Prokaryotic Expression and Identication on the 20kDa Protein Gene of Mycobacterium paratuberculosis

Abstract:

The gene encoding 20kDa protein gene from Mycobacterium paratuberculosis C-2, chromosomal DNA was amplified by using polymerase chain reaction (PCR), the PCR product was approximately 520bp DNA segment. The PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-20 was constructed successfully. The purified 20kDa protein gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression plasmid pET28a-20 was constructed. Plasmid containing pET28a-20 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 23kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity of M. paratuberculosis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of 20kDa protein gene in their prevention against bovine paratuberculosis.

• Periodical: Applied Mechanics and Materials (Volume 319)
• Pages: 146-150
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.319.146
• Online since: May 2013
• Authors: Yun Hang Gao, Chun Fang Wang, Xiu Yun Jiang, Hong Xia Ma, Fan Li Zeng, Yu Qing Hu, Xin Yu Liu, Xiaoai Zhu, Yang Hong Dong, Lei Liu
• Keywords: 20kDa Protein Gene, Cloning, Mycobacterium paratuberculosis, Prokaryotic Expression

References

[1] U. Jark, I. Ringena, B. Franz, G. F. Gerlach, M. Beyerbach and B. Franz: Vet. Microbiol. Vol.57(1997), p.189

DOI: 10.1016/s0378-1135(97)00125-9

[2] K. T. Park, J. Ahn, W. C. Davis, H. C. Koo, N. H. Kwon, W. K. Jung, J. M. Kim, S. K. Hong and Y. H. Park: J. Vet. Sci. Vol.7(2006), p.349

[3] R. J. Chiodini, H. J. Van Kruiningen, W. R. Thayer, R. S. Merkal and J. A. Coutu: Dig. Dis. Sci. Vol.29(1984), p.1073

[4] M. T. Moss, E. P. Green, M. L. Tizard, Z. P. Malik and J. Hermon-Taylor: Gut Vol.32(1991),p.395

[5] J. D. Sanderson, M. T. Moss, M. L. Tizard and J. Hermon-Taylor: Gut Vol.33(1992), p.890

[6] S. Q. Zheng, D. G. Fu, C. Y. Li and W. M.Yu: Chin. J. Vet. Sci. Technol. Vol. 18(1988), p.34

[7] T. Wang, D. P. Long and H. Q. Zhang: Chin. J. Vet. Sci. Technol. Vol. 23(1993), p.20

[8] E. Spangler, L. E. Heider, S. Bech-Nielsen and C. R. Dorn: Am. J. Vet. Res. Vol.52(1991),p.1197

[9] P. Valentin-Weigand and K. M. Moriarty: J. Vet. Med. B Vol. 39(1992), p.762

[10] J. Mullerad, A.H. Hovav, R. Nahary, Y. Fishman and H. Bercovier: Microb. Pathog.Vol.34 (2003), p.81

DOI: 10.1016/s0882-4010(02)00209-7

[11] H. Cai, J. L. Li, J. Wu, F. M. Zhang and F. Y. Chen: Acta Microbiologica Sinica Vol.39(1999), p.121

[12] J. Sambrook, E. F. Fritsch and T. Maniatis, in: Molecular Cloning: A Laboratory Manual,2nd Translated by D. Y. Jin and M. F. Li, Science press, BJ（China）(1992)