Foot and mouth disease virus-like particle vaccine and preparation method thereof

A foot-and-mouth disease virus and particle technology, applied in the field of biomedicine, can solve the problems of high production cost, low yield of virus-like particles, incomplete structure of virus-like particles, etc., and achieves the effects of easy preparation and easy purification

Inactive Publication Date: 2015-12-09
吕宏亮 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most FMD virus-like particles are expressed and assembled in host cells with P12A3C. Due to the toxicity of 3C to host cells, the yield of virus-like particles is low and the structure of virus-like particles is incomplete, resulting in low yield of virus-like particles and high production costs.

Method used

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  • Foot and mouth disease virus-like particle vaccine and preparation method thereof
  • Foot and mouth disease virus-like particle vaccine and preparation method thereof
  • Foot and mouth disease virus-like particle vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 foot-and-mouth disease virus infectious cDNA clone

[0063] 1.1 Cell lines and viruses

[0064] The BHK-21 cell line is preserved by Inner Mongolia Biwei Antai Biotechnology Co., Ltd., and the culture medium is MEM+10% bovine serum. Primary bovine thymocytes (Primarybovinethyroid, BTY) come from the thymus of a 1-year-old calf, and are maintained and cultured with MEM+10% bovine serum medium. Cell culture domesticated foot-and-mouth disease virus OSFS2014 (FMDV / O / Mya98-XJ-2010 vaccine strain), which was obtained from the Quality Control Department of Inner Mongolia Biwei Antai Biotechnology Co., Ltd., was used to prepare cDNA clones.

[0065] 1.2 OSFS2015 full-length cDNA clone

[0066] Carry out 4 rounds of site-directed mutagenesis with QuickChangeSite-DirectedMutagenesis Kit (purchased from American Stratagene box), and form NdeI, NheI, NotI, SpeI restriction at the multiple cloning site of plasmid pGEMR-7Zf (-) (purchased from American Promega Company...

Embodiment 2

[0103] Embodiment 2 foot-and-mouth disease virus P12AcDNA clone

[0104] 2.1 Materials and methods

[0105] 2.1.1 Materials

[0106] Serum-free F17 medium was purchased from Invitrogen, USA; BHK-21 cell suspension culture conditions: 37°C, 5% CO 2 , rotating speed 120rpm; BHK-21 cells (purchased from ATCC, USA) were incubated with DMEM medium containing 1% calf serum at 37°C, 5% CO 2 Incubator culture.

[0107] FMDV / O / Mya98-XJ-2010 vaccine strain (current vaccine production strain, derived from Lanzhou Veterinary Research Institute of China), was cultured in serum-free suspension, inactivated with diethyleneimine, purified by sucrose density gradient centrifugation, and prepared as a control vaccine and Positive control for protein assays. The non-inactivated virus is passaged in BHK-21 cells and used for virus infection and challenge in the present invention, and all operations related to infectious foot-and-mouth disease virus are carried out in facilities with a biosafe...

Embodiment 3

[0125] Example 3 Mammalian cell BHK-21 transfection prepares stably expressing foot-and-mouth disease virus-like particles

[0126] 3.1 Transfection and screening, library construction

[0127] BHK-21 cells (working cell bank cells, BHK-LZ-sf-2012) were transfected with 25KD linear polyethylene (PEI): the cells were diluted with fresh cell culture medium to 0.5×10 6 cells / ml, the density transfection cell survival rate ratio is 1.5-2×10 6 The cells / ml is high, and the transfection survival rate is as high as 95%. Dilute the pTT5-mP12A plasmid and PEI concentration of Example 2 to 1 μg / ml and 2.5 μg / ml respectively with serum-free F17 medium, shake for 4 seconds at room temperature, incubate for 3 minutes, and add to a 96-well plate for culture. A single clone was obtained by screening with G418 and Zeocin (lepomycin).

[0128] The primers in Example 2 were used to amplify and confirm the cell-integrated P12A sequence, changes in amino acid positions, and gene stability by P...

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Abstract

The invention discloses a foot and mouth disease virus-like particle vaccine and a preparation method thereof. By expressing P12A (three amino acid site mutation, VP1K210E, VP1E83K and VP1C134S) and serum-free suspension culture, a virus-like particle (VLP) standard similar to natural 75S FMDV is built, and VLP yield is increased. The invention further provides serum-free suspension cultured and high pH tamed insect cells, the insect cells can stably and efficiently produce FMDV VLP, the size and structure of the VLP produced by the insect cells is similar to those of the VLP produced by standard BHK-21, and the yield of the VLP produced by the insect cells is 11 times of that of the VLP produced by female parent cells Sf21. The vaccine prepared by the VLP produced by the insect cells can immunize animals to generate IgG antibody response. The IgG antibody titer, foot and mouth disease virus neutralizing antibody titer and antibody titer of immunization of the VLP vaccine produced by the insect cells are not evidently different from those of immunization of the VLP vaccine prepared by the BHK-21, so that the high-pH-adaptive insect cells are applicable to FMDV VLP vaccine production.

Description

technical field [0001] The invention relates to a foot-and-mouth disease virus-like particle vaccine and a preparation method thereof, belonging to the technical field of biomedicine. Background technique [0002] The pathogen of foot-and-mouth disease is foot-and-mouth disease virus (Foot-and-mouth disease virus, FMDV), which belongs to Picornaviridae. C, SAT1, SAT2, SAT3 and ASIA1) and multiple subtypes. The main popular ones in our country are type O, type C and subtype 1. The immunization of each serotype has no cross-protection, and the preparation of the vaccine often changes with the epidemic. [0003] The currently used foot-and-mouth disease vaccine is prepared by suspension culture of BHK-21 cells, inactivated by diethyleneimine, and emulsified. Risk of escape, difficulty in differentiating infection and immunization of animals, problems with adaptability of many vaccine master seed lots to cell culture processes. To overcome the above problems, it is inevitabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/135C12N15/85A61K9/16A61P31/14
Inventor 张澍吕宏亮
Owner 吕宏亮
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