concentrated, the purifying of embodiment 5:EV71 virus strain 1 and deactivation
Ultrafiltration and concentration virus: by the virus harvest liquid prepared according to the method described above, the ultra-filtration membrane being 100-1000KD through molecular weight cut-off carries out ultrafiltration and concentration, cycles of concentration is about 20-200 doubly.
The purifying of virus
Gel permeation chromatography: viral concentration liquid is added to the Sepharose 4FF medium balanced with pH6.0-8.0PBS and carries out gel permeation chromatography, collect viral peak.
Ion exchange chromatography: be added in the DEAE-Sepharose FF medium balanced with pH6.0-8.0PBS carry out ion exchange chromatography by collecting liquid after gel permeation chromatography, elutriant is the pH6.0-8.0PBS containing 0.1M ~ 0.5M NaCl, collects viral peak.
Inactivation of virus: adopt formaldehyde as inactivator, in 1: 2000 ratio in 35 DEG C of deactivations 7 days.Also can use beta-propiolactone deactivation, in 1: 2000 ratio in 2-8 DEG C of deactivation 2 days, then 37 DEG C are hydrolyzed 2 hours.After removing inactivator, be vaccinogen liquid 5 ~ 40 μ g/ml.
The preparation of embodiment 6:EV71 vaccine 1
The preparation of EV71 inactivated vaccine work in-process and finished product (aluminum hydroxide adjuvant adsorbs)
EV71 inactivated vaccine stoste embodiment 5 obtained mixes with aluminum hydroxide solution (aluminium content is 10-20mg/ml), make that aluminium hydroxide content is 0.5 ~ 1.5mg/ml, protein content is 1 ~ 4 μ g/ml, EV71 inactivated vaccine 1 work in-process can be obtained.The work in-process made are divided and is filled in aseptic cillin bottle (or precharging type syringe), EV71 inactivated vaccine 1 finished product can be obtained.
The preparation of EV71 inactivated vaccine work in-process and finished product (adsorbing without adjuvant)
By the EV71 inactivated vaccine stoste dilution obtained, diluent is 50mM PBS damping fluid (pH is 7.0), makes protein content be 1 ~ 4 μ g/ml, can obtain EV71 inactivated vaccine 1 work in-process.The work in-process made are divided and is filled to aseptic cillin bottle (or precharging type syringe), EV71 inactivated vaccine 1 finished product can be obtained.
The mass analysis of embodiment 7:EV71 vaccine 1
According to the relevant requirements of " Chinese Pharmacopoeia (three) version in 2010 ", EV71 inactivated vaccine 1 finished product is measured, result shows, the outward appearance of vaccine is the muddy suspension body of oyster white, the loading amount of vaccine is not less than 0.5ml/ bottle, the pH value of vaccine is 6.0 ~ 8.0, telling test conforms with the regulations, aluminium hydroxide content≤2mg/ml, sterility test is negative, bacterial endotoxin≤10EU/ agent, abnormal toxicity test conforms with the regulations, bovine serum protein residual content≤50ng/ agent, free formaldehyde content≤10 μ g/ agent, Vero cell DNA residual quantity < 100pg/ agent etc., all be not less than national standard, comply with relevant regulations.
Embodiment 8:EV71 vaccine 1 Analysis of Immunogenicity
Measure serum NAT method: test serum carries out 2 times of serial dilutions on 96 hole microtiter plates, neutralization virus (virus that embodiment 4 obtains) is diluted to 100CCID respectively
50/ 50 μ l, drop to (except serum, cell controls) on microtiter plate, 50 μ l/ holes.To 36 DEG C of incubators, in and 2 hours.After neutralization terminates, be prepared into cell suspension, 1.5 × 10 by after the trysinization of RD cell
5/ ml, adds in 96 orifice plates, 100 μ l/ holes, 36 DEG C, 5%CO
2continue cultivation after 7 days, observation of cell pathology, result of determination, carry out viral residual titration after neutralization terminates, set up virus control, serum control and cell controls.Result judges: in 2 holes of most high dilution serum, have 1 hole to occur cytopathy, cytopathy does not appear in another hole, and this dilution inverse meter is NAT of this serum specimen; When the complete pathology in high dilution 2 hole, adjacent low extent of dilution 2 hole not pathology completely, then the inverse of both Average dilutions is the NAT of this serum specimen; When 1 porocyte pathology all appears in two adjacent extent of dilution serum, there is not cytopathy in another 1 hole, then the inverse of both Average dilutions is the EV71 NAT of this serum specimen.
Mouse immune: EV71 inactivated vaccine is divided into various dose group: 5,2.5,1.25,0.625,0.31,0.15,0.07 μ g/ml, carries out random packet by laboratory animal Balb/c mouse by vaccination doses, often organizes 10.Get EV71 inactivated vaccine, immune Balb/c mouse, abdominal injection, only, immunity 2 times, 2 ~ 4 weeks, interval, blood sampling in 3 ~ 4 weeks after booster immunization, separation of serum, detects serum NAT to 0.5ml/ as stated above.
Result shows, there is obvious dose-effect relationship in serum neutralizing antibody level and vaccine dose, 5,2.5,1.25,0.625,0.31,0.15,0.07 μ g/ml group, serum neutralizing antibody Conversion rate except 0.07 μ g/ml group be except 90%, other all reach 100%, control group does not produce neutralizing antibody, and serum neutralizing antibody Conversion rate is 0%.Each group of serum neutralizing antibody GMT reaches 1: (32 ~ 1024), control group GMT is lower than 1: 8.Positive judgement standards: Neutralizing titer is greater than 1: 8.
The analysis of embodiment 9:EV71 vaccine 1 protected effect
The vaccine of the aluminium hydroxide adjuvant obtained for embodiment 6, adopts the protected effect of EV71 infecting mouse model of the present invention to vaccine to analyze.EV71 inactivated vaccine 1 is divided into high, medium and low three dosage groups (2,1,0.5 μ g/ml).To be grown up ICR (often only join 1 male mouse, divide nest after female mouse pregnancy by male mouse) 8, and 0 week initial immunity, post-coitum 2-3 week booster immunization before mating, intramuscular injection, 0.1-1.0ml/ only.Female mouse gives birth to 1-7 days after suckling mouse, virus strain 2 nutrient solution that EV71 virus strain 2 nutrient solution that 10 μ l are prepared by the method in following embodiment 10/only or abdominal injection 100 μ l prepared by the method in following embodiment 10/is only injected by encephalocoele, day by day observe incidence, calculate survival rate.
After virus attack the 1st day, 2 days and 3 days vaccine group and vehicle control group all without exception, after virus attack there is significantly paralysis, paralysis symptom in 4th ~ 5 days control group suckling mouse hind legs, and vaccine group is without exception.After virus attack, 5th ~ 6 days vehicle control group suckling mouses are all dead; and vaccine group is without any exception; survival rate curve as shown in Figure 2; result shows; EV71 vaccine 1 has significant protective effect; after adopting EV71 vaccine 1 immunity of high, medium and low dosage group, mouse all can be protected to infect from EV71.Without significant difference, Normal-weight, there is not the action such as limb paralysis unusual phenomenon in vaccine inoculation group and Normal group mouse, survival rate rate reaches 100%, and vehicle control group suckling mouse after virus attack 4 ~ 5 days phase secondary diseases until death.
The Isolation and Identification of embodiment 10:EV71 virus strain 2
The separation method of 1.EV71 virus strain 2 is as follows: collect hand foot mouth disease patient's (being separated from Ningbo of Zhejiang 3 years old severe hand foot mouth disease infant Gong ××) oropharyngeal swab specimen, under 4 DEG C of conditions, the centrifugal 30min of 2000 ~ 4000rpm, get supernatant degerming with 0.2 μm of frit, the supernatant after filtering is inoculated Vero cell or human diploid cell.Before inoculation sample, outwell growth media, every bottle of cell (8 × 10
6individual/bottle) inoculate the sample suspension of 0.2 ~ 1ml, culture temperature is 36 ± 1 DEG C; Adsorb after 1 ~ 2 hour, the DMEM substratum 10ml changed containing 2% bovine serum continues to cultivate.Use inverted microscope observation of cell pathology day by day, if the appearance of 7 days acellular pathology effects (CPE), in blind passage 1 generation, continues observation 7 days, until the cell of 75% ~ 90% changes, is then stored at-20 DEG C and goes down to posterity in order to secondary.The s-generation is cultivated when seeing suspicious cells pathology and is continued to go down to posterity, until cytopathy is stable occur after-70 DEG C frozen, feminine gender is then discarded.Thus obtain EV71 virus strain 2, deliver to center (CGMCC) preservation of China Microbiological bacterium kind preservation management committee's common micro-organisms, preserving number is: CGMCC No.5539.
The cultural method of 2.EV71 virus strain 2 is as follows:
Freeze-stored cell is taken out, transplanted cells suspension 1ml (concentration: 8 × 10 from liquid nitrogen container
6individual/ml) in 10ml 199 cell growth medium, cultivate under being placed in the temperature of 36 ± 1 DEG C, change nutrient solution after 4 hours, cultivate under continuing to be placed in the temperature of 36 ± 1 DEG C, go down to posterity after growing into fine and close individual layer.Discard original 199 cell growth mediums in bottle, add 10m10.25% trysinization, take off after wall until cell, supplement 6ml 199 cell growth medium, piping and druming to cell is dispersible suspension shape, get 1ml cell suspension inoculation in square vase, carry out cell amplification, cultivate after 4 ~ 7 days, observation of cell growth conditions, determine the virus inoculation time, the ratio being M.O.I=0.001 ~ 1 in virus inoculation amount adds the DMEM substratum of 10ml containing 2% bovine serum, mixing, be seeded to and grow up in the cell of individual layer, continue to cultivate and observe pathology, 48-120 hour after virus to be seeded, after there is obvious pathology in cell, results virus-culturing fluid.
The authentication method of 3.EV71 virus strain 2 is as follows:
(1) morphological observation: observe the pathology of EV71 virus strain 2 on cell under an optical microscope, the contracting of cell circle, dispersion, endochylema endoparticle increase.After ultrafiltration and concentration, after 2% Salkowski's solution negative staining, under putting Electronic Speculum, can be observed spherical virus particle.
(2) immunological testing qualification: by the anti-EV71 immune serum of the virus-culturing fluid of EV71 virus strain 2 and equivalent in 36 ± 1 DEG C with 1 ~ 2 hour after, be inoculated in Tissue Culture Plate, continue cultivation 7 days, observe sick cell hole count, establish serum and cell controls, the titre of virus control should be not less than 500CCID simultaneously
50/ ml.All there is cytopathy in result display virus control group hole, serum control group, cell controls group cell all do not occur pathology; Candidate's strain all can be neutralized by anti-EV71 immune serum, proves that they are really EV71 virus strain 2.
(3) molecular biology identification: get virus liquid 0.2-0.5ml, extracts viral RNA, adopts EV71 Auele Specific Primer to carry out full genome amplification, sequencing and gene type, and above step can be matched genome research center by Beijing promise and carry out.Result shows, and its full-length genome 7404bp, belongs to C4 gene hypotype.Complete genome sequence is as shown in SEQ ID No.:2.
Embodiment 11: prepare EV71 vaccine 2 by EV71 virus strain 2
The strain of attacking that EV71 virus strain 2 can be used as animal model uses, and similarly also can be used for preparation EV71 inactivated vaccine.
Preparation method and EV71 virus strain 1 similar, concrete steps can see embodiment 2-6, uniquely unlike, EV71 virus strain 1 used in each step is replaced with EV71 virus strain 2.Such as, EV71 inactivated vaccine 2 is prepared for Vero cell, set up and examine and determine seed culture of viruses three grades of seed lot storehouses, and by bio-reactor or cell factory Cultivation of Vero, grow up to fine and close individual layer or cell when grown on microcarriers is to proper density at cell, virus inoculation, cultivate after 4-7 days for 36 ± 1 DEG C and gather in the crops viral suspension, after the steps such as concentrated, purifying deactivation, prepare vaccinogen liquid.Can be passed through conversion dilution to add such as Al (OH)
3and so on adjuvant absorption be mixed with containing adjuvant work in-process, also can not add adjuvant Cheng Buhan adjuvant work in-process.Packing 0.5ml/ props up and namely can be enterovirns type 71 vaccine 2.
The mass analysis of embodiment 12:EV71 vaccine 2
According to the relevant requirements of " Chinese Pharmacopoeia (three) version in 2010 ", EV71 inactivated vaccine 1 finished product is measured, result shows, the outward appearance of vaccine is the muddy suspension body of oyster white, the loading amount of vaccine is not less than 0.5ml/ bottle, the pH value of vaccine is 6.0 ~ 8.0, telling test conforms with the regulations, aluminium hydroxide content≤2mg/ml, sterility test is negative, bacterial endotoxin≤10EU/ agent, abnormal toxicity test conforms with the regulations, bovine serum protein residual content≤50ng/ agent, free formaldehyde content≤10 μ g/ agent, Vero cell DNA residual quantity < 100pg/ agent etc., all be not less than national standard, comply with relevant regulations.
Embodiment 13:EV71 vaccine 2 Analysis of Immunogenicity
Measure serum NAT method: test serum carries out 2 times of serial dilutions on 96 hole microtiter plates, neutralization virus (virus that embodiment 11 obtains) is diluted to 100CCID respectively
50/ 50 μ l, drop to (except serum, cell controls) on microtiter plate, 50 μ l/ holes.To 36 DEG C of incubators, in and 2 hours.After neutralization terminates, be prepared into cell suspension, 1.5 × 10 by after the trysinization of RD cell
5/ ml, adds in 96 orifice plates, 100 μ l/ holes, 36 DEG C, 5%CO
2continue cultivation after 7 days, observation of cell pathology, result of determination, carry out viral residual titration after neutralization terminates, set up virus control, serum control and cell controls.Result judges: in 2 holes of most high dilution serum, have 1 hole to occur cytopathy, cytopathy does not appear in another hole, and this dilution inverse meter is NAT of this serum specimen; When the complete pathology in high dilution 2 hole, adjacent low extent of dilution 2 hole not pathology completely, then the inverse of both Average dilutions is the NAT of this serum specimen; When 1 porocyte pathology all appears in two adjacent extent of dilution serum, there is not cytopathy in another 1 hole, then the inverse of both Average dilutions is the EV71 NAT of this serum specimen.
Mouse immune: EV71 vaccine is divided into various dose group: 5,2.5,1.25,0.625,0.31,0.15,0.07 μ g/ml, carries out random packet by laboratory animal Balb/c mouse by vaccination doses, often organizes 10.Get EV71 inactivated vaccine, immune Balb/c mouse, abdominal injection, only, immunity 2 times, 2 ~ 4 weeks, interval, blood sampling in 3 ~ 4 weeks after booster immunization, separation of serum, detects serum NAT to 0.5ml/ as stated above.
Result shows, there is obvious dose-effect relationship in serum neutralizing antibody level and vaccine dose, 5,2.5,1.25,0.625,0.31,0.15,0.07 μ g/ml group, serum neutralizing antibody Conversion rate except 0.07 μ g/ml group be except 85%, other all reach 100%, control group does not produce neutralizing antibody, and serum neutralizing antibody Conversion rate is 0%.Each group of serum neutralizing antibody GMT reaches 1: (24 ~ 1024), control group GMT is lower than 1: 8.Positive judgement standards: Neutralizing titer is greater than 1: 8.
The analysis of embodiment 14:EV71 vaccine 2 protected effect
The vaccine of the aluminium hydroxide adjuvant obtained for embodiment 11, adopts the protected effect of EV71 infecting mouse model of the present invention to vaccine to analyze.EV71 vaccine 2 is divided into high, medium and low three dosage groups (2,1,0.5 μ g/ml).To be grown up ICR (often only join 1 male mouse, divide nest after female mouse pregnancy by male mouse) 8, and 0 week initial immunity, post-coitum 2-3 week booster immunization before mating, intramuscular injection, 0.1-1.0ml/ only.Female mouse gives birth to 1-7 days after suckling mouse, virus strain 2 nutrient solution that EV71 virus strain 2 nutrient solution that 10 μ l are prepared by the method in embodiment 10/only or abdominal injection 100 μ l prepared by the method in embodiment 10/is only injected by encephalocoele, day by day observe incidence, calculate survival rate.
After virus attack the 1st day, 2 days and 3 days vaccine group and vehicle control group all without exception, after virus attack there is significantly paralysis, paralysis symptom in 4th ~ 5 days control group suckling mouse hind legs, and vaccine group is without exception.After virus attack, 5th ~ 6 days vehicle control group suckling mouses are all dead; and vaccine group is without any exception; survival rate curve as shown in Figure 9; result shows; EV71 vaccine 2 has significant protective effect; after adopting EV71 vaccine 2 immunity of high, medium and low dosage group, mouse all can be protected to infect from EV71.Without significant difference, Normal-weight, there is not the action such as limb paralysis unusual phenomenon in vaccine inoculation group and Normal group mouse, survival rate rate reaches 100%, and vehicle control group suckling mouse after virus attack 3 ~ 4 days phase secondary diseases until death.
Embodiment 15: the establishment method of intracranial injection EV71 infected animal model
Comprise the following steps: get 1-14 age in days ICR suckling mouse, intracranial injection attacks the virus-culturing fluid (concrete preparation method can see embodiment 10, lower with) of poison strain, and concentration is 10
5cCID
50/ ml, 10-30 μ l/ only, namely obtains EV71 infected animal model.
Concrete operation step is as follows:
(1) animal grouping
Get 1-14 age in days suckling mouse two groups (often organizing 10), be set to viral group and Normal group respectively, virus group intracranial injection attacks poison strain nutrient solution, Normal group injection DMEM nutrient solution.
(2) intracranial injection
Draw with asepsis injector and attack poison strain virus-culturing fluid, intracranial injection suckling mouse, only, Normal group injecting virus nutrient solution, 10-30 μ l/ only, observes suckling mouse incidence to 10-30 μ l/ day by day.
(3) suckling mouse incidence is observed
After intracranial injection the 1st day, 2 days and 3 days viral group and Normal group all without exception, after virus attack there is significantly paralysis, paralysis symptom in 4th ~ 5 days virus group suckling mouse hind legs, and Normal group is without exception.After virus attack, 5th ~ 6 days virus group suckling mouses are all dead, and Normal group is without exception, and survival rate curve as shown in Figure 3, proves Animal Model success.
Embodiment 16: the establishment method of abdominal injection EV71 infected animal model
Comprise the following steps: get 1-14 age in days ICR suckling mouse, abdominal injection attacks the virus-culturing fluid (concrete preparation method can see embodiment 10, lower with) of poison strain, and concentration is 10
5cCID
50/ ml, 100 μ l/ only, namely obtain the abdominal cavity infection animal model of enterovirus type 71 viruses.
Concrete operations are as follows:
(1) animal grouping
Get 1-14 age in days suckling mouse two groups (often organizing 10), be set to viral group and Normal group respectively, virus group intracranial injection attacks poison strain virus-culturing fluid, Normal group injection DMEM nutrient solution.
(2) abdominal injection
Draw with asepsis injector and attack poison strain virus-culturing fluid, abdominal injection virus group suckling mouse, 100 μ l/ only; Normal group injection DMEM nutrient solution, 100 μ l/ only.Day by day suckling mouse incidence is observed.
(3) suckling mouse incidence is observed
After abdominal injection the 1st day, 2 days and 3 days viral group and Normal group all without exception, after virus attack there is significantly paralysis, paralysis symptom in 4th ~ 5 days virus group suckling mouse hind legs, and Normal group is without exception.After virus attack, 5th ~ 6 days experimental group suckling mouses are all dead, and Normal group is without exception, as shown in Figure 5.Survival rate curve as shown in Figure 4, proves Animal Model success.
The application of embodiment 17:EV71 infected animal model
(1) preparation of enterovirns type 71 inactivated vaccine
EV71 inactivated vaccine (aluminum hydroxide adjuvant absorption) finished product using embodiment 6 to prepare.
(2) application of EV71 infected animal model in EV71 inactivated vaccine protected effect is evaluated
1) vaccine prepares
By the EV71 inactivated vaccine be prepared into, dilute for high, medium and low three dosage (2,1,0.5 μ g/ml), contrast as aluminum hydroxide adjuvant, concentration is 1mg/ml.
2) animal immune and attack poison
The female mouse of ICR that will grow up (often only joins 1 male mouse, nest is divided by male mouse) after female mouse pregnancy, before mating, (0 week) carries out initial immunity, post-coitum 2-3 week booster immunization with the EV71 vaccine prepared by the present invention, and intramuscular injection, 0.1-1.0ml/ only.Female mouse gives birth to 1-7 days after suckling mouse, by intracranial injection 10 μ l virus liquid/only or abdominal injection 100 μ l virus liquid/only, observes incidence day by day, calculates survival rate.
3) observation after poison is attacked
After virus attack the 1st day, 2 days and 3 days vaccine group and vehicle control group all without exception, after virus attack there is significantly paralysis, paralysis symptom in 4th ~ 5 days control group suckling mouse hind legs, and vaccine group is without exception.After virus attack, 5th ~ 6 days vehicle control group suckling mouses are all dead; and vaccine group is without any exception; survival rate curve as shown in Figure 6; result shows; EV71 inactivated vaccine has significant protective effect; after adopting the EV71 inactivated vaccine immunity of high, medium and low dosage group, mouse all can be protected to infect from EV71.Without significant difference, Normal-weight, there is not the action such as limb paralysis unusual phenomenon in vaccine inoculation group and Normal group mouse, survival rate rate reaches 100%, and vehicle control group suckling mouse after virus attack 4 ~ 5 days phase secondary diseases until death.
4) NAT is measured
Gather control group respectively, the female mouse of vaccine inoculation group, 1 age in days suckling mouse, 14 age in days suckling mouse serum carry out neutralizing antibody detection, and (detection method can see with Publication about Document: Mao Qunying, He Peng, Yu Xiang, Li Nan, Hao Chunsheng etc. the laboratory evaluation of human enterovirus 71 neutralizing antibody detection method. Products in China magazine .2010,23 (8): 885-888).As shown in Figure 7, after the immunity of EV71 inactivated vaccine, female mouse, 1 age in days, 14 age in days suckling mouse serum neutralizing antibodies are apparently higher than control group for result.
5) each histoorgan virus load detects
(detection method can see with Publication about Document: piece build English to adopt fluorescence quantifying PCR method, Wang Wei, clump Zhe, Liu Qiang, the .SYBR Green I real-time fluorescence quantitative RT-PCRs such as Weiqiang measure the foundation of enterovirns type 71 (EV71) RNA copy number method. Chinese comparative medicine magazine .2010,20 (7): 27-31.) relative content of the EV71 RNA in vaccine inoculation group and each organ of aluminum hydroxide adjuvant control group mice (brain, the heart, liver, spleen, lung, kidney, muscle, spinal cord, intestines), is detected.Found that, in vaccine inoculation group suckling mouse histoorgan, virus load is starkly lower than control group.For spleen, as shown in Figure 8.In the above-mentioned each internal organs of control group suckling mouse, EV71RNA content is apparently higher than aluminum hydroxide adjuvant control group, after this shows EV71 vaccine inoculation, obviously can reduce the EV71 virus load in each organ-tissue, thus reaches the effect protecting suckling mouse to infect from EV71.
Claims (12)
1. enterovirns type 71 (Enterovirus 71) virus strain, the deposit number of described virus strain is CGMCC No.5539.
2. virus strain according to claim 1, the complete genome sequence of wherein said virus strain is the sequence shown in SEQ ID No.:2.
3. virus strain according to claim 1 is for the preparation of the purposes prevented and/or treated in the medicine of hand foot mouth disease.
4. purposes according to claim 3, wherein, described medicine is vaccine.
5. for preventing and/or treating a vaccine for hand foot mouth disease, wherein, described vaccine contains: 1) through deactivation claim 1 or 2 described in virus strain; And optional 2) adjuvant.
6. vaccine according to claim 5, wherein, with viral protein content meter, the content of described virus strain is 1 ~ 4 μ g/ml; Described adjuvant is aluminium hydroxide, and the content of described aluminium hydroxide is 0.5 ~ 1.5 μ g/ml.
7. a preparation method for the vaccine according to claim 5 or 6, it comprises: the virus strain described in claim 1 or 2 cultivated, and carries out deactivation, purifying, thus prepares described vaccine.
8. virus strain according to claim 1 and 2 is being set up for evaluating the purposes in the animal model of enterovirns type 71 vaccine effect.
9. purposes according to claim 8, described virus strain is used as to attack strain.
10., for evaluating an establishment method for the animal model of enterovirns type 71 vaccine effect, it comprises:
1) virus strain according to claim 1 is cultivated, obtain virus-culturing fluid; And
2) by step 1) virus-culturing fluid of gained bestows animal, thus prepares described animal model;
Wherein, described animal is mouse.
11. preparation methods according to claim 10, wherein, described mouse is the suckling mouse of 1-14 age in days.
12. preparation methods according to claim 11, wherein, described suckling mouse obtains by the following method: will often only grow up female mouse and 1 Female odor mating, before described mating, enterovirns type 71 vaccine to be evaluated is adopted to carry out initial immunity, adopt this enterovirns type 71 vaccine to carry out booster immunization in described post-coitum 2-3 week to described female mouse, and nest is divided by itself and described male mouse after described female mouse pregnancy, the suckling mouse given birth to by described female mouse cultivates 1-14 days.
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| CN101717754A (en) * | 2009-12-30 | 2010-06-02 | 北京科兴生物制品有限公司 | Enterovirus type 71 and use 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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