HIV多抗原位点同源序列合成基因的表达及活性鉴定  

Expression and preliminary activity identification of several linked antigen epitopes of consensce genes of HIV

作　　者：杨怀宁[1] 徐卉[2] 浦昀[1] 于湘晖[3] 张煜[3] 赵大力[4] 孙志伟[1] 

机构地区：[1]吉林大学公共卫生学院卫生毒理学教研室,吉林长春130021 [2]吉林大学第一医院电诊科,吉林长春130021 [3]吉林大学生命科学学院疫苗研究中心,吉林长春130012 [4]吉林出入境检验检疫局,吉林长春130062

出　　处：《吉林大学学报（医学版）》2008年第2期221-225,共5页Journal of Jilin University：Medicine Edition

基　　金：国家质检总局科研基金资助课题(2007IK202)

摘　　要：目的:在原核表达载体系统中对HIV-1gp41/gp120和HIV-2gp125/gp36外膜蛋白多个抗原位点同源序列合成基因进行表达、纯化并鉴定其活性。方法:人工合成含HIV-1gp41的3个抗原位点、HIV-1gp120的2个抗原位点、HIV-2gp125的3个抗原位点和HIV-2gp36的1个抗原位点的串联基因,克隆到原核表达载体pRSETB中,构建重组表达质粒pRSETB-env,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的基因在大肠埃希菌BL21(DE3)中高效表达,采用金属离子亲和层析技术纯化表达蛋白,逐渐降低尿素浓度使目的蛋白复性,免疫印迹和ELISA法分别对表达产物进行鉴定。结果:目的基因在BL21中有较高表达率,纯化后表达蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)可见相对分子质量约44000的蛋白带,与设计相对分子质量相符。免疫印迹、ELISA试验显示pRSETB-env质粒的HIV-1/2表达蛋白与HIV-1及HIV-2患者血清都能较好地结合,与其他患者血清无交叉反应。结论:成功构建了由HIV-1/2外膜蛋白多个抗原位点串联基因片段的表达载体pRSETB-env,并在原核细胞中高效表达,表达蛋白经纯化后纯度较高,并具有良好特异性和活性。Objective To express the several linked antigen epitopes of consence genes of HIV-1gp41/gp120 and HIV-2gp125/gp36 in the system of prokaryotic expression vector;to purify and identify the expression products. Methods In order to construct the recombinant expression plasmid pRSETB-env, the linked genes contained three antigen epitopes of gp41, two antigen epitopes of gp120, one antigen epitope of gp36 and three antigen epitopes of gp125 were inserted into pRSETB vector. After transformed into F.coli BL21 （DE3） cells, the recombinant protein was expressed with induction of isopropyl/3-D-thiogalactopyranoside （IPTG）, and was purified by immobilized metalion affinity chromatograph and was recoveried by decreasing the concentration of urea. The immunoactivity was analyzed by Western blotting and enzyme-linked immunosorbent assay （ELISA）. Results An expected expressing protein band （about 44 000） was seen with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The aim genes revealed higher expressive rate in F.coli BL21 （DE3） cells. The results of Western blotting and ELISA showed specific reactions with HIV patients＇ sera, and no cross-reaction with other patients＇ sera using the expression protein. Conclusion The recombinant expression plasmid pRSETB env which contained the several linked antigen epitopes of envelop genes of HIV is successfully constructed. After purification, the expression protein possesses higher pureness, good specificity and activity.

关 键 词：人免疫缺陷病毒 合成基因 基因表达 活性鉴定 

分 类 号：Q78[生物学—分子生物学] R446.6[医药卫生—诊断学]