Cloning and expression of P67 protein of Mycoplasma leachii

Research (Published online: 21-09-2017)

15. Cloning and expression of P67 protein of Mycoplasma leachii

Sabarinath Thankappan, Rajneesh Rana, Arun Thachappully Remesh, Valsala Rekha, Viswas Konasagara Nagaleekar and Bhavani Puvvala

Veterinary World, 10(9): 1108-1113

ABSTRACT

Aim: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii.

Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein.

Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis.

Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent.

Keywords: cloning, dot blot, expression, Mycoplasma leachii, P67 protein, western blot.