Detection Of Canine Parainfluenza Virus And Study On Cloning And Expression Of CPIV-NP Gene And Immunocompetence Of Its Recombinant Protein

Canine infectious respiratory disease(CIRD)in dogs,referred to respiratory illness kennel cough.The main clinical symptoms of including fever,cough and nose running,which were caused by such as multiplex,pathogens,virus and bacterium,and canine parainluenza virus(CPIV) was main cause of CIRD.CPIV was a single-strand negative RNA virus which belongs to the paramyxoviridae family.The total genome of CPIV is approximately 15245nt,which contain NP, P, M, F, HN and L genes.Binn and his colleges first isolated a strain of CPIV from sick dogs with respiratory syndrone using Madine-Darby Canine kidney (MDCK) cells in 1967.The investigation indicate CPIV was one of the main infectious disease in dogs.So three study were conducted to research CPIV in the present article as followings:1.A RT-PCR method was developed to detect of CPIV.The result of specific,sensitive and reproducibility tests was shown that established RT-PCR method was a good method for detection of CPIV.When RT-PCR was used to detect samples was clinically collected,17 were positive from 32.The positive percentage was 53.12%.2.The cloned CPIV NP gene was 1658bp.The NP gene was amplified by RT-PCR from extracted total RNA of CPIV in the nasal cavity secretion of illness dogs.This gene was subcloned into PMD18-T simple vector for identification and sequencing.The result was shown that there were three mutant base pairs among the nucleotide sequence of NP gene which cause three matant amino acid in its encode protein, comparing with that of type strain published in GeneBank.3.Using the PCR technique to amplify expression gene sequence of NP protein from plasmid of NP total gene.The cloned gene was subcloned into expression vector PGEX-6P-1 to construct the Prokaryotic expression vector PGEX-6P-1-NP.The fusion protein was expression smoothly in the E.coli BL21(DE3).The result of wastern blot analysis were shown the fusion protein (GST-NP) had strong immunological competence.