弓形虫RH株表面抗原SAG3去信号肽基因的蛋白原核表达及鉴定

摘要

根据GenBank中弓形虫表面抗原SAG3基因序列,以弓形虫总RNA反转录的cDNA为模板,扩增出SAG3去除信号肽基因并进行原核表达.将重组pET-28a(+ )-SAG3阳性表达质粒转入大肠杆菌BL(DE3)中,用IPTG进行诱导表达.通过SDS- PAGE和Western blotting对重组蛋白进行分析和鉴定.结果表明:成功扩增了不合信号肽的SAG3基因,构建的原核表达质粒在大肠杆菌中得到了高效表达,能够与鼠抗弓形虫阳性血清发生特异性反应,去信号肽的SAG蛋白具有反应原性.%The research was conducted to clone, express and identify the SAG3 gene containing no signal peptide of Toxoplasma gondii. The SAG3 gene containing no signal peptide was amplified by RT - PCR. The prokaryotic expression plasmid pET-28a( + )-SAG3 was constructed by sub-cloning the SAG fragment into the prokaryotic expression vector pET-28a( + ). The expression of pET-28a-SAG3 was induced by IPTG in E. Coli BL21(DE3)system; then the fusion protein was identified by SDS- PAGE and Western blotting. The results indicated that the fusion protein was expressed in E. Coli BL21 (DE3) and could be specially recognized by polyclonal antibody against the SAG3 of Toxoplasma gondii. The results were useful for further studies on the diagnosis of toxoplasmosis by SAG3 gene containing no signal peptide.