Cloning Of Foot-and-Mouth Disease Virus 2C3ABC Gene And Its Expression In Pichia Pastoris System

In this study, Pichia Pastoris System had been utilized for expression of FMDV 2C3ABC gene which aimed for establishing a sensitive and specific molecular dignosis method.First,2C and 3ABC genes were amplified individually from P2 and 3ABC postive clones and ligated together using PCR method,then this 2C3ABC product was Cloned into pGEM-T Easy vector and transformed E.coli DH5a competent cell.A postive recombinant plasmid which contained whole 2C3ABC gene had been confirmed by PCR ,enzyme digestion and sequencing.After that, the 2C3ABC gene was sub-cloned into pPICZaA expression vector and transformed E.coli DH5 a competent cell and selected by Zeocin?antibiotic.The postive recombinant expression vector was linerized and electro-transformed Pichia Pastoris SMD1168 competent cell.A recombinant Pichia Pastoris had been obtained by Zeocin?antibiotics selection and induced with 0.5% methanol for target protein expression.The expression product was analysised by SDS-PAGE and Western blotting assay.The result showed that 2C3ABC gene Was expressed successfully in Pichia Pastoris and the product was a 95ku fusion protein which could be recognized by anti-FMDV serum.The amount of target protein was over 15% of the total bacteria protein by gel thin layer scanning analysis.This research had supplied materials for establishing a FMD diagnosis method to differentiate infected animals from vaccinated animals .