细粒棘球蚴Eg95重组表位蛋白的免疫特性研究

A study of the immune response generated by recombinant Echinococcus granulosus 95 proteins tagged with epitopes

目的研究Eg95-1、Eg95-2、Eg95-3表位蛋白的抗原性,为细粒棘球蚴多表位疫苗的研制奠定基础。方法构建细粒棘球蚴Eg95-1、Eg95-2、Eg95-3抗原原核表达载体并体外诱导表达重组蛋白,纯化后免疫家兔,制备Eg95抗原多克隆抗体,采用Western blot检测Eg95-1、Eg95-2、Eg95-3抗原与Eg95多抗的反应性;分别用重组蛋白Eg95-1、Eg95-2、Eg95-3免疫BALB/c小鼠,采用ELISA检测小鼠血清特异IgG水平,MTT法检测脾淋巴细胞增殖。结果用Western blot检测重组蛋白Eg95-1、Eg95-2、Eg95-3均能与Eg95多抗血清反应,以Eg95-2和Eg95-3反应性较强。用Eg95-1、Eg95-2、Eg95-3重组抗原免疫小鼠后,随着免疫次数的增加和时间的延长,小鼠血清IgG水平升高,小鼠脾淋巴细胞在体外经ConA和Eg95抗原刺激诱导发生增殖。结论构建的3个表位均为Eg95的有效抗原表位,用重组抗原Eg95-1、Eg95-2、Eg95-3免疫小鼠后均可诱导以产生IgG为主的体液免疫应答,因此构建的3个表位蛋白抗原可用于细粒棘球蚴多表位肽疫苗的研制。

Objective To investigate the antigenicity of Eg95-1,Eg95-2,and Eg95-3protein epitopes in order to lay the foundation for the development of an epitope-based vaccine against Echinococcus granulosus. Methods Vectors for the prokaryotic expression of the antigens Eg95-1,Eg95-2,and Eg95-3were constructed;expression of the antigens was induced in vitro and the recombinant proteins were purified.The recombinant proteins were used to immunize rabbits to obtain polyclonal antibodies against Eg95 antigens.The specific immune response of polyclonal antibodies against Eg95 to the antigens Eg95-1,Eg95-2,and Eg95-3 was determined with Western blotting.The recombinant proteins Eg95-1,Eg95-2,and Eg95-3were used to immunize BALB/c mice and ELISA was used to determine IgG levels in serum.The proliferation of lymphocytes in the spleen was detected with MTT. Results The recombinant proteins Eg95-1,Eg95-2,and Eg95-3reacted to Eg95 antiserum according to Western blotting.Eg95-2and Eg95-3in particular reacted strongly,suggesting that maybe they are effective as epitopes of the Eg95 antigen.After mice were immunized with the recombinant antigens Eg95-1,Eg95-2,and Eg95-3,the level of IgG increased（P〈0.05）.When splenic lymphocytes were stimulated with ConA and Eg95 antigen in vitro,they proliferated（P〈0.05）,and that proliferation increased over time. Conclusion The 3constructed epitopes were all effective as epitopes of the Eg95 antigen.Eg95-2and Eg95-3have stronger antigenicity and could be used to develop an epitope-tagged peptide vaccine against E.granulosus.Immunization with the recombinant antigens Eg95-1,Eg95-2,and Eg95-3induced a humoral immune response primarily in the form of IgG production.