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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、实验研究奠定了基础
Objective: To construct a retroviral vector carrying human Trx (thioredoxin) gene. Methods: The human Trx gene was cloned into the retroviral vector pLXSN by DNA recombination technology and identified by restriction endonucleases EcoRⅠ and BamHⅠ. The recombinant retroviral vector was transfected into the packaging cell line PA317 by calcium phosphate transfection and the titer of the virus in the supernatant of transfected cells was determined by G418 screening. Results: The recombinant retroviral vector was constructed and double-digested with EcoRⅠ and BamHⅠ. Two bands appeared, which were respectively 0.3kb and 5.9kb. The virus titer in the transfected cells was 5.5 × 10 9 cfu / L. It suggested that the construction of retroviral vector carrying human Trx gene was successful. CONCLUSIONS: The retroviral vector carrying human Trx gene was successfully constructed as a therapy for Trx gene, which laid the foundation for experimental study