Open Access
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

Sabarinath Thankappan: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Rajneesh Rana: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Arun Thachappully Remesh: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Valsala Rekha: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Viswas Konasagara Nagaleekar: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Bhavani Puvvala: Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.

doi: 10.14202/vetworld.2017.1108-1113

Share this article on [Facebook] [LinkedIn]

Article history: Received: 29-04-2017, Accepted: 18-07-2017, Published online: 21-09-2017

Corresponding author: Rajneesh Rana


Citation: Thankappan S, Rana R, Remesh AT, Rekha V, Nagaleekar VK, Puvvala B (2017) Cloning and expression of P67 protein of Mycoplasma leachii, Veterinary World, 10(9): 1108-1113.

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.


1. Hum, S., Kessell, A., Djordjevic, S., Rheinberger, R., Hornitzky, M., Forbes, W. and Gonsalves, J. (2000) Mastitis, polyarthritis and abortion caused by Mycoplasma species bovine group 7 in dairy cattle. Aust. Vet. J., 78: 744-750. [Crossref] [PubMed]

2. Shiel, M.J., Coloe, P.J., Worotniuk, B. and Burgess, G.W. (1982) Polyarthritis in a calf associated with a group 7 mycoplasma infection. Aust. Vet. J., 59: 192-193. [Crossref] [PubMed]

3. Simmons, G.C. and Johnston, L.A.Y. (1963) Arthritis in calves caused by Mycoplasma sp. Aust. Vet. J., 39: 11-14. [Crossref]

4. Tardy, F., Maigre, L., Poumarat, F. and Citti, C. (2009) Identification and distribution of genetic markers in three closely related taxa of the Mycoplasma mycoides cluster: Refining the relative position and frontiers of the Mycoplasma sp. Bovine group 7 taxon (Mycoplasma leachii). Microbiology, 155: 3775-3787. [Crossref] [PubMed]

5. Wang, H., Ni, L., Yang, H., Xu, L., Ma, N. and Ding, H. (2014) Isolation and identification of Mycoplasma mycoides cluster strains from goats in Chongqing, China. Bull. Vet. Inst. Pulawy, 58: 11-15. [Crossref]

6. Manso-Silvan, L., Perrier, X. and Thiaucourt, F. (2007) Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group. Int. J. Syst. Evol. Microbiol., 57: 2247-2258. [Crossref] [PubMed]

7. Manso-Silvan, L., Vilei, E.M., Sachse, K., Djordjevic, S.P., Thiaucourt, F. and Frey, J. (2009) Mycoplasma leachii sp. Nov. As a new species designation for Mycoplasma sp. Bovine group 7 of leach, and reclassification of Mycoplasma mycoides subsp. Mycoides LC as a serovar of Mycoplasma mycoides subsp. Capri. Int. J. Syst. Evol. Microbiol., 59: 1353-1358. [Crossref]

8. Fischer, A., Santana-Cruz, I., Hegerman, J., Gourle, H., Schieck, E., Lambert, M., Nadendla, S., Wesonga, H., Rachel A.M., Vashee, S., Weber, J., Meens, J., Frey, J. and Jores, J. (2015) High quality draft genomes of the Mycoplasma mycoides subsp. Mycoides challenge strains Afade and B237. Stand. Genomic Sci., 10: 89. [Crossref] [PubMed] [PMC]

9. Krieg, N.R., Ludwig, W., Whitman, W.B., Hedlund, B.P., Paster, B.J. and Staley, J.T. (2010) Bergey's Manual of Systematic Bacteriology. 2nd ed. Springer Publishing, New York. p948. [Crossref]

10. Chang, J.T., Liu, H.J. and Yu, L. (2011) Mycoplasma leachii sp. Nov. In calves, China. Emerg. Infect. Dis., 17: 1772-1773. [Crossref] [PubMed] [PMC]

11. Alexander, P.G., Slee, K.J., McOrist, S., Ireland, L. and Coloe, P.J. (1985) Mastitis in cows and polyarthritis and pneumonia in calves caused by Mycoplasma species bovine group 7. Aust. Vet. J., 62: 135-136. [Crossref] [PubMed]

12. Al-Aubaidi, J.M. and Fabricant, J. (1971) Characterization and classification of bovine Mycoplasma. Cornell Vet., 61: 490-518. [PubMed]

13. Langford, E.V. (1975) Mycoplasma species recovered from the reproductive tracts of Western Canadian cows. Can. J. Comp. Med., 39: 133-138. [PubMed] [PMC]

14. Pal, B.C., Singh, P.P. and Pathak, R.C. (1984) Mycoplasmas from the genital tract of aborting buffaloes. Indian Vet. J., 61: 1-3.

15. Katoch, R.C., Sodhi, S.S. and Baxi, K.K. (1984) Distribution of Mycoplasmas in the genital tract of dairy bulls. Indian Vet. J., 61: 89-95.

16. Atalaia, V., Machado, M. and Frazao, F. (1987) Patologia dos pequenos ruminantes infeccoes em ovinos e caprinos, originadas pelo micoplasma do Grupo 7, Leach. Repositorio de Trabalhos do Laboratorio Nacional de Investigacao Veterinaria. Vol. 19. p50, 55-60.

17. Djordjevic, S.P., Forbes, W.A., Forbes-Faulkner, J., Kuhnert, P., Hum, S., Hornitzky, M.A., Vilei, E.M. and Frey, J. (2001) Genetic diversity among Mycoplasma species bovine group 7: Clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle. Electrophoresis, 22: 3551-3561.

18. Bertin, C., Pau-Roblot, C., Courtois, J., Manso-Silvan, L., Tardy, F., Poumarat, F., Citti, C., Sirand-Pugnet, P., Gaurivaud, P. and Thiaucourt, F. (2015) Highly dynamic genomic loci drive the synthesis of two types of capsular or secreted polysaccharides within the Mycoplasma mycoides cluster. Appl. Environ. Microbiol., 81: 676-687. [Crossref]

19. Cottew, G.S. and Leach, R.H. (1969) Mycoplasmas of cattle, sheep and goats. In: Hayflick, L., editor. The Mycoplasmatales and the L-Phase of Bacteria. 1st ed. Appleton-Century-Crofts, New York. p232-234.

20. Citti, C. and Blanchard, A. (2013) Mycoplasmas and their host: Emerging and re-emerging minimal pathogens. Trends Microbiol., 21: 196-203. [Crossref] [PubMed]

21. Wise, K.S., Michael, J.C., Mark, F.F, Ramana, M., Robert, T.D., Kerstin, R., Miranda, L.H., Tara, R.M., Scott, A.D., John, I.G. and Barbara, A.M. (2012) Complete genome sequences of Mycoplasma leachii strain PG50T and the pathogenic Mycoplasma mycoides subsp. Mycoides small colony biotype strain gladysdale. J. Bacteriol., 194(16): 4448-4449. [Crossref] [PubMed] [PMC]

22. Yamao, F., Muto, A., Kawauchi, Y., Iwami, M., Iwagami, S., Azumi, Y. and Osawa, S. (1985) UGA is read as tryptophan in Mycoplasma capricolum. Proc. Natl. Acad. Sci. USA, 82: 2306-2309. [Crossref]

23. Larsen, J.E., Lund, O. and Nielsen, M. (2006) Improved method for predicting linear B-cell epitopes. Immunome Res., 2: 2. [Crossref] [PubMed] [PMC]

24. Saha, S. and Raghava, G.P.S. (2006) Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins., 65(1): 40-48. [Crossref] [PubMed]

25. Frey, J., Cheng, X., Monnerat, M.P., Abdo, E.M., Krawinkler, M., Bolske, G. and Nicolet, J. (1998) Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp. Bovine group 7. Res. Microbiol., 149: 55-64. [Crossref]

26. Bhanuprakash, V. and Srivastava, N.C. (1996) Partial purification and electrophoretic profile of M. arginini antigenic preprations. Indian J. Anim. Sci., 66: 542-544.

27. Fischer, A., Shapiro, B., Muriuki, C., Heller, M., Schnee, C., Rudloff, E.B., Vilei, E.M., Frey, J. and Jores, J. (2012) The origin of the "Mycoplasma mycoides cluster" coincides with domestication of ruminants. PLoS One, 7(4): e36150. [Crossref]

28. Deneke, Y., Sabarinath, T., Gogia, N., Lalsiamthara, J., Viswas, K.N. and Chaudhuri, P. (2014) Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India. Mol. Cell. Probes, 28(4): 141-146. [Crossref] [PubMed]

29. Hoelzle, K., Grimm, J., Ritzmann, M., Heinritzi, K., Torgerson, P., Hamburger, A., Wittenbrink, M.M. and Hoelzle, L.E. (2007) Use of recombinant antigens to detect antibodies against Mycoplasma suis, with correlation of serological results to hematological findings. Clin. Vaccine Immunol., 14(12): 1616-1622. [Crossref] [PubMed] [PMC]

30. Wawegama, N.K., Browning, G.F., Kanci, A., Marenda, M.S. and Markham, P.F. (2014) Development of a recombinant protein-based enzyme-linked immunosorbent assay for diagnosis of Mycoplasma bovis infection in cattle. Clin. Vaccine Immunol., 21(2): 196-202. [Crossref] [PubMed] [PMC]

31. Sun, Z., Fu, P., Wei, K., Zhang, H., Zhang, Y., Xu, J., Jiang, F., Liu, X., Xu, W. and Wu, W. (2014) Identification of novel immunogenic proteins from Mycoplasma bovis and establishment of an indirect ELISA based on recombinant E1 beta subunit of the pyruvate dehydrogenase complex. PLoS One, 9(2): e88328. [Crossref]

32. Rocha, T.S., Tramuta, C., Catania, S., Matucci, A., Giuffrida, M.G., Baro, C., Profiti, M., Bertolotti, L. and Rosati, S. (2015) Cloning, expression and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli. Poult. Sci., 94(4): 621-627. [Crossref] [PubMed]

33. Khan, F.A., Faisal, M., Chao, J., Liu, K., Chen, X., Zhao, G., Menghwar, H., Zhang, H., Zhu, X., Rasheed, M.A., He, C., Hu, C., Chen, Y., Baranowski, E., Chen, H. and Guo, A. (2016) Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection. Oncotarget, 7: 39376-39395. [Crossref] [PubMed] [PMC]

34. Wawegama, N.K., Markham, P.F., Kanci, A., Schibrowski, M., Oswin, S., Barnes, T.S., Firestone, S.M., Mahony, T.J. and Browning, G.F. (2016) Evaluation of an IgG enzyme linked immunosorbent assay as a serological assay for detection of Mycoplasma bovis infection in feedlot cattle. J. Clin. Microbiol., 54: 1269-1275. [Crossref]

35. Fodor, L., Janosi, K., Makrai, L. and Gyuranecz, M. (2017) Screening of Hungarian cattle herds for seropositivity to Mycoplasma bovis. Acta Vet. Hung., 65(2): 166-172. [Crossref] [PubMed]