丙型肝炎病毒包膜蛋白E2的DNA免疫  

DNA Immunization with Recombinant HCV E2 Expression Plasmids

作　　者：朱珺[1] 王春林[1] 朱立新[1] 孔玉英[1] 汪垣[1] 李光地 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室,上海200031

出　　处：《生物化学与生物物理学报》2002年第4期445-451,共7页

基　　金：国家高科技"863"计划资助项目 (No .2 0 0 1AA2 15 171)~~

摘　　要：为了比较启动子及分泌信号对丙型肝炎病毒包膜蛋白E2 (HCVE2 )的DNA免疫效果的影响 ,构建了 4个不同的HCVE2 (384～ 6 6 0 )融合表达质粒 :CMV启动子控制下和EF1α启动子控制下的分泌表达质粒pCMVSec S1E2t6 6 0和pEF1αSec S1E2t6 6 0以及非分泌表达质粒 pCMV S1E2t6 6 0和 pEF1α S1E2t6 6 0。4个表达质粒在HeLa细胞中进行了暂时表达 ,免疫印迹分析表明 ,表达产物都同时具有HBVpreS1及HCVE2蛋白的抗原性 ,夹心ELISA测定结果表明 ,分泌表达质粒的表达量明显高于非分泌表达质粒 ,带CMV启动子的质粒表达量高于EF 1α ;并且只有分泌型质粒转染细胞后 ,才能在细胞培养上清液中检测到融合蛋白。4种表达质粒免疫C5 7BL/ 6小鼠 ,可产生 preS1及E2的抗体 ,比较了抗体转阳率、抗体滴度和维持时间等 ,发现带CMV启动子的E2分泌表达质粒 pCMV S1E2t6 6 0明显优于其他 3组。对 4种质粒产生体液免疫反应不同的可能原因进行了分析。DNA based immunization, the delivery of plasmid DNA by direct inoculation, is a newly developed method of vaccination. Besides of many advantages, DNA immunization was demonstrated to generate weaker antibody and CTL responses than did protein and live attenuated vaccines, respectively. To circumvent this shortage, several methods were tested such as using different vector, changing injection mode, and co expressing cytokines, etc. The studies showed that many factors could greatly affect the immune responses to DNA vaccine. The aim of this study is to compare different vectors for the presentation of the HCV E2 to generate immune responses by using DNA based immunization. Four expression plasmids, with different promoter types and with or without signal sequences were constructed, which encode C terminally truncated E2 (384—660) with HBV preS1(21—47) tag fused to its N termini. Transient expression in HeLa cells showed that only recombinant plasmids with signal sequence could be expressed and properly processed, and the product could be secreted into medium. Protein expression level is slightly higher in plasmids with CMV promoter than with EF 1α promoter. After immunization of C57BL/6 mice, all the recombinant constructs could elicit both anti preS1 and anti E2 antibodies. But only pCMV Sec S1E2t 660 with both CMV promoter and signal sequence could induce high level and long lasting antibody, showing that it is a good vaccine candidate. Possible reasons for the different immune responses to these constructs were discussed.

关 键 词：丙型肝炎病毒 包膜蛋白E2 DNA免疫 启动子 信号序列 

分 类 号：R392[医药卫生—免疫学]