Farshadpour Fatemeh, Taherkhani Reza, Makvandi Manoochehr, Rajabi Memari Hamid, Samarbafzadeh Ali Reza
Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran ; Department of Medical Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran.
Department of Agronomy and Plant Breeding, Faculty of Agriculture, Ahvaz Shahid Chamran University, Ahvaz, IR Iran.
Jundishapur J Microbiol. 2014 Jul;7(7):e11261. doi: 10.5812/jjm.11261. Epub 2014 Jul 1.
Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential.
The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate.
The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media.
In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified protein was obtained, which can be a potential vaccine candidate and as an antigen in ELISA to diagnose HEV infections.
戊型肝炎病毒（HEV）是不同年龄人群急性肝炎的病原体，在孕妇中的死亡率高达30%。因此，戊型肝炎病毒感染的一级预防至关重要。
本研究旨在获得高度纯化的截短开放阅读框2（ORF2）蛋白，其可能是未来戊型肝炎病毒疫苗的候选物。
编码戊型肝炎病毒衣壳蛋白序列第112 - 660个氨基酸的截短orf2基因（orf2.1）经优化、合成后克隆至pBluescript II SK(+)载体。亚克隆至表达载体pET - 30a(+)后，从构建体中缺失一个193核苷酸片段，构建重组质粒pET - 30a - ORF2.2（orf2.2编码戊型肝炎病毒衣壳蛋白第112 - 608个氨基酸序列），并用于转化大肠杆菌BL21细胞。用异丙基 - β - D - 硫代半乳糖苷（IPTG）诱导并优化表达条件后，目标蛋白得以高效表达，并通过镍离子螯合亲和层析进行纯化。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法对表达并纯化的蛋白进行分析。
通过PCR、限制性内切酶消化和重组质粒pET30a - ORF2.2的DNA测序确认了亚克隆。优化表达条件的结果表明，在37℃下加入终浓度为1 mM的IPTG孵育4小时可使蛋白获得最高表达量。通过SDS - PAGE和蛋白质印迹法确认了截短ORF2蛋白的表达和纯化。SDS - PAGE分析显示一条约55 kDa的蛋白条带。纯化蛋白的SDS - PAGE显示，在pH为4.5的洗脱缓冲液中获得的目标蛋白量最高。纯化蛋白的产量约为1 mg/L培养基。
本研究成功在大肠杆菌中表达了优化的截短ORF2蛋白，并获得了高度纯化的蛋白，该蛋白可作为潜在的疫苗候选物以及用于酶联免疫吸附测定（ELISA）诊断戊型肝炎病毒感染的抗原。
