弓形虫棒状体蛋白的免疫原性及其DNA疫苗的研制  

Studies on immunogenicity and proparation of DNA vaccine of Toxoplasma gondii rhoptry protein

作　　者：魏庆宽[1] 肖婷[1] 崔勇[1] 胡颖新[1] 史世俊[1] 李桂玲[1] 傅婷霞[1] 李瑾[1] 黄炳成 韩广东[1] 

机构地区：[1]山东省寄生虫病防治研究所,世界卫生组织淋巴丝虫病,绦囊虫病合作中心,山东济宁272033

出　　处：《中国热带医学》2009年第1期1-5,共5页China Tropical Medicine

基　　金：山东省自然科学基金(NO.022130133)

摘　　要：目的研制抗弓形虫DNA疫苗,评价其免疫原性及免疫保护作用,进而推广应用。方法首先用RH株弓形虫速殖子提取基因组DNA,应用PCR、酶切、连接、测序等技术,扩增棒状体蛋白2(ROP2)基因片段,将其克隆于真核表达载体pc-DNA3质粒中,制备抗弓形虫棒状体蛋白2DNA疫苗。再应用该疫苗免疫小鼠,取其血清、脾和淋巴结培养液检测免疫学指标评价其免疫原性,最后应用弓形虫攻击感染实验评价其免疫保护作用。结果PCR扩增出1.7kbROP2基因片段,克隆、构建出pc-DNA3-ROP2重组DNA疫苗能诱发小鼠产生强烈的细胞免疫和体液免疫反应,小鼠血清抗体滴度高且能正确识别由基因重组体外诱导表达的ROP2蛋白抗原;实验组小鼠CD4+T细胞增殖明显,体液中各细胞因子含量均有不同程度的升高,尤其是血清中升高最为显著。弓形虫攻击感染180h后,疫苗免疫保护率为88.9%,小鼠存活时间明显延长。结论pc-DNA3-ROP2重组DNA疫苗具有较强的免疫原性,安全可靠,能产生良好的免疫保护作用。Objective To prepare a DNA acid vaccine for control of Toxoplasmosis in human and animals and evaluate its the immunogenicity and the immunization. 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 nuclei acid vaccine. Immunogenicity of the vaccine was evaluated through detecting immunity indexes. Challenge experiment with Toxoplasm gondii RH strain was conducted. The safety and protective effect of this vaccine was appraised. Results The specific gene fragment of ROP2 about 1.7kb in length was amplified. Eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. ROP2 nuclei 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 nuclei acid vaccine was safe, its immunogenicity was 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 DNA疫苗 免疫保护作用 

分 类 号：R382.5[医药卫生—医学寄生虫学]