Isolation And Identification And Hereditary Variation Study On The Membrane Protein Gene Of Avian Infectious Bronchitis Virus In Shaanxi Province

Recently, avian infectious bronchitis (IB) has been a serve disease that just second to ND, MD and IBD to demestic fowl, and caused significant economic losses to poultry industry. Membrane protein (M protein) is one of the structural proteins of avian conoravirus infectious bronchitis virus (AIBV). M gene encodes a protein of 224~225 amino acids, and approximately 40% of viroprotin is M protein. To the glycosylation degree, the molecular weight of M protein is vary from 23ku to 36ku. The glycoproteins of M protein contain N-linked oligosaccharides, and the main functions of M protein is related with virion information, accumulation of viron in the Golgi region. Morever, the glycoproteins are also indispensable for viral infectivity, and induce viral nutrilizational antibody when the complements is apparent, and also augments the specific responses induced by nucleocapsid DNA immunization. So the study of M protein molecular epidemiology to know the genetic variation of IBV M protein can give useful theory foundation for the prevention and therapy of IB. 13 IBV isolates were isolated from suspected flocks in Shaanxi province, and the releventive study on the M protein gene were conducted.Firstly, 18 isolates were isolated successively from 20 suspected flocks with respiratory infection symptoms, and 13 of them were identified to be of IBV, a member of the family Coronaviridae, 3 of the rest were of NDV, the last 2 were of AIV H9, through serology tests (HA, HI, NDV interference test, NVT in embryonated eggs), EID50 test, molecular biology tests and animal infection tests. The results of animal infection test showed that 8 isolates, including BJ2, FF2, YL2, B01, G5, YX, WH, WG, had strong nephropathogencity. Moreover, the results of NVT in embryonated eggs revealed the control strain W118 could neutralize the 8 isolates completely, while Gray and W93 could not do that, though Gray and W93 were able to neutralize W118 to a considerable high degree. Results convinced that the main prevalent strain of IBV in Shannxi was NIBV, and it had stong nephropathogencity and infectivity.Secondly, to study on the genetic variation of the IBV M protein, we designed a pair of primers according to the nucleotide sequence stowed by NCBI, then the M gene of the 8 nephropathogenic isolates were amplified, and the clonal vectors were constructed, and they were sequenced after identification by PCR and restriction endonuclease digestion. The sequence distance of nucleotide and predicted amino acid, phylogenetic tree, the predicted secondary structure and the membrane-spanning regions of M protein were analysed through DNAStar, AnthePro5.0 and PRED-TMR biological softstares. The results showde that the 8 isolates had BamHI cleavage sit, excepted YX and WH isolates. And they shared 87.4%~ 99.5%and 89.4%~99.5% homology with the control strains in sequence of nucleotide and predicted amino acid, respectively; and they shared 92.3%~ 99.8%, 95.4%~ 100% homology with the all each. Theα-helix regions mainly located in the first 100aa of the isolates M protein amino acid sequences, and the rests comprisedβ-sheets and coils regions.The M protein of all isolates had a triple-spanning region in theα-helix regions of the N end. Thirdly, to analyse the homology of M protein gene seguence of 8 isolates from Shaanxi province through MegAlign module of DNAStar biologic software, and predicted the B cell epitopes and secondary structure of M protein, including Kyte-Doolittle hydrophility, Jameson-Wolf antigenic index, Karplus-schulz flexible regions, Plot-Emini surface probability, Gamier-Robsonβ-tuner regions and coil regions based on the sequence of M gene . The results showed that the mutations of the predicted amino acid sequence of isolates M protein mainly localized in the N-termianl No.10~40aa and 90~175aa, but it was just a ruleless single-letter code mutation. Moreover, B cell epitopes of M proteins of 4 deputy isolates were possibly situated in the shared regions ( N-termianl No. 159~164aa and 181~193aa ) of hydrophilicty, antigenic index, flexible regions, surface probability,β-tuner regions and coil regions or about them. All in all, the gene of AIBV M protein was considerable conserved, and the gene variations had no effect on the stability of B cell epitopes of M protein.Forthly, M protein of isolate G5 (W118) was expressed, and the expression conditions were optimized after the pET28a-M expression vector transformed with E.coli BL21(DE3), and a high expression method was disgussed. Then the expression products were identified through SDS-PAGE and Western-blotting. The results showed that the target protein was expressed successively in the condition that to add a 100μl sample to 8 ml TB + 80μg/ml kanamycin and grow the culture at 37℃until OD600≈1.5, then collect the cells by centrifugation at 1000g for 5min and resuspend in fresh TB + 80μg/ml kanamycin containing 0.5 mmol/L IPTG, and incubate at 25℃for 2~4h before harvest. Morever, the moleculal weight of the fusion protein is 30.5ku, and the expressed fusion protein is twenty percent of total protein of E.coli BL21. The high expression success give a base for the further study of IBV M protein.Through isolation and identification of IBV Shaanxi isolates, and analysis of the M gene sequence homology, B cell epitopes, secondary structure and the high level expression of M protein, the research gives a new and valuble material and theory foundation for prevention and therapy of avian infectious bronchitis.