Establishment Of MDCK Cell Line Stably Expressing Chicken Aminopeptidase N And Its Preliminay Characterization

Avian infectious bronchitis virus(IBV) is single-stand RNA virus of Coronviridae family.Clinicaly, infectious bronchitis(IB) could result in serious decrease of egg production and slow growth in broilers, theses brought great economic loss in chicken industry. IBV has great number of different serotype strains even in the same district, this gave rise to low cross protection with vaccine immunization and brought tremendous difficulities in the prevention and treatment of the disease. The illustration of IB pathogenesis mechanism, especially the study on the IBV infection of host cell, could provide solid basis for its comprehensive control. It was reported that chicken aminopeptidas N(c APN) played important role in the IBV infection. In order to make further investigation of Capn biofunctions and its effect on IBV infection of host cell, this study was undertooken to establish the MDCK cell line stably expressing cAPN and its preliminary characterization.In this paper, p ET30a-c APN prokaryotic recombinant plasmid was constructed successfully and transformed into Rosetta cell for induction. SDS-PAGE demonstrated that the recombinant protein was expressed in the form of inculsion with the molecular weight of 113 kDa. Then, the recombinant expressed protein was purified and renatured, and use as immunogens to immunized rabbit for the preparation of polyclonal antibody.For the stabilized expression cell line, MDCK cells were transfected with recombinant eukaryotic expression plasmid of pc DNA3.1-c APN, and then screened with G418 and serial coloning. Reverse transcription polymerase chain reaction(RT-PCR) and indirect immunofluorescence assay indicated that the c APN stabilized expression MDCK cell was successfully established and the protein could be expressed continuously. Virus infection experiment demonstrated IBV could infect the acquired MDCK cells and produce typic CPE,which could not be observed in the control cells. This illuminated that c APN might acted as cell surface receptor to exert its crucial biological function for IBV infection, and provided important basis for the study of IB pathogenesis mechanism and IBV field strain isolation and characterization.