具有抗HIV活性的天花粉蛋白在大肠杆菌中的表达及纯化

Cloning Expression and Purification of Trichosanthin in E.coli

目的:天花粉蛋白(TCS)有较强的抗HIV活性。利用基因工程技术在大肠杆菌中表达TCS并进行纯化。方法:从新鲜栝楼叶片中获取TCS基因组DNA,利用PCR技术扩增其全长基因,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)得到工程菌,经IPTG诱导表达后,对表达产物进行SDS-PAGE及Western印迹鉴定;用Ni-NTA柱对所获目的蛋白进行纯化。结果:获得了目的蛋白的可溶性高效表达,并通过了Western印迹鉴定。经Ni-NTA柱纯化后,得到大量均一的6His-TCS融合蛋白。结论:TCS在大肠杆菌中的表达与纯化,为通过基因工程方法研制具有抗HIV活性的药物奠定了基础。

Objective： To explore the method for producing trichosanthin（TCS） by gene engineering techniques. Methods： TCS gene was amplified from Trichosanthes kirilowii M. genomic DNA by using PCR, and then cloned into expression vector pRSET-A. After sequence analysis, the acquired recombinant plasmid was transformed into E.coli BL21（DE3） and the 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 plasmid containing TCS gene was constructed successfully, and expressed at high level in soluble form in E.coli BL21（DE3） and purified easily to homogeneity. Conclusion： The highly efficient expression and purification of TCS lay a foundation of further studying the biological and pharmacological activities of TCS.