一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性  

Expression, purification and immunogenicity of a novel hepatitis E virus-like particle

作　　者：董晨[1] 孟继鸿[1] 

机构地区：[1]东南大学医学院病原生物学与免疫学系,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2006年第3期339-342,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No.30271231;30271212);江苏省自然科学基金资助项目(No.BK2002053);江苏省卫生厅医学科技发展基金资助项目(No.H200115);教育部留学回国人员科研启动基金资助项目(No.2004-176)

摘　　要：目的：以非包涵体形式表达含戊型肝炎病毒（HEV）中和抗原表位的新型HEV重组蛋白,并对其进行鉴定和分析.方法：将HEV开放阅读框架2（ORF2）编码452～617位氨基酸的基因片段连接到载体pET28a（＋）,转化大肠杆菌,获取表达克隆.以Ni-NTA层析柱纯化表达的蛋白,并用SDS-PAGE、Western blot和直接电镜负染等方法分析鉴定,最后免疫小鼠检测特异性抗体的产生水平.结果：表达的重组蛋白天然可溶,相对分子质量（Mr）约为22 000,并可形成直径为20 nm左右的病毒样颗粒,能与戊型肝炎患者血清发生Westem blot阳性反应,免疫小鼠后可诱导产生高滴度的特异性抗体.体外中和试验显示产生的抗体具有中和HEV的活性.结论：原核表达的、含HEV中和抗原表位的、长度仅166个氨基酸的HEV ORF2近3＇端编码蛋白能够形成病毒样颗粒,而且该新型病毒样颗粒具有良好的抗原性和免疫原性.AIM： To express and characterize a novel hepatitis E virus （HEV） recombinant protein which contains HEV neutralization epitope（s）. METHODS： The gene fragment encoding for amino acid 452 - 617 of HEV open reading frame 2 protein （pORF2） was inserted into the plasmid pET28a（ ＋ ）. The recombinant plasmid was used to transform the E. coli BL21 （ DE3 ） strain. The recombinant protein was expressed by IPTG induction, purified by Ni-NTA chromatography and analyzed by SDS-PAGE, Western blot and electromicroscopy. Immune responses to the recombinant protein were determined by the immunization of mice. RESULTS： The expressed HEV recombinant protein was in a naturally-soluble form with a molecular weight of 22 000. The protein assembled into a virus like particle （VLP） with a diameter of approximate 20 nanometer. Moreover, this novel protein was reactive to serum samples obtained from the patients with HEV infection. After the mice were immunized with the protein, they developed anti-HEV antibodies which could neutralize HEV infection in cell culture. CONCLUSION： An E, coli-expressed recombinant protein containing 166 amino acid of HEV pORF2 can form VLP with good immunogenicity and antigenicity. This novel HEV VLP is valuable for the development of HEV vaccine and new diagnostic kit.

关 键 词：戊型肝炎病毒 重组蛋白 中和抗原表位 病毒样颗粒 

分 类 号：R512.6[医药卫生—内科学]