CAJ | 학술논문

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ORF2 p154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200μg· 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 ORF2 p154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。
목적：이용필적효모계통표체무형간염병독（HEV）ORF2편마단백제452～605안기산다태（p154），탐토필적효모표체계통용우연발무형간염기인공정역묘적가능성。방법：채집무형간염환자분편，진행RT-PCR검측화측서감정，병의차위모판확증ORF2 p154기인，극륭도표체재체pPICZαA상，전전화입필적효모숙주균GS115，경사선감정후장휴대목적기인적중조극륭균진행유도표체，대표체산물진행SDS-PAGE화단백질인적법분석。결과：이용기인중조기술구건료함HEV p154편단적pPICZαA중조질립，병재필적효모균주GS115중실현료분비성표체，표체적목적단백p154분자질량약위22 kDa，상청중p154표체량가체도200μg· ml -1。 p154가여HEV감염토회복기혈청급HEV단극륭항체6F9진행반응，표명효모표체적p154구유량호적항원성。결론：HEV ORF2 p154재필적효모표체계통중득도료성공표체，위진일보연발진핵표체적무형간염기인공정역묘전정료실험기출。
Objective: To investigate a new expression system to develop recombinant hepatitis E vaccine . Methods:The recombinant plasmid was transformed into GS 115 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 p 154 could be specifically recognized by monoclonal antibody against HEV and the recovery serum of rabbit infected by HEV .Conclusion:The successful expression of HEV p 154 protein in Pichia pastoris provides foundation for the further development of recombinant vaccine against hepatitis E .