Expression And Protein Activity Analysis Of Avain Infectious Bronchitis Virus M Gene In Pichia Pastoris

IBV is a fast and highly—touched infectious disease which has been variating frequently.It has much serological type and was difficult to prevent. IBV M protein was ratherconservative, concerned with replication of viruses it was an important immunogen gene.In addition, M gene holds a largest proportion in IBV particle and has typical feature ofmembrane protein. The characteristics of M protein provided molecular biology andimmunology base for M protein becaming IB diagnostic antigen.A pair of M gene expression fragment PCR primers were designed according toisolated IBV's M gene sequence which was accepted by Genbank and the sequence ofPichia pastoris expression vector pPIC9K's Multiple Cloning Site(MCS). Virus wereobtained by chicken embryo cultivating and the RNA of IBV virus was extracted. M genewas amplified by RT-PCR and cloned into the vector pMD18.Then the cloned vectorpMD18-M was identified by colony-PCR and enzyme digestion. The M fragment,producing from pMD18-M by EcoR I and Not I, was ligated with the yeast expressingvector pPIC9K which was also digested by EcoR I and Not I,then the ligation wastransformed to E.coli. The results of PCR and enzyme digestion of plasmid proved thatrecombination vector was obtained (named pPIC9K-M). The expressing plasmidpPIC9K-M was linearized with Sac I, then it was transformed into Pichia pastoris GS115by electroporation. After the transformants were selected by PCR,Methanol-utilization-plus and different G418 concentrations YPD medium, the positiverecombination yeast was obtained(named GS115/ pPIC9K-M His~+Mut~+). GS115/pPIC9K-M His~+Mut~+was induced with 1% methanol at 28℃for target protein expression. Expressed M protein of different time section was analysised by SDS-PAGE,the resultsshowed that the target protein was up to the highest yield after 3 days induced, and theproduct was about 33KD. The amount of target protein shares about 13.3% of the totalprotein which consist in the supematant fluid of yeast culture medium.Western blotting assay showed that the expressed protein could act with the positiveserum of IBV. The protein was initially purifed and acted as the envelope antigen of ELISAdetecting, the results proved expressed protein acted well with the specific antibody andthe expressed M protein has powerful biologic activity.Now there had no report about expressing M protein with Pichia pastoris. The expressedM protein may act as diagnostic antigen meterial of IB.The result has important theoreticaland practical value to prevention and cure of IB.