褐黄血蜱HSC70的抗凝机制及免疫原性研究

The aims of this study are to illuminate the anticoagulant mechanism and immunogenicity of HSC70 in Haemaphysalis flava, further to provide a new clue for controlling the tick infestations. Based on our previous proteomic and transcriptomic datasets of salivary gland of H. flava, we cloned the cDNA of hsc70. It has been proved by us that the expression of HSC70 would increase during blood feeding, and HSC70 possessed anticoagulant activity. However, the exact anticoagulant mechanism, immunogenicity and anti-tick efficacy of HSC70 are not clear. In view of this, we will produce recombinant proteins of HSC70 in eukaryotic expression system. The anticoagulation mechanism of HSC70 will be clarified by analyzing the composition of rHSC70-hemagglutination factor complex. The rabbits will be immunized by rHSC70, then the antibody titer and the CD4+/CD8+ T cells will be detected to assess the immunogenicity of HSC70. Base mutation will be induced in hsc70, the recombinant proteins fused with TKD will be produced, then the recombinant proteins fused with TKD will be used to immunize the rabbits to observe the effect of enhancing immunogenicity. Finally, the ticks will be fed on the high antibody titer rabbits to assess the anti-tick efficacy of HSC70 and the recombinant proteins fused with TKD. By this study, we will clarify the anticoagulant mechanism of HSC70. At the same time, it also will be help to evaluate the availability of HSC70 candidates for universal anti-tick vaccine development.

本项目旨在探明褐黄血蜱HSC70的抗凝机制和免疫原性，为探索控蜱防病提供新材料和策略。基于对褐黄血蜱唾液腺蛋白质组学和转录组学的前期研究，课题组克隆出了hsc70的cDNA；探明了褐黄血蜱吸血与HSC70表达的关系；证实了HSC70的抗凝活性。但HSC70确切的抗凝机制、免疫原性和抗蜱效果强弱还不明确。本研究拟以真核表达系统制备rHSC70，通过分析rHSC70-血凝因子复合物组成，明确HSC70的抗凝机制；rHSC70免疫家兔，测定抗体滴度和CD4+、CD8+ T细胞百分比，评估HSC70的免疫原性；突变hsc70的相应碱基，制备具TKD肽的融合蛋白并免疫家兔，观测增强免疫原性的效果；饲蜱于高抗体效价兔，考察HSC70和具TKD肽融合蛋白的抗蜱效果。通过本项目的研究，理论上将明确HSC70的抗凝机制，实践上对于明确HSC70能否作为优质保护性抗原开发抗蜱疫苗具有重要意义。

项目围绕化药灭蜱贻害无穷，免疫灭蜱疫苗缺失，研究通过重叠延伸法，成功将HSP70-b2和HSC70中编码类TKD肽的核酸序列突变为了编码TKD肽的核酸序列，获得了HSP70-b2M、HSC70TKD ORF框序列；通过原核表达系统制备了rHSP70-b2和rHSP70-b2M、rHSC70和rHSC70TKD，经测定，上述四种重组蛋白均对PT、APTT无影响，但均可显著延长TT和降低血浆中FIB含量，说明重组蛋白具有一定抗凝活性，且在体外可通过延长凝血酶时间和降低纤维蛋白原含量而发挥抗凝血作用；通过真核表达系统制备了rHSC70和rHSC70TKD，经测定，rHSC70和rHSC70TKD仍具有显著延长TT和降低血浆中FIB含量的作用，且rHSC70对TT时间的抑制作用极显著强于rHSC70TKD，rHSC70TKD对降低血浆中纤维蛋白原含量的作用极显著强于rHSC70；通过将rHSP70-b2和rHSP70-b2M、rHSC70和rHSC70TKD-弗氏完全佐剂和弗氏不完全佐剂乳剂免疫家兔后，rHSP70-b2和rHSP70-b2M第二次免疫的抗体水平明显高于首次，第三次免疫抗体水平增长不明显；且rHSP70-b2的免疫原性明显高于rHSP70-b2M，表明含有类TKD肽段的HSP70-b2免疫原性高于含TKD肽段的HSP70- b2M。rHSC70和rHSC70TKD在首次和第二次免疫后均产生了强烈的体液免疫反应，且rHSC70TKD血清抗体水平低于rHSC70。在第三次免疫后，rHSC70和rHSC70TKD血清抗体水平均得到了进一步的加强，其血清抗体效价均达到了1:124000，表明rHSC70、rHSC70TKD均可刺激动物机体产生较高的抗体水平；同时，原核表达获得rHSC70和rHSC70TKD-弗氏完全佐剂和弗氏不完全佐剂乳剂免疫家兔后，rHSC70和rHSC70TKD组的血清IFN-γ浓度显著高于阴性组，rHSC70免疫家兔的血清IFN-γ浓度显著低于rHSC70TKD免疫家兔，rHSC70组的血清IL-4浓度高于rHSC70TKD免疫家兔，表明rHSC70和rHSC70TKD可刺激机体产生一定的细胞免疫。研究为实现以疫苗控蜱目标奠定了基础，为开发人类抗血栓新型药物提供了思路。