新型抗病毒蛋白CVN的构建及原核表达  

Construction and prokaryotic expression of a new antiviral protein

作　　者：刘玉生[1] 李昌[1] 金宁一[2] 于芳[2] 金洪涛[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《免疫学杂志》2007年第4期395-398,共4页Immunological Journal

基　　金：吉林省应用基础项目资助(20060570)

摘　　要：目的构建重组抗病毒蛋白CVN原核表达载体,并进行表达和鉴定。方法采用全基因合成的方法合成目的基因CVN(Cyanovirin-N),将该片断插入到pBluescriptⅡSK(+)载体中后,亚克隆至原核表达载体pET28a(+),构建pET-CVN表达载体,转入大肠杆菌BL21(DE3),IPTG诱导表达。用SDS-PAGE,Western-blot等方法分析鉴定表达产物。结果合成的目的基因全长303bp,重组载体pET-CVN经PCR和双酶切鉴定,证实构建成功。将其导入大肠杆菌BL21(DE3)中表达,表达产物相对分子量为17 000左右,与理论预期值完全相符。凝胶成像分析表明最高表达量可占菌体总蛋白的46.4%。SDS-PAGE,West-ern-blot分析表明目的蛋白得到了很好的表达。结论成功构建了pET-CVN原核表达载体,并得到了大量的表达,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 gene, and identify the expression of CVN protein using Western blot assay. Methods The whole sequence of CVN gene was synthesized and inserted into pSK（ ＋ ） plasmid, and then subcloned into pmkaryofic expression vector pET28a （ ＋ ）. The vector was transformed into BL21 （DF3） to construct a pmkaryofic expression system. The expressed product was identified by SDS-PAGE and Western blot assay.Results A DNA fragment about 303 bp was obtained and the recombinant plasmid was constructed, which named pET-CVN. Restriction endonuclease digestion and PCR identification proved that the CVN was correcdy cloned into expression vector. SDS-PAGE and Western-blot analysis showed that CVN was expressed in Ecoli BL21（DE3） successfully. The relative molecular mass （Mr） of the expression protein was 17 000, which was accorded with the predicted Mr value. And the expression yield was about 46.4% of total bacterial protein. Conclusion The successful expression of CVN gene in Ecoli provides a support for further study of the biological function of CVN.

关 键 词：抗病毒蛋白 原核表达 CYANOVIRIN-N 

分 类 号：R373[医药卫生—病原生物学]