弓形虫pc-DNA3-ROP2核酸疫苗的研制  

Studies of pc-DNA3-ROP2 nucleic acid vaccine of Toxoplasma gondii

作　　者：李瑾[1] 魏庆宽[1] 徐秀来[1] 傅婷霞[1] 崔勇[1] 肖婷[1] 王洪法[1] 张佃波[1] 王用斌[1] 黄炳成 闫歌[1] 韩广东[1] 

机构地区：[1]山东省寄生虫病防治研究所,山东济宁272033

出　　处：《中国病原生物学杂志》2008年第5期374-378,共5页Journal of Pathogen Biology

基　　金：山东省自然科学基金项目(No.022130133)

摘　　要：目的研究廉价有效的抗弓形虫核酸疫苗,以用于人类和畜类弓形虫病的防治。方法收集、纯化RH株弓形虫速殖子,提取基因组DNA;根据ROP2基因序列,设计合成ROP2基因引物,应用PCR、酶切、连接、测序等技术,扩增ROP2基因片段,克隆于真核表达载体pc-DNA3质粒中,制备抗弓形虫pc-DNA3-ROP2核酸疫苗。应用该疫苗免疫小鼠,通过ELISA检测血清抗体,Western blot鉴定该疫苗的免疫原性;应用RH株弓形虫进行动物攻击实验,评价其安全性及免疫保护性。结果以弓形虫基因组DNA为模板,PCR扩增出1.7 kb ROP2基因片段,克隆于pc-DNA3质粒中,成功构建了pc-DNA3-ROP2重组质粒。测序结果显示,重组质粒包含了ROP2蛋白基因读码框内的完整序列,能完整表达ROP2的抗原蛋白。应用该疫苗免疫小鼠,能诱导产生强烈的细胞、体液免疫反应,使实验组小鼠攻虫感染后的存活时间明显延长,攻虫感染192 h后的存活率为77.8%,空质粒及PBS对照组存活率为0,差异有统计学意义(P<0.001);实验全程免疫小鼠未发生毒性及异常反应。结论该核酸疫苗具有很强的免疫原性,安全可靠,能诱导小鼠产生良好的免疫保护作用,具有很好的开发应用价值。Objective To study an cheap and effective nucleic acid vaccine for control of Toxoplasmosis in human and animals. Methods Tachyzoites of Toxoplasma gondii RH strain were collected and depurated to obtain genome. Primers were designed and synthesized and ROP2 gene was amplified, then recombined into an eukaryotic expression vector of pc-DNA3, pc-DNA3-ROP2 was then constructed, The insert was identified by PCR amplification, enzyme digestion, conjunction and sequence analysis. Mice were immunized with this pc-DNA3-ROP2 nucleic acid vaccine. Immunogenicity of the vaccine was evaluated through detecting immunity indexes. Challenge experiment with T. gondii RH strain was conducted. The safety and protective effect of this vaccine was appraised, Results The specific gene fragment of ROP2 about 1.7 kb in length was amplified, The gene fragment cloned into pc-DNA3 was correct, Eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. ROP2 nucleic acid vaccine could induce strong cellular and humoral immune response. The titer of antibodies in serum was high after inoculating. The antibody could recognize ROP2 protein antigen expressed in vitro, The death and survival time of mice in experiment was clearly delayed and prolonged compared with that of control group, The difference was statically significant（P〈0.01）. The harmfulness of this vaccine inoculated to mice was not observed during the experiment. Conclusion The recombinant ROP2 nucleic acid vaccine is safe, its immunogenicity is potent. It could produce good immunoprotection and possesses significant value for development and application.

关 键 词：刚地弓形虫 棒状体蛋白2(ROP2) 基因重组 核酸疫苗 免疫保护作用 

分 类 号：R382.5[医药卫生—医学寄生虫学]