Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli

• DOI: 10.5812/jjm.11261
• Publication Date: 2014-07-06T04:27:48Z
• Authors: Fatemeh Farshadpour; Reza Taherkhani; Manoochehr Makvandi; Hamid Rajabi Memari; Ali Reza Samarbaf...
• Keywords: Subcloning; Hepatitis E Virus; Hepatitis E

Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people different age groups and has high mortality rate up to 30% pregnant women. Therefore, primary prevention HEV infection essential.The aim this study was obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be future vaccine candidate.The orf2 gene (orf2.1), encoding 112-660 amino acid capsid protein sequence, optimized, synthesized, cloned into pBluescript II SK(+) vector. After subcloning expression vector pET-30a (+), 193-nucleotide fragment deleted from construct recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 sequence protein) constructed used for transformation Escherichia coli BL21 cells. induction with isopropyl-β-D-thiogalactopyranoside (IPTG) optimizing conditions expression, target expressed by Ni(2+)-chelate affinity chromatography. The analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting.The confirmed PCR, restriction enzyme digestion, DNA sequencing pET30a-ORF2.2. results obtained showed that highest adding IPTG at final concentration 1 mM 37℃ four hours. purification ORF2 SDS-PAGE western blotting. analysis band about 55 kDa. revealed amount in elution buffer pH 4.5 obtained. yield mg/L culture media.In study, optimized E. successfully obtained, can potential candidate as an antigen ELISA diagnose infections.