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.
Keywords:
Mycoplasma agalactiae; chimeric protein; P48; P40
HIGHLIGHTS
• Chimeric protein P48-40 of Mycoplasma agalactiae was designed and expressed in E.coli BL21 (DE3).
• P48-40 protein was highly expressed in soluble form and purified with yield of 33mg/Lby using Nickel resin.
• Novel P48-40 protein showed high performance for detection specific antibodies with 100% sensitivity and specificity.