CN104694486B - A kind of bigeminy hand-foot-and-mouth disease inactivated vaccine and preparation method thereof - Google Patents
A kind of bigeminy hand-foot-and-mouth disease inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to biological technical field, discloses two kinds of hand-foot-and-mouth disease Strain and the bigeminy hand-foot-and-mouth disease inactivated vaccine prepared by two kinds of Strain.The 71 type Strain CC 09 of people's enteron aisle that deposit number is CGMCC No.7753 and the viruses of Coxsack A16 type Strain CC 16 that deposit number is CGMCC No.7752 are inoculated in the Vero cells for expanding secondary culture respectively and cultivated；Two kinds of virus-culturing fluids are harvested respectively, except concentration, inactivation, purifying obtain two kinds of virus stock solution useds after impurity；Two kinds of virus stock solution useds are degerming, and mixing, adsorbs adjuvant, bigeminy hand-foot-and-mouth disease inactivated vaccine is prepared.Bigeminy hand-foot-and-mouth disease inactivated vaccine immunogenicity of the present invention is strong, and without inhibitory action is intersected between two kinds of viruses, can effectively prevent domestic popular hand-foot-and-mouth disease at present.Bigeminy hand-foot-and-mouth disease inactivated vaccine preparation process of the present invention is simple, and production cost is low, is adapted to the large-scale production of vaccine.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of vaccine and preparation method thereof.
Background technology
Hand-foot-and-mouth disease (Hand foot and mouth disease HFMD) is a kind of children's infectious disease, also known as eruptive
Vesicular stomatitis.Occurred in less than 5 years old children, the bleb at the positions such as hand, foot, oral cavity can be caused, the disease is with hand, foot and mouth
It is main clinic symptoms that ulcer is formed after transmucosal bleb or ulceration.A small number of infants can cause myocarditis, pulmonary edema, aseptic brain
The complication such as meningocephalitis.If indivedual children with serious disease progression of the disease are fast, lead and also result in death.
Hand-foot-and-mouth disease is the infectious disease as caused by enterovirus, and enterovirus infectiousness is strong, and route of transmission is extensive, popular model
Enclose extensively, propagate soon, the prevalence of its disease may occur in which at all seasons, and have the characteristics of periodic epidemic.People is enterovirus
Unique natural reservoir (of bird flu viruses), major source of infection are patient, virus carrier and subclinical infection person.Trigger the enteron aisle disease of hand-foot-and-mouth disease
Poison has kind more than 20 (type), wherein coxsackievirus A16 (Coxsachievirus, CA16) and Enterovirus 71 with A groups
Type (Enterovirus 71, EV71) is most commonly seen.Australia occurred within 1972~1973 years, 1986 and 1999
EV71 is popular, and for critically ill patient mostly with central nervous system symptom (CNS), some patients also have serious respiratory system disease
Shape.The 1970s mid-term, Bulgaria, Hungary break out popular as the EV71 of main clinical characteristics using CNS in succession, only protect
Add the Leah just morbidity more than 750,149 people cause paralysis, and 44 people are dead.Japan is the more country of hand-foot-and-mouth disease morbidity, in history
Had repeatedly extensive popular, based on 1969~1970 years popular is infected with CA16,2 prevalences of 1973 and 1978 are
EV71 causes, and main clinic symptoms are hand-foot-and-mouth disease, and the state of an illness is generally relatively gentle, but are also observed at the same time with aseptic meningitis
Case.Hand-foot-and-mouth disease comes back in Japan within 1997~2000 years, and EV71, CoxA16 have separation, the genotype of EV71 strains
Also with conventional difference.Later stage the 1990s, EV71 start to wreak havoc East Asia Region.1997 Malaysia there occurs mainly by
Hand-foot-and-mouth disease caused by EV71 is popular, and 4~August shares 2628 morbidities, just there is 29 patient deaths only 4~June.The dead is averaged
At 1.5 years old age, the course of disease only 2 days, 100% fever, 62% brothers' fash, 66% canker sore, 28% ongoing disease is rapid, and 17%
Limb collapses from physical exhaustion, 17 chest films showed pulmonary edema.
Hand-foot-and-mouth disease, later Beijing, Hebei, Tianjin, Fujian, Jilin, Shandong, peace were seen from 1981 in the Shanghai beginning by China
The provinces and cities such as emblem, Guangdong have been reported that.Outbreak of epidemic hand-foot-and-mouth disease is concentrated in Fuyang, Heze City, Shandong Province, Henan in 2008 more, and
Cause many cases infant dead.From 2 days Mays in 2008, hand-foot-and-mouth disease included Class C communicable disease control by country.China in recent years
Hand-foot-and-mouth disease incidence and the death rate are counted in trend is gradually increasing according to the Ministry of Public Health:Report number of the infected is in the whole nation in 2011 altogether,
1619706, morbidity number 2198442, death toll 569 are reported in death 509, the whole nation in 2012 altogether.EV71 and
Hand-foot-and-mouth disease caused by CoxA16 is there are subclinical infection and mild cases are more, and the infection sources is difficult to find and controls, and children are generally easy
Sense, route of transmission are more, it is difficult to the effectively feature such as blocking, and hand-foot-and-mouth disease is mostly only by common acyclovir, cuts down former times at present
The antiviral drugs of the high-efficiency broad spectrums such as Luo Wei, famciclovir shortens fever and skin lesion healing time, mitigates herpes of mouth pain,
So far also without for the prevention sick specific treatment medicine.Therefore hair of the vaccine for hand-foot-mouth disease to prevention hand-foot-and-mouth disease is developed
Life is of great significance.
The content of the invention
The bigeminy brothers prepared the object of the present invention is to provide two kinds of hand-foot-and-mouth disease Strain and by two kinds of Strain
Stomatosis inactivated vaccine, the bigeminy hand-foot-and-mouth disease inactivated vaccine prevent hand-foot-and-mouth disease, and epidemic prevention effect is good, and security is good.
71 type Strain CC-09 of a kind of people's enteron aisle provided by the invention, is deposited in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, deposit number is CGMCC No.7753.
Another kind Coxsack A16 type Strain CC-16 provided by the invention, are deposited in Chinese microorganism strain preservation management
Committee's common micro-organisms center, deposit number are CGMCC No.7752.
The present invention injects 71 type Strain CC-09 of the people's enteron aisle viruses and Coxsack A16 type Strain CC- by cranial cavity
The methods of 16 viruses to 1 age in days ICR suckling mouses attack poison, and it is bad that the results show can find that the state of mind occurs in mouse for 3 days, OK
The clinical symptoms such as slow, drowsiness are moved, the typical brothers mouthful clinical symptoms of quadriplegia occur in 4 days individual mices, and then other are small
Mouse also all shows the symptom of quadriplegia, and 6 days whens start to occur dead, and the 7th day mouse is without survival, and control group mice is without facing
Bed Symptoms.Show 71 type Strain CC-09 of people's enteron aisle of the present invention and Coxsack A16 type Strain two plants of diseases of CC-16
Poison is velogen strain.
The present invention also provides the vaccine for hand-foot-mouth disease prepared by above two hand-foot-and-mouth disease Strain.
The vaccine for hand-foot-mouth disease, preferably, being bivalent inactivated vaccine.
The present invention also provides a kind of preparation method of bigeminy hand-foot-and-mouth disease inactivated vaccine, comprise the following steps：
Step 1：It is by 71 type Strain CC-09 of people's enteron aisle and deposit number that deposit number is CGMCC No.7753
The Coxsack A16 type Strain CC-16 of CGMCC No.7752 is inoculated in the Vero cells or human embryo lung (HEL) for expanding secondary culture respectively
Cultivated in diploid cell；
Step 2：Two kinds of virus-culturing fluids are harvested respectively, except concentration, inactivation, purifying obtain two kinds of virus stock solution useds after impurity；
Step 3：Take two kinds of virus stock solution useds prepared by step 2 degerming, mix up to the bigeminy hand-foot-and-mouth disease inactivated vaccine.
Preferably, when culture described in step 1 is that 33 DEG C~36 DEG C cultures 72~120 are small.
Preferably, inactivation described in step 2 is to be inactivated with formalin or beta-propiolactone solution.
Preferably, purifying described in step 2 is anion-exchange chromatography, molecular sieve gel filtration chromatography, composite mode are situated between
One or more in matter chromatography.
Further, purifying described in step 2 is first using anion-exchange chromatography again using molecular sieve gel filtration chromatography.
Preferably, degerming described in step 3 is degerming through 0.2 μm of filter membrane.
Preferably, be mixed into described in step 3 will be degerming after two kinds of virus stock solution useds mixing after adsorb adjuvant；Or to incite somebody to action
Two kinds of virus stock solution useds after degerming adsorb adjuvant and then remix respectively.Wherein, the adjuvant is preferably aluminum hydroxide solution.
The present invention proves that bigeminy hand-foot-and-mouth disease inactivated vaccine of the present invention is directed to two kinds by potency test in Mice Body
Virus generates higher antibody titer, and immunogenicity is strong, and without intersection inhibitory action between two kinds of viruses.
It is of the present invention be mixed by 71 type Strain CC-09 of people's enteron aisle and Coxsack A16 type Strain CC-16 two
Join hand-foot-and-mouth disease inactivated vaccine, generate higher antibody titer for two kinds of viruses, immunogenicity is strong, and nothing between two kinds of viruses
Intersect inhibitory action, and have extensive neutralization protective effect to different regions virus.Domestic popular hand at present can effectively be prevented
Sufficient stomatosis.Its preparation process of bigeminy hand-foot-and-mouth disease inactivated vaccine of the present invention is simple, effectively removes host protein and
DNA, viral purity is high, and vaccine safety is good, and production cost is low, is adapted to the large-scale production of vaccine, before having good application
Scape.
Biological deposits information：
Strain CC-09：Classification And Nomenclature：Enterovirns type 71, Chinese microorganism strain is deposited on June 13rd, 2013
Preservation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the Chinese Academy of Sciences is micro-
Biological study institute, deposit number are CGMCC No.7753.
Strain CC-16：Classification And Nomenclature：Coxsack A16 types virus, China Microbiological is deposited on June 13rd, 2013
Culture presevation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, deposit number are CGMCC No.7752.
Embodiment：
The invention discloses people's enteron aisle 71 type Strain CC-09 and Coxsack A16 type Strain CC-16 and with described two
Bigeminy hand-foot-and-mouth disease inactivated vaccine prepared by Strain and preparation method thereof.In particular, all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The product of the present invention,
Method is described by preferred embodiment, and related personnel can substantially not depart from present invention, spirit and scope
It is interior that method described herein and application are modified or suitably change with combining, to realize and using the technology of the present invention.
For the fashion trend of current domestic hand-foot-and-mouth disease, for the outburst of preferably prevention hand-foot-and-mouth disease, inventor passes through
Unremitting effort for many years, finally filters out 71 type Strain CC-09 of people's enteron aisle and Coxsack with extensive cross-protection
A16 type Strain CC-16, establish three-level virus seed bank, and production of vaccine seed bank is used as after assay approval.For above-mentioned
Strain, applicant were deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 13rd, 2013,
The deposit number of wherein 71 type Strain CC-09 of people's enteron aisle is CGMCC No.7753, the guarantor of Coxsack A16 type Strain CC-16
It is CGMCC No.7752 to hide numbering.
On this basis, present inventors have developed the vaccine for preventing hand-foot-and-mouth disease.A kind of hand-foot-and-mouth disease epidemic disease
Seedling, is CGMCC No.7752 by 71 type Strain CC-09 of people's enteron aisle and deposit number that deposit number is CGMCC No.7753
Coxsack A16 type Strain CC-16 be prepared.Vaccine for hand-foot-mouth disease of the present invention can be more broadly useful for preventing
Hand-foot-and-mouth disease caused by EV71 viruses and CA16 viruses.Vaccine is the important substance basis of immunization campaign work.Drawn by its property
Point, inactivated vaccine, attenuated live vaccine, component vaccine, recombination engineered vaccine etc. can be divided into, preferably, of the present invention
Vaccine for hand-foot-mouth disease is bivalent inactivated vaccine, more suitable for the immunoprophylaxis of infant.
Preferably to produce high quality bivalent inactivated vaccine, present invention also offers the bigeminy hand-foot-and-mouth disease to inactivate epidemic disease
The preparation method of seedling.
A kind of preparation method of bigeminy hand-foot-and-mouth disease inactivated vaccine, comprises the following steps：
Step 1：It is by 71 type Strain CC-09 of people's enteron aisle and deposit number that deposit number is CGMCC No.7753
The Coxsack A16 type Strain CC-16 of CGMCC No.7752 is inoculated in the Vero cells or human embryo lung (HEL) for expanding secondary culture respectively
Cultivated in diploid cell；
Step 2：Two kinds of virus-culturing fluids are harvested respectively, except concentration, inactivation, purifying obtain two kinds of virus stock solution useds after impurity；
Step 3：Take two kinds of virus stock solution useds prepared by step 2 degerming, mix up to the bigeminy hand-foot-and-mouth disease inactivated vaccine.
Two kinds of Strain are inoculated in expansion first and passed by the preparation method of bigeminy hand-foot-and-mouth disease inactivated vaccine of the present invention
Cultivated in the Vero cells or human embryonic lung diploid fibroblast of being commissioned to train foster to obtain virus-culturing fluid.
Vero cells are that African green monkey kidney cell is a kind of heteroploid cell, are produced after RhMK cell culture derivation,
It is common production of vaccine cell line, frequently as the host cell of culture virus.
Human embryonic lung diploid fibroblast is common production of vaccine cell line.Preferably, the human embryonic lung diploid fibroblast is
The cell lines such as MRC-5,2BS.
Preferably, the expansion secondary culture of the Vero cells or human embryonic lung diploid fibroblast be using bioreactor or
Multi-layered cell factory is enlarged secondary culture.Using bioreactor or multi-layered cell factory culture cell, cell density is high,
After virus inoculation, viral yield is high, so as to improve vaccine yield.
Preferably, culture medium used during the Virus culture is 199 culture mediums, and the culture medium does not contain serum, connects
The virus-culturing fluid bovine serum albumin content obtained after kind virus is low, and vaccine safety is high.
Preferably, inoculation is in 0.001MOI~0.1MOI ratio virus inoculations described in step 1.Wherein, MOI is
The abbreviation of multiplicity of infection, Chinese are translated into infection multiplicity, are commonly used to the research of virus infected cell
In.It is generally acknowledged that MOI is a ratio, without unit, its implicit unit is pfu number/cell in fact, is meant that sense
Virus and the ratio of cell quantity during dye.Inoculation is 1 preferably according to virus and cell quantity i.e. described in step 1:10~1:
1000 ratio inoculation.
Further, when culture described in step 1 is that 33 DEG C~36 DEG C cultures 72~120 are small.The culture of i.e. two kinds Strain
When condition is that 33 DEG C~36 DEG C cultures 72~120 are small.
Two kinds of virus-culturing fluids are harvested after the preparation method culture virus of bigeminy hand-foot-and-mouth disease inactivated vaccine of the present invention
Afterwards, except impurity, concentration, inactivation, purifying obtain two kinds of virus stock solution useds.
Wherein, the removal of impurities matter is preferably by centrifugation or multistage filter filtering and impurity removing matter.
Further, the centrifugation centrifuges 30min for 6000rpm；Filter column pore size is used in the multistage filter
0.1 μm~0.5 μm.
Virus-culturing fluid concentration of the present invention is is concentrated by ultrafiltration, and further, the ultrafiltration membrane of the ultrafiltration concentration is cut
It is 100KD~300KD to stay molecular weight.
Inactivation is so that it loses fertility while immunogenicity is retained after virus-culturing fluid is concentrated.As excellent
Choosing, the inactivation are to be inactivated with formalin or beta-propiolactone solution.
Further, the actual conditions that is inactivated with formalin is that the concentration ratio of formaldehyde in formalin and water is
1: 2000~1: 4000, inactivation temperature is 35 DEG C~37 DEG C, and inactivation time is 6 days~12 days.
Further, described with beta-propiolactone solution inactivation actual conditions is beta-propiolactone in beta-propiolactone solution with going out
The concentration ratio of live virus liquid is 1: 2000~1: 4000, and inactivation temperature is 4 DEG C of inactivation times 2 days~5 days.
Preparation method of the present invention purifies virus after inactivation of viruses, wherein the purifying is preferably anion
One or more in displacement chromatography, molecular sieve gel filtration chromatography, composite mode medium chromatography, to obtain the EV71 of high-purity
With CA16 virus stock solution useds.
Further, purifying described in step 2 is first using anion-exchange chromatography again using molecular sieve gel filtration chromatography.
Preliminary purification is carried out to inactivation of virus liquid using anion exchange, to remove host cell DNA, reapplies molecular sieve gel filtration
Purification process carry out polishing purification, remove various impurity proteins, obtain the higher virus stock solution used of purity.
Preparation method of the present invention takes two kinds of virus stock solution useds made from purifying degerming, mixes up to the bigeminy brothers mouthful
Inactivated vaccine.
Preferably, it is described degerming to be degerming through 0.2 μm of filter membrane.
To strengthen inactivated vaccine immunogenicity, it usually needs add adjuvant.Adjuvant is also known as nospecific immunity proliferant agent, this
Body does not have antigenicity, but synantigen is expelled to body interior energy enhancing immunogenicity or changes immune response type together or in advance.
Therefore bigeminy hand-foot-and-mouth disease inactivated vaccine of the present invention preparation method step 3 described in be mixed into will be degerming after two kinds of viruses
Adjuvant is adsorbed after stoste mixing；Or adjuvant is adsorbed respectively for two kinds of virus stock solution useds after will be degerming and is then remixed.
Preferably, the adjuvant is aluminum hydroxide solution.Aluminium hydroxide uses in vaccine can not only improve body
Immune response and there is preferable security.
Preferably, the ratio of the mixing is the 71 type Strain CC-09 of people's enteron aisle that deposit number is CGMCC No.7753
Virus stock solution used and deposit number be CGMCC No.7752 Coxsack A16 type Strain CC-16 virus stock solution used press 1:0.5
~1:2.5 mixing.
For a further understanding of the present invention, with reference to embodiment, the present invention is described in detail.Embodiment 1：Virus
The screening of strain and the measure of the virulence of strain
1st, Strain is screened：
Positive totally 20 plants totally 15 plants viral, positive CA16 of the viruses of EV71 are isolated from brothers mouthful patient's throat swab,
Carry out adapting to Vero cultures respectively, and analyze and research one by one to its biological characteristics, select and big plaque and typical case, sense occur
2~3 plants of the virus that metachromia titre is high, cross protection scope is wide is used as first run vaccine candidate strain.
Plaque Clone purifying is carried out to the strain of first run vaccine candidate, the multiple clone strains of picking carry out complete sequence determinations, choose with
The consistent primordial seed of establishing of original strain sequence is criticized, and carries out tentative vaccine art research, finally definite 71 type disease of people's enteron aisle
Strain CC-09 and Coxsack A16 type Strain CC-16 are as production of vaccine strain.
2nd, Strain toxicity test
Toxicity test method：7.0LgCCID is chosen respectively50The 71 type Strain CC-09 of people's enteron aisle of/ml,
6.0LgCCID50The Coxsack A16 type Strain CC-16 viruses of/ml, by the method that cranial cavity is injected to (the purchase of 1 age in days ICR suckling mouses
From preclinical medicine institute of Jilin University animal experimental center) carry out attacking poison, while pressed using PBS as control, whole experiment process
Carried out according to animal protection and instruction policy.Every group is attacked malicious 10 suckling mouses, and 10 μ L/ are only.Observe and record suckling mouse clinical condition daily
Shape, is observed 21 days altogether.
Toxicity test result：71 type Strain CC-09 viruses of cranial cavity injection people enteron aisle and Coxsack A16 type Strain CC-
It is bad that 16 viruses can find that the state of mind occurs in mouse for 3 days, and the clinical symptoms such as be slow in action, be drowsiness, and individual mice occurs within 4 days
The typical brothers of quadriplegia mouthful clinical symptoms, other subsequent mouse also all show the symptom of quadriplegia, and 6 days whens start
Existing death, the 7th day mouse is without survival, and control group mice occurs without above-mentioned clinical symptoms.71 type of people's enteron aisle is judged accordingly
Strain CC-09 and two strain virus of Coxsack A16 type Strain CC-16 are velogen strain.
Above two Strain CC-09 and CC-16 are subjected to biological deposits, its relevant information is as follows：
Strain CC-09：Classification And Nomenclature：Enterovirns type 71, Chinese microorganism strain is deposited on June 13rd, 2013
Preservation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the Chinese Academy of Sciences is micro-
Biological study institute, deposit number are CGMCC No.7753.
Strain CC-16：Classification And Nomenclature：Coxsack A16 types virus, China Microbiological is deposited on June 13rd, 2013
Culture presevation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, deposit number are CGMCC No.7752.
Embodiment 2：The preparation of bigeminy brothers mouthful inactivated vaccine of the present invention
(1) prepared by vaccine：The African green monkey kidney cell (Vero) for the spinner culture for covering with individual layer is taken, through 0.25% tryptose
Enzyme (containing EDTA) digests, and is inoculated with bioreactor by certain cell number, cultivates 3-5 days, takes EV71 or CA16 strains work seed
Batch seed culture of viruses is through appropriate dilution, the Vero cells being inoculated with bioreactor, when 35 DEG C of ± 0.5 DEG C of cultures 72~120 are small, harvest disease
Malicious nutrient solution, puts -20 DEG C of freezer freeze thawing, and cell fragment, 300KD retention molecules are removed through 0.1 μm~0.5 μm aperture filter filtering
The ultrafiltration membrane of amount concentrates 50 times, final concentration of 1: 4,000 37 DEG C of formaldehyde inactivation 10 days is aseptically added, after inactivation
Virus liquid, with 0.02mol/L PB buffer solutions (pH5.0~7.0) balance after, purified using anion-exchange chromatography, use
0.02mol/L PBS buffer (pH 7.0) elutes, and collects viral peak, for the virus liquid slightly purified；0.01mol/L PBS are used again
After buffer solution (pH 7.0) balance, using the method polishing purification of molecular sieve gel filtration, with 0.01mol/L PBS buffer
(pH 7.0) is eluted, and is collected viral peak, is polishing purification virus liquid, then through 0.2 μm of filter membrane aseptic filtration.According to virus antigenicity
Titre levels do appropriate dilution, make the whole content of EV71, CA16 virus protein between the μ g of 5 μ g~10, by volume 1:1 ratio
Two kinds of virus liquids are mixed, adsorb aluminum hydroxide adjuvant to obtain the final product by example.After further assay approval, semi-finished product are sub-packed in sterile
In cillin bottle or precharging type syringe, finished product vaccine is made.
(2) the bigeminy hand-foot-and-mouth disease inactivated vaccine component prepared：Less than EV71, CA16 viral antigen of 10 μ g/mL,
0.6mg/mL aluminium hydroxides, 0.1mol/L sodium chloride, 0.1mol/L disodium hydrogen phosphates, 0.1mol/L sodium dihydrogen phosphates, glucose.
(3) vaccine detects：Sterility test, mycoplasma, abnormal toxicity test are examined and determine by 2010 editions States Pharmacopoeia specifications；Ox blood
The examination criteria of pure protein residue amount, host cell proteins residual quantity and host cell DNA residual quantity is thin with reference to other Vero
The national standard of born of the same parents' vaccine product is examined and determine.The bigeminy brothers mouthful inactivated vaccine of preparation is through calibrating, sterility test, undue toxicity
The calibrating project such as experiment is qualified, meets the requirements；Bovine serum protein residual content, host protein remain, host DNA remains,
Endotoxin content is below the standard verification of other existing Vero cell culture process vaccines, EV71 after aluminium hydroxide absorption,
CA16 viruses are without dissociation.
Embodiment 3：The preparation of bigeminy brothers mouthful inactivated vaccine of the present invention
The African green monkey kidney cell (Vero) for the spinner culture for covering with individual layer is taken, through 0.25% Trypsin Induced, by one
Determine the cell factory that cell number is inoculated with 40 layers, cultivate 3 days, take EV71 or CA16 strain working seed lots seed culture of viruses to be diluted through appropriate,
Vero cells in inoculating cell factory, when 35 DEG C of ± 0.5 DEG C of cultures 72~96 are small, harvest virus-culturing fluid, put -20 DEG C of freezers
Freeze thawing, cell fragment is removed through 0.1 μm~0.5 μm aperture filter filtering, and the ultrafiltration membrane of 300KD molecular cut offs concentrates 50 times,
Final concentration of 1: 4,000 4 DEG C of beta-propiolactone inactivation 4 days is aseptically added, the virus liquid after inactivation is used
After 0.02mol/L PB buffer solutions (pH 5.0~7.0) balance, purified using anion-exchange chromatography, with 0.02mol/L PBS
Buffer solution (pH 7.0) elutes, and collects viral peak, for the virus liquid slightly purified；0.01mol/L PBS buffer (pH is used again
7.0) after balancing, using the method polishing purification of molecular sieve gel filtration, washed with 0.01mol/L PBS buffer (pH7.0)
It is de-, viral peak is collected, is polishing purification virus liquid, then through 0.2 μm of filter membrane aseptic filtration.According to virus antigenicity, titre levels do
Appropriate dilution, makes the whole content of EV71, CA16 virus protein then adsorb aluminum hydroxide adjuvant respectively between the μ g of 5 μ g~10,
By volume 1:2 ratio mixes two kinds of virus liquids to obtain the final product.After further assay approval, semi-finished product are sub-packed in sterile west
In woods bottle, finished product vaccine is made.
(2) vaccine detects：Sterility test, mycoplasma, abnormal toxicity test are examined and determine by 2010 editions States Pharmacopoeia specifications；Ox blood
The examination criteria of pure protein residue amount, host cell proteins residual quantity and host cell DNA residual quantity is thin with reference to other Vero
The national standard of born of the same parents' vaccine product is examined and determine.The bigeminy brothers mouthful inactivated vaccine of preparation is through calibrating, sterility test, undue toxicity
The calibrating project such as experiment is qualified, meets the requirements；Bovine serum protein residual content, host protein remain, host DNA remains,
Endotoxin content is below the standard verification of other existing Vero cell culture process, EV71, CA16 disease after aluminium hydroxide absorption
Poison is without dissociation.
Embodiment 4：Bigeminy hand-foot-and-mouth disease inactivated vaccine immunogenicity determining of the present invention
Bigeminy brothers mouthful inactivated vaccine finished product prepared by the embodiment of the present invention 2, embodiment 3 carries out Mouse immunogenicity
Experiment, calculates the neutralization titer of corresponding antibodies, observes the immunogenicity of corresponding virus.
Take Female ICR mice, weight 16-18g, random packet.Experiment is divided into 4 groups, every group 20, negative control group：Note
Penetrate aluminium hydroxide thinner for vaccine；2 groups of 1 group of test seedling and test seedling：Vaccine prepared by embodiment 2 and embodiment 3 is injected respectively,
Test seedling to be serially diluted with thinner for vaccine, every mouse peritoneal injects 0.5mL, and after being immunized 3 weeks, non-execution takes blood,
Serum is centrifuged, -20 DEG C save backup.Same dose booster immunization after 2 days, puts to death and takes blood, centrifugation serum, -20 DEG C
Save backup.Using fixed virus, dilute serum method detection virucidin potency, calculates according to Reed-Muench methods
Neutralize antibody titers.
The serum that 2 groups of 1 group of test seedling and test seedling are gathered carries out neutralizing antibody detection, comprises the following steps that：
1) experimental method is neutralized：After virus infection sensitive cells, cause cytomorphology to change, cytopathogenic effect effect occur
Answer (CPE), after specific neutralizing antibody is combined with virus, but virion loses infectivity, suppresses CPE and occurs.
2) operating procedure of virucidin's measure：
(1) by the inactivation 30 minutes of 56 DEG C of test serum, it is serially diluted with sterile PBS by 2 times, is respectively 1: 8,1: 16,1:
32、1∶64、1∶128、1∶256、1∶512、1∶1024；
(2) it is 100CCID by viral dilution according to virus titer50/50μL；
(3) above-mentioned serum and each 50 μ L of virus are added into 96 orifice plates, each serum dilution at least does 8 multiple holes；
(4) 37 DEG C of CO are put into2When incubation 2 is small in incubator；
(5) cell control well, serum control hole and virus control wells are done while；
(6) digestive juice vitellophag is used, prepares cell suspension, the concentration of cell suspension is 2 × 105A/mL；
(7) each test serum hole, serum control hole and virus control wells and cell control well add respectively after being incubated
Enter 0.1mL cell suspensions, be put into 37 DEG C of CO2Culture is incubated in incubator；
(8) CPE is observed daily using inverted microscope, and record titration of virus as a result, not produce the blood of cytopathy
The inverse of clear highest dilution is endpoint titers.Work as 100CCID50When complete lesion occur in the virus control wells of/0.05mL, judge
Final result (7~10d)；Pay attention to：If virus control result is not in 32~320CCID50In the range of/0.05mL, nothing is tested
Effect is tested it is necessary to repetition.
(9) result judgement：When having 1 hole cytopathy occur in 2 holes of highest dilution serum, another hole occurs without cell
Lesion, the inverse of the dilution factor are the neutralize antibody titers for being calculated as the serum specimen；When the complete lesion in 2 hole of highest dilution, phase
Adjacent low 2 hole of dilution factor not lesion completely, then the inverse of both Average dilutions is the neutralize antibody titers of the serum specimen；When
Occurs 1 hole cytopathy in two adjacent dilution factor serum, another 1 hole occurs without cytopathy, then both Average dilutions
Inverse is the neutralize antibody titers of the serum specimen.Testing result is shown in Table 1.
1 vaccine immunogenicity of table measures
After the visible two groups of tentative inactivated vaccine booster immunizations of 1 result of table, test group animal Conversion rate is 100%,
And higher neutralizing antibody is generated for EV71, CA16 virus, there is very high humoral immunogenicity, can prevent well
Hand-foot-and-mouth disease poison is popular to be propagated.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of 71 type Strain CC-09 of people's enteron aisle, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, deposit number are CGMCC No.7753.
2. a kind of Coxsack A16 types Strain CC-16, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center, deposit number are CGMCC No.7752.
3. a kind of vaccine for hand-foot-mouth disease, it is characterised in that by the 71 type Strain of people's enteron aisle that deposit number is CGMCC No.7753
CC-09 and the Coxsack A16 type Strain CC-16 that deposit number is CGMCC No.7752 are prepared.
4. vaccine for hand-foot-mouth disease as claimed in claim 3, it is characterised in that the vaccine is bivalent inactivated vaccine.
5. a kind of preparation method of bigeminy hand-foot-and-mouth disease inactivated vaccine, it is characterised in that comprise the following steps：
Step 1：It is CGMCC by 71 type Strain CC-09 of people's enteron aisle and deposit number that deposit number is CGMCC No.7753
The Coxsack A16 type Strain CC-16 of No.7752 is inoculated in the Vero cells for expanding secondary culture or human embryo lung (HEL) diploid respectively
Cultivated in cell；
Step 2：Two kinds of virus-culturing fluids are harvested respectively, except concentration, inactivation, purifying obtain two kinds of virus stock solution useds after impurity；
Step 3：Take two kinds of virus stock solution useds prepared by step 2 degerming, mix up to the bigeminy hand-foot-and-mouth disease inactivated vaccine.
6. preparation method as claimed in claim 5, it is characterised in that described in step 1 culture for 33 DEG C~36 DEG C culture 72~
120 it is small when.
7. preparation method as claimed in claim 5, it is characterised in that inactivation described in step 2 is with formalin or β-the third
Ester solution inactivates.
8. preparation method as claimed in claim 5, it is characterised in that purifying described in step 2 is anion-exchange chromatography, molecule
Sieve the one or more in gel permeation chromatography, composite mode medium chromatography.
9. preparation method as claimed in claim 5, it is characterised in that be mixed into described in step 3 will be degerming after two kinds of viruses
Adjuvant is adsorbed after stoste mixing；Or adjuvant is adsorbed respectively for two kinds of virus stock solution useds after will be degerming and is then remixed.
10. preparation method as claimed in claim 9, it is characterised in that adjuvant described in step 3 is aluminum hydroxide solution.
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Citations (1)
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| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
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| CN101695570A (en) * | 2009-11-03 | 2010-04-21 | 中国人民解放军军事医学科学院微生物流行病研究所 | Univalent and bivalent inactivated vaccine for hand-foot-and-mouth disease and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| A combination vaccine comprising of inactivated enterovirus 71 andcoxsackievirus A16 elicits balanced protective immunity against both viruses;Yicun Cai et al;《Vaccine》;20140318;2406–2412 * |
| Prospect and challenges for the development of multivalent vaccines against hand, foot and mouth diseases;Chia-Chyi Liu et al;《Vaccine》;20140913;6177–6182 * |
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