抗弓形虫感染ROP2核酸疫苗的研究  

Studies on ROP2 gene/vaccine for control of toxoplasma infection

作　　者：韩广东[1] 魏庆宽[1] 张佃波[1] 李瑾[1] 崔勇[1] 李桂萍[1] 王洪法[1] 刘玉冰[1] 刘克义[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：《中国热带医学》2005年第6期1161-1167,共7页China Tropical Medicine

基　　金：山东省科技发展计划项目(编号:022130133)

摘　　要：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 carry out study on nucleic acid vaccine for control of toxoplasmosis in human and animals. Method Primers were designed and synthesized and ROP2 gene was amplified , then cloned into plasmid PUCm - T, again recombined into an eukaryotic expression vector of pc - DNA3 and named pc - DNA3 - ROP2; The insert was identified by enzyme digestion,PCR amplification,sequence analysis and the naked pc - DNA- ROP2 as a vaccine was inoculated into mice; blood was collected one day before and 2 weeks after each inoculation; 4 mice in each group were killed to get lymph notes for lymphocyte phenotype analysis and cytokines detection 14 days after third immunization; challenge experiment with Toxoplasma gondii RH strain was conducted; the protective effect and safety of DNA vaccine , its application value for use in the human and animals were also evaluated. Result The specific gene fragment of ROP2 was amplified by PCR from genomic DNA of RH T. gondii isolated, the fragment was about 1.7kb in length. The insert was succsesfully cloned into plasmid PUCm - T and then subeloned into pc - DNA3 vector. Sequence analysis showed that the insert fragment include 1683bp of open reading frame and code 561 amino acid. After inoculation, the Lymphocyte phenotype was analyzed and CD4 ＋ proliferated sharply （ P〈0.01 ） and CD4＋/CD8＋ ratio apparently increased （ P〈0.01 ）. The level of the cytokines （ IL-2, IL-4, IL-6, IL-12, IFN-γ and TNF） detected in blood and in culture supernatant of spleen cell and lymph cell were higher than that in same sample of control group. The antibody was detected by ELISA using a recombinant expressed ROP2 coating plate and the antibody generated by pc- DNA3 - ROP2 immunization, it was shown that the antibody could recognize ROP2 expressed, the level of antibody and the number of mice with positive antibody was sharply increased following three time inoculation, especially after the third immunization, the GMRT reached 1：〉1194.23. After challenge with 500 tachyzoites of th

关 键 词：弓形虫 ROP2基因重组 核酸疫苗 免疫保护作用 

分 类 号：R531.8[医药卫生—内科学]