大鼠TCR Vβ8.2基因真核表达质粒的构建及表达

目的：构建含大鼠TCR Vβ8．2基因的重组真核表达质粒，并检测其在体内外的表达。方法：以RT-PCR法分离Lewis大鼠脾淋巴细胞中的TCR Vβ8．2基因片段，插入到真核表达载体pTARGET中，构建重组表达载体pTARGET-TCR Vβ8．2，并转化E．coli JM109，经蓝白斑筛选阳性菌落，进行菌落PCR和测序鉴定。将重组质粒以肌注法免疫BALB／c小鼠，采集注射部位股四头肌处的肌肉，用RT-PCR及免疫组化染色法检测TCRVB8．2基因在注射部位的转录和表达：通过脂质体介导，将再组质粒导入COS-7细胞中进行瞬时表达，用免疫细胞化学染色法检测靶基因在真核细胞内的表达。结果：测序鉴定证实，TCR Vβ8．2基因已被正确插入到pTARGET载体中，并检测到在肌肉组织和COS-7细胞内重组蛋白的表达。结论：成功地构建重组真核表达质粒pTARGET-TCR Vβ8．2，并在体内体外均可检测到其转录和表达，为进一步进行TCR VβDNA疫苗的研究奠定了基础。

Construction and expression of eukaryotic expression plasmid containing rat TCR Vβ 8.2 gene

AIM: To construct the recombinant eukaryotic expression vector pTARGET-TCR Vbeta8.2 and detect its their expression. METHODS: 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. RESULTS: 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. CONCLUSION: 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.