Identification, Characterization and Purification of MSC_0265, a Potential Immunogenic Antigen Homologue of Mycoplasma mycoides subsp. mycoides in Mycoplasma capricolum subsp. capripneumoniae
M. Orwe *
Department of Production, Kenya Veterinary Vaccines Production Institute, P.O.Box 53260-00200, Nairobi, Kenya
J. Kinyua
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P.O.Box 62000-00200, Nairobi, Kenya
J. Ngaira
Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology, P.O.Box 62000-00200, Nairobi, Kenya
H. Wesonga
Division of Bacteriology, Kenya Agricultural and Livestock Research Organization, P.O.Box 32-00902, Kikuyu, Kenya
J. Naessens
Division of Bacteriology, Kenya Agricultural and Livestock Research Organization, P.O.Box 32-00902, Kikuyu, Kenya
*Author to whom correspondence should be addressed.
Abstract
In silico identification and characterization of vaccine antigens has opened up new frontiers in the field of reverse vaccinology to mitigate the effects of livestock diseases by development of new subunit vaccines. This study aims to characterize, express and purify MSC_0265 for eventual use in immunoassays and inoculation in goats. Mycoplasma mycoides subs. mycoides (Mmm) and Mycoplasma capricolum capripneumoniae (Mccp) are similar pathogens on the genomic level and are the causative agents of Contagious Bovine Pleuropneumonia (CBPP) in cattle and Contagious Caprine Pleuropneumonia (CCPP) in goats respectively. In this study, BLAST was used to identify the homology of MSC_0265 in Mycoplasma capricolum capripneumoniae genome and the protein it is similar to. Characterization of MSC_0265 was also done using I-TASSER to predict secondary structure, solvent accessibility, normalised B-factor, 3D models and function. With cut off points of 0.0 for E-value, 100% for Query coverage and 90% for Identity, MSC_0265 a pyruvate dehydrogenase enzyme gave a high homology score on tBLASTn and BLASTp. It had earlier been cloned in pGS21a vector before proceeding with expression and purification of the His-tagged protein by Ni-NTA affinity chromatography. This study identified the homologue of MSC_0265 as protein WP_029333261.1 in the Mycoplasma capricolum capripneumoniae genome (Accession NZ_LN515398.1) using tBLASTn and BLASTp. Additionally, MSC_0265 was characterized and its optimal expression profile and estimated molecular weight verified.
Keywords: Mmm, CBPP, Mccp, CCPP, BLAST, I-TASSER, expression, purification