非复制重组痘苗病毒表达人免疫缺陷病毒Bostwana C亚型Nef蛋白及免疫效果的观察  

The Expression of nef Gene of HIV-1 Subtype C Bostwana in Non-replicative Vaccinia Virus and Its Immune Effect in Mice

作　　者：辛伟[1] 郭斐[1] 陆柔剑[1] 王世峰[1] 邓瑶[1] 王文玲[1] 张相民[1] 李仁清[1] 吕亦晨[2] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]哈佛大学公共卫生学院免疫和传染病室

出　　处：《病毒学报》2004年第2期97-103,共7页Chinese Journal of Virology

基　　金：国家八六三计划新型艾滋病(HIV/AIDS)疫苗的研究项目(2001AA215031)

摘　　要：从含人类免疫缺陷病毒Ⅰ型(HIV 1)BostwanaC亚型全基因的质粒pJM4 HIV中克隆了nef基因,并利用非复制型痘苗病毒载体构建表达Nef蛋白的重组病毒NTVJ1175nef,经PCR和Southernblot鉴定,nef基因正确整合到痘苗病毒基因组的J片段上;感染人源细胞后,经Westernblot和免疫荧光检测表明,重组病毒能很好地表达Nef蛋白,并定位于细胞质中。NTVJ1175nef免疫BALB/c小鼠后,经Pep IFN ′γ Assay法检测,可诱导产生针对表位肽P1特异的可分泌IFN ′γ的CD+8T细胞(占脾细胞总数0 20%);经乳酸脱氢酶(LDH)法检测证实,诱导的细胞毒性T细胞(CTL)可特异性地杀伤表位肽P1特异P815靶细胞。这些结果表明,NTVJ1175nef具有良好的细胞免疫原性,为下一步构建表达包含HIV早期抗原的多组分重组痘苗病毒候选疫苗奠定了免疫学基础。A nef gene was cloned from plasmid containing the whole gene of HIV-1 subtype C Bostwana strain.A recombinant vaccinia virus NTVJ1175nef with nef gene above was constructed using a viral vecter of non-replicative vaccinia virus.By means of PCR and Southern blot,the nef gene was proved to be integrated into vaccinia virus genome.Detecting by Western blot and immunofluorescence,the Nef protein was well expressed in the recombinant vaccinia virus NTVJ1175nef after infecting cells derived from human,and the expressed Nef protein located in the cytoplasm. After immunizing Balb/C mice, NTVJ1175nef could induce peptide(P1)-specific and IFN-′γ-secreting CD~ +_8T cell(accounting for 0.20% of the total spleen cells)by Pep-IFN-′γ-Assay,and also could induce CTL which could specifically kill the peptide(P1)-specific P815 target cells by LDH-Assay.It could be concluded that NTVJ1175nef has good immunogenicity in mice.The immunologic base has been established for next construction of candidate HIV-1 recombinant vaccinia virus vaccine including HIV early proteins.

关 键 词：人类免疫缺陷病毒 HIV-1 NEF蛋白 细胞毒性T细胞 疫苗 

分 类 号：R373.9[医药卫生—病原生物学] R512.91[医药卫生—基础医学]