携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

发布时间：2018-01-17 19:12

本文关键词：携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定　出处：《生物工程学报》2015年03期 　论文类型：期刊论文

【摘要】：利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。
[Abstract]:Using bacterial artificial chromosome technique to connect the HIV-1 gp160. Gag and protease genes and expression elements were inserted into Herpes simplex virus type 1 of herpes simplex virus type 1. HSV-1) internal reverse repeat region to obtain herpes simplex virus vaccine carrying HIV-1 antigen. First, HIV-1 gp160(B and C). Gag and protease genes were cloned into PC DNA3 to obtain the recombinant plasmids PC DNA/g Bgp and PC DNA/g Cgp. The recombinant plasmid was transfected into 293FT cells. Western blotting was used to detect the expression of HIV antigen. Then PC DNA/g Bgp and PC DNA/g were detected. Recombinant shuttle plasmids p KO5/BN/g Bgp and p KO5/BN/g were obtained by cloning into p KO5/BN the expression frame containing HIV-1 antigen gene and expression element in Cgp. Cgp. The shuttle plasmid was electrotransferred to Escherichia coli containing BAC-HSV to screen the recombinant bacteria, extract the recombinant DNA and transfect it into Vero cells, and select the virus plaque to purify the recombinant virus. Southern blotting was used to identify the expression of HIV antigen in the cells infected with recombinant virus. The characteristics of virus proliferation were analyzed. Western blotting in PC DNA/g Bgp and PC DNA/g. Expression of gp160 and gag proteins. P KO5/BN/g Bgp and p KO5/BN/g were detected in 293FT cells transfected with Cgp. The recombinant bacteria were obtained by electroporation of Cgp. The recombinant Vero cells transfected with recombinant DNA were purified and purified by Southern blotting. The results of Southern blotting analysis showed that the recombinant HSV genome was specifically recombined. Gp120 and gp41 were detected in the cells infected with recombinant virus, and the recombinant HSV retained the ability of replication in mammalian cells. In this study, HIV-1 gp160 was obtained. The recombinant HSV gene of gag and protease could be used as the vector vaccine of HIV-1 replication virus, and reserved the ability of replication in mammalian cells.
【作者单位】：新疆大学生命科学与技术学院;
【基金】：新疆维吾尔自治区高技术研究发展项目(No.2010016)资助~~
【分类号】：R752.11
【正文快照】： 人类免疫缺陷病毒(Human immunedeficiency virus,HIV)感染机体后主要侵犯CD4+T细胞,导致机体免疫缺陷并继发各种机会性感染和肿瘤。自首例艾滋病报道以来,全球科学家一直致力于艾滋病疫苗的研制[1]。针对HIV这类高度变异、攻击免疫系统且能引起持续感染和产生免疫病理反应的

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