弓形虫ROP5基因真核表达载体的构建及其免疫保护性的研究  

Construction of eukaryotic expression vector of Toxoplasma gondii ROP5 gene and its immunoprotective effect

作　　者：赵焕阁[1] 王华[1] 黄用豪[1] 周松林[1] 郭峻莉[1] 黄凤迎[1] 林映莹[1] 谭光宏[1] 

机构地区：[1]海南医学院海南省热带病重点实验室,海口571101

出　　处：《中国人兽共患病学报》2012年第9期947-950,共4页Chinese Journal of Zoonoses

基　　金：海南省自然科学基金(No.809018)资助~~

摘　　要：目的构建弓形虫pcDNA-ROP5真核表达载体,观察弓形虫ROP5基因在小鼠体内诱导的免疫反应。方法克隆弓形虫ROP5基因,将其插入真核表达载体pcDNA-3.1中构建重组质粒pcDNA-ROP5,脂质体法转染Hela细胞体外表达蛋白,蛋白质印迹(Western blotting)分析鉴定。将45只小鼠随机均分为3组,分别为PBS组、空质粒组、pcDNA3.1-ROP5组,每2w肌注免疫(100μg/只)1次,共3次,于免疫前及每次免疫前1d和末次免疫后2w收集小鼠血清,用间接ELISA法检测血清抗弓形虫的IgG。各组小鼠腹腔注射RH株弓形虫速殖子1 000个/每鼠,观察小鼠受攻击感染后的存活时间。结果真核表达载体构建成功,Western blotting分析结果显示,重组质粒pcDNA3.1-ROP5能在Hela细胞表达,蛋白相对分子质量为61kDa。pcDNA3.1-ROP5末次免疫后血清中IgG水平显著高于其他组,pcDNA-ROP5免疫的小鼠与对照组相比,实验组小鼠产生了明显的体液免疫应答。攻击感染试验中,实验组的小鼠比对照组的存活时间有明显延长。结论构建的真核表达重组质粒pcDNA-ROP5对抗击弓形虫攻击感染能够激发一定的免疫保护性。The purpose of this study was to construct eukaryotic expression pcDNA-ROP5 and evaluated the immune responses induced by recombinant plasmid pcDNA-ROP5 of T.gondii in Kunming mice.The gene sequence encoding ROP5 of T.gondii was amplified by PCR and inserted into the eukaryotic expression vector pcDNA-3.1,and the recombinant plasmid pcDNA-ROP5 was constructed.Then the recombinant plasmid pcDNA-ROP5 was transfected into Hela cells to test its expression and the recombinant protein characterized by Western blotting.Forty-five mice were divided into 3 groups randomly: pcDNA-ROP5 as experimental group,and pcDNA3.1 and NS as control.Each mouse was injected intramuscularly by 100 μg recombinant plasmid for 3 times every two weeks.Mice were bled on day 0,13,27 and 41 for 5 mice of each group to detect the level of IgG.And the rest mice were challenged with 1 000 tachyzoites of the virulent T.gondii RH strain at the 14th day after the last immunization to observe the survival time.In this study,the recombinant plasmid pcDNA-ROP5 was successfully constructed by PCR and restriction analysis,and the ROP5 gene of T.gondii was successfully expressed in Hela cells(Mr.61 kDa).The pcDNA-ROP5 vaccine could induce humoral immunity in Kunming mice.The level of IgG antibodies in pcDNA-ROP5 vaccine injected mice serum was higher than those in mice with other groups.After infected with Toxoplasma tachyzoites,the survival time of pcDNA-ROP5 immunized mice in experimental group was prolonged comparing with control groups.The ROP5 of T.gondii could trigger immuno-protection against T.gondii challenge in Kunming mice.

关 键 词：弓形虫 ROP5 真核表达质粒 免疫保护 

分 类 号：R382.5[医药卫生—医学寄生虫学]