戊型肝炎病毒ORF2片段与ORF3嵌合重组抗原的纯化与免疫学性质

目的 考察具有多个优势原表位区段嵌合表达的戊型肝炎病毒（HEV）重组抗原的免疫学性质。方法 将构建的含有3个不同HEV抗原表位基因（ORF2.1:6287-6403nt,ORF2.2:6743-7126nt,ORF3）的表达质粒pThioHisORF(2.1 2.2 3）在大肠杆菌中进行异丙基硫代半乳糖苷（IPTG）诱导表达,其表达产物P（2.1 2.2 3）在8mol/L尿素下经阳离子柱SP Sepharose FF层析纯化,透析复性,以P(2.1 2.2 3）免疫家兔和小鼠,通过ELISA检测实验动物血清及戊型肝炎患者阳性血清IgG抗体,以Western蛋白印迹检测动物免疫血清对杆状病毒系统表达的抗原片段及嵌合抗原P（2.1 2.2 3）对患者阳性血清的免疫反应性。结果 纯化的抗原纯度达90％以上;免疫动物血清中检测到特异性抗体的滴度分别为1：25600（家兔）和1：12800（小鼠）以上,抗血清能够特异识别杆状病毒系统表达的HEV抗原片段,P（2.1 2.2 3）能与患者阳性血清特异反应。结论 嵌合表达的HEV重组抗原P（2.1 2.2 3）具有良好的免疫原性和免疫反应性,为进一步研究开发HIV诊断试剂盒及基因工程疫苗提供了基础。

Purification and immunological characterization of hepatitis E virus recombinant chimeric antigen encoded by ORF2 fragments and ORF3

OBJECTIVE: To study immunological characteristics of recombinant chimeric HEV antigen. METHODS: Constructed recombinant plasmids pThioHisORF(2.1 + 2.2 + 3), which contains three HEV antigen gene fragments (ORF2.1:6287-6403nt, ORF2.2:6743-7126nt, ORF3), was transformed into E. coli and induced with IPTG. Expressed product P(2.1 + 2.2 + 3) existed in inclusion bodies, was purified by denature SP Sepharose FF cation exchange chromatography. Rabbits and rats were immunized with renatured P(2.1 + 2.2 + 3). The level of IgG in sera from experimental animals and clinical patients were examined with P(2.1 + 2.2 + 3) by ELISA. The characteristics of IgG of immunized animals interacted with recombinant antigen expressed by baculovirus system as well as recombinant chimeric antigen interacted with clinical patients sera were evaluated by Western-blotting. RESULTS: High titer of IgG antibodies, 1:25,600 in rabbits and 1:12,800 in rats, were detected after immunized with P(2.1 + 2.2 + 3). Furthermore, recombinant antigen expressed by baculovirus system was specifically recognized by IgG of experimental animal immunized with P(2.1 + 2.2 + 3), and the purified recombinant chimeric antigen P(2.1 + 2.2 + 3) was specifically reacted with the IgG of clinical patients. CONCLUSIONS: Recombinant chimeric antigen appears a promising strategy for detection of and prevention from HEV infection.