猪前胸腺素的分子克隆与原核表达初步研究

Cloning and Prokaryotic Expression of Porcine Prothymosin α Gene

应用RT-PCR技术从猪肾脏中扩增到猪前胸腺素基因,测序结果表明,猪前胸腺素完整开放阅读框(ORF)共333 bp,编码109 aa,与已公布的2个猪前胸腺素基因核苷酸同源性均为99.4%。构建了重组质粒pET-32-mProTα,并转化大肠杆菌(E.coli)Rosetta 2(DE3)。SDS-PAGE电泳和免疫印迹分析显示,表达产物约占菌体总蛋白的40%,相对分子质量约为12.07 ku。MTT法试验证实,表达产物初步纯化、透析复性后,可明显增强淋巴细胞的增殖。

The porcine prothymosin α（ProTα） cDNA fragment was amplified from kidney of porcine by reverse transcription-polymerase chain reaction（RT-PCR）.Sequence analysis showed that the complete open reading frame（ORF） of ProTα gene includes 333 bp and encodes 109 aa.The sequence shared 99.4% homology with the two published ProTα genes.pET32-mProTα was constructed by gene rearrangement,then it was transformed into E.coli Rosetta 2（DE3）.SDS-PAGE electrophoresis analysis showed that the fusion protein of ProTα molecular mass was about 12 070 of molecular weight.MTT essay confirmed that it can enhance lymphocyte proliferation obviously.