hTrx基因重组逆转录病毒载体的构建及鉴定

Construction and identification of recombinant retrovirus vector containg hTrx gene

目的 :构建携带人Trx(硫氧还蛋白 )基因的逆转录病毒载体。方法 :用DNA重组技术 ,将人Trx基因定向克隆入逆转录病毒载体pLXSN ,用限制性内切酶EcoRⅠ和BamHⅠ进行酶切鉴定。以磷酸钙转染法 ,将重组的逆转录病毒载体转染入包装细胞系PA317,并用G4 18筛选的方法测定转染细胞上清中病毒的滴度。结果 :构建了重组逆转录病毒载体 ,经EcoRⅠ和BamHⅠ双酶切 ,电泳出现两个条带 ,分别为 0 .3kb和 5 .9kb。转染细胞上清中病毒滴度为 5 .5× 10 9cfu/L。提示构建携带人Trx基因的逆转录载体成功。结论 :携带人Trx基因的逆转录病毒载体成功构建为Trx基因的治疗。

AIM: To construct a recombinant retrovirus vector carrying hTrx gene. METHODS: hTrx gene was cloned directionally into a retroviral vector pLXSN by DNA recombinant technique and the recombinant vector was identified Eco RⅠ and Bam HⅠ digestion analysis. The recombinant was transfected into packaging cells PA317 via CaPO 4 based transfection method, and viral titer in culture supernatant of transfected cellswas detected. RESULTS: The recombinant pLTrxSN had been constracted. Eco RⅠ and Bam HⅠ digestion analysis displayed two positive bands of 0.3 kb and 5.9 kb respectively. The viral titer in supernatant of transfected cells was 5.5×10 9 cfu/L. CONCLUSION: pLhTrxSN has been constructed successfully, which will be useful for Trx gene therapy and experiment study.