新型抗病毒蛋白2CVN融合基因的构建、表达与纯化  

Construction and Expression of Fusion Gene 2CVN Encoding A Novel Antiviral Protein and Purification of Expressed Product

作　　者：刘玉生[1] 金宁一[1] 李昌[1] 于芳[1] 佟敬山[1] 胡宁宁[1] 金洪涛[1] 李臻[1] 

机构地区：[1]军事医学科学院十一所病毒室

出　　处：《中国生物制品学杂志》2007年第5期327-329,共3页Chinese Journal of Biologicals

基　　金：吉林省应用基础项目(20060570).

摘　　要：目的构建新型抗病毒蛋白2CVN融合基因,增强CVN对病毒的结合性及中和活性,为进一步研究抗病毒药物奠定基础。方法采用PCR法,以pBluescriptⅡSK(+)-CVN质粒为模板,扩增CVN片段,在引物中引入Linker序列,将扩增的CVN片段与载体pET-CVN连接,构建pET-2CVN原核表达载体,转化大肠杆菌BL21(DE3),进行表达及产物纯化。结果表达产物经SDS-PAGE和Western blot分析,相对分子质量为29000,与理论预期值完全相符。凝胶成像分析表明目的蛋白表达量可占菌体总蛋白的31.48%,纯化后的目的蛋白纯度达95%以上。结论已成功构建了2CVN融合基因,并获得高效表达。Objective To construct fusion gene 2CVN encoding a novel antiviral protein, enhance the binding and neutralizing activities of CVN to viruses and lay a foundation of further development of antiviral drugs. Methods Amplify CVN gene fragment by PCR with the primers with linker sequence introduced, using plasmid pBluescrip Ⅱ SK（ ＋ ）-CVN as template. Ligate the amplified CVN gene fragment to plasmid pET-CVN, and transform the constructed prokaryotic expression vector pET-2CVN to E. coli BL21 （DE3） for expression. Purify the expressed product by affinity chromatography and identify by SDS-PAGE and Western blot. Results The expressed product, with a relative molecular mass of 29 000, contained 31.48% of total somatic protein, and reached a purity of more than 95% after purification. Conclusion 2CVN fusion gene was successfully constructed and highly expressed.

关 键 词：抗病毒蛋白 融合基因 原核表达 

分 类 号：Q786[生物学—分子生物学]