戊型肝炎病毒ORF2截短蛋白p154在毕赤酵母中的分泌性表达及其抗原性-东南大学学报医学版

戊型肝炎病毒ORF2截短蛋白p154在毕赤酵母中的分泌性表达及其抗原性

单位：东南大学医学院, 江苏南京 210009

分类号：R373.21;G782;Q784

出版年·卷·期（页码）：2014·33·第五期（564-568）

摘要：

目的：利用毕赤酵母系统表达戊型肝炎病毒（HEV）ORF2编码蛋白第452~605氨基酸多肽（p154），探讨毕赤酵母表达系统用于研发戊型肝炎基因工程疫苗的可能性。方法：采集戊型肝炎患者粪便，进行RT-PCR检测和测序鉴定，并依此为模板扩增ORF2p154基因，克隆到表达载体pPICZαA上，电转化入毕赤酵母宿主菌GS115，经筛选鉴定后将携带目的基因的重组克隆菌进行诱导表达，对表达产物进行SDS-PAGE和蛋白质印迹法分析。结果：利用基因重组技术构建了含HEV p154片段的pPICZαA重组质粒，并在毕赤酵母菌株GS115中实现了分泌性表达，表达的目的蛋白p154分子质量约为22 kDa，上清中p154表达量可达到200 μg·ml^-1。p154可与HEV感染兔恢复期血清及HEV单克隆抗体6F9进行反应，表明酵母表达的p154具有良好的抗原性。结论：HEV ORF2p154在毕赤酵母表达系统中得到了成功表达，为进一步研发真核表达的戊型肝炎基因工程疫苗奠定了实验基础。

Objective: To investigate a new expression system to develop recombinant hepatitis E vaccine.Methods: The recombinant plasmid was transformed into GS115 by electroporation.The transformants were cultured in selection media and screened for the existence of foreign gene by PCR. Then the positive transformant was induced and the expression product was detected by SDS-PAGE and Western blotting assays.Results: The p154 gene of HEV was cloned into the Pichia pastoris expression vector pPICZαA.The p154 could be secreted from yeast and its molecular weight was 22 kDa,in supernatant the p154 was accumulated up to 200μg·ml^-1.The result of Western blotting demonstrated that the p154 could be specifically recognized by monoclonal antibody against HEV and the recovery serum of rabbit infected by HEV.Conclusion: The successful expression of HEV p154 protein in Pichia pastoris provides foundation for the further development of recombinant vaccine against hepatitis E.

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