人类免疫缺陷病毒HIV结构蛋白Gag在家蚕杆状病毒表达系统中的表达  

Expression of Structural Protein Gag of Human Immunodeficiency Virus in Bombyx mori Baculovirus Expression System

作　　者：陈贝妮[1] 何丹枫[1] 章杰[1] 黄梦园 邹日泽 麦丽丽[1] 李司[1] 

机构地区：[1]浙江理工大学生命科学学院生物化学研究所,浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2015年第1期87-91,共5页ACTA SERICOLOGICA SINICA

基　　金：大学生科技创新项目(No.2014R406041;2013R406018);浙江省自然科学基金项目(No.Y3110354);国家高技术发展计划"863"项目(No.2012ZX09102301-009;2011AA100-603);浙江省高校生物学重中之重(一级)学科项目

摘　　要：人类免疫缺陷病毒(HIV)是导致艾滋病的主要病原之一,HIV病毒的核心结构蛋白Gag有自我装配功能,并能激发体液免疫和细胞免疫,是理想的HIV疫苗靶抗原。将Gag基因克隆至质粒p Bac PAK8中构建重组质粒,并与线性化的家蚕杆状病毒基因组DNA共转染Bm N细胞,得到重组家蚕杆状病毒Bm NPV-Gag。将该重组病毒感染Bm N细胞和家蚕5龄幼虫,SDS-PAGE、Western blotting检测显示在重组病毒感染的Bm N细胞和家蚕5龄幼虫血淋巴中均有与Gag蛋白大小(55 k D)相符的特异性条带出现,证明重组Gag蛋白成功表达。用ELISA法测定重组病毒感染后不同时间目的蛋白质的表达变化:Bm N细胞中的Gag蛋白含量在感染后的第4天达到最大值6.995 pg/个,在感染第5天后出现下降趋势;5龄幼虫血淋巴中的Gag蛋白含量在感染后的第7天最高,为790.1 ng/m L。重组Gag蛋白在家蚕杆状病毒表达系统中的成功表达,为艾滋病疫苗的研究探索了新的途径。Human immunodeficiency virus（ HIV） is one of the main pathogens causing acquired immune deficiency syndrome（ AIDS）. The core structural protein Gag of HIV,which can self-assemble and stimulate both humoral and cellular immunity,is an ideal HIV vaccine target antigen. In this experiment,Gag gene of HIV was cloned into plasmid p Bac PAK8-VP6 and co-transfected into silkworm Bm N cells with genomic DNA of linearized Bombyx mori baculovirus to construct the recombinant baculovirus Bm NPV-Gag. After Bm NPV-Gag was used to infect Bm N cells and the 5th instar silkworm larvae,SDS-PAGE and Western blotting detected a specific band of 55 k D both in Bm N cells and in hemolymph of the 5th instar silkworm larvae,indicating that the recombinant protein Gag was successful expressed and the expressed protein was consistent with the size of Gag protein. Expressional variations of target protein at different time points after infection with recombinant virus were determined by ELISA method. The results show that the content of Gag protein in Bm N cells reached the maximum value of 6. 995 pg / cell at day 4,and showed a downward trend from day 5. The content of Gag protein in hemolymph of the 5th instar silkworm larvae reached the highest value of 790. 1 ng / m L at day 7 after infection.The successful expression of recombinant Gag protein in Bombyx mori baculovirus expression system provides anew way to develop novel AIDS vaccines.

关 键 词：人类免疫缺陷病毒 GAG蛋白 杆状病毒表达系统 BM N细胞 家蚕幼虫 

分 类 号：R512.91[医药卫生—内科学]