具有抗HIV活性的突变型天花粉蛋白TCSYFF-KR的构建及原核表达

目的：构建具有抗HIV活性的突变型天花粉蛋白（TCS），并将其在原核系统内进行表达与纯化。方法：借助计算机预测TCS分子上可能的抗原决定簇（YFF81-83和KR173-174），并依此设计适当的突变引物；以栝楼基因组DNA为模板，利用重组PCR技术扩增双突变型TCS全长基因，经BamHI和EcoRI双酶切后与原核表达载体pRSET-A连接，转化感受态大肠杆菌DH5α，提取质粒进行酶切鉴定及测序；将所获阳性重组质粒转化感受态大肠杆菌BL21（DE3），经IPTG诱导表达后，对表达产物进行Western印迹鉴定；用Ni-NTA亲和层析柱对所获突变型TCS蛋白进行纯化。结果：构建了突变型TCSYFY-KR，并获得了该蛋白在大肠杆菌内的可溶性高效表达；经Ni-NTA亲和层析柱纯化，产生大量均一的突变型TCS蛋白。结论：TCS的定点突变及其在原核系统内的表达，为基因工程方法改造TCS提供了一条新途径。

Site-Directed Mutagenesis and Prokaryotic Expression of Trichosanthin

Objective： To construct site-directed mutagenesis, and obtain expression and purification of trichosanthin（TCS） in E.coli. Methods： By computer modeling, sites YFF81-83 and KR173-174 were identified as potential antigenic sites of TCS. Then, TCS mutant namely TCSYFF-KR was constructed by PCR using appropriate mutagenic primers, and cloned into expression vector pRSET-A. After sequence analysis, the acquired recombinant plasmid was transformed into E.coli BL21 （DE3） and target protein was expressed by inducing with IPTG. Then, the protein was identified by Western blotting and purified with Ni-NTA purification system. Results： The recombinant expression vector containing TCS mutant gene was constructed successfully. The protein was expressed at high level in soluble form in E.coli BL21 （DE3） and purified easily to homogeneity. Conclusion： The site-directed mutagenesis, expression and purification of TCS provide a new approach for reconstructing TCS.