猪源戊型肝炎病毒ORF2蛋白生物信息学分析及真核表达研究

Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus

【目的】分析猪源戊型肝炎病毒(Swine hepatitis E virus,sHEV)4型ORF2蛋白的结构和功能,筛选具有保护性抗原的基因片段并表达蛋白,为研究其作为潜在保护性抗原提供候选蛋白。【方法】对sHEV 4型ORF2蛋白进行生物信息学分析,选取具有潜在保护性抗原蛋白的基因片段ORF 2(128/140),通过PCR扩增、酶切后克隆至昆虫细胞表达载体,转染sf9细胞,通过Western blotting鉴定分析表达蛋白p128/p140。【结果】生物信息学分析显示,sHEV 4型ORF2蛋白存在2个稳定蛋白:p128和p140,分别含有496和476个氨基酸,二者皆为稳定蛋白,无跨膜区,蛋白二级结构主要以无规则卷曲为主。p128蛋白有11个T细胞表位、21个B细胞表位;p140蛋白有14个T细胞表位、18个B细胞表位。成功构建ORF 2(128)、ORF 2(140)2个基因的昆虫细胞表达载体,转染sf9细胞后经Western blotting检测发现,表达蛋白可被His标签单抗、sHEV抗体阳性血清识别。【结论】试验发现2个sHEV具有潜在保护性抗原的p128和p140蛋白,均可在sf9细胞中表达,且具有免疫活性。结果为进一步研究sHEV ORF2蛋白的功能和新型疫苗的研发提供了材料。

【Objective】The purpose of this study was to analyze the structure and function of ORF2 protein of Swine hepatitis E virus(sHEV)genotype 4,screen the gene fragment with protective antigen and express,so as to provide a candidate protein for the study of its potential protective antigen.【Method】The bioinformatics analysis of sHEV genotype 4 ORF2 protein was carried out.The gene fragment ORF 2(128/140)with potential protective antigen protein was selected,amplified by PCR and digested,which cloned into insect cell expression vector,and transfected into sf9 cells.The expressed protein p128/p140 were analyzed by Western blotting.【Result】Bioinformatics analysis showed that the ORF2 protein of sHEV genotype 4 had two stable proteins(p128 and p140),which contained 496 and 476 amino acids,respectively.Both of them were stable proteins,without transmembrane regions,and the secondary structure of the protein was mainly random coil.p128 protein had 11 T cell epitopes and 21 B cell epitopes,and p140 protein had 14 T cell epitopes and 18 B cell epitopes.Insect cell expression vector of ORF 2(128)and ORF 2(140)genes were successfully constructed,and transfected into sf9 cells,Western blotting results showed that the expressed proteins could be recognized by His-tag monoclonal antibody and positive serum of sHEV antibody.【Conclusion】p128 and p140 proteins with potential protective antigen of sHEV were found in this study,and the two proteins were expressed in sf9 cells with immune activity,which provide materials for further study of the function of sHEV ORF2 protein and the development of new vaccines.