Cloning, Expression Of Spikeprotein And Nucleiuprotein Gene Of Infectious Bronchitis Virus And Its Preliminary Application In Detecting Antibodies Against IBV

Avian infectious bronchitis (IB) is an acute, highly contagious respiratory disease caused by Infectious bronchitis virus(IBV) to which all ages, types of chickens are susceptible. In most areas of world this disease happened as one of the most important infectious diseases which seriously hinder the development of poulty industry. IBV is the typical virus of Coronavirus in the family Coronaviridae with a single-strand positive-sense RNA genome, and include no less than three main structure proteins: Spike glycoprotein (S protein), Membrane glycoprotein (M protein) and Nucleocapsid protein (N protein). S protein is the most variable one in evolution and the most important antigen that could induce neutralizing antibody comparied with M protein and N protein. It acts essentially in the process that virus absorbs onto cell and in the serological classification of virus. It is important in vaccine immunization against IBV too. N protein is the most conservative one in evolution of IBV of which the main function is packaging nucleotide, duplicating RNA of IBV genome and activing cell-mediated immunity. From the year 1931 by now more than twenty nine serotype of IBV is reported with occurrence of new variant serotypes. For weak cross protection between different serotypes and monitoring immunological level of antibodies against IBV, a ELISA method was developed with the conservative recombinated N protein expressed in E.coli that provide dependable and available technological safeguard for controlling of IBV . And in the same time S1 gene charactered serology was cloned from genome of IBV and S1 protein was expressed in E.coli to contribute to the development of vaccine against IBV. So following researchs were explored: 1. Cloning and sequence analysis of S1 protein gene and N protein geneAccording to published S1 protein gene and N protein gene sequence of IBV in Genebank, specific primers were designed and synthesized. A 1685bp fragments of S1 gene and 1700bp fragments of N gene were amplified from RNA of Infectious Bronchitis virus preservatived in our lab by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid was proved to be true by restriction-enzyme analysis and sequenced. The sequence BLAST showed that S1 gene have the most homology 98% with the strain GX1-98, N gene most 97% with the strain H52 LKQ3. 2.Expression of S1 , N gene of IBV in E.coliAccording to analysis of amino acids translated from the S1 gene, a pair of primers were designed and used to amplify the S1 gene lacked signal peptide. Then the subcloned fragment of S1 gene and the fragment of N gene were each recombinated into prokaryotic...