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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MAKVANDI MANOCHEHR; TEIMOORI ALI; NEISI NILOOFAR; SAMARBAFZADEH ALIREZA

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Journal: Jundishapur Journal of Microbiology Year:2016 | Volume:9 | Issue:11 Page(s): 0-0

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Title

Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

Author(s)

MAKVANDI MANOCHEHR | TEIMOORI ALI | NEISI NILOOFAR | SAMARBAFZADEH ALIREZA | Issue Writer Certificate

Keywords

HEV ORF2

NSP4 Protein

Rotavirus

pFastBac1

sf9 Cell

Abstract

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 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.

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APA: Copy

MAKVANDI, MANOCHEHR, NEISI, NILOOFAR, & SAMARBAFZADEH, ALIREZA. (2016). Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System. JUNDISHAPUR JOURNAL OF MICROBIOLOGY (JJM), 9(11), 0-0. SID. https://sid.ir/paper/317749/en

Vancouver: Copy

MAKVANDI MANOCHEHR, NEISI NILOOFAR, SAMARBAFZADEH ALIREZA. Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System. JUNDISHAPUR JOURNAL OF MICROBIOLOGY (JJM)[Internet]. 2016;9(11):0-0. Available from: https://sid.ir/paper/317749/en

IEEE: Copy

MANOCHEHR MAKVANDI, NILOOFAR NEISI, and ALIREZA SAMARBAFZADEH, “Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System,” JUNDISHAPUR JOURNAL OF MICROBIOLOGY (JJM), vol. 9, no. 11, pp. 0–0, 2016, [Online]. Available: https://sid.ir/paper/317749/en

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