天花粉蛋白突变体的构建及其在原核系统中的表达

Site-directed mutagenesis and expression of trichosanthin in E.coli

目的:构建天花粉蛋白(TCS)突变体,并将其在原核系统内进行表达及纯化。方法:借助计算机预测TCS可能的抗原决定簇(YFF81-83)并设计出适当的突变引物。以栝楼基因组DNA为模板,利用重组PCR技术扩增TCS突变体全长基因,经BamHⅠ和EcoRⅠ双酶切后与原核表达载体pRSET-A连接,转化感受态E.coliDH5α,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E.coliBL21(DE3),经IPTG诱导表达后,对表达产物进行W estern b lot鉴定。最后用N i-NTA亲和层析柱对所获突变体蛋白进行纯化。结果:成功构建了TCS突变体(TCSYFF81-83ACS),并获得了突变体蛋白在大肠杆菌内的可溶性高效表达,经N i-NTA亲和层析柱纯化后,产生出大量均一的TCS突变体蛋白。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提供了新的途径。

Objective：To construct site-directed mutant of trichosanthin （TCS）, express and purify of TCS in E. coll. Methods：By computer modeling, YFF81-83 was identified as potential antigenic site of TCS. TCS mutant namely TCSYFF81-83ACS 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 BI221 （DE3）. Conclusion：The site-directed mutagenesis, expression and purification of TCS provide a new approach for reconstructing TCS.