介导人硫氧还蛋白基因转移的重组腺病毒构建

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。

Construction of recombinant adenovirus vector coding the human thioredoxin gene

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 adenovirnses 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.