Recombinant baby hamster kidney (BHK) cell line capable of expressing encephalitis B virus PrM/M-E protein and application thereof

A Japanese encephalitis virus and cell line technology, applied in the fields of biomedical genetic engineering and immunology, can solve problems such as difficult suspension and high-density fermentation culture, and achieve the effect of easy mass production, easy culture, and increased antigen expression

Active Publication Date: 2012-02-01
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although foreign countries have used mammalian cell systems to express JEV structural protein antigens, the current report shows that the expression effect in RK13 cells is better (Journal of Virology, August 2003, p.8745-8755, Vol.77, No.16 ), but RK13 cells are rabbit kidney cells, which are adherent cells, which are not easy to carry out suspension high-density fermentation culture, and ATCC data show that the cells also have BVDV virus contamination, and BVDV contamination is strictly controlled in cell culture in biological products of

Method used

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  • Recombinant baby hamster kidney (BHK) cell line capable of expressing encephalitis B virus PrM/M-E protein and application thereof
  • Recombinant baby hamster kidney (BHK) cell line capable of expressing encephalitis B virus PrM/M-E protein and application thereof
  • Recombinant baby hamster kidney (BHK) cell line capable of expressing encephalitis B virus PrM/M-E protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction and detection of recombinant cell lines stably expressing Japanese encephalitis virus PrM / M-E protein

[0042] 1 Materials and methods

[0043] 1.1 Plasmids, strains and cells

[0044] The expression vector plasmid pCAG-neo, DH5α competent cells and BHK21 cells were preserved by the State Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute, the plasmid extraction kit and RNA extraction kit were produced by QIAGEN, and the gel recovery kit was purchased from Shanghai Huashun Biotechnology Co., Ltd., G418 was purchased from Gibco Company, trypsin was purchased from Hyclone Company, Reverse Transcriptase M-MLV, PrimeSTARTM HS DNA Polymerase, Sac I, Xho I, BamH I, T4 DNA Ligase were purchased from TaKaRa Company, anti- The monoclonal antibodies of JEV PrM protein and anti-JEV E protein were prepared by our research group, goat anti-mouse IgG antibody labeled with infrared fluorescent dye was purchased from KPL Company, ...

Embodiment 2

[0075] Embodiment 2 cell line expresses recombinant protein to the immunization experiment of BALB / c mouse

[0076] Vaccine preparation: When the recombinant cell line BHK-JEV-ME (No. 3) cells constructed and screened in Example 1 were normally passaged and grew to 90% full, the serum-free medium was changed to continue to cultivate for 4-6 days, and the cell culture was harvested at Freezing and thawing at -20°C for three times, and adding a final concentration of 0.02% thimerosal to the cell-containing culture stock solution (containing JEVE protein content greater than 3 μg / ml) after repeated freezing and thawing three times was used as the vaccine antigen solution. The oil adjuvant vaccine was prepared by mixing the vaccine antigen solution with the French SEPPIC ISA 50 V2 oil adjuvant at a volume ratio of 1:1, fully emulsified and stored at 4°C for later use. The preparation method of the aluminum gel adjuvant vaccine is as follows: the adjuvant is aluminum phosphate adju...

Embodiment 3

[0082] Embodiment 3 cell lines express recombinant protein to the immune test of pig

[0083] Vaccine preparation: when the recombinant cell line BHK-JEV-ME (No. 3) cells constructed and screened in Example 1 grow to 90% full after normal passage, change the serum-free medium to continue culturing for 4-6 days, and harvest the cell culture supernatant liquid or all cell cultures were frozen at -20°C, and the cell supernatant was concentrated by ultrafiltration with a molecular weight cut-off of 30kD 10 times in volume or the culture stock solution containing cells that was repeatedly frozen and thawed three times (the content of JEV E protein contained was greater than 3 μg / ml.) was added with a final concentration of 0.02% thimerosal as the vaccine antigen solution. The vaccine antigen solution and the white oil adjuvant are mixed and fully emulsified at a volume ratio of 1:1.5 to obtain whole cell culture oil shoots of the recombinant cell line. The attenuated vaccine of th...

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Abstract

The invention discloses a recombinant baby hamster kidney cell line (BHK 21) capable of stably expressing encephalitis B virus PrM/M-E protein, and more particularly, the recombinant cell line capable of expressing the PrM/M-E protein is BHK-JEV-ME and is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the culture preservation number of CGMCC No. 5263. The invention also discloses a method for preparing the recombinant cell line and the application of the recombinant cell line in the preparation of encephalitis B prevention vaccine.

Description

technical field [0001] The present invention relates to a recombinant mammalian expression cell line, specifically, the present invention relates to a recombinant BHK cell line expressing Japanese encephalitis virus PrM / M-E protein, more specifically, the recombinant BHK cell line is BHK-JEV-ME, the bacteria The species deposit number is: CGMCC No.5263. The invention also discloses a method for preparing the recombinant BHK cell line and the application of the recombinant cell line in preparing a vaccine for preventing Japanese encephalitis. It belongs to the field of biomedical genetic engineering and immunology. Background technique [0002] Japanese encephalitis, also known as Japanese encephalitis or Japanese encephalitis, is a viral zoonotic disease that affects the central nervous system and is caused by the Japanese encephalitis virus. [0003] Epidemic Encephalitis B (Epidemic Encephalitis B) is also known as Japanese B Encephalitis (Japanese BEncephalitis), referr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85A61K39/12A61P31/14G01N33/569
CPCY02A50/30
Inventor 华荣虹步志高
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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