Asian-Australasian Journal of Animal Sciences

  • 제30권5호
  • /
  • Pages.736-742
  • /
  • 2017
  • /
  • 1011-2367(pISSN)
  • /
  • 1976-5517(eISSN)
아세아태평양축산학회 (Asian Australasian Association of Animal Production Societies)
권호 표지
DOI QR코드

DOI QR Code

Cloning and sequence analysis of Wild Argali short palate, lung and nasal epithelium clone 1 cDNA

  • Shen, Wen (College of Animal Science and Technology, Shihezi University) ;
  • Chen, Kaili (College of Life Science, Shihezi University) ;
  • Sun, Yanming (College of Animal Science and Technology, Shihezi University) ;
  • Guo, Haiying (College of Animal Science and Technology, Shihezi University) ;
  • Chen, Dongmei (College of Animal Science and Technology, Shihezi University) ;
  • Cao, Yang (College of Life Science, South China Agricultural University)
  • 투고 : 2015.07.04
  • 심사 : 2016.09.10
  • 발행 : 2017.05.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Objective: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. Methods: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by $Ni^{2+}$ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. Results: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). Conclusion: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.

키워드

참고문헌

  1. Yu YQ , Ji MZ, Liu CG, Li KC, Guo SG. Geographical distribution and vicissitude of argali, Ovis ammon, in China. Biodivers Sci 2008;16:197-204. https://doi.org/10.3724/SP.J.1003.2008.07035
  2. Sun YM, Chen KL, Shen W, Cui RP, Lu HF. Cloning and sequence analysis of wild argali ISG15 cDNA. Asian-Australas J Anim Sci 2014;27:561-6. https://doi.org/10.5713/ajas.2013.13455
  3. Di YP, Harper R, Zhao Y, et al. Molecular cloning and characterization of spurt, a human novel gene that is retinoic acid-inducible and encodes a secretory protein specific in upper respiratory tracts. J Biol Chem 2003;278:1165-73. https://doi.org/10.1074/jbc.M210523200
  4. Liu Y, Bartlett JA, Di ME, et al. SPLUNC1/BPIFA1 contributes to pulmonary host defense against Klebsiella pneumoniae respiratory infection. Am J Pathol 2013;182:1519-31. https://doi.org/10.1016/j.ajpath.2013.01.050
  5. McGillivary G, Bakaletz LO. The Multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway. PloS ONE 2010;5:e13224. https://doi.org/10.1371/journal.pone.0013224
  6. Sambrook J, Russell WD. Molecular cloning: A laboratory manual. 3rd edition. Cold Spring Harbor: NY : Cold Spring Harbor Laboratory Press; 2001.
  7. Marion MB. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248-54. https://doi.org/10.1016/0003-2697(76)90527-3
  8. Chu HW, Gally F, Thaikoottathil J, et al. SPLUNC1 regulation in airway epithelial cells: role of toll-like receptor 2 signaling. Respir Res 2010;5:155-62.
  9. Xiao DL, Zhong LL, Liang M, et al. Expression of SPLUNC1 in human bronchial epithelial cells induced by Mycoplasma peumoniae lipid-associated membrane proteins in human bronchial epithelial cells. J Clin Res 2013;30:1041-5.
  10. Di YP. Functional roles of SPLUNC1 in the innate immune response against Gram-negative bacteria. Biochem Soc Trans 2011;39:1051-5. https://doi.org/10.1042/BST0391051
  11. Chu HW, Thaikoottathil J, Rino JG, et al. Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation. J Immunol 2007;179:3995-4002. https://doi.org/10.4049/jimmunol.179.6.3995
  12. Gally F, Di YP, Smith SK, et al. SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice. Am J Pathol 2011;178:2159-67. https://doi.org/10.1016/j.ajpath.2011.01.026
  13. Hao RX, Hao YQ, Xu CG, Liu B, Zhang C. Isolation of Mycoplasma ovipneumoniae a sheep identification and culture characteristics. J Inner Mongolia Agric Univ 2013;34:10-3.