The objective of this study was to prepare polyclonal antibodies (pAb) against the Mycoplasma hyopneumoniae (Mhp) membrane protein Mhp271 and systematically evaluate their immunological characteristics and potential applications. The mhp271 gene (3 156 bp) was amplified by PCR and cloned into the prokaryotic expression vector pMAL-c5x to construct the recombinant plasmid pMAL-c5x-mhp271. After verification by PCR and DNA sequencing, the construct was transformed into Escherichia coli BL21(DE3) competent cells, and the expression of recombinant Mhp271 (rMhp271) was induced with IPTG. Western blotting revealed a specific band at approximately 160 kDa, confirming successful expression of rMhp271. Purified rMhp271 was emulsified and used to immunize BALB/c mice three times. Serum samples were collected one week after the final immunization, and anti-Mhp271 specific pAb was isolated. To assess the reactivity of the anti-Mhp271 pAb, we used porcine alveolar macrophages (PAMs) to establish a cell model of Mhp infection. After Mhp infection for 12 h, Western blotting and indirect immunofluorescence assay (IFA) were employed to assess protein expression. Western blotting results showed a specific band at approximately 118 kDa in the lysate from Mhp-infected PAMs, while no corresponding band was detected in the uninfected control group. IFA demonstrated distinct green fluorescence signals in infected cells, whereas no fluorescence was observed in the uninfected control group. Furthermore, the potential of anti-Mhp271 pAb to inhibit Mhp infection was evaluated through in vitro blocking assays. Mhp was pre-incubated with either 100-fold diluted anti-Mhp271 serum (experimental group) or negative serum (control group) for 30 min before being inoculated into PAMs for 12 h. TaqMan real-time quantitative PCR indicated a significant reduction in Mhp load in the experimental group compared with the control group, which was further confirmed by the weakened fluorescence in IFA. Overall, the prepared anti-Mhp271 pAb demonstrated good reactogenicity and anti-infective activity, being suitable for immunological detection methods such as Western blotting and IFA.

Keywords: Mhp271; Mycoplasma hyopneumoniae; polyclonal antibody; prokaryotic expression.