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目的:构建天花粉蛋白(TCS)突变体基因,并在原核系统进行表达及纯化。方法:应用计算机预测TCS分子上可能的抗原决定簇(FYY163-165),并进行定点突变。以栝楼基因组DNA为模扳,经PCR扩增突变基因,与pRSET-A表达载体连接,转化感受态E.coli DH5α,提取质粒进行酶切鉴定及测序。将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法鉴定和Ni-NTA层析纯化。结果:构建了带有TCS突变体基因(TCSFYY163-165CSA)的重组表达质粒,使目的蛋白在大肠杆菌中获得高效可溶性表达,表达产物经纯化后,得到均一的TCS突变体蛋白。结论:成功地构建了TCS突变体基因TCSFYY163-165CSA,并获得高效表达,为基因工程方法改造TCS提供了新的途径。
Objective: To construct the TCS mutant gene and express it in prokaryotic system. Methods: The possible antigenic determinants of TCS (FYY163-165) were predicted by computer and subjected to site-directed mutagenesis. The mutant was amplified by PCR and ligated with pRSET-A expression vector. The recombinant plasmid was transformed into competent E. coli DH5α, and the plasmid was digested and sequenced. The positive recombinant plasmid was transformed into competent E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by Western blot and purified by Ni-NTA chromatography. Results: The recombinant plasmid with TCS mutant gene (TCSFYY163-165CSA) was constructed, and the target protein was efficiently and solublely expressed in E. coli. The expressed product was purified to obtain the uniform TCS mutant protein. CONCLUSION: TCS mutant TCSFYY163-165CSA was successfully constructed and highly expressed, which provided a new approach for gene engineering to transform TCS.