甲/戊型肝炎重组抗原的表达、纯化及与传统甲肝疫苗免疫原性的比较

Expression,Purification and Immunogenicity of the Recombinant Antigen of the Hepatitis A and Hepatitis E Viruses and Comparison with the Traditional Vaccine against HAV

目的比较基因工程重组抗原与传统疫苗的免疫原性;探讨同一载体表达不同抗原的能力和抗原间的相互作用。方法将编码甲型肝炎病毒Vp1aa24-171和戊型肝炎病毒ORF2aa431-615的重组表达质粒pBV220-EAAg342及pBV220-AEAg342转化入大肠杆菌BL-21,筛选出高效表达的菌种进行目的蛋白的表达。用DEAE-sepharose FF和CM-sepharoseFF离子层析交换柱纯化表达重组蛋白,采用SDS-PAGE、Western blotting方法进行分析鉴定。用纯化的重组蛋白及传统甲肝疫苗灭活疫苗与减毒活疫苗免疫小鼠,比较特异性抗体的产生水平。结果表达的重组蛋白相对分子质量约为36000,表达量约占菌体总量的25%;且重组嵌合抗原EAAg342可以形成直径10～20nm的类病毒(virus-like particles,VLP)颗粒。Western blotting证实2种重组蛋白均能分别与甲型肝炎和戊型肝炎患者阳性血清发生特异性反应。免疫小鼠后可诱导产生高滴度的抗戊肝特异性抗体;而抗甲肝特异性抗体水平与传统甲肝疫苗相比较低。结论使用原核系统表达的甲/戊肝重组抗原能够高效表达,2种不同的融合蛋白均分别具有良好的抗原性和免疫原性,具有开发双价疫苗及同时诊断甲型戊型肝炎试剂盒的前景。

Objective To compare the immunogenicity between the genetically engineered recombinant antigen and the traditional vaccine against hepatitis A virus（HAV） and study the ability of expressing different antigens by the same vector and the interaction among the different antigens. Methods Plasmid pBV220-EAAg342 and pBV220-AEAg342 constructed previously which code for HAV vp1 aa24-171 and hepatitis E virus（HEV） ORF2 aa431-615 genes were amplified and identified,then transformed into E.coliBL21 and induced to express the target protein. The expressed proteins were purified by DEAE-Sepharose and CM-Sepharose,identified by SDS-PAGE and Western blotting. KM mice were immunized with the purified recombinant antigen and the traditional HAV inactivated vaccine and the attenuated live vaccine by subcutaneous route and the specific antibody level was determined by ELISA. Results The molecular mass of the expressed recombinant antigen was about 36 000; the expressed product was about 25% in total bacterial proteins. The recombinant EAAg342 could assemble into virus-like particles（VLP） of 10~20 nm radiuses. It indicated that the fusion protein could have specific reaction with HAV and HEV positive sera by Western blotting. The geometric mean titers（GMT） of anti-HEV IgG antibody induced by both different recombinant antigens were high. However the levels of anti-HAV IgG antibody induced by the two recombinant antigens were lower than the one induced by the traditional HAV vaccines. Conclusion The recombinant antigen of the hepatitis A and hepatitis E viruses could be expressed in high yield by procaryotic expression system. The two different fusion proteins both have good antigenicity and immunogenicity respectively. The data provided experimental confirmation for development of bivalent vaccine and serologically diagnostic kit for HAV and HEV.