Mycoplasma pneumoniae is thought to be the simplest and smallest cell wall-deficient bacterium which can cause chronic respiratory infections. Recently vaccination has been a possible and reliable way to reduce the spreading and infection effectively. In this study, the transmembrane proteins P116N (the N-terminal of P116), P1C (the C-terminal of P1), P30, and P116N-P1C-P30 (MP559 for short), a chimeric protein were expressed using prokaryotic expression system. The four purified recombinant proteins were synergized with freund's adjuvant and immunized New Zealand White rabbits respectively for three times. The IgG antibodies collected from immunized rabbits and mouse were analyzed by ELISA to analyze the immunogenicity and antigenicity. The results showed the four different recombinant proteins could induce strong humoral immune response. Protein MP559 could react with antisera from rabbit immunized with P1C, P30, and P116N, indicating MP559 was well designed and presented antigen epitopes of all the three antigens. Antibodies against P116N, P1C, and P30 could be stimulated by MP559 immunization, indicating MP559 has a potential to replace the three antigens as a vaccine candidate. This study laid a substantial foundation for the vaccine development of M. pneumoniae, and at the same time provided a essential strategy for the vaccine research of other pathogen.