介导入硫氧还蛋白基因转移的重组腺病毒构建

Construction of recombinant adenovirns vector coding the human thioredoxin gene

目的 应用基因重组方法构建携带人硫氧还蛋白（hTRX）基因的复制缺陷型重组腺病毒。方法 用内切酶、RT-PCR、测序方法获得目的基因片段，然后将其cDNA克隆至表达载体pShuttle构建hTRX的重组真核细胞表达载体pShuttle—hTRX，在HeLa细胞中进行瞬时表达检测。从真核表达载体pShuttle—hTRX切下目的基因，并插入至腺病毒载体构建hTRX的重组腺病毒载体Adeno—hTRX，经PCR方法亦证明了成功构建腺病毒重组体。重组腺病毒在293细胞中扩增、纯化。结果 成功构建了携带人硫氧还蛋白基因的复制缺陷型重组腺病毒，并以多种方法证实了构建载体的正确性。结论 正确构建的人硫氧还蛋白重组腺病毒载体，可为基因治疗心血管系统疾病打下良好基础。

Objective To replicate defective recombinant adenoviruses coding the human thioredoxin （hTRX） gene by means of recombination. Methods The interest gene fragment was obtained by RT-PCR and sequencing. Its cDNA was cloned into the expression vector pshuttle to construct recombinant eukaryotic expression vector pshuttle-hTRX, whose transient expression in HeLa cell line was detected by immunochemistry and Western blot, recombinant adenoviruses adeno-hTRX was then constructed and amplified. Results The structure of the recombinant adenoviruses had been constructed and confirmed by light microscopy of 293 cell, PI-Sce I and I-Ceu I enzyme restriction analysis and PCR amplification. Conclusions The recombinant adenoviruses carrying thioredoxin can be used in the gene therapy for cardiovascular diseases.