Cloning Of Nucleocapsid Gene Of Novel Variant Infectious Bronchitis Viruses And It S Expression In E.coli

Infectious brochitis virus (IBV) causes a highly contagious respiratorydisease in chickens and is considerably economic importane. Failure of vaccinesto protect against IBV is attributed to emergency of novel variam tris, changesof tissue tropism and serotype of IBV. So it is theoretically important to researchIBV antigen genes and their products on molecular level for controlling avianinfectious bronchitis.In this research, the viral RNAs were extracted from allantoic fluid ofchicken embryos inoculated with IBV strain ZJ97l, N and H52 respectively byTrizol Reagent. Based on the reported nucleotide sequence of nucleocapsid geneof Beaudette strain of IBV two specific primers were synthsized. Thenucleocapsid genes were amplified by reverse transcription and Polymerase cyclereaction using the viral RNAs as the templates. Then the cDNAs were cloned intopBluescript SK(+) vector and sequenced respectively The sequences and thehydrophilicity of the deduced proteins were analyzed using Lasergene SequenceAnalysis software. And then nucleocapsid gene of IBV strain ZJ97l was clonedinto the exptess vector pBV220. The nucleocapsid protein was expressed in Ecoli. DH5α and detected by Westem blotting. The results show as followings:The open reading frame of nucleocapsid genes of ZJ97l strain, N strain andH52 strain was consist of l230 nucleotide acids, coding the nucleocapsidpolypeptides of 409 amino acids.Homologies of nucleotides between the proventriculopathogic IBV strainZJ971 and other IBV strains from Genebank were 86.4% to 98.6%. Andaccordingly, amino acid homologies were 90.0% to 98. 1%. The hydrophilicity ofthe protein increased markdly due to the mutaion of 171^st amino acid.Hydrophobilic peak of the protein moved from 197^th to 282^cd due to hydrophilicdrop of 282^cd amino acid caused by mutation of 283^nd amino acid. Phylogenetictree showed that the relative relationship among IBV strains ZJ97l and D1466and Cu/T2 was more adjacent. So they might have coancestor.The nucleotide homologies between nephropathogenic IBV strain N andother IBV strains from Genebank were 86.6% to 87.4%. Accordingly, amino acidhomologies were 90.5% tO 91.7%. The hydrophilicity of l89-l90^th, 223^ird, 236^th,286", 335-336th sites markedly decreased due to the mUtaion of the aInin acidsof the same sites of the Protein. The average hydrophilicity of the whole proteinwas lower than tha of others. Phylogenetic tree showed tha N sbon divempdmarked1y hom Others, including the nephropathogenic IBV strain Gray So it migh beseparated frOm other strains of IBVThese findings indicated tha lBV strains ZJ97l and N wer novel variansof IBVA protein band wh expected molecular weigh of 45 kD was detected bySDS-PAGE and westem bloning. It indicated tha the nucleocaPsid protein ofZJ97l staln with sPecific reactivities of monoclonai antibody againsnucleocaPsid protein of IBV was exPressed efficientiy in E coli It ndgh be abasis fOr developing a gene engineering vaccine against IBV...