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Cloning and expression of P67 protein of Mycoplasma leachii
Sabarinath Thankappan1, Rajneesh Rana1, Arun Thachappully Remesh1
1Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
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.
Insights
The P67 protein from Mycoplasma leachii was successfully cloned and expressed. This Mycoplasma leachii P67 protein demonstrated immunogenicity in diagnostic tests, suggesting potential for diagnostic applications.
Area of Science:
- Veterinary Microbiology
- Molecular Biology
- Immunology
Background:
- Mycoplasma leachii is a significant pathogen in livestock.
- The P67 protein is a potential virulence factor and diagnostic target.
Purpose of the Study:
- To clone and express the P67 protein of Mycoplasma leachii.
- To evaluate the immunogenicity of the recombinant P67 protein.
Main Methods:
- Polymerase chain reaction (PCR) amplification of the P67 gene.
- Cloning into pRham N-His SUMO Kan vector and expression in Escherichia coli.
- Purification using nickel nitrilotriacetic acid affinity chromatography.
- Assessment of immunoreactivity via Western blot and dot blot analysis.
Main Results:
- Successful amplification and cloning of the P67 gene (1500 bp).
- Expression of a 79 kDa recombinant P67 fusion protein.
- Demonstrated immunogenicity of the recombinant P67 protein through blot analyses.
Conclusions:
- The P67 protein of Mycoplasma leachii is immunogenic.
- Further research is warranted to explore P67 as an immunodiagnostic agent for Mycoplasma leachii infections.
