戊型肝炎病毒ORF3和标签His融合真核表达载体的构建与表达

doi:10.3969/j.issn.1672-5069.2016.02.010

实用肝脏病杂志 ›› 2016, Vol. 19 ›› Issue (2): 164-167.doi: 10.3969/j.issn.1672-5069.2016.02.010

戊型肝炎病毒ORF3和标签His融合真核表达载体的构建与表达

张丹, 于浩, 荣义辉

124000辽宁省盘锦市传染病医院（张丹）; 盘锦市中医院（于浩）; 解放军第302医院肝衰竭诊疗与研究中心（荣义辉）

收稿日期:2015-07-30 出版日期:2016-03-10 发布日期:2016-03-04
作者简介:张丹,女,42岁,大学本科。E-mail：zhangdanwz_1973@sina.com

Hepatitis E virus ORF3 and His tag fusion eukaryotic expression vector construction and expression in vitro

Zhang Dan, Yu Hao, Rong Yihui

Infectious Disease Hospital,Panjin 124000,Liaoning Province

Received:2015-07-30 Online:2016-03-10 Published:2016-03-04

摘要/Abstract

摘要： 目的体外构建HEV ORF3蛋白融合His标签的真核表达载体及其初步鉴定和蛋白表达。方法从1例男性31岁戊型肝炎患者血清中分离HEV RNA,逆转录合成cDNA,特异性引物扩增HEV ORF3片段,将其定向连接到pcDNA3.1(-）-His真核表达载体中,经抗生素筛选后,酶切及测序鉴定其序列正确性。采用提取质粒转染HepG2细胞,培养96 h后裂解细胞,采用抗His-Tag抗体进行Western blot检测目的蛋白。结果从戊型肝炎患者血清中成功分离出I型HEV RNA,经抗生素筛选、酶切鉴定和测序确认pcDNA3.1(-）-HEF3-His真核表达载体构建成功,转染HepG2细胞,经Western blot检测显示15 kDa位置有明显融合蛋白条带。结论成功构建了HEV ORF3与His标签融合的真核表达载体,体外转染HepG2细胞后鉴定其工作良好,为后续HEV ORF3蛋白的研究奠定了基础。

关键词: 戊型肝炎病毒, 真核表达, 融合蛋白

Abstract: Objective To construct the eukaryotic expression vector,to identify and to investigate its expression of HEV ORF3 fusion protein His label. Methods HEV RNA was isolated from the serum of a male patient with hepatitis E, and the HEV ORF3 cDNA was synthesized by reverse transcription and specific primers,then directionally ligated into pcDNA3.1（-） his in the eukaryotic expression vector,and the sequence of the enzyme digestion and sequencing was detected. The vector plasmid was transfected into HepG2 cells,which was used to detect the target protein 4 days later,and the target protein was detected by Western blot with anti-His. Results The HEV RNA type I were successfully isolated;after antibiotic screening and enzyme digestion,the pcDNA3.1（-）-HEF3-His eukaryotic expression vector was successfully constructed. The vector was transfected into HepG2 cells,and after 96 h incubation, an 15 kDa protein was demonstrated suggesting the fusion protein existed. Conclusion The eukaryotic expression vector of HEV ORF3 and His tag fusion are successfully constructed,and transfected into HepG2 cells,which might lay a foundation for further study of HEV ORF3 protein.

Key words: Hepatitis E virus, Eukaryotic expression, Fusion protein

张丹, 于浩, 荣义辉. 戊型肝炎病毒ORF3和标签His融合真核表达载体的构建与表达[J]. 实用肝脏病杂志, 2016, 19(2): 164-167.

Zhang Dan, Yu Hao, Rong Yihui. Hepatitis E virus ORF3 and His tag fusion eukaryotic expression vector construction and expression in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(2): 164-167.

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戊型肝炎病毒ORF3和标签His融合真核表达载体的构建与表达

Hepatitis E virus ORF3 and His tag fusion eukaryotic expression vector construction and expression in vitro

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