【作者】 洪艳； 陈勇； 杨连华； 王怡婷； 经络； 胡华军；

【Author】 HONG Yan, CHEN Yong, YANG Lian-hua, Wang Yi-ting, Jing Lou, Hu Hua-jun (Zhejiang Academy of Medical Sciences, Hangzhou 310013, China)

【机构】 浙江省医学科学院生物工程研究所；

【摘要】 目的对含有HEV主要抗原表位的嵌合蛋白进行表达与纯化,并对其免疫原性进行研究.方法将含有一段HEV ORF2基因片段(394aa-660aa)和HEVORF3全基因的表达质粒pHEV23在大肠杆菌中进行异丙基硫代半乳糖苷(IPTG)诱导表达,其表达产物经Ni2+ -NTA树脂亲和纯化,透析复性.纯化蛋白免疫小鼠,用ELISA检测小鼠血清IgG抗体,T细胞增殖反应检测细胞免疫应答.以Western-blot检测纯化蛋白对患者阳性血清的免疫反应性. 蛄果纯化蛋白的分子量为55KDa,纯度达90%以上;实验组特异性抗体水平明显高于对照组;T细胞增殖反应实验组与对照组比较有极显著差异.纯化蛋白能与患者阳性血清呈特异反应.结论嵌合蛋白具有良好的免疫原性和免疫反应性,为进一步研究开发HEV诊断试剂盒及基因工程疫苗提供了基础.更多还原

【Abstract】 Objective To explore the immunogenicity of a HEV recombinant chimeric protein expressed in E. coli. Methods Recombinant plasmids pHEV23 , which contains a fragment of HEV ORF2 gene and a full length of ORF3 gene, was transformed into E. coli B1-21. Expressed chimeric protein was analyzed by SDS-PAGE and Western -blot. Blab/c mice were immunized intramuscularly with purified protein twice. Antibody responses and T cell proliferation were then detected. Results The SDS-PAGE showed a dominant additional protein with molecular weight of 55KDa which could specially react with the sera of patients with hepatitis E.Expressed chimeric protein could induced high specific anti HEV IgG and T lymphocyte proliferation. Conclusion Expressed chimeric protein have a good immunogenicity and may be the promising candidate for a HEV vaccine.更多还原