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A Kazal-type serine proteinase inhibitor from Cyclina sinensis is involved in immune response and signal pathway initiation

Ren, Yipeng, Zhang, Hao, Pan, Baoping, Yan, Chuncai
Fish & shellfish immunology 2015 v.47 no.1 pp. 110-116
RNA interference, Toll-like receptor 2, Vibrio anguillarum, amino acids, bacteria, cDNA libraries, complementary DNA, gene expression, genes, hemocytes, immune response, innate immunity, isoelectric point, messenger RNA, molecular weight, open reading frames, proteinase inhibitors, quantitative polymerase chain reaction, serine proteinases, signal transduction, tissues
Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis.