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Transcript
Search for the potential antigens present in the outer membrane of Pasteurella multocida by immunoproteomics Hsiao-Ping Chou1, Keng-Hung Liu1, Cheng-Hao Huang1, Wei-Chen Yang1, Ya-Sheng Yu1, Chia-Ching Lin1, Yeou-Guang Tsay3* Ting-Feng Wu1,2* 1 3 Department of biotechnology and 2Research Center of Biotechnology, Southern Taiwan University, Tainan, Taiwan Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei, Taiwan Gram-negative coccobacillus pathogen which is Pasteurella multocida result in fowl cholera and hemorrhagic septicaemia. Current vaccines against fowl cholera including the live naturally attenuated and the killed whole cells, provide only limited protection. In this study, we tried to find the possible antigens in outer membrane, which may serve as the potential subunit vaccines. Two dimensional (2-D) western blotting with the elicited-sera, which were taken from the formalin-killed bacteria-administrated mice was exploited to found out the possible antigens present in the outer membrane of P. multocida. Western blotting demonstrated that 23 proteins were able to interact with the sera. These 23 proteins were further identified using high performance liquid chromatography electro-spray tandem mass spectrometry. One of these 23 proteins was recognized as pm1992 which is an outer membrane protein and other recognized proteins were involved in energy metabolism. Owing to pm1992 protein’s specific membrane location and unknown function, we anticipated that pm1992 may be a possible subunit vaccine candidate. Therefore, pm1992 gene was cloned into the pPAL7 procaryotic expression vector and purified with Profinity eXact™ Protein Purification System (Bio-Rad). The results of western blotting showed that the purified pm1992 could be recognized by induced-sera, indicating that pm1992 protein may be a potential subunit vaccine candidate.