摘要
目的构建旋毛虫Ts43基因原核表达载体,并且在大肠埃希菌中表达。方法将构建的pMD-Ts43克隆质粒酶切回收目的片段定向克隆到pET-28a(+)载体上,构建原核表达载体pET-28a-Ts43,酶切鉴定正确后,导入菌株Rosetta中,用IPTG诱导表达,并经SDS-PAGE及Westernblot鉴定。结果成功构建了含目的基因的原核表达质粒pET-28a-Ts43,IPTG诱导表达分子质量单位约为43ku的融合蛋白,与预测值相符。以0.9mmol/LIPTG诱导4.5h后表达量最高,薄层扫描表达的融合蛋白占菌体蛋白总量的20%。Westernblot显示表达产物可被抗旋毛虫的多克隆血清识别。结论原核细胞融合表达旋毛虫Ts43基因获得成功,为旋毛虫重组苗的研究奠定了基础。
Objective To construct prokaryotic expression vector of the Ts43 gene and express it in Escherichia coli. Methods The Ts43 gene was subcloned into pET-28a（＋） and then expressed in E. coli DE3 inducing by IPTG. The fusion product was identified by SDS-PAGE and Western blot. Results The Ts43 gene was expressed in E. coli by inducing with IPTG. The peak of the target protein was presented at 4.5 h after adding 0. 9 mmol/L IPTG. And a special reaction band to anti-Ts43 sera has been observed by Western blot. Conclusion The fusion protein of Ts43 gene expressed successfully in prokaryotic cells and it has a potential candidate for the recombinant subunit vaccines.
出处
《中国病原生物学杂志》
CSCD
2007年第1期41-43,共3页
Journal of Pathogen Biology
基金
吉林省科技厅基础研究项目(No.20030550-5)。
关键词
旋毛虫
Ts43基因
克隆
原核表达
Trichinella spiralis
Ts43 gene
clone
prokaryotic expression
