Cloning and expression of ompH gene of type a Pasteurella multocida of pig origin

Abstract

A study was undertaken to clone, express and purify the OmpH recombinant protein of Pasteurella multocida type A of pig origin. OmpH is a highly immunogenic porin protein found on the outer surface of P. multocida. For cloning and expression of ompH gene belonging to most pathogenic isolates of P. multocida capsular type A, pET303CT/His plasmid vectors were used. The amplified products of ompH and plasmid vector were digested with Xba1 and Xho1 at 37 °C for 5 hours separately. The restriction enzyme (RE) digested PCR product was ligated with the vector by using T4 ligase enzyme which was confirmed by performing T7 plasmid specific PCR. The ligated product was transformed into E. coli DH5α cell. Recombinant plasmid was extracted from positive clone and confirmed by T7 plasmid specific PCR, ompH gene specific PCR, double RE digestion and nucleotide sequence. Nucleotide sequence of the subjected recombinant plasmids showed 97-98% homology with ompH gene specific of porcine P. multocida, available in the GeneBank, NCBI. The recombinant OmpH protein was expressed in E. coli BL21 host in the insoluble fraction, which was purified by using His-Trap columns. The purified protein was confirmed by SDS-PAGE and western blotting by using anti His tag antibody which showed dark intense band at 37 kDa position.