内脏利什曼病多表位DNA疫苗和多表位抗原疫苗的构建及免疫策略研究

In this study, the Amastin, A2 and FML genes of Leishmania donovani were analyzed by the DNA star soft and their antigenic epitopes were calculated. To increase the protective immunity of the recombinant vaccine, the fragments containing antigenic epitopes of T cell and CTL of the Amastin, A2 and FML genes and linker were linked with pCDNA3.1 to construct pCDNA3.1-amastin/A2, pCDNA3.1-amastin/fml, pCDNA3.1-A2/fml and pCDNA3.1-amastin/A2/fml with PCR.The immunogenicity and protective efficacy of 4 muti-epitopes DNA vaccine of Leishmania donovani were analyzed in animal experiments to determent the best muti-epitopes DNA vaccine. The best muti-epitopes DNA vaccine in immunogenicity and protective efficacy were linked with pET32a(+) to construct pET32a(+)-AAF with PCR，expressed in E.coli to obtain AAF muti-epitopes fusion antigens. The effect of immunifaction about immunization with DNA vaccine firstly and to enhance immunization with muti-epitopes fusion antigens secondly was analyzed in animal experiments. Our results of this project would lay good foundation for study of kala-azar vaccine development and to immunize dog for control the source of kala-azar infection.

本研究采用免疫信息学对利什曼原虫无鞭毛体蛋白（amastin）、A2蛋白和岩藻糖-甘露糖配合体（FML）进行T细胞抗原表位和CTL抗原表位分析，剔除抗原基因中不含T细胞表位和CTL表位片段，保留富含T细胞表位和CTL表位片段。选择上述3个抗原中富含T细胞表位和CTL表位的片段，再加上分子佐剂CpG，采用重叠PCR引入柔性连接肽(linker)和PCR加酶切技术构建利什曼原虫amastin/A2多表位DNA疫苗、A2/fml多表位DNA疫苗、amastin/fml/多表位DNA疫苗以及pCDNA3.1-amastin/A2/fml/多表位DNA疫苗，对4种多表位DNA疫苗进行免疫原性及免疫保护性评估以确定最佳的多表位DNA疫苗。并挑选具有最佳免疫效果的表位多肽序列，连接质粒pET32a(+)以构建真核表达质粒，表达纯化多表位抗原，通过接种犬来验证“核酸初免，蛋白加强”免疫策略的效果。

本研究采用免疫信息学对利什曼原虫无鞭毛体蛋白（amastin）、A2蛋白和岩藻糖-甘露糖配合体（FML）、amastin、kmp11和gp63进行T细胞抗原表位和CTL抗原表位分析，剔除抗原基因中不含T细胞表位和CTL表位片段，保留富含T细胞表位和CTL表位片段。选择上述6个抗原中富含T细胞表位和CTL表位的片段，再加上分子佐剂CpG，采用重叠PCR引入柔性连接肽(linker)和PCR加酶切技术构建8个利什曼原虫多表位DNA疫苗。通过体外实验和动物实验筛选出最佳免疫原性和免疫保护性的利什曼原虫CaNA2-kmp11多表位DNA疫苗和amastin-gp63多表位DNA疫苗，并对2种多表位DNA疫苗进行免疫原性及免疫保护性评估。并通过基因克隆构建利什曼原虫CaNA2-kmp11多表位抗原疫苗和amastin-gp63多表位抗原疫苗，通过动物实验来验证“核酸初免，蛋白加强”免疫策略的效果。本研究构建了2种内脏利什曼病疫苗，为通过免疫犬来阻断黑热病流行提供了实验数据。