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Baculovirus Expression of the Bovine Coronavirus Nucleocapsid Protein in Spodopetra Frugiperda Insect Cells

Baculovirus Expression of the Bovine Coronavirus Nucleocapsid Protein in Spodopetra Frugiperda Insect Cells

Amer, H.M.; Hussein, H. A.; El-Sabagh, I. M.; El-Sanousi, A. A. Saber,M.S. and Shalaby, M. A.

Department of Virology, Faculty of Veterinary Medicine, Cairo University, 11221 Giza, Egypt

ABSTRACT

In the current study, cloning and expression of the bovine coronavirus (BCV) nucleocapsid protein was carried out in a baculovirus expression system. The specific RT-PCR product of N gene was cut and extracted from gel using DNA gel extraction kit (Millipore). Eluted DNA was successfully cloned in pBlueBac4.5N5-His TOPO TA baculovirus transfer vector and transformed in chemically competent E. coli. A modified colony PCR assay was utilized to identify the positive bacterial colonies that harbor the recombinant plasmids carrying N gene in correct orientation. Generation of recombinant baculoviruses was achieved by co-transfection of Spodopetra frugiperda (Sf-9) insect cells with a linearized replication-defective baculovirus DNA (Bac-N-BlueTM) and the transfer vector. The recombinant baculoviruses were plaque purified; verified for the presence of target sequences using PCR and propagated for generation of high-titer viral stocks. In vitro expression studies utilizing the recombinant baculoviruses were conducted and revealed high-level expression of N protein as indicated by its distinct reactivity in immunofluoresence, solid phase ELISA and Western blot.

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Journal of Virological Sciences

July

Vol. 3, Iss. 1

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