Prokaryotic Expression And Some Biological Properties Of Enolase From Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae (Mhp) can cause chronic respiratory disease of swine, it damaged pork production seriously. The study of the Pathogenic virulence factor will provide basic for the prevention and control of the disease. In this experiment we tested the prokaryotic expression and functional analysis of Mycoplasma hyopneumoniae enolase.According to the gene sequences of Mycoplasma hyopneumoniae enolase in GenBank, we designed the primers by the primer 5.0 software. Due to one rare codon in this sequence at 730bp. Then anterior 516bp and posterior segment 773bp sequence by using the SOE-PCR method respectively and connected. Then the gene fragment of Mycoplasma hyopneumoniae enolase was inserted into the expression vector pGEX-4T-1 by restriction enzymes and T4 ligase to construct the recombinant expression plasmid pGEX-4T-1/Eno. Then, we transformed the recombinant expression plasmid pGEX-4T-1/Eno into E.coli BL21 (DE3). The fusion protein was induced and expressed with 1mM IPTG after 24 h. The recombinant protein was purified by using GST fusion protein purification kit. The result of Western-blot confirmed that the fusion protein of Mycoplasma hyopneumoniae enolase has a good reactionogenicity.The purified Mycoplasma hyopneumoniae Eno recombinant protein was immunized to BALB/c mice, and was used as the antigen in indirect ELISA to detect the mice antibody level after Mycoplasma hyopneumoniae rEno protein immunization. The Western-blot and ELISA were used to detect the antibody response and antibody level of Mycoplasma hyopneumoniae rEno protein of mice. The results indicated that Mycoplasma hyopneumoniae rEno protein could induce higher levels of antibody in mice. It showed that the rEno protein has a good antigenicity. Then, the swine tracheal epithelial cells was incubated with the purified protein into for the adhesion and the damage essay. The results showed that the Mycoplasma hyopneumoniae rEno protein induced damage in STEC cells after 16 hours, and it can effectively inhibit Mycoplasma hyopneumoniae adhering to STEC cells.In summary, the Mycoplasma hyopneumoniae Eno recombinant bacteria was constructed, and the purified protein of Mycoplasma hyopneumoniae Eno was obtained. The protein has antigenic reactivity and can damage the cell and inhibit Mhp adhering to STEC cells. It will provided the necessary theoretical and experimental basis for study of pathogenic mechanism and diagnosis methods of Mycoplasma hyopneumoniae.