Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System
| العنوان: | Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System |
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| المؤلفون: | Ali Teimoori, Manoochehr Makvandi, Niloofar Neisi, Alireza Samarbafzadeh |
| المصدر: | Jundishapur Journal of Microbiology |
| بيانات النشر: | Briefland, 2016. |
| سنة النشر: | 2016 |
| مصطلحات موضوعية: | Rotavirus, 0301 basic medicine, Microbiology (medical), animal diseases, viruses, Sf9, Biology, medicine.disease_cause, Microbiology, law.invention, NSP4 Protein, 03 medical and health sciences, 0302 clinical medicine, Plasmid, Hepatitis E virus, law, medicine, 030212 general & internal medicine, Gene, Escherichia coli, sf9 Cell, pFastBac1, HEV ORF2, virus diseases, biochemical phenomena, metabolism, and nutrition, Hepatitis E, medicine.disease, Virology, Fusion protein, 030104 developmental biology, Infectious Diseases, Recombinant DNA, Research Article |
| الوصف: | Background: The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives: The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods: The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5 alpha and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results: The lengths of the subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions: The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine. |
| تدمد: | 2008-4161 2008-3645 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2c3f11dd23da2dedfa6bd385b7ebbe70 https://doi.org/10.5812/jjm.40303 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....2c3f11dd23da2dedfa6bd385b7ebbe70 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 20084161 20083645 |
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