Insulin-like Growth Factor Binding Protein 3 Gene Of Golden Pompano(TroIGFBP3) Involved In Antimicrobial Immune Response

Insulin-like growth factor binding protein 3(IGFBP3)is an important member of the IGFBP family.At present,studies on IGFBP3 in mammals mainly focus on the regulation of cell proliferation,differentiation,growth,apoptosis and innate immunity,however,the studies concerning IGFBP3 in teleosts are very limited and its function still remains unclear.Therefore,in this study,we cloned and identified the whole length of the golden pompano(Trachinotus ovatus)IGFBP3 gene,analyzed its sequence structure,and analyzed its main functions in vivo and in vitro.The contents and results are as follows:1.The c DNA sequence of Tro IGFBP3 was cloned from golden pompano liver.Tro IGFBP3 c DNA contained an ORF of 861 bp,encoding a predicted polypeptide of 286 amino acid residues.The predicted polypeptide sequence contained a putative signal peptide of 21 amino acid residues.The putative molecular mass was 29 k Da,and the theoretical isoelectric point was 8.802.Sequence homology analysis showed that Tro IGFBP3 had high homology with other teleosts IGFBP3(58.1%?93.71%),and the homology with Amphiprion ocellaris was the highest,reaching 93.71%.The results of phylogenetic tree analysis showed that Tro IGFBP3 and A.ocellaris IGFBP3 were both a clade,which belongs to Perciformes.2.The results of tissue specificity analysis showed that Tro IGFBP3 was expressed in all tissues of healthy T.ovatus,and had a high expression level in immune organs such as liver,spleen,and head kidney,among which the expression level was the highest in liver,and the lowest in skin and muscle.Furthermore,the expression of Tro IGFBP3 was significantly upregulated in liver,spleen and head kidney under Vibrio harveyi infection.The maximum expression levels of Tro IGFBP3 after infection 24 h in the liver,spleen and head kidney were ?20.21-fold greater,?13.87-fold greater and ?13.91-fold greater than control levels respectively.3.The prokaryotic expression vectors of Tro IGFBP3 were constructed.Then,the fusion protein r Tro IGFBP3 was successfully induced and expressed.The results of in vitro assays showed that r Tro IGFBP3 incubated with macrophages and peripheral blood leukocytes(PBLs)could stimulate macrophage activation and promote peripheral blood leukocytes proliferation.Meanwhile,r Tro IGFBP3 could inhibit cellular proliferation and promote apoptosis of murine liver cancer cells.4.The results of subcellular localization showed that IGFBP3 was expressed in both nucleus and cytoplasm of golden pompano snout cells;In addition,the results of in vivo experiments showed overexpressing IGFBP3 in golden pompano could significantly improve the antibacterial ability,and the number of bacteria was significantly reducedcompared with the control group.Consistent with this,when the expression of Tro IGFBP3 in golden pompano was disturbed,the antibacterial ability of the body was significantly reduced,and the amount of bacteria in these tissues was significantly increased.In summary,our results indicate that the Tro IGFBP3 can improve the body's antimicrobial immune response and promote the apoptosis of liver cancer cells in mice.The results of this study will enrich our understanding of the anti-bacterial immune mechanism of golden pompano,and provide new research ideas for the healthy and sustainable development of artificial culture of golden pompano.In addition,its anti-tumor effect provides a new research perspective for biology,medicine and other fields.