CN103182079A - Respiratory syncytial virus vaccine and preparation method thereof - Google Patents
Respiratory syncytial virus vaccine and preparation method thereof Download PDFInfo
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- CN103182079A CN103182079A CN 201110458973 CN201110458973A CN103182079A CN 103182079 A CN103182079 A CN 103182079A CN 201110458973 CN201110458973 CN 201110458973 CN 201110458973 A CN201110458973 A CN 201110458973A CN 103182079 A CN103182079 A CN 103182079A
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- respiratory syncytial
- syncytial virus
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Abstract
The invention discloses a respiratory syncytial virus vaccine and a preparation method thereof. The vaccine contains membrane proteins obtained by cracking of whole virus particles of respiratory syncytial virus, wherein the respiratory syncytial virus vaccine contains 100-200 Mug of viral proteins per dose and 0.5 mg/dose of aluminum phosphate adjuvant. Compared with the existing viral purified stock solution, the respiratory syncytial virus vaccine for human, disclosed by the invention, has a greatly-reduced content of foreign proteins, is better in immunogenicity and potency, is safer in use, and is more suitable for large-scale production.
Description
Technical field
The invention belongs to biological technical field, specifically, relate to a kind of human respiratory syncytial virus's vaccine and preparation method thereof.
Background technology
Respiratory syncytial virus is the RNA viruses of the non-segmental of a kind of sub-thread minus strand, belongs to myxovirus section, and pneumonitis virus belongs to.Internal core is the RNA of capsid protein parcel, and skin is coated with the lipid bilayer that comes from the host, and transmembrane protein.11 kinds of protein of the main coding of virus, three kinds of transmembrane proteins are relevant with immunoprotection and the virulence of causing a disease, and are respectively fusion protein F, adhesion protein G and little hydrophobin SH.
Respiratory syncytial virus (Respiratory syncytial virus, RSV), be the one of the main reasons that worldwide causes the infant lower respiratory infection, its cause of disease as the pneumonia that causes not enough one-year-old baby and bronchiolitis is all more serious than other all microbial pathogens.In fact, all children all can be infected in two years old, and it is also higher in bigger child and adolescence the frequency that infects again to take place on one's body.Although the adult of most of health does not have the disease of serious rsv infection, the individuality of Lao Nian patient and non-responsiveness can suffer serious infection even possibility threat to life usually slightly.Present stage, antiviral drugs can only be alleviated clinical symptoms to a certain extent and shorten sick time.Therefore, the preventative antiviral drugs that gives remains the most effective clinical measure.And RSV does not also have desirable specific aim vaccine at present.
Summary of the invention
Technical problem to be solved by this invention is, in order to overcome defective of the prior art, provide a kind of and can in human body, produce faster, higher immune response, and side reaction few the novel respiratory syncytial virus vaccines of human.
The invention discloses a kind of Vero of utilization cell comes large-scale industrialization to produce the method for human respiratory syncytial virus's inactivated vaccine.The cellular matrix of virus breeding is the Vero cell in the inventive method, is that the cellular matrix that is used for the human biological product is granted by The World Health Organization (WHO).Compare with diploid cell, have easy cultivation, and can satisfy with clear superiorities such as fermentation tank (microcarrier) large-scale production, the Vero cell has been widely used in now both at home and abroad produces the refining rabies vaccine of human, upgraded products such as Vaccinum Encephalitidis Epidemicae.
The invention discloses a kind of method of the Vero of utilization cell preparation human respiratory syncytial virus inactivated vaccine, comprise the following steps:
1. static or three-dimensional absorption Cultivation of Vero in culture medium obtains the Vero cell concentration and reaches 10
5-10
7The cellular matrix culture fluid of/ml;
2. the patient throat swab collection of human respiratory syncytial virus from falling ill 2 days separates through routine, with the infective dose (M.O.I.) of 0.01-0.2, virus inoculation in the cellular matrix culture fluid of step 1, cultivated 6-10 days;
3. obtain virus-cell culture suspension with pancreas enzyme-EDTA digestion;
4. purified virus prepares vaccine.
The invention also discloses a kind of vaccine combination, comprise the human respiratory syncytial virus vaccines and the 0.5mg/ agent aluminum phosphate adjuvant that utilize method for preparing to obtain of virus protein content 100-200 μ g/ agent.
The specific embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect is easy to understand, the present invention is further elaborated below in conjunction with embodiment.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method and the material similar or impartial to the record content all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1
In liquid nitrogen container, take out Vero cell strain (source, Nat'l Pharmaceutical ﹠ Biological Products Control Institute), centrifugal 1000rpm, 10 minutes, discard former preservation liquid, add and contain 10% new-born calf serum, MEM nutritional solution (the pH 7%NaHCO of 0.20% lactoalbumin hydrolysate
3Transfer to 6.8) put 37 ℃ of cultivations, treat that cell grows up to monolayer, with the PBS washing, pancreas enzyme-EDTA digestion adds above-mentioned nutritional solution then, with 1: 2 minute kind rate, goes down to posterity 1 time in per 5 days, until obtaining to produce in batches the enough cells that need usefulness.
Reach 10 in above-mentioned cell culture concentration
6During/ml, the branch kind rate by 0.05 M.O.I. in the culture fluid of pH7.4 adds human respiratory syncytial virus's strain, and static absorption was cultivated 8 days.Change 1 time therebetween and keep liquid.After cultivating expiration, obtain virus-cell culture suspension with pancreas enzyme-EDTA digestion.This virus-cell culture suspension is through ultrasonic Treatment and crash cells, reaches more than 95% to cell crashing ratio.Carry with chloroform afterwards and take out 4 times, vibrated 35 minutes at every turn, centrifugal 3000rpm extracted supernatant, and removed foreign protein in 40 minutes.The gained supernatant NaC l solution concentration of 10%PEG (molecular weight 6000) and 0.4M, centrifugal 10000rpm, 2 hours, collecting precipitation.The precipitation of collecting is suspended the different gradient centrifugations of glycerol sucrose, concentration range: begin 0.5ml from bottom with PBS, 80% glycerol (100mM NaCl, 100Mm TrispH7.4), 1.8ml 30% sucrose, 1.8ml 20% sucrose,, 1.8ml 10% sucrose (containing 10%SDS).Centrifugal 37000rpm, 6 hours.The Sepharose-4FF gel is crossed post and is further gone out host's residual DNA and foreign protein.With 1: 4000 formalin-inactivated 12 days, the every dosage of packing finished product was for containing virus protein 100-200 μ g, aluminum phosphate adjuvant 0.5mg/ agent with the virus behind the purification.
Embodiment 2
Aluminum phosphate adjuvant with above-described embodiment 1 method, inactivated virus vaccine and vaccine combination (inactivated virus vaccine+aluminum phosphate adjuvant) be immune mouse respectively, each immune 0.5ml, 0 day, 7 days immune twice respectively, observe blood sampling after 14 days, detect the serum neutralizing antibody and tire.
Table 1
| The vaccine kind | (IU) tires/agent |
| The aluminum phosphate adjuvant | 0.08 |
| Inactivated virus vaccine | 2.48 |
| Vaccine combination | 3.21 |
Therefore; illustrate that the aluminum phosphate adjuvant can obviously increase the effect of tiring of human respiratory syncytial virus's inactivated vaccine; play the effect that strengthens immunity, in the preparation of vaccine, can significantly reduce human respiratory syncytial virus's inactivated vaccine purification stock solution antigen amount, have excellent protection and render a service.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the description just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Every piece of list of references mentioned above is listed this paper in as a reference all in full.
Claims (5)
1. a method for preparing human respiratory syncytial virus's inactivated vaccine comprises the following steps:
A. static or three-dimensional absorption Cultivation of Vero in culture medium obtains the Vero cell concentration and reaches 10
5-10
7The cellular matrix culture fluid of/ml;
B. the patient throat swab collection of human respiratory syncytial virus from falling ill 2 days separates through routine, with the infective dose (M.O.I.) of 0.01-0.2, virus inoculation in the cellular matrix culture fluid of step a, cultivated 6-10 days;
C. obtain virus-cell culture suspension with pancreas enzyme-EDTA digestion;
D. purified virus prepares vaccine.
2. method according to claim 1 is characterized in that described cellular matrix culture fluid is the MEM nutritional solution that contains 2%-10% new-born calf serum and 0.20-0.30% lactoalbumin hydrolysate, and this MEM nutritional solution pH is 6.8-7.4.
3. the human respiratory syncytial virus's inactivated vaccine for preparing according to the described method of claim 1.
4. a vaccine combination comprises human respiratory syncytial virus's inactivated vaccine as claimed in claim 3 and aluminum phosphate adjuvant.
5. vaccine combination according to claim 4, the content that it is characterized in that described human respiratory syncytial virus's inactivated vaccine is 100-200 μ g/ agent, the content of aluminum phosphate adjuvant is the 0.5mg/ agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110458973 CN103182079A (en) | 2011-12-30 | 2011-12-30 | Respiratory syncytial virus vaccine and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110458973 CN103182079A (en) | 2011-12-30 | 2011-12-30 | Respiratory syncytial virus vaccine and preparation method thereof |
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| CN103182079A true CN103182079A (en) | 2013-07-03 |
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| CN 201110458973 Pending CN103182079A (en) | 2011-12-30 | 2011-12-30 | Respiratory syncytial virus vaccine and preparation method thereof |
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2011
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| PB01 | Publication | ||
| C53 | Correction of patent for invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 201203 building 1690, 9 Zhang Heng Road, Shanghai, Pudong New Area Applicant after: Shanghai Wison-Bio Co., Ltd. Address before: 201203, Edison Road 333, Zhangjiang hi tech park, Shanghai, Pudong New Area Applicant before: Shanghai Wison-Bio Co., Ltd. |
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| C05 | Deemed withdrawal (patent law before 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130703 |