TR基因重组腺病毒载体的构建和鉴定

Construction and identification of recombinant adenovirus vector containing thioredoxin reductase gene

目的 :构建含人硫氧还蛋白还原酶 (TR)基因的重组腺病毒载体 ,探讨TR的抗氧化功能与神经退行性疾病的相关性。方法 :从重组质粒pGEM TR上用内切酶切下编码 5 0 0个氨基酸的全长TRcDNA片段 ,并连接穿梭质粒pShuttle,再双酶切pShuttle TR。将带有CMV启动子的目的片段 ,插入E1、E3缺失的Adeno X病毒DNA中 ,以Adeno TRDNA通过脂质体转染HEK2 93细胞 ,获得重组腺病毒Adeno TR进行PCR鉴定及病毒滴度测定。用重组腺病毒感染CV1细胞 ,通过免疫荧光染色与Westernblot分别检测重组腺病毒感染的细胞上和裂解液中TR蛋白的表达。结果 :重组腺病毒Adeno TR的病毒滴度为 4 .4× 10 11pfu/L。PCR、荧光显微镜证实 ,以及Westernbolt分析 ,在相对分子质量 (Mr)约 5 5 0 0 0处均出现特异性条带。结论 :成功地构建了重组腺病毒载体 ,并能介导外源基因TR表达 ,为进一步研究TR的功能及其与疾病的相关性奠定了基础。

AIM: To construct recombinant adenovirus vector containing human thioredoxin reductase (TR) gene and to explore the correlation between antioxdant activity of TR and the degenerative neuropathy. METHODS: Full length TR cDNA was obtained from recombinant plasmid pGEM TR via digestion with Apa I and Not I and was cloned into pShuttle vector and pShuttle TR was recut with I Ceu I and PI Sce I. Fragment containing TR gene and CMV promoter was inserted into E1 and E3 deficient adeno X virus DNA, and then the recombinant adenovirus vector was transfected into HEK 293 cells through lipofectamine and identified by PCR. The TR expression on and in cell lysate CV1 cells infected with recombinant adenovirus was by immuno fluorescentce assay and Western blot analysis. RESULTS: After replication of recombinant adenovirus Adeno TR the virus titer was about 4.4×10 11 pfu/L. The TR expression on CV1 cells was proved by fluorescent microscopy. Western blot analysis showed a band with relative molecular mass ( M r) being 55 000 . CONCLUSION: A recombinant adenovirus vector have been successfully constructed and TR is expressed on CV1 cells. This result lays the foundation for further study on function of TR and its correlation with degenerative neuropathy.