微小牛蜱Bm86/Bm91双基因重组蛋白和DNA疫苗联合免疫效果及机制研究

Boophilus microplus causes serious harm to pillar industries of cattle industry and sheep farming in Xinjiang. It is an urgent problem to prevent Boophilus microplus and its borne diseases effectively. The immune prevention method shows great application prospect and other studies have shown that combined immunization of protein and DNA vaccine can enhance the immune effects. However, the research of combining immune effect and mechanism of recombinant protein and DNA vaccine control Boophilus microplus is unknown. Thus further research is needed. In order to construct Bm86 and Bm91 digenic DNA vaccine and co-expresse digenic recombinant protein, Bm86 and Bm91 which have high immunogenicity antigen genes of Boophilus microplus were selected and obtained to take the advantage of common expression under the basis of breeded Boophilus microplusby the laboratory. Experimental animals will be vaccinated with digenic DNA vaccine and recombinant protein. The differences in cellular immunity, humoral immunity, immune protection ratio and damage of Boophilus microplus with control groups would be applied to evaluate combined immune efficacy and elucidate combined immune mechanism. Consequently, the study has theoretical significance and practical value for development of new immune preparation that could effectively prevent and control Boophilus microplus and its borne diseases, moreover, it may provide theoretic and experimental basis to new immune prevention strategies of other ticks and parasites.

微小牛蜱对新疆地区两大支柱产业-奶牛业和养羊业危害严重，有效防制微小牛蜱及其蜱传病已成为迫切需要解决的问题。免疫防制具有很强的应用前景，已有研究表明蛋白与DNA疫苗联合免疫可增强免疫效果，而对微小牛蜱双基因重组蛋白和DNA疫苗联合免疫效果及机制研究尚属空白。本项目在前期已人工饲养微小牛蜱和获得微小牛蜱免疫原性较好的2个基因Bm86和Bm91的基础上，拟利用共表达的优势，构建微小牛蜱Bm86和Bm91双基因DNA疫苗，同时进行原核共表达Bm86和Bm91重组蛋白，将构建的双基因DNA核酸疫苗和双基因重组蛋白疫苗联合免疫实验动物。通过测定体液免疫、细胞免疫、免疫保护率以及对微小牛蜱的损伤等指标与对照组的差异，进而对联合免疫效果进行评价并阐明其免疫机制。对研制新型抗微小牛蜱免疫制剂并有效预防和控制微小牛蜱及其蜱传病具有重要的理论意义与实际价值，为其他蜱及寄生虫免疫防制新策略提供理论与实验依据。

项目研究过程中发表各类科研论文12篇，其中SCI期刊刊出4篇和CSCD期刊刊出4篇，SCI期刊录用2篇；授权实用新型专利5项，申请发明专利2项；培养青年骨干教师1名，该教师在此研究过程中发现科学问题、申报并立项国家自然科学基金进行后续研究；培养3名硕士研究生和30余名本科生，毕业后大部分工作于新疆南疆生产一线。具体研究成果概述：获得了新疆微小牛蜱样品及其12S、Bm86和Bm91基因及其序列，并登录GenBank数据库获得了登录号；利用原核表达系统分别成功表达、纯化了微小牛蜱Bm86和Bm91蛋白；利用含有增强子和佐剂单元的pVAXl-CpG载体，成功构建了微小牛蜱Bm86和Bm91双基因真核表达载体pVAXl-CpG-Bm；进行了微小牛蜱两种蛋白和双基因DNA疫苗混合免疫动物试验，探索了免疫效果。其次，在研究过程中20个县市60多个采样点采集2万余只蜱，掌握了新疆南疆地区蜱分布、种类（2科5属12种）及优势蜱种，了解了蜱携带部分病原种类和基因特征；同时，采样过程中获得了新疆羊蜱蝇并掌握了其分布，鉴定与分析了其携带几种病原的特征。另外，在研究期间在新疆南疆基层开展了科技服务和疾病防控培训工作，为当地老百姓诊断疾病解决棘手问题，提出可靠防制方案减少经济损失，为当地疫病的防控和群众家庭增收做了贡献。