Bi Yanli, Shen Xiaoyan, Cong Guozheng, Liu Xiangtao, Chang Huiyun, Cai Xuepeng
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.
Wei Sheng Wu Xue Bao. 2008 Aug;48(8):1115-20.
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.
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.
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.
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.
本研究旨在建立具有稳定且有效3Dpol基因表达的体外细胞系，以研究口蹄疫病毒（FMDV）3Dpol的生物学功能及口蹄疫（FMD）基因工程疫苗。
从pMD18-T-3D中扩增FMDV 3D基因，并将其插入pGEM-Teasy载体。通过NotI/BamHI酶切，将带有Not I/BamH I位点的3D基因插入pBPSTR1逆转录病毒载体的Not I/BamH I克隆位点，以获得重组逆转录病毒载体pBPSTR1-3D。使用Lipofectamine 2000将pBPSTR1-3D和pVSV-G包膜载体导入Gp2-293包装细胞，获得人工逆转录病毒。用8μg/mL聚凝胺处理人工逆转录病毒颗粒感染BHK-21细胞。使用嘌呤霉素持续筛选基因组中含有3Dpol基因的阳性细胞克隆，并通过四环素调控12天。将细胞接种到96孔板中，每孔一个细胞，获得高效表达3D融合蛋白的单克隆。
利用逆转录病毒基因转移技术，将3Dpol基因整合到BHK-21细胞染色体中，然后在选择压力下，建立了稳定表达3D的细胞系。最终，建立了稳定表达3D融合蛋白的细胞系。通过Western-blot证实融合蛋白表达正确。在宿主细胞传代35次过程中，细胞系中的转染基因持续表达。
成功构建了在后续传代中稳定携带外源基因的转基因细胞株。它为FMDV 3Dpol的生物学功能及FMD基因工程疫苗研究提供了良好的实验工具。
