摘要
目的构建CTxB-Eg95(TP)融合基因原核表达载体,并诱导表达CTxB-Eg95(TP)融合蛋白。方法采用PCR技术分别克隆CTxB基因和141bp的Eg95基因片段序列Eg95(TP)并鉴定。分别将两个基因片段定向克隆到大肠杆菌表达载体pET28a(+)中,构建pET-CTxB-Eg95(TP)重组质粒。将鉴定为阳性的重组质粒转化E.coliBL21(DE3)感受态细胞,经IPTG诱导后,进行SDS-PAGE电泳鉴定。结果测序结果表明已成功构建CTxB-Eg95(TP)融合基因的原核表达载体,SDS-PAGE电泳结果显示,转染pET-CTxB-Eg95(TP)的E.coliBL21(DE3)菌经IPTG诱导可表达大小约23.5kDa的特异蛋白条带,与预期结果一致。结论CTxB-Eg95(TP)融合基因获得了高水平的表达,为Eg95的表位研究和应用于口服疫苗奠定了基础。
To construct a recombinant prokaryotic expression vector for gene encoding fusion protein CTxB-Eg95（TP）, the sequences of cholera toxin B subunit（CTxB）gene and the gene for Eg95 of 141 bps were cloned respectively and identified by PCR. These two gene fragments were inserted into a prokaryotic vector pET28a（＋）to construct the recombinant plasmid pET-CTxB-Eg95 （TP）. Then, the recombinant plasmid identified to be positive was transfected into susceptible cells of E. coli BL21 （DE3）, and the expressed protein obtained after IPTG induction was identified by SDS-PAGE electrophoresis. As demonstrated by the results of sequencing,it was indicated that the prokaryotic expression vector for fusion gene pCTxB-Eg95 （TP） had been successfully constructed and the result in SDS-PAGE elctrophoresis revealed the presence of specific bands with molecular mass of 23.5 kDa could be observed in the expressed product from E. coli BL21 （DE3）cells trasfected with plasmid pET-CTxB-Eg95 （TP）after induction with IPTG. The molecular mass of the special protein was just the same as the expected result of the fusion protein CTxB-Eg95 （TP）. From these observations,it is concluded that the fusion protein CTxB-Eg95 （TP） has already been expressed,and the results obtained in the present study can provide a basis for the development of the oral vaccine for Eg95 in the future.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2008年第4期360-364,共5页
Chinese Journal of Zoonoses
基金
甘肃省农牧厅生物技术专项(GNSW-2004-12)资助
