الفهرس 
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Abstract
Haemagglutinin-Esterase (HE) and integral membrane (M) genes of bovine coronavirus (BCoV), Mebus reference strain (EBCV), were individually cloned into baculovirus expression vector, pBlueBac4.5/V5-His TOPO® utilizing the TA cloning technology, and then expressed in Spodopetra frugiperda (SF-9) insect cells. Homologous recombination had efficiently redirected the target genes to be inserted in the cloning cassette of Bac-N-Blue™ defective linearized baculovirus under the regulation of hyperactive very late polyhedron gene promoter. Prior to its genetic manipulation, BCoV was propagated in MDBK cells for four serial passages, titrated, and characterized by plate HA and indirect FAT. BCoV was concentrated by; ultracentrifugation, PEG-6000, then purified on sucrose density gradient. Rabbit polyclonal anti-BCoV were prepared, titrated, and evaluated by indirect ELISA, HA/HI, AGPT, and SDS-PAGE assays followed by western immunoblot against BCoV antigen. Viral RNA was extracted from the infected MDBK cells. RT-PCR assays were employed to amplify M and HE ORF of BCoV genome. The identified PCR products were cloned into baculovirus transfer plasmids. Colony PCR assays were carried out to check positive recombinant plasmids. Transfection processes applied homologous recombination of the recombinant plasmid and linear baculovirus. Recombinant baculoviruses were selected by plaque purification; checked for the presence of M and HE genes by PCR, then propagated to obtain high-titer viral stocks, and finally titrated by plaque assay to calculate recombinant virus concentration/MOI inoculated during expression studies. High-level expression of recombinant BCoV proteins had been produced in the inoculated SF-9 cells. Indirect FAT by rabbit anti-BCoV, indirect solid phase ELISA utilizing three anti-BCoV anti-sera (mouse, rabbit, and bovine), SDS-PAGE followed by western immunoblot assays with the same antiserum species were indebted to characterize the expressed BCoV glycoproteins. In conclusion, this study is the first detailed report on cloning and expression of HE and M genes of BCoV in Egypt. Which, the recombinant expressed proteins, will be utilized in further studies as a tool for development of new diagnostic measure and/or novel vaccine candidate to improve diagnosis and control of BCoV infection in young calves in Egypt.