CLONING AND EXPRESSION OF GENE ENCODING P46 ANTIGEN OF MYCOPLASMA HYOPNEUMONIAE IN ESCHERICHIA COLI

Abstract

Enzootic pneumonia is a highly contagious chronic respiratory disease in pigs caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) worldwide. P46 of M. hyopneumoniae is considered one of the main surface antigen proteins. This study aims to successfully clone and express P46 of M. hyopneumoniae in Escherichia coli M15 cells. The gene encoding P46 of M. hyopneumoniae was isolated from lung samples of infected pigs raised in Thua Thien Hue province, Vietnam, and cloned into vector pGEM®-T Easy. The PCR product digested by KpnI and BamHI enzymes was cloned into the pQE30 and expressed in E. coli M15 cells. The results of sequencing show that the nucleotide sequence of the cloned gene encoding P46 antigen protein has a length of 456 bp, corresponding to 152 amino acid residues and 99.34% similarity to the polypeptide chain of recorded M. hyopneumoniae P46 protein in GenBank (accession number: AAZ53879.1). The results of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) analysis show that the molecular mass of the expressed protein is approximately 20 kDa, and anti-histag ELISA confirms that the expressed protein is the 6xHis-P46 recombinant protein.