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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 a cheap and effective anti-Toxoplasma nucleic acid vaccine for the prevention and treatment of toxoplasmosis in humans and livestock. Methods Toxoplasma gondii RH strains were collected and purified for genomic DNA extraction. According to the ROP2 gene sequence, ROP2 gene primers were designed and synthesized. The ROP2 gene fragment was amplified by PCR, restriction enzyme digestion, ligation, sequencing and cloned into eukaryotic expression vector Vector pc-DNA3 plasmid, prepared anti-toxoplasma pc-DNA3-ROP2 nucleic acid vaccine. The vaccine was used to immunize mice and the serum antibody was detected by ELISA. The immunogenicity of the vaccine was identified by Western blot. Toxoplasma gondii RH strain was used in animal experiments to evaluate its safety and immunoprotection. Results Toxoplasma gondii genomic DNA was used as a template to amplify the 1.7 kb ROP2 gene fragment and cloned into pc-DNA3 plasmid. The pc-DNA3-ROP2 recombinant plasmid was successfully constructed. The sequencing results showed that the recombinant plasmid contains the complete sequence in the reading frame of ROP2 protein gene and can express the antigen protein of ROP2 completely. Immunization of mice with this vaccine induced a strong cellular and humoral immune response, significantly prolonging the survival time of the mice in the experimental group after infection with the helminth worm, and the survival rate was 77.8% after 192 h of infection by the helminth. The empty plasmid and PBS The survival rate of the control group was 0, the difference was statistically significant (P <0.001); experimental mice immunized without toxicity and abnormal reaction. Conclusion The nucleic acid vaccine has strong immunogenicity, safe and reliable, can induce mice to produce good immune protection, has good development and application value.