人巨细胞病毒抗原表位的重组表达及免疫原性分析  

Recombinant Expression of Antigen Epitope Genes of Human Cytomegalovirus and Immunogenicity Detection of the Fusion Protein

作　　者：王蒙[1] 孙文辉[1] 姬芳玲[1] 蒲中机 李寅生 包永明[1] WANG Meng SUN Wen-hui JI Fang-ling PU Zhong-ji LI Yin-sheng BAO Yong-ming(School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China Dalian Institute for Drug Control, Dalian 116029, China)

机构地区：[1]大连理工大学生命科学与技术学院,大连116024 [2]大连市药品检验所,大连116029

出　　处：《中国生物工程杂志》2017年第7期27-33,共7页China Biotechnology

摘　　要：目的:用大肠杆菌表达系统表达人巨细胞病毒(HCMV)pp65蛋白和gB蛋白的优势抗原表位基因,制成HCMV亚单位疫苗,探究其在Balb/c小鼠体内的体液免疫和细胞免疫活性。方法:选取pp65蛋白的490~508aa和gB蛋白的607~621aa的基因片段,经PCR扩增目的片段,连接表达载体pET-32a(+),转化BL21(DE3)plys菌株,十二烷基硫酸钠-聚丙烯酰胺凝胶(SDSPAGE)分析蛋白质表达,金属螯合亲和镍柱法纯化目的蛋白。重组蛋白免疫Balb/c小鼠,Western blotting法和间接ELISA法检测重组蛋白的体液免疫活性;通过流式细胞仪和双抗夹心ELISA法检测重组蛋白的细胞免疫活性。结果:获得分子质量约为22kDa的融合蛋白,Western blotting检测显示抗体有特异性,间接ELISA法检测其效价为1∶102 400。与对照组相比,实验组小鼠外周血中CD4^+T细胞和CD8^+T细胞的数量,以及IFN-γ、IL-2、IL-12的含量有显著提高(P<0.01)。结论:制备的具有免疫优势抗原表位的重组蛋白能诱导小鼠产生较强的体液免疫和细胞免疫反应。Objective： To obtain the recomninant protein as HCMV subunit vaccine, the dominant antigen epitope gene of protein pp65 and gB of human cytomegalovirus （HCMV） were expressed in E. coli, and the humoral and cellular immune responses of the recombinant protein in Balb/c mice were detected. Methods ： The coden genes of 490 - 508 aa in pp65 protein and 607 - 621 aa in gB protein were selected as gene segments and cloned into expression vector pET32a （ ＋ ） after amplified by PCR. The protein expressed was analyzed by SDS- PAGE, after the recombinant plasmid transformed into E. coli BL21 （DE3） plys. Balb/c mice were immunized with the recombinant protein purified by nickel metal chelate affinity column. The humoral immune activity of recombinant proteins was detected by Western blotting and indirect ELISA. The cellular immune activity was detected by flow cytometry and sandwich ELISA. Results ： The fusion protein about 22kDa was obtained. Western blotting test showed that the multiclonal antibody for HCMV has specificity and the titers reach to 1：102 400 detected by indirected ELISA. The number of CD4 ＋ T ceils and CD8 ＋ T cells in peripheral blood and the quantities of IFN-γ, IL-2, IL-12 were significantly higher than control group （ P 〈 0.01 ）. Conclusions ： The recombinant protein with dominant antigen epitopes can induce significant humoral immunity and cellular immunity in mice, and is a potential vaccine for HCMV.

关 键 词：人巨细胞病毒 PP65蛋白 gB蛋白 体液免疫 细胞免疫 

分 类 号：Q816[生物学—生物工程]