Journal of Molecular Biology Research; Vol. 8, No. 1; 2018 ISSN 1925-430X E-ISSN 1925-4318 Published by Canadian Center of Science and Education 61 Comparative Study of p40 Gene Expression of Mycoplasma Agalactiae Isolated from Iranian Provinces Fereshteh Yavari 1 , Seyed Ali Pourbakhsh 2 , Hossein Goudarzi 2 & Ramezan Ali Khavarinejad 1 1 Department of biology, Science and Research branch, Islamic Azad University, Tehran, Iran 2 Mycoplasma Reference Laboratory, Razi Vaccine& Serum Research Institute, Karaj, Iran Correspondence: Seyed Ali Pourbakhsh, Mycoplasma Reference Laboratory, Razi Vaccine& Serum Research Institute, Karaj, Iran. E-mail: a.pourbakhsh@chmail.ir Received: November 30, 2017 Accepted: January 2, 2018 Online Published: July 11, 2018 doi:10.5539/jmbr.v8n1p61 URL: https://doi.org/10.5539/jmbr.v8n1p61 Abstract Mycoplasma agalactiae pathogen is the main cause of agalactia in small ruminants and as such, results in some economic losses in Iran. Contagious agalactia disease should be regarded as a syndrome, caused by various Mycoplasmas which infect several organs. In the present study, 30 suspected samples from 11 provinces of Iran were isolated from mammary gland, joint and eyes of sheep and goats and subjected to genus and species detection via PCR technique for 2 genes. These Mycoplasma strains and three Iranian vaccinal strains of Mycoplasma agalactiae were submitted to sequence analysis of a surface lipoprotein called p40 protein. Based on a comparative study between Iranian strains and PG2 Spanish strain of Mycoplasma agalactiae, most Iranian strains presented 97% homology, whereas some strains showed 80-88% and three vaccinal strains were associated with 99% homology. According to PCR and bioinformatics analysis outcomes, 6 provinces of Iran were recognized as infection areas with different expressions of this effecter protein, suggesting that finding the individual characteristics of a particular effecter may require empirical testing for vaccination. Finally, the selected sequence of p40 gene was cloned into pGEMB1cloning vector and subsequently expressed in Escherichia coli by pET-22b+ expression plasmid under the control of the T7 promoter. The expression of this fusion protein was absorbed and confirmed by SDS-PAGE. The recombinant P40 protein was expressed with a molecular mass of 37 kDa on SDS- PAGE. The sera taken from rabbits infected with Mycoplasma agalactiae produced polyclonal antibody which was then used for westernblotting. The rabbits were bled 10 days after the booster immunization using cardiac puncture. Significantly, the use of recombinant specific antigens instead of other tools for diagnosis of a disease could improve the discrimination and separation of positive and negative animals in the area under investigation and therefore, it can be applied to control infected animals and reduce economic losses. Keywords: P40 lipoprotein, Mycoplasma agalactiae, gene expression, Iranian isolate, western blot, polyclonal antibod. 1. Introduction Mycoplasma agalactiae is the cause of small ruminant syndrome that infects he main target organs of animals, including mammary glands, joints, respiratory system and eyes (Bergonier and Berthelo,1997). Agalactia, which is induced by other mastitis-causing Mollicutes like Mycoplasma putrefaciens, Mycoplasma mycoides and Mycoplasma capricolum (Nouvel, 2010), has been reported in the Mediterranean countries of Asia Minor like Iran, Iraq, the United Arab Emirates, Afghanistan and Pakistan with serious economic losses in shepherding due to the reduction in milk, cheese production and also mortality of lambs. Although Contagious agalactia has been observed in different continents, the major foci of stable and long-standing infection in goats has been found in Iran, Spain, Switzerland, Iraq, France and Italy (Bergonier and Berthelo, 1997). While the serological response to Mycoplasma agalactiae has been detected through various strategies such as growth inhibition test, ELISA and immunoblotting (Fusco and Coronal, 2007), these protocols need a proper knowledge of surface proteins of this pathogen. Many surface lipoproteins including P30, P40, P80, P55, Vpma, P48, etc. which have been found in Mycoplasma agalactiae, are able to switch their gene expression during immune evasion and adherence to epithelium cells underc host physiologic conditions (Nouvel, 2009). Genomic and proteomic analyses have provided powerful immunological insights into a few proteins of this pathogen (including P40, Vpma and P48) as suitable agents for determination kits or detection reports. Surface lipoproteins of Mycoplasma agalactiae play