猪囊尾蚴磷蛋白基因P2的克隆及在大肠杆菌中的表达

Cloning and identification of phosphoprotein P_2 of Cysticercus cellulosae and expression in E.coli

本研究从猪囊尾蚴虫体中提取总RNA ,用磷蛋白特异性引物 ,通过RT -PCR扩增出 - 36 6bp的片段 ,扩增产物连接到PGEM -Teasy载体中 ,经酶切鉴定、PCR分析以及确证性测序后证明 ,所克隆的 36 6bp的片段含P2蛋白基因完整的开放阅读框。将重组质粒PGEM -P2和表达载体pProEX -HTb分别酶切后 ,亚克隆至原核表达载体 pProEX -HTb中 ,筛选阳性克隆 ,用IPTG诱导表达 ,收集不同时间的菌液进行SDS -PAGE电泳、Westernblot分析 ,结果表明 ,磷蛋白基因 p2在大肠杆菌中成功表达 ,其表达产物为分子量 15kDa的融合蛋白 ,并能被猪囊虫阳性血清所识别。

Total RNA was extracted from Cysticercus cellulosae,then mRNA was isolated by using oligo(dT)as a probe.A 366bp specific fragment was amplified by RT-PCR and ligated into PGEM-Teasy vector.It was identified by restriction endonuclease analysis and PCR and sequencing that this fragment contained the complete open reading frame(ORF)of the P 2 gene.In comparison with GenBank data,the homologies of the nucleotide sequence and amino acid sequence Wene 99.45% and 98.36%,respectively.After restriction digest the PGEM-P2 and pProEX-HTb,respectively,subcloned the interested gene P2 into expression vector pProEX-HTb.Positive clones were selected by restriction endonuclease analysis and PCR identification.The expression was induced by IPTG,then collected the culture in different times and tested them by SDS-PAGE.It showed that the P2 gene was expressed successfully in E.coli.The 15kDa fusion protein can be recognized by the positive serum of Cysticercus cellulosae.