猴D型逆转录病毒P27基因的表达、纯化及重组蛋白的鉴定

根据GenBank上已发表的猴D型逆转录病毒SRV-2株基因组序列设计合成了一对特异性引物,通过PCR扩增出P27基因。将扩增出的片段克隆到原核表达载体pBAD/Thio-TOPO上,通过序列分析证实该片段与猴D型逆转录病毒SRV-2株P27基因序列一致。将阳性重组质粒转化至大肠杆菌T0P010中,用阿拉伯糖诱导表达,表达的融合蛋白进行SDS-PAGE和WesternBlot分析,并用proBondTM柱在天然状态下进行纯化。结果表明,表达的融合蛋白分子量约为50KDa,其大小与预期相符。

Expression,Purification and Identification of Simian Type-D Retrovirus p27 Gene

The simian type-D retrovirus p27 gene was PCR amplified with a pair primers designed according to the published genomic sequence of SRV-2 strain.The PCR fragment was cloned into prokaryotic expression vector Pbad/Thio-TOPO vector and sequenced.The amplified p27 sequence was identical to that of SRV-2 strain.The positive recombinant explasma was tranfered into host cell TOP10 and expressed under Arabinose induction.The expressed recombinant product was identified by SDS- PAGE and Western Blot,and purified by proBond^? column under native conditions.The results demonstrated that recombinant protein had a molecular weight of 50 KDa and its size in line with expectation.