Designing a P48-40 Chimeric Protein of Mycoplasma agalactiae and Highly Expression in E. coli, Applicable for Indirect ELISA

Abstract Mycoplasma agalactiae is an economically important pathogen and the main etiological agent of contagious agalactiae (CA) in small ruminants with a relatively high prevalence in sheep and goat, mainly characterized by mastitis, conjunctivitis and arthritis as predominant symptoms of a localized infection. Two genes encoding p48 and p40 major surface lipoprotein are played a fundamental role in the pathogenesis of Mycoplasma agalactiae. In this study a chimeric protein P48-40 of Mycoplasma agalactiae was designed, expressed, purified and evaluated in indirect ELISA. Coding sequence of antigenic regions of two p48 and p40 proteins was cloned into the expression vector Pet32a+ then expressed successfully into the E. coli BL21 (DE3) at optimal temperature 22oC after 4 hours with 0.1mM IPTG as an inducer. Affinity batch formation method was done for purification of recombinant P48-40 by using Nickel resin. The protein was highly expressed in soluble form and purified with yield of 33mg/L. Function of recombinant protein in indirect ELISA was evaluated and the results showed that 125ng of novel P48-40 protein could well detect specific antibodies with 100% sensitivity and Specificity by indirect ELISA. We concluded that the recombinant p48-40 protein could be applied for serodiagnosis of Mycoplasma agalactiae or evaluation of antibody levels in vaccinated animal.