基于组学的田鼠巴贝虫分泌蛋白预测分析及其诊断抗原免疫高通量筛选

Babesia microti is a kind of protozoa transmitted by ticks and can parasitize in the erythrocytes of human beings or other mammals and cause babesiosis. Babesiosis is an emerging parasitic disease which is seriously threatening the public health. The identification of B. microti by morphology is difficult, meanwhile the molecular diagnosis is restricted in application on field. Relatively, serological diagnostics are the most convenient diagnostic tools to be carried out. But, so far, the antigens applied in serological diagnosis of babesiosis were insufficient. What’s more, the sensitivities and specificities of antigens of B. microti currently used are limited. Secreted proteins of parasites can contact with the immune system of the hosts directly and regulate the immune reactions of hosts at different infection stages. So the secreted proteins can be the immunologically important antigens or other targets. The researches on omics of B. microti are deficient and need to be improved. Based on the reasons above, this research is carried on by the following steps: 1) downloading the database of B. microti from Pubmed and PiroplasmaDB to complete the original database of B. microti; 2) making use of the bioinformatics methods, such as softwares of SignalP4.1 Server, TMHMM Server v.2.0 and big-PI Predictor etc. to predict the signal peptides, transmembranes regions and GPI-anchored respectively and construct a local database of secreted proteins of B. microti ; 3) applying the high-throughput systems combined by In-fusion cloning, Wheat germ cell-free expression system and protein chips to hight-hroughput screening and analysis the candidates from secreted proteins database and carrying on further evaluations of these candidates in diagnosis of babesiosis on fields. By the studies above, some antigens and drug candidates will be screened and identified. It will improve the prevention and treatment of babesiosis.

田鼠巴贝虫是寄生于人或其它哺乳动物红细胞内一重要蜱媒原虫，可引起巴贝虫病并严重威胁人类健康。田鼠巴贝虫形态学鉴定困难，分子生物学诊断受现场应用限制，血清学诊断相对方便快捷，然其诊断抗原有限，敏感性与特异性尚不能满足实际诊断需要。虫体分泌蛋白与宿主免疫系统直接接触、影响宿主免疫调节，是免疫学上重要的抗原与靶点，目前田鼠巴贝虫组学研究基础薄弱，本研究通过1) Pubmed和梨浆虫PiroplasmaDB等公共数据库整理田鼠巴贝虫原始数据库；2) 采用SignalP4.1 Server、TMHMM Server v.2.0、Big-PI Predictor等生物信息学软件预测分析信号肽、跨膜区和GPI锚定点等构建田鼠巴贝虫分泌蛋白本地数据库；3) 无缝克隆、麦胚无细胞蛋白表达系统和蛋白芯片等高通量技术筛选鉴定若干关键候选分子；评价其应用于巴贝虫病现场血清学诊断意义，为巴贝虫病诊断和治疗提供靶标。

田鼠巴贝虫是寄生于人或其它哺乳动物红细胞内一重要蜱媒原虫，可引起巴贝虫病并严重威胁人类健康。田鼠巴贝虫形态学鉴定困难，分子生物学诊断受现场应用限制，血清学诊断相对方便快捷，然其诊断抗原有限，敏感性与特异性尚不能满足实际诊断需要。虫体分泌蛋白与宿主免疫系统直接接触、影响宿主免疫调节，是免疫学上重要的抗原与靶点，目前田鼠巴贝虫组学研究基础薄弱，本研究通过Pubmed和梨浆虫PiroplasmaDB等公共数据库整理田鼠巴贝虫原始数据库；结合SignalP4.1 Server、TMHMM Server v.2.0、Big-PI Predictor等生物信息学软件预测分析信号肽、跨膜区和GPI锚定点等构建田鼠巴贝虫分泌蛋白本地数据库；并据此筛选出204个序列扩增得到200个序列，对于扩增得到的序列再进行In-fusion无缝克隆、麦胚无细胞蛋白表达系统表达得到169个蛋白，通过蛋白芯片高通量技术筛选应用不同感染时期BALB/C小鼠血清筛选高效克隆表达的B.microti蛋白。通过蛋白芯片筛选得到10个高度免疫反应候选分子（5.9%，10/169），其中大多数（80%，8/10）以前没有被鉴定过，而这些候选分子有可能成为巴贝虫病诊断候选抗原或药物靶标、免疫靶标等候选分子。. 由于本实验室缺乏临床发热病人血清资源等不足，对于以上蛋白芯片筛选蛋白除了进行感染不同时期小鼠血清评价其诊断效能外对其免疫功能进行了评价。本课题选取其中一个蛋白（BmSP44）用大肠杆菌融合表达系统进行克隆表达，成功表达得到该蛋白，纯化后该蛋白约24kD，ELISA结果表明感染田鼠巴贝虫1-4周小鼠血清均可被该蛋白识别，结果与蛋白芯片结果一致。BmSP44主动免疫小鼠4周后，攻击感染田鼠巴贝虫，经外周血血涂片或荧光定量PCR两种方法监测巴贝虫虫浓度，结果表明该蛋白可诱导一定的免疫保护力，免疫组小鼠感染田鼠巴贝虫后其体重、体温和血红蛋白含量较对照组无明显改变。田鼠巴贝虫感染血涂片经体外间接荧光抗体IFA试验结果表明，该蛋白BmSP44主要分布在巴贝虫胞质中，与其分泌蛋白属性相符，也为该蛋白诱导免疫保护作用的体外实验证据，而其免疫小鼠体内相应细胞因子与抗体动态变化正在检测中。