弓形虫AMA1蛋白的原核表达载体的构建及体外表达

Construction of Prokaryotic Expression Vector of Toxoplasma gondii AMA1 Protein and Expression in vitro

[目的]构建截短型AMA1的原核表达载体,并进行体外诱导表达,为在体外表达弓形虫AMA1重组蛋白。[方法]设计去掉弓形虫AMA1信号肽的PCR引物,以弓形虫cDNA为模板进行PCR扩增,PCR扩增产物酶切后与pGEX-4T-3原核表达载体连接,并转化到BL21感受态细胞内。对经PCR鉴定为阳性的重组质粒pGEX-4T-3-AMA1进行体外诱导表达。[结果]成功构建了弓形虫AMA1的原核表达质粒pGEX-4T-3-AMA1,通过体外诱导表明,AMA1融合蛋白是以包涵体的形式存在。[结论]弓形虫AMA1蛋白的体外表达为进一步制备抗AMA1血清及免疫学实验奠定了基础。

[Objective] The aim was to construct recombinant prokaryotic expression vector of truncated AMA1 fragment and induced expression in vitro was carried out.[Methods] The PCR was performed using T.gondii cDNA as templates,and then the production of PCR was connected with pGEX-4T-3 prokaryotic expression vector.The recombinant vector with AMA1 was transferred into BL21 competent cells,and was introduced in vitro.[Result] We successfully constructed recombinant prokaryotic expression vector pGEX-4T-3-AMA1 which could express the AMA1 fusion protein in vitro.It confirmed that the AMA1 fusion protein was the inclusion body exist in E.coli.[Conclusion] These results provided foundation for the further studies on the immunologic tests for T.gondii.