Cloning, Expression Of The R1 Region Of P97 Gene Of Mycoplasma Hyopneumoniae In Escherichia Coli And Identification Of Its Biological Activities

Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. Attachment to host tissue is essential for colonization by most mucosal pathogens, such as Mycoplasma hyopneumoniae. According to the reported sequence of P97 gene in strain 168, a pair of primers was designed and the Rl region of P97 gene in strain 168F485 was amplicated by PCR. In this case, two restriction sites(EcoR I , Hind III) were added to the primers (P1,P2) separately. The PCR product was inserted into expression plasmid pET-32a(+) after restriction digest. Then the recombinant plasmid was identified by endonuclease analysis, PCR ampliation and DNA sequencing. The report showed that the recombinant plasmid had right open reading frame. It was transformed into competent cells of BL21(DE3), then was induced to express by 0.2mmol/L IPTG at 37℃ for 4 hours .The recombinant protein was nearly 23.3% of the total products. It was proved that the recombinant protein had immune activity through Western blot and ELISA analysis. This provided the basis for the future preparation of Mycoplasma hyopneumoniae test kit and the new vaccine against Mycoplasma hyopneumoniae.