Biological Activity Analysis And Protein Interaction Screening Of Fish β-defensin

Defensin is a cysteine-rich cationic small peptide with broad-spectrum of antimicrobial activity. Itis ubiquitously distributed in various organisms including animals, plants or insects. In spite ofantimicrobial activity, defensins directly interact with the bacterial membrane, so that target cells haveno resistance. Moreover, defensin can regulate the immune system as an immunomodulator whichmakes it a valuable gene to study.Fish defensins (BD) have been studied at only a rudimentary level and study on scaleless freshwater fish defensin is blank. In this study, we obtained the full-length cDNA and gemomic DNA of twodefensin genes from a scaleless fish--loach, respectively named pdBD-1and pdBD-2. Sequencealignment revealed that pdBD-1and pdBD-2contained three exons divided by two introns, which wasconsistent with other fish BDs. The full-length cDNA of pdBD-1and pdBD-2were204bp and189bprespectively, which encoded67and62animo acids. Signal P predicted that the front24and19animoacids belong to signal peptides. The pI of mature peptides of pdBD-1and pdBD-2was7.8and8.9,while the molecular weight was4.7KDa and5.1KDa, respectively; pdBD-1and pdBD-2had thehighest identity with carp defensin-1and trout defensin-2.To study antibacterial activity of loach defensin in vitro, we cloned pdBD-1and pdBD-2intoplasmid pcDNA3.1(+) and transfected to HEK293T cell line. After screening for15days by G418, theexpression level of mRNA and protein were detected by RT-PCR and western blot in HEK293T celllines, and the recombinant protein showed antibacterial activity against Aeromonas hydrophila andBacillus subtilis.For further study on the function information of defensin, we used quantitative real-time PCR toinvestigate the expression pattern of the Chinese loach β-defensin gene, the mRNA of which could beobserved in various tissues. Interestingly, highest expression of pdBD-1and pdBD-2were both detectedin eye which may partly explain why some eye-tissue (e.g iris) rarely gets microbial infections. Then weused A. hydrophila (a pathogen to loach) to challenge loach and analysed the relative mRNA level ofdefensin. Our results showed A. hydrophila could cause up-regulation of both pdBD-1and pdBD-2infour tissues that we examined, including skin, gill, spleen and liver. A different trend of change wasobserved in different tissues, or at different times, indicating a possible relationship between defensinand A. hydrophila. Finally, spleen primary cell was also challenged with poly I:C、LPS or A. hydrophila,and defensin showed upregulation towards these three stimulants.In this study, we also analysed the sub-cellular distribution of pdBD-1and pdBD-2. We clonedpdBD-1and pdBD-2into RFP (Red Fusion Protein) plasmid pDsRed2-C1, and then transientlytransfected them to zebrafish cell line ZF4. The result showed that after72h transfection, pdBD-1andpdBD-2was exclusively distributed in the nucleus.In order to study the possible immune pathway defensin may be involved in, we used zebrafishdefensin-1and-3as bait protein to conduct yeast two-hybrid (Y2H) assay. We have conducted zebrafishspleen cDNA library to screen the interacting proteins. This is also the first time to conduct a Y2H assay of fish defensin. We then preliminarily tested in vivo interaction between defensin and interestingproteins (elastase, lysosomal protein transmembrane-5and granulin) screened from Y2H. We cloneddefensin and the candidate genes into GFP (Green Fusion Protein) and RFP plasmids respectively andcotransfected to HEK293T cell line. Our preliminary data showed the fluorescence of defensin-1andelastase, as well as defensin-1and LAPTM5, merged together, indicating that these two pairs of proteinsmay interact together in HEK293T cells. Futher studies need to be done in the future to elucidate thepossible mechanism and pathways.