Identification of An Antibacterial Gene by Differential Display from Lipopolysaccharide-Stimulated Dung Beetle, Copris tripartitus

  • 발행 : 2008.12.31

초록

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

키워드

참고문헌

  1. Almeida, M.S., K. Cabral, L. N. D. Medeiros, A. P. Valente, F. C. Almeida and E. Kurtenbach (2001) cDNA cloning and heterologous expression of functional cystein-rich antifungal protein Psd1 in the yeast Pichia pastoris. Arch Biochem Biophys 395, 199-207 https://doi.org/10.1006/abbi.2001.2564
  2. Bang, H. S., K. G. Wardhaugh, S. J. Hwang and O. S. Kwon (2003) Development of Copris tripartitus (Coleoptera: Scarabaeidae) in two different rearing media. Korean J. Entomol. 33, 201-204 https://doi.org/10.1111/j.1748-5967.2003.tb00070.x
  3. Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61-92 https://doi.org/10.1146/annurev.iy.13.040195.000425
  4. Bulet, P., R. Stocklin and L. Menin (2004) Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169-184 https://doi.org/10.1111/j.0105-2896.2004.0124.x
  5. De Lucca, A. J. and T. J. Walsh (1999) Antifungal peptides: novel therapeutic compounds against emerging pathogens. Antimicrob. Agents Cemother. 43, 1-11
  6. Hancock, R. E. and R. Lehrer (1998) Cationic peptides: a new source of antibiotics, Trends Biothecnol. 16, 82-88 https://doi.org/10.1016/S0167-7799(97)01156-6
  7. Hoffman, B. J., J. A. Brodwater, P. Johnson, J. Harper, B. G. Fox and W. R. Kenealy (1995) Lactose fed-batch overexpression of recombinant metalloprotein in Escherichia coli BL21 (DE3): process control yielding high levels of metalincorporated, soluble protein. Protein Expr. Purif. 6, 646-654 https://doi.org/10.1006/prep.1995.1085
  8. Jin, F. L., X. X. Xu, W. Q Zhang and D. X. Gu (2006) Expression and characterization of a housefly cecropin gene in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 49, 39-46 https://doi.org/10.1016/j.pep.2006.03.008
  9. Koczulla, A. R. and R. Bals (2003) Antimicrobial peptidescurrent status and therapeutic potentials. Drugs 63, 389-406 https://doi.org/10.2165/00003495-200363040-00005
  10. Lehrer, R. I., A. K. Lichtenstein and T. Ganz (1993) Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annu. Rev. Immunol. 11, 105-128 https://doi.org/10.1146/annurev.iy.11.040193.000541
  11. Lehrer, R. I., M. Rosenman, S. S. Harwig, R. Jackson and P. Eisenhauer (1991) Ultrasensitive assay for endogenous antimicrobial polypeptides. J. Immunol .Methods 137, 167-173 https://doi.org/10.1016/0022-1759(91)90021-7
  12. Li, L., J. X. Wang, X. F. Zhao, C. J. Kang, N. Liu, J. H. Xiang, F. H. Li, S. Sueda and H. Kondo (2005) High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr. Purif. 39, 144-151 https://doi.org/10.1016/j.pep.2004.09.006
  13. Maruyama, K. and S. Sugano (1994) Oligo-capping : a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174 https://doi.org/10.1016/0378-1119(94)90802-8
  14. McMaster, G. K. and G. G. Carmichael (1977) Analysis of single and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. 74, 4835-4838
  15. Nicolas, P. and A. Mor (1995) Peptides ans weapons against microorganisms in the chemical defense system of vertebrates. Annu. Rev. Microbial. 49, 277-302 https://doi.org/10.1146/annurev.mi.49.100195.001425
  16. Skosyrev, V. S., E. A. Kulesskiy, A. V. Yakhnim, Y. V. Temir and L. M. Vinokurov (2003) Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli. Protein Expr. Purif. 28, 350-356 https://doi.org/10.1016/S1046-5928(02)00697-6
  17. Steiner, H., D. Hultmark, A. Engster, H. Bennich and H. G. Boman (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246-248 https://doi.org/10.1038/292246a0
  18. Xu, X., F. Jin, X. Yu, S. Ji, J. Wang, H. Cheng, C. Wang and W. Zhang (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr. Purif. 53, 293-301 https://doi.org/10.1016/j.pep.2006.12.020
  19. Yamada, K., Y. Nakajima and S. Natori (1990) Production of recombinant sarcotoxin IA in Bombyx mori cells. Biochem. J. 272, 633-636 https://doi.org/10.1042/bj2720633
  20. Zasloff, M. (2002) Antimicrovial peptides of multicelluar organisms. Nature 415, 389-395 https://doi.org/10.1038/415389a
  21. Zelezetsky, I. And A. Tossi (2006) Alpha-helical antimicrobial peptides - Using a sequence template to guide structure - activity relationship studies. Biochim. Biophys. Acta. 1758, 1436-1449 https://doi.org/10.1016/j.bbamem.2006.03.021