携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

Construction and identification of HSV-1 vector vaccine carrying HIV-1 antigen

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pcDNA3获得重组质粒pcDNA/gBgp和pcDNA/gCgp，重组质粒转染293FT细胞，Western blotting检测HIV抗原表达。继而将pcDNA/gBgp和pcDNA/gCgp中包括HIV-1抗原基因和表达元件的表达框克隆入pKO5/BN获得重组穿梭质粒pKO5/BN/gBgp和pKO5/BN/gCgp，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pcDNA/gBgp和pcDNA/gCgp转染的293FT细胞中检测到表达的gp160和gag蛋白。pKO5/BN/gBgp和pKO5/BN/gCgp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:

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus’ DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus’ replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.