[Establishment of BHK-21 cell lines stably expressing FMDV 3Dpol gene by retroviral-mediated gene transfer technique].
OBJECTIVE
The aim of the study is to establish in vitro cell line with stable and effective 3Dpol gene expression, so as to study the biological function of foot-and-mouth disease virus (FMDV ) 3Dpol and foot-and-mouth disease (FMD) gene engineering vaccine.


METHODS
FMDV 3D gene was amplified from pMD18-T-3D and inserted into pGEM-Teasy vector. By NotI/BamHI digestion, 3D gene with Not I/BamH I site was inserted into Not I/BamH I cloning site of the pBPSTR1 retroviral vector in order to obtain recombinant retroviral vector pBPSTR1-3D. The artifical retroviral viruses were obtained by both pBPSTR1-3D and pVSV-G envelope vector into the Gp2-293 package cells using Lipofectamine 2000. The BHK-21 cells were infected by artificial retroviral particles with 8 microg/mL Polybrene. The positive cell clones which genomes contained the 3Dpol gene were continually selected using puromycine and was regulated by tetracycline for 12 days .The single clone highly effective expressing 3D fusion protein was obtained by seeding the cells into 96-well plates with one cell per well.


RESULTS
By using retroviral gene transfer technology, the 3Dpol gene was integrated into the chromosome of BHK-21 cells, then under selection pressure, the cell lines stably expressing 3D were established. Finally, a cell line stably expressing the 3D fusion protein was established. The fusion protein was confirmed to be expressed correctly by Western-blot. The transfected genes in the cell line were consistently expressed during 35 passages of the host cells.


CONCLUSIONS
Transgene cell strain stably carrying exogenous gene in subsequent passaging was successfully constructed. It provide a good experimental tool for the biological function of FMDV 3Dpol and FMD gene engineering vaccine research.
Bi
Yanli
Y
State Key Lab of Vet Etiological Biol, Key Laboratory of Animal Virology of Ministry of Agriculture, National FMD Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China. biyanli81@163.com
Shen
Xiaoyan
X
Cong
Guozheng
G
Liu
Xiangtao
X
Chang
Huiyun
H
Cai
Xuepeng
X
D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D019084:Fluorescent Antibody Technique, Indirect; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology
D011691:Puromycin; D012324:RNA-Dependent RNA Polymerase; D013752:Tetracycline