Expression Of Nucleoprotein Gene Fragment Of Equine Coronavirus And Development Of ELISA Kit For Detecting Antibody To Equine Coronavirus

According to the nucleocapsid protein (NP) gene sequence of Equinecoronavirus from NCBI and a pair of primers was designed and the NP gene wasamplified by PCR. PCR products was cloned into pGEX-6p-1 to constructpGEX-ECV-N. The plasmid pGEX-ECV-N was transformed into the BL21 E.colicompetent cells. Induced by IPTG, the fusion protein 42KD GST-ECV-N wasexpressed in the BL21 E. coli cells and identified by SDS-PAGE.The ECV-N gene from pGEX-ECV-N was subcloned into transfer vectorpFastBacl. The recombinant transfer vector pFastBac-ECV-N was transformed intothe DH10Bac E.coli competentcells which contain the bacmid with a mini-attTn7target site and the helper plasmid. Recombinant Bacmid-ECV-N was generated bytransposing the mini-Tn7 element located in pFastBac- ECV-N to the mini-attTn7attachment site on the bacmid. Subsequently the recombinant Bacmid-ECV-N wastransfected into the Sf9 insect cells mediated by lipofectin to produce recombinantbaculovirus rBac-ECV-N. The result of PCR showed ECV-N gene was successfullyinserted into the rBac-ECV-N. The expression of rBac-ECV-N was determined byindirect immunofluorescence assay (IFA) at 5 days post-infection with mouse serumanti-GST-ECV-N. It proved that protein ECV-N was expressed in Sf9 cells infectedwith recombinant baculovirus rBac-ECV-N.Two monoclonal antibodies (mAbs) to equine serum IgG was developed byfusion berween SP2/0 and spleen cells of BalB/c mice immunized with the IgG ofequine serum, which was extracted and purified by saturation (NH₄)SO₄. The mAbsnamed as EQ-IgG-3D10 and EQ-IgG-6G6. The immunogulobin subtype of the two mAbs are IgG; These mAbs worked very well in indirect Enzyme-LinkedImmunosorbent Assay (ELISA) and didn't react with any other animal serums; MabEQ-IgG-6G6 could react with lgG of equine in western blot.The ELISA kit for detecting Equine coronavirus antibody was developed withprotein ECV-N expressed by Sf9 cells infected with recombinant baculovirus and mAbaganist equine serum lgG. The optional conditon of ELISA was that antigen ofrecombinant ECV-N was at the amount protein of 10μg/ml, equine serumdilutionl:100 in PBS and mAb cell supematant to equine serum. 1064 samples ofequine serum from China, Australia and Sweden were detected. The positive serumswas 0.98% (7/711) from China, 6.87% (23/335) from Australia and 0 (0/18) fromSweden.