期刊
FISH & SHELLFISH IMMUNOLOGY
卷 42, 期 2, 页码 430-438出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.fsi.2014.11.027
关键词
Murrel; Protease inhibitor; Antimicrobial protein; Gene expression; Kinetic activity
资金
- DBT's Prestigious Ramalingaswami Re-entry Fellowship - Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi [BT/HRD/35102/2006, BT/RLF/Re-entry/27/2011]
Kazal-type serine protease inhibitor (KSPI) is a pancreatic secretary trypsin inhibitor which involves in various cellular component regulations including development and defense process. In this study, we have characterized a KSPI cDNA sequence of freshwater striped murrel fish Channa striatus (Cs) at molecular level. Cellular location analysis predicted that the CsKSPI was an extracellular protein. The domain analysis showed that the CsKSPI contains a Kazal domain at 47-103 along with its family signature between 61 and 83. Phylogenetically, CsKSPI is closely related to KSPI from Maylandia zebra and formed a sister group with mammals. The 2D structure of CsKSPI showed three a-helical regions which are connected with random coils, one helix at signal sequence and two at the Kazal domain region. The relative gene expression showed that the CsKSPI was highly expressed in gills and its expression was induced upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and poly I:C (a viral analogue) challenge. The CsKSPI recombinant protein was produced to characterize and study the CsKSPI gene specific functions. The recombinant CsKSPI strongly inhibited trypsin compared to other tested proteases. The results of the kinetic activity of CsKSPI against trypsin was V(max)s = 1.62 nmol/min, K(M)s = 0.21 mM and K(i)s = 15.37 nM. Moreover, the recombinant CsKSPI inhibited the growth of Gram-negative bacteria A. hydrophila at 20 mu M and Gram-positive bacteria Bacillus subtilis at the MIC50 of 15 mu M. Overall, the study indicated that the CsKSPI was a potential trypsin inhibitor which involves in antimicrobial activity. (C) 2014 Elsevier Ltd. All rights reserved.
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