Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells.
- Author:
Guozheng CONG
1
;
Jianhua ZHOU
;
Shandian GAO
;
Junzheng DU
;
Junjun SHAO
;
Tong LIN
;
Huiyun CHANG
;
Qingge XIE
Author Information
1. State Key Laboratory of Veterinary Etiological Bilogy, Key Laboratory of Animal Virology of Ministry of Agriculture, National Foot-and-mouth Disease MD Reference Laboratory, Chinese Academy of Agriculture Science, Lanzhou 730046, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cattle;
Cell Line;
Cloning, Molecular;
Endopeptidases;
biosynthesis;
genetics;
Foot-and-Mouth Disease Virus;
genetics;
Genetic Vectors;
genetics;
Puromycin;
pharmacology;
Recombinant Proteins;
biosynthesis;
genetics;
Retroviridae;
genetics;
metabolism;
Tetracycline;
pharmacology;
Transfection
- From:
Chinese Journal of Biotechnology
2008;24(5):740-745
- CountryChina
- Language:Chinese
-
Abstract:
In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.
