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Adaptive culture method of rotavirus P(2) G3 strain and P(8) G1 strain on KMB17 cells and immunogenicity

A rotavirus and culture method technology, applied in biochemical equipment and methods, inactivation/attenuation, instruments, etc., can solve the problems of unestablished immunogenicity and low infectivity titer of rotavirus, and achieve good results. The effect of immunogenicity

Active Publication Date: 2011-09-28
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

Human diploid cell KMB17 has been successfully used in the production of hepatitis A for many years, and it is a safe and reliable human-derived cell. Based on this idea, in the previous study, we selected rotavirus P[2]G3 strain in The adaptive culture of the virus and the biological properties and immunogenicity of the adapted strain were carried out on the human embryonic lung diploid cell KMB17, but it was limited to the rotavirus P[2]G3 strain, and no P[8] was established and studied. ] G1 strain immunogenicity, and the obtained rotavirus infectivity titer is low (10 5.5 PFU / mL)

Method used

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  • Adaptive culture method of rotavirus P(2) G3 strain and P(8) G1 strain on KMB17 cells and immunogenicity
  • Adaptive culture method of rotavirus P(2) G3 strain and P(8) G1 strain on KMB17 cells and immunogenicity
  • Adaptive culture method of rotavirus P(2) G3 strain and P(8) G1 strain on KMB17 cells and immunogenicity

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Embodiment Construction

[0030] Materials used in the following examples include:

[0031] Monkey rotavirus P[2]G3 strain (18th passage, multiplied on Vero cells),

[0032] Human rotavirus P[8]G1 strain (10th passage, propagated on Vero cells).

[0033] (1) Adaptive culture of rotavirus on KMB17 cells

[0034] Use MEM medium containing 10% newborn bovine serum to culture KMB17 cells in a spinner bottle. After the cells grow into a dense monolayer, inoculate P[2]G3 and P[8]G1 strains of virus at a MOI of 0.05, and observe the pathological changes. When the CPE reaches +++-++++, the virus liquid is harvested. In this way, the virus was serially passaged for 10 passages.

[0035] 20ug / ml of acetylated trypsin and 600ug / ml of CaCl can be used before virus inoculation 2 Activate in a water bath at 37°C for 60 minutes, inoculate the virus and adsorb at 37°C for 90 minutes, discard the unadsorbed virus liquid, and add the MEM maintenance solution with a final concentration of 0.5ug / ml acetylated trypsin ...

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Abstract

The present invention relates to an adaptive culture method of a rotavirus P(2)G3 strain and a P(8)G1 strain on KMB17 cells and immunogenicity, belonging to the field of virus culture methods. 10 generations of adaptive culture of the two strains of rotavirus are continuously carried out on the KMB cells, thereby gradually increasing the cytopathy along with the virus generation, gradually increasing the infective titers and stabilizing the infective titers at the higher level. The virus titers respectively achieve 7.75CCID50 / ml and 7.33CCID50 / ml. The virus is applicable to the KMB17 cells and has a stable genome, and the neutralizing antibody titers in serum of mice immunized with the copied virus are 1:16384 and 1:8192 respectively. The rotavirus P(2)G3 strain and the P(8)G1 strain formed by the adaptive culture can be stabilized copied in the KMB17 cells, and the produced virus can keep the basic biological property of parent virus strains and have better immunogenicity.

Description

technical field [0001] The invention belongs to a virus culture method, in particular to the culture of rotavirus on human cells. Background technique [0002] The safety and effectiveness of any vaccine are the most important basic elements, and it is very important to consider safety to the maximum extent when developing a vaccine. There are many factors affecting the safety of vaccines, among which the cell matrix used to prepare vaccines is one of the important factors. As the raw material for preparing vaccines, the quality of cell matrix directly affects the quality and yield of vaccines, especially the safety of vaccines. Rotavirus (RV) vaccines have played an important role in the prevention of RV infection, but up to now, RV vaccines at home and abroad are mainly prepared in animal-derived cells. Because animal-derived primary cells may carry potential viruses and other transmissible factors, there is a great safety hazard for human vaccines; secondly, the residua...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70C12N7/08G01N33/569
Inventor 孙茂盛李鸿钧易山马应霞张光明吴晋元
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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