人类免疫缺陷病毒-2 gag重组鸡痘病毒疫苗的构建及实验免疫研究  

Construction of HIV-2 gag recombinant fowlpox virus and its immune reactivity in mice

作　　者：张立树[1] 金宁一[1] 李子健[2] 江文正[3] 王宏[4] 宋英今[5] 

机构地区：[1]军事医学科学院军事兽医研究所,全军基因工程重点实验室,长春130062 [2]北京大学第三医院血管医学研究所 [3]第二军医大学免疫学研究所 [4]暨南大学生命科学院生物系 [5]天津大学农业与生物工程学院

出　　处：《中华传染病杂志》2006年第4期220-224,共5页Chinese Journal of Infectious Diseases

基　　金：国家"863"计划资助项目(2003AA219051);吉林省科技发展计划项目(20030550-1)

摘　　要：目的构建并探讨人类免疫缺陷病毒(HIV)-2 gag重组鸡痘病毒(FPV)在小鼠体内的免疫应答,为开发HIV-2基因重组活载体疫苗提供实验依据。方法将HIV-2结构基因gag插入到转移载体pUTA2复合启动子ATI-P7．5×20下游．构建出鸡痘病毒重组转移质粒pUTA2-gag;经转染和BrdU加压筛选,以基因组聚合酶链反应(PCR)和Western blot法鉴定重组病毒;将获得的重组病毒大量制备并纯化后,肌内注射免疫Balb/c小鼠,酶联免疫吸附实验(ELISA)检测小鼠血清HIV-2抗体,流式细胞仪测定CD4^+、CD8^+T淋巴细胞亚类数量,乳酸脱氢酶(LDH)释放法检测脾特异性细胞毒性T淋巴细胞(CTL)杀伤活性。结果构建出重组鸡痘病毒转移质粒pUTA2-gag;从重组病毒的基因组中可扩增出大小为766bp的目的基因,其表达产物可与人HIV-2阳性血清发生反应,目的蛋白的相对分子质量约为55000;重组病毒免疫组小鼠的血清出现HIV-2抗体．脾T细胞亚类的数量增加,并产生针对HIV-2靶细胞的特异性CTL杀伤活性。结论获得一株重组鸡痘病毒,该病毒可稳定表达HIV-2结构蛋白Gag,并能诱导小鼠产生特异性细胞和体液免疫应答。Objective To provide experimental evidences for developing a safe and effective re eombinant fowlpox virus which can prevent the infection of HIV-2. Methods A fowlpox virus（FPV） transferring vector was constructed by inserting HIV 2 gag gene to the downstream of a synthetic complex promoter ATI-p7.5 × 20 of vector pUTA2. Transfeetion was then carried out, and reeombinant FPV （rFPV） was screened by 5＇-bromo deoxyuridine （BrdU）, genome PCR and western blot detection. Balb/e mice were immunized with rFPV by muscular injection. Anti HIV-2 antibody, CD4^＋ and CD8^＋ T-cell count and specific target killing activity of spleen CTL in immunized mice were analyzed by ELISA, FACS and LDH release assay, respectively. Results A transferring vector pA gag was constructed and confirmed by amplifying a fragment of 766 bp from the rFPV genome. Meanwhile, HIV-2 multi-antibody-specific protein blot （55 000） was detected from the recombinant virus and the HIV-2 specific antibody was detected from the immunized Balb/e mice. HIV 2-specific targetkilling activity of spleen CTL was observed in immunized mice. Conclusion A recombinant fowlpox virus expressing HIV-2 structural protein Gag has been obtained, and it can stimulate HIV-2-specific celluar and humoral immune reactions in mice.

关 键 词：HIV-2 基因 gag 重组 遗传 鸡痘病毒 免疫 细胞 抗体生成 

分 类 号：R392[医药卫生—免疫学]