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目的研究弓形虫核酸疫苗,以用于弓形虫病的防治。方法根据ROP2基因序列,设计合成能表达ROP2完整蛋白基因的扩增引物,扩增ROP2靶基因并克隆于PUCm-T载体,然后再亚克隆于真核表达载体pc-DNA3质粒中,制备弓形虫pc-DNA3-ROP2核酸疫苗;应用裸pc-DNA3-ROP2免疫小鼠,通过免疫学指标的检测,评价小鼠的免疫应答反应,最后应用弓形虫RH株攻击实验,评价出该疫苗的有效保护率、安全性和在人类及畜类的应用价值。结果以ROP2基因为模板,PCR扩增出1.7kbDNA条带,与预想结果一致;将扩增DNA回收并成功的克隆了ROP2(PUCm-T-ROP2),然后亚克隆并构建了pc-DNA-ROP2重组体,通过酶切、PCR扩增和基因测序证明亚克隆的重组子正确,ROP2扩增基因包含了ROP2蛋白基因读码框内的完整序列;在应用疫苗免疫接种小鼠结果显示,疫苗接种能使小鼠产生强烈的细胞、体液免疫反应;攻击实验结果显示,实验组小鼠的存活时间明显延长,并且开始死亡时间明显延迟(P<0.001),实验组9只小鼠中有1只长期存活;在实验的整个过程中,没有发现小鼠对免疫接种的毒性和异常反应。结论该疫苗免疫原作用强,具有很好的开发应用价值,目前可在动物群体中试验应用。
Objective To study Toxoplasma nucleic acid vaccine for the prevention and treatment of toxoplasmosis. Methods According to the sequence of ROP2 gene, we designed and synthesized the amplification primer that can express the complete ROP2 gene, amplified the target gene of ROP2, cloned it in PUCm-T vector and subcloned it into the eukaryotic expression vector pc-DNA3 to prepare Toxoplasma gondii pc-DNA3-ROP2 DNA vaccine. Immunized mice with naked pc-DNA3-ROP2 were used to evaluate the immunological response of mice by immunological detection. Finally, the Toxoplasma gondii RH strain challenge experiment was carried out to evaluate the effective protection of the vaccine Rate, safety and value in human and livestock applications. Results The 1.7 kb DNA band was amplified by PCR using the ROP2 gene as template, which was consistent with the expected results. The amplified DNA was cloned and successfully cloned ROP2 (PUCm-T-ROP2), then subcloned into pcDNA- ROP2 recombinants, subclones were confirmed by restriction enzyme digestion, PCR amplification and gene sequencing. The ROP2 gene contained the complete sequence within the reading frame of ROP2 protein gene. Immunization of mice with the vaccine showed that the vaccine Inoculation can make the mice produce strong cellular and humoral immune responses. The experimental results showed that the survival time of the mice in the experimental group was significantly prolonged, and the time to start the death was significantly delayed (P <0.001). One of the mice in the experimental group had 1 Only long-term survival; in the whole process of the experiment, did not find the mice on the immunization of toxicity and abnormal response. Conclusion The vaccine has strong immunogenicity and good development and application value. It is currently used in animal populations.