【摘要】 目的:构建含大鼠TCRVβ8.2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RTPCR法分离Lewis大鼠脾淋巴细胞中的TCRVβ8.2基因片段,插入到真核表达载体pTARGET中,构建重组表达载体pTARGETTCRVβ8.2,并转化E.coliJM109,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RTPCR及免疫组化染色法检测TCRVβ8.2基因在注射部位的转录和表达。通过脂质体介导,将重组质粒导入COS7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,TCRVβ8.2基因已被正确插入到pTARGET载体中,并检测到在肌肉组织和COS7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pTARGETTCRVβ8.2,并在体内体外均可检测到其转录和表达,为进一步进行TCRVβDNA疫苗的研究奠定了基础。更多还原

【Abstract】 AIM: To construct the recombinant eukaryotic expression vector pTARGET-TCR Vβ8.2 and detect its their expression. METHODS: Gene encoding TCR Vβ8.2 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 Vβ8.2 gene was detected by RT-PCR and immunohistochemistry. Immunocytochemical staining was used to detect the expression of pTARGET-TCR Vβ8.2 gene after the recombinant plasmid was transfected into COS-7 cells by lipofectamine. RERULTS: DNA sequencing demonstrated that TCR Vβ8.2 gene was successfully inserted into pTARGET. RT-PCR demonstrated that TCR Vβ8.2 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 Vβ8.2 was successfully constructed and expressed in vivo and vitro, which would lay foundation for further studies on the protective effects of TCR Vβ DNA vaccine on CIA.更多还原