天花粉蛋白突变体的构建及其在原核系统中的表达

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.coli 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E.coli BL21（DE3）,经IPTG诱导表达后,对表达产物进行Western blot鉴定。最后用Ni-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Ni-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。

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

Objective:To construct site-directed mutant of trichosanthin(TCS),express and purify of TCS in E.coli. 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 BL21(DE3).Conclusion:The site-directed mutagenesis,expression and purification of TCS provide a new approach for reconstructing TCS.