【作者】 魏庆宽； 李瑾； 傅婷霞； 柏雪莲； 崔勇； 王用斌； 张佃波； 王洪法； 付斌； 刘玉冰； 宰德福； 黄炳成； 刘克义； 韩广东；

【Author】 Wei Qing - Kuan, Zhang Dian - Bo, Li Jin, Fu Ting - Xia, Bai Xue - Lian, Cui Yong, Wang Yong - Bin, Wang Hong - Fa, Fu Bin, Liu Yu - Bing, Zai De - Fu, Huang Bing - Cheng, Liu Ke - Yi, Han Guang - Dong Molecular medicine department ,Shandong Institute of Parasitic Disease, Jining 272033, China

【机构】 山东省寄生虫病防治研究所分子医学部；

【摘要】 目的研究一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基因的引物,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,以制备抗弓形虫pc-DNA3 -ROP2核酸疫苗。应用该疫苗免疫小鼠,通过免疫学指标的检测,来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。来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． 7kb 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。应用该疫苗免疫接种小鼠, 能产生强烈的细胞、体液免疫反应,使实验组小鼠的存活时间明显延长,开始死亡时间也明显延迟(P<0． 001);实验全程未发现免疫接种对小鼠的毒性及异常反应。结论该核酸疫苗具有很强的免疫原性,安全可靠,能产生良好的免疫保护作用,具有很好的开发应用价值。更多还原

【Abstract】 Objective: To carry out study on an cheap and effective nucleic acid vaccine for control of Toxoplas-mosis 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 eu-karyotic 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. The gene fragment cloned into pc - DNA3 was correct. 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.更多还原