抗AIDS新型导向毒素CVN-LP1融合基因的构建及原核表达  

Construction and Prokaryotic Expression of a New Anti-HIV Targeted Toxin

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

机构地区：[1]军事医学科学院军事兽医研究所基因工程实验室

出　　处：《中国生物工程杂志》2007年第3期100-104,共5页China Biotechnology

基　　金：吉林省应用基础项目(20060570)

摘　　要：目的:构建重组抗病毒融合蛋白CVN-LP1原核表达载体,并进行表达和鉴定。方法:将已经构建好的pET-CVN和pET-LP1重组质粒,EcoRI和HindⅢ同时进行双酶切,保留LP1片段前端的linker序列。将所得的CVN片段连入pET-LP1载体上,构建pET-CVN-LP1融合表达载体,转化入大肠杆菌BL21(DE3),IPTG诱导表达。SDS-PAGE、Western-blot鉴定表达产物。结果:重组载体pET-28a(+)-CVN-LP1经PCR及XhoI/EcoRI和EcoRI/HindIII双酶切鉴定,证实构建成功。将其转化入大肠杆菌BL21(DE3)中表达,表达产物相对分子量约为20kDa,与理论大小一致。凝胶成像分析表明最高表达量可占菌体总蛋白的49.58%。SDS-PAGE、Western-blot分析表明目的蛋白得到了很好的表达。结论:构建了pET-CVN-LP1原核表达载体,并得到了目的融合蛋白,为进一步研究其生物学功能奠定了坚实的基础。Objective ： To construct prokaryofic expression vector containing CVN-LP1 gene , and identify the expression of CVN-LP1 protein. Methods： pET-CVN and pET-LP1 was restriction endonuclease digested by EcoPd and HindIII, and the CVN gene was inserted into pET-LP1 plasmid. The vector was transformed into BI2？.I （DE3） to construct a prokaryotic expression system. The expressed product was identified by SDS-PAGE and Western blot assay. Results： The recombinant plasmid pET-CVN-LP1 was constructed, restriction endonuclease digestion and PCR identification proved that the CVN was correctly cloned into vector pET-LP1. SDS-PAGE and Western-blot analysis showed that CVN-LP1 was successfully expressed in E. coli BL21 （DE3） , The relative molecular mass （Mr） of the expression protein was 20kDa, according with the predicted Mr value. And the expression yield was about 49.58% of total bacterial protein. Conclusion ： The successful expression of CVN-LP1 gene in E. coli provides a support for further study of the anti-HIV agent.

关 键 词：核糖体失活蛋白 原核表达Cyanovirin-N LuffinP1 

分 类 号：Q786[生物学—分子生物学]