Li Yun, Ma Li-ping, Zhao Wen-ming, Liu Zhen-long
Department of Immunology, Capital University of Medical Sciences, Beijing 100069, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Sep;22(5):560-3.
To construct the recombinant eukaryotic expression vector pTARGET-TCR Vbeta8.2 and detect its their expression.
Gene encoding TCR Vbeta8.2 was amplified by RT-PCR from spleen cells of Lewis rats, and then cloned into eukaryotic expression vector pTARGET. Recombinant clones were identified by blue/white screening on indicator plates after transformed into E.coli strain JM109, and then by bacteria colonies PCR and DNA sequencing. Recombinant plasmid was injected into BALB/c mice intramuscularly. Then the injected skeletal muscle was isolated, and expression of TCR Vbeta8.2 gene was detected by RT-PCR and immunohistochemistry. Immunocytochemical staining was used to detect the expression of pTARGET-TCR Vbeta8.2 gene after the recombinant plasmid was transfected into COS-7 cells by lipofectamine.
DNA sequencing demonstrated that TCR Vbeta8.2 gene was successfully inserted into pTARGET. RT-PCR demonstrated that TCR Vbeta;8.2 gene was successfully expressed in the injected muscle. Immunohistochemistry staining showed the expression of recombinant plasmid in the transfected COS-7 cells.
The eukaryotic expression vector pTARGET-TCR Vbeta8.2 was successfully constructed and expressed in vivo and vitro, which would lay foundation for further studies on the protective effects of TCR Vbeta DNA vaccine on CIA.
构建重组真核表达载体pTARGET-TCR Vβ8.2并检测其表达。
采用RT-PCR从Lewis大鼠脾细胞中扩增编码TCR Vβ8.2的基因，然后克隆至真核表达载体pTARGET中。转化大肠杆菌JM109菌株后，通过在指示平板上进行蓝/白筛选，随后进行菌落PCR和DNA测序来鉴定重组克隆。将重组质粒肌肉注射到BALB/c小鼠体内。然后分离注射部位的骨骼肌，通过RT-PCR和免疫组化检测TCR Vβ8.2基因的表达。利用脂质体将重组质粒转染至COS-7细胞后，采用免疫细胞化学染色检测pTARGET-TCR Vβ8.2基因的表达。
DNA测序表明TCR Vβ8.2基因成功插入pTARGET。RT-PCR表明TCR Vβ8.2基因在注射的肌肉中成功表达。免疫组化染色显示重组质粒在转染的COS-7细胞中有表达。
成功构建了真核表达载体pTARGET-TCR Vβ8.2，并在体内和体外实现了表达，这为进一步研究TCR Vβ DNA疫苗对胶原诱导性关节炎（CIA）的保护作用奠定了基础。
