人硫氧还蛋白基因克隆及其重组逆转录病毒载体的构建和鉴定

Cloning of Human Thioredoxin Gene and Construction and Identification of Its Recombinant Retrovirus Vector

目的克隆人硫氧还蛋白(hTRX)基因,并构建含有该目的基因的重组逆转录病毒载体。方法利用RT-PCR法,以PHA活化的人外周血单个核细胞总RNA为模板,扩增hTRX编码蛋白的cDNA基因,并亚克隆至逆转录病毒载体pSIV-1中进行PCR、双酶切和测序鉴定。结果将所得的序列与GenBank(BC003377)报道的序列比较,其酶活性中心(Trp-Cys-Gly-Pro-Cys)与已知序列一致,第132、136、170、264位碱基与已知序列不同,其中第136、170位相应密码子编码的氨基酸发生了变化(Phe→Leu,Ile→Thr),成功获得了pSIV-1-hTRX重组逆转录病毒载体。结论hTRX基因的克隆及其重组逆转录病毒载体pSIV-1-hTRX的构建,为进一步探讨hTRX的生物学活性和应用奠定了基础。

Objective To clone human thioredoxin（hTRX） gene and construct recombinant retrovirus vector carrying the target gene. Methods The cDNA gene of human thioredoxin encoding protein was amplified by RT-PCR from PHA-stimulated human peripheral blood mononuclear cells（PBMCS）. Then the amplification product was subcloned into retrovirus vector pSIV-1 identified by PCR, double endonuclease digestion and DNA sequencing. Results By comparing with the hTRX cDNA sequence reported in GenBank（BC003377）, we found that the highly conserved enzymatic active site （Trp-Cys-Gly-Pro-Cys） of hTRX gene cloned was identical with that had been reported. However, there were four differences at bp132, 136, 170, 264 and the corresponding amino acids of bp136, 170 were changed（Phe→Leu, Ile→Thr） . The pSIV-1-hTRX recombinant retrovirus vector has been obtained successfully. Conclusion Human thioredoxin gene cloning and construction of its recombinant retrovirus vector pSIV-1-hTRX paves the way for future study on biological activities and application of hTRX.