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CA-193 virus strain and application thereof to preparation of inactivated vaccine

A Coxsackie virus and virus strain technology, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of slow replication, increased difficulty in the prevention and control of severe HFMD, and insufficient research on CA16 to achieve immunogenicity. Good sex and immune protection, good immune protection effect, perfect preparation method

Active Publication Date: 2017-02-01
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, it was believed that the cases of CA16 virus infection only had mild symptoms such as cough and runny nose. The research on CA16 was not enough. Now it is found that CA16 infection can also cause severe symptoms such as myocarditis, brainstem encephalitis, aseptic meningitis and pneumonia. Death cases have also been reported in the United States, France, Japan, mainland China, and Taiwan, China. At the same time, analysis of clinical patients with HFMD found that CA16 virus also co-infects and recombines with enterovirus 71 (Enterovirus71, EV71). The difficulty of prevention and control of severe HFMD
[0005] Vaccines are one of the most important means to prevent and control hand, foot and mouth disease. At present, the stromal cells used in the production of CA16 vaccine are mainly Vero cells. Although Vero cells have the advantages of easy standardization and large-scale production, because they are passaged cells, there may be Tumorogenicity; human embryonic lung diploid fibroblast MRC-5 is derived from the human body, has no foreign protein allergens, and has no tumorigenicity, so it is more favored by vaccine researchers, and human diploid MRC-5 has not been used so far. A report on preparation of CA16 vaccine by 5 cells
At the same time, the replication speed of CA16 virus in human diploid cells is slow, and the virus titer is not high, which also restricts the application of human diploid cells in the preparation of CA16 vaccines. At present, there is still no specific vaccine against CA16 virus on the market worldwide.

Method used

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  • CA-193 virus strain and application thereof to preparation of inactivated vaccine
  • CA-193 virus strain and application thereof to preparation of inactivated vaccine
  • CA-193 virus strain and application thereof to preparation of inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CA-193

[0024] Isolation and identification of embodiment 1CA-193 virus strain

[0025] In 2010, throat swabs and herpes fluid samples were obtained from a 12-month-old male patient with hand, foot and mouth disease in the Sixth People's Hospital of Hangzhou. The samples were filtered through a 0.22 μm filter membrane, and the filtrate was inoculated into a single layer of green monkey kidney Passage cells (Vero, purchased from the American Type Culture Collection (ATCC CCL-81)) 25cm 2 In the square bottle, collect the cell suspension after more than 80% of the Vero cells have lesions, freeze and thaw three times, centrifuge at 1000rpm for 10 minutes, take the supernatant as the virus liquid, and take 1ml of the virus liquid to inoculate the Vero cells (25cm 2 square bottle), and after repeated passage three times, the CA-193 virus strain was obtained by the plaque method, and was determined to be the original strain of the first generation. figure 1 Middle A is the situation of Vero...

Embodiment 2

[0052] The preparation of embodiment 2 vaccine

[0053] (1) Recovery and culture of diploid cells

[0054] The cryopreserved human embryonic lung diploid fibroblast MRC-5 (purchased from the American Type Culture Collection (ATCCCCL-171)) was thawed in a 37°C water bath, and inoculated into MEM medium containing 20% ​​fetal bovine serum ( Invitrogen), transfer to cell culture incubator, 37 ℃, 5% CO 2 After culturing until the cells cover the surface of the culture flask, digest with 0.25% trypsin for 3-5 minutes, gently pipette the cells to a single-cell suspension, subculture at a ratio of 1:2, pass to the sixth generation, and obtain the sixth generation Generation of MRC-5 cells.

[0055] (2) Virus cultivation and amplification

[0056] Preparation of CA-193 virus seed: wait for step (1) the sixth generation of MRC-5 cells to cover 75cm 2 Culture flask, discard the culture solution, inoculate the CA-193 virus strain in Example 1 to MRC-5 cells, the inoculum amount is MO...

Embodiment 3

[0062] The mensuration of embodiment 3 vaccine potency

[0063] The titer of the pure vaccine prepared in Example 2 was determined by conventional ELISA method. 100 μl of vaccine diluted with PBS was added dropwise to a 96-well plate, and coated overnight at 4°C. The specific anti-CA16 polyclonal antibody was diluted with PBS at a concentration of 1:2000-1:10000 and added to a 96-well plate, incubated at 37°C for 60 minutes, and then incubated with HRP-linked secondary antibody (1:5000-1:10000, diluted in PBS) for 1 hour , Detect the absorbance at a wavelength of 450nm, if the absorbance is greater than 0.1, it is considered positive, and the vaccine titer with the maximum positive dilution is set as 400U.

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Abstract

The invention discloses a CA-193 virus strain (Coxackievirus A16) and application thereof to preparation of an inactivated vaccine. For the application, MRC-5 cells are used for proliferating the CA-193 virus strain; virus proliferation liquid is subjected to centrifugation, concentration and column chromatography filtration and purification after inactivation by formaldehyde or beta-propiolactone; and a CA16 inactivated vaccine is obtained. The invention provides the vaccine virus strain capable of being used for preparing a human CA16 inactivated vaccine; the subtype of the vaccine virus strain belongs to the main epidemiological subtype B1 in Chinese Mainland; the vaccine virus strain has good growth characteristics on the MCR-5 cells; and the virus titer (7.5 to 8.251gTCID50 / ml) with high stability can be obtained. A preparation method of the vaccine is complete; the impurity content is low; the immunogenicity and the immunizing protection performance are good; meanwhile, cell culture substrates are MRC-5 cells; the safety is high; and the international and CFDA specifications and requirements on the vaccine research and development can be well met.

Description

[0001] (1) Technical field [0002] The invention relates to a novel human Coxsackievirus A group 16 virus strain and a preparation method of an inactivated vaccine thereof, belonging to the field of biotechnology. [0003] (2) Background technology [0004] Human Coxsackievirus A16 (Coxsackievirus A16, CA16) belongs to the Picornaviridae Enterovirus genus and is one of the main pathogens that cause hand, foot and mouth disease (HFMD). It is transmitted by oral route and close contact, and it is easy to break out and become popular among children under 5 years old. The structure of CA16 is a 20-sided stereosymmetric spherical particle with a diameter of 23-30nm and no envelope and protrusions. Its nucleic acid is a single-stranded positive-strand RNA with a total length of about 7410bp, encoding four structural proteins and seven non-structural proteins. Through the nucleotide sequence analysis of the VP1 structural protein, CA16 can be divided into three subtypes, A, B, and C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/125A61P31/14
CPCA61K39/12A61K2039/5252C12N7/00C12N2770/32321C12N2770/32334
Inventor 朱函坪孙一晟陈直平姚苹苹徐芳杨章女朱智勇卢杭景周小龙
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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