日本血吸虫dysferlin基因的克隆及重组蛋白免疫原性的分析  

Cloning of dysferlin gene of Schistosoma japonicumand immunogenicity analysis of recombinant dysferlin protein

作　　者：熊雅念[1] 魏梅梅[1] 洪炀[1] 黄莉妮[1] 孟培培[1] 艾德宙[1] 韩艳辉[1] 傅志强[1] 林矫矫[1] 

机构地区：[1]中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室,上海200241

出　　处：《中国兽医科学》2013年第6期599-604,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31172315);中国博士后科学基金项目(2012M510630);公益性行业(农业)科研专项(200903036);上海科技发展基金项目(12140902700);中国农业科学院院长基金项目(2013JB18)

摘　　要：为评估日本血吸虫dysferlin重组蛋白(rSjDF)的免疫原性,利用PCR技术扩增SjDF基因,构建重组表达质粒pET-28a(+)-SjDF,诱导表达重组蛋白rSjDF,并用重组蛋白免疫BALB/c小鼠制备抗体。应用间接免疫荧光技术对SjDF进行蛋白组织定位。应用Western-blot、ELISA及Bio-Plex悬液芯片技术检测重组蛋白诱导的免疫反应。间接免疫荧光试验表明SjDF主要分布于血吸虫体被表膜。Western-blot结果显示rSjDF能被该重组蛋白免疫小鼠血清识别,具有良好的免疫原性。rSjDF能诱导小鼠产生高水平的特异性IgG、IgG1、IgG2a抗体和IL-2、IL-4、IL-12p70、IL-10、IFN-g等细胞因子。结果表明,制备的重组抗原能刺激小鼠产生较强的免疫反应,为筛选新的血吸虫疫苗候选分子奠定了基础。To evaluate immunogenicity of recombinant Schistosoma japonicum dysferlin protein （rSjDF） ,SjDF gene was amplified by PCR and then subcloned into the plasmid pET-28a（＋） to construct a recombinant expression plasmid pET-28a （＋）-SjDF. The recombinant protein rSjDF was expressed in Escherichia coli. The purified recombinant protein was used to immunize BALB/c mice emulsified with ISA206 adjuvant three times to produce polyclonal antibodies. Indirect immunofluorescent analysis revealed that SjDF was mainly distributed on the tegument surface. Western-blot analysis showed that rSjDF was recognized by murine serum specific to this recombinant protein,indicating rSjDF had good immunogenicity. A high level of specific IgG, IgG1 and IgG2a antibodies as well as IL-2 ,IL-4 ,IL-10, IL-12p70 and IFN-g cytokines were detected after the vaccination of rSjDF. The results suggest that the recombinant protein can induce stronger immune responses in mice. The study provides the foundations for screening the vaccine candidate for schistosomiasis.

关 键 词：日本血吸虫 DYSFERLIN 表膜蛋白 基因克隆 免疫应答 

分 类 号：S852.735[农业科学—基础兽医学]