Cloning and expression of H gene of PPRV in baculovirus expression system
Subject Areas : Virology
Mehdi Taheri Asl
1
(Department of Microbiology, Islamic Azad University, Jahrom Branch, Jahrom, Iran)
majid moghbeli
2
(Professor, Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.)
Mohammad Kargar
3
(Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran)
mohsen lotfi
4
(Razi vaccine and serum research institute, agricultural research, education and extension organization(AREEO), Karaj, Iran.)
Farshid Kafilzadeh
5
(Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran)
Keywords: Sf9 Cell, Recombinant, Peste des petits ruminants,
Abstract :
Background and Objectives: Small ruminant plague is one of the most economically important pathogens in sheep and goats caused by morbilli viruses. Protein H is one of the major proteins for immunity against PPRV. The aim of the present study was to clone and express the H gene of this virus by the baculovirus expression system.Materials and Methods: H gene was amplified by RT-PCR and specific primers and then cloned into pFastBac Dual plasmid. The recombinant vector with the gene was transferred to the DH10Bac host cell. By transfection Sf9 cell with recombinant vector, its expression and characteristics were evaluated by SDS-PAGE, western blotting and Bradford methods.Results: The H gene was amplified using specific primers from the PPR virus genome and the specific band was obtained. Cloning of the H gene on the pFastBac Dual vector and baculovirus genome was proved with PCR and enzymatic digestion. By transfection the recombinant vector into Sf9 cells and performing sequential passages, the recombinant protein was shown to be completely pure and specific using SDS-PAGE, Western blotting and Bradford methods. The expression level of this gene was obtained 218 µg/ml.Conclusion: Considering the production of an appropriate amount of recombinant H protein, this protein can be a suitable candidate for the production of recombinant vaccine against PPRV.
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