Challenging for Expression Bovine Rotavirus (RF Strain) Full-Length VP۷ Protein in Prokaryotic System

Background and Aims: Rotavirus enteritis is an acute viral infectious disease among infants. VP۷ protein has a key role in attachment and entry virus into the target cell. The VP۷ protein is involved in inducing the production of neutralizing antibodies that protect infants against reinfection of the virus. The aim of this study was to heterologous expression of the VP۷ gene of bovine rotavirus as a fusion protein of Trx–VP۷ in a genetically engineered bacteria. Materials and Methods: Total RNA was extracted from MA۱۰۴ cells infected with bovine rotavirus strain RF. BRV VP۷ gene was amplified using polymerase chain reaction. Another gene was the VP۷ synthetic that made in the pBSK plasmid. The pBSK-VP۷ plasmid was digested using BamHI and SacI restriction endonucleases, then recombined into the prokaryotic expression vector pET۳۲a. The pET۳۲a-VP۷ amplified and pET۳۲a-VP۷ synthetic were transformed into BL۲۱ (DE۳) competent cells of Escherichia coli, respectively, and induced by different concentrations of Isopropyl β-D-۱-thiogalactopyranoside at ۳۰°C and ۳۷°C in luria bertani and terrific broth media, then analyzed using SDS-PAGE and western blotting. Results: SDS-PAGE analysis showed that the both pET۳۲a-VP۷ amplified and pET۳۲a-VP۷ synthetic were not expressed in the BL۲۱ (DE۳) cells, But the expression of pET۳۲a-VP۷ synthetic was weak at ۳۰° C in luria bertani media. Conclusions: This is the first report of the production of bovine rotavirus (RF strain) Full-Length VP۷ in prokaryotic system expression. VP۷ is a membrane protein and has toxic domains that show high toxicity in this expression system.

کلیدواژه ها:

Expression system ، Rotavirus ، Toxicity ، VP۷