Introduction: Introduction: Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. This study aims is to obtain the optimum conditions for high expression and purification of PvpA-pMGA1.2 recombinant protein from Mycoplasma gallisepticum.
Methods: Methods: The PvpA-pMGA1.2 gene was cloned into a pET-32a (+) expression vector. BL21(DE3) was used as expression host for transformation.The expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration.
Results: Results: SDS-PAGE analysis showed that the production of rPvpA-pMGA protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1M and 16h in 2xTY medium respectively. The purification of the rPvpA-pMGA
protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, three times washing process and elution buffer with a pH 8.
Conclusion: Conclusion: Based on the results of this study, optimizing the expression and purification process for over production of rPvpA-pMGA protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for avian mycoplasmosis.
Keywords: Key words: Mycoplasma gallisepticum, rPvpA-pMGA1.2, Recombinant protein