Construction Of CDNA Library, Identification And Functional Research On Four Important Immune-related Proteins In Abalone(Haliotis Diversicolor Supertexta)

Abalone is a type of important mollusc species for commercial production in the world. But in recent years, the global industry of abalone has been gradually decreased due to diseases and environmental pollution. Therefore, more investigation is required to aid in understanding the innate immune system of abalone for aquacμLture development. In this thesis, a cDNA library has been constructed and four novel immune-related genes have been selected in abalone (Haliotis diversicolor supertexta), including histidine triad nucleotide binding protein, cytidine deaminase, ubiquitin ligase RING boxl andβ-thymosin. Furthermore, functional analyses of these four genes have been carried out by bioinformatics methods, molecμLar biotechnology, immunology technology, cell biology and so on.1. A cDNA library from abalone (H. diversicolor supertexta) was constructed310 EST sequences were selected from the cDNA library including 18 novel genes. Based on bioinformatics analysis, these genes were annotated to be involved in different biological processes including cellular metabolic processes, cellular component organization and biosynthesis, signal transduction, immune defense and so on. The related genes sequences have been submitted to the GenBank.2. Identification, function and mechanism research of ab-HINTThe full length cDNA of abalone (H. diversicolor supertexta) histidine triad nucleotide binding protein (ab-HINT) is 616 bp. It has higher similarity with mammalian HINT homologues and has three conserved domains. The results of the tissues distribution showed that the ab-HINT was ubiquitously expressed in abalone (H. diversicolor supertexta) tissues indicating that ab-HINT is involved in a wide variety of biological processes. Moreover, relatively higher amount of ab-HINT was observed in hemocyte and gills, which suggest that ab-HINT may be involved in the immune response of abalone (H. diversicolor supertexta). In addition, expression levels of ab-HINT increased quickly after LPS and Poly I:C challenge, which futher support our hypothesis. To further study the biological activity and physiological function of ab-HINT, the ab-HINT recombinant protein was expressed and purified, which was further identified by LC-MS/MS analyses. In addition, the structure of ab-HINT was studied by analytical ultracentrifμgation, which showed that ab-HINT is a homodimer. Analysis by HPLC demonstrates that the ab-HINT has the ability to hydrolyze AMP-NH2. To investigate the expression localization of ab-HINT in abalone tissues, we performed immunohistochemical staining and immunofluorescent staining with prepared anti-ab-HINT antibody. Based on the studies above, we examined the effects on cell apoptosis of ab-HINT. The results showed that ab-HINT can trigger hemocytes apoptosis and P53 is involved in this process. Moreover, ab-HINT can also induce human hepatocellular carcinoma (HCC) cells apoptosis via inducing an up-regulation of P53 expression coinciding with an up-regulation of the proapoptotic factor Bax and a concomitant down-regulation of the apoptosis inhibitor Bcl-2.3. Identification and functional research of ab-CDAThe full length cDNA of abalone (H. diversicolor supertexta) cytidine deaminase (ab-CDA) is 1752 bp. It contains two conserved active site domains and conserved cysteine/histidine residues, which are the characteristics of CDA protein family. After sequence and phylogenetic tree analysis, we suggest that the ab-CDA is a homotetramer protein that belongs to the homotetrameric class of CDA superfamily. The results of the expression distribution revealed that ab-CDA was ubiquitously expressed in all examined tissues of abalone (H. diversicolor supertexta), which is consistent with the fact that ab-CDA is involved in a wide variety of biological processes. Moreover, the expression level in hemocyte is higher than that of other tissues, suggesting that ab-CDA may be involved in the immune response of abalone (H. diversicolor supertexta). The results that both LPS and Poly I:C can significantly up-regulate the expression level of ab-CDA further supported this point. To further study the biological activity and function of ab-CDA, the ab-CDA protein was expressed and purified, which was further confirmed by MS analyses. The enzymatic activity of ab-CDA studied by a spectrophotometric assay demonstrated that the ab-CDA fusion protein can deaminate cytidine effectively. Immunohistochemistry was performed to detect the intracellular localization of native ab-CDA in abalone (H. diversicolor supertexta) tissues. It was sμggested that ab-CDA is largely concentrated in the cytoplast and partially in the nuclei.4. Identification and functional research of ab-Rbx1The full length cDNA of abalone (H. diversicolor supertexta) ubiquitin ligase RING box1 (ab-Rbx1) is 509-bp. It has higher similarity with other Rbxl homologues and contains conserved cysteine/histidine residues. Study of the tissues distribution showed that ab-Rbx1 was ubiquitously expressed in abalone (H. diversicolor supertexta) tissues and a relatively high amount of ab-Rbx1 transcript was observed in hemocyte. Moreover, the expression levels of ab-Rbxl in hemocyte were increased quickly after PHA challenge. These results indicate that the ab-Rbx1 may be involved in a variety biological process in abalone (H. diversicolor supertexta) and may play a role in abalone (H. diversicolor supertexta) immune response. To further study the function of ab-Rbx1, the ab-Rbx1 was expressed snd purified. Immunohistochemical and immunofluorescent staining were performed with prepared antibody to investigate the expression localization of ab-Rbxl in abalone (H. diversicolor supertexta) tissues. The ab-Rbx1 was expressed predominantly in epithelial cells and localized both in the cytoplasmic and nuclear compartment. In addition, the activity of ab-Rbx1 was examined and the results showed that ab-Rbx1 had ubiquitin ligase activity, which was dependent on ATP.5. Identification and expression analysis of ab-TMSBThe full length cDNA of abalone (H. diversicolor supertexta)β-thymosin (ab-TMSB) is 499 bp. It showed higher similarity with other TMSB homologous and contains the conserved actin-binding motif which is the characteristic ofβ-thymosin protein family. The tissue distribution profile revealed that the ab-TMSB was ubiquitously expressed in all examined tissues of abalone (H. diversicolor supertexta), which suggests that the ab-TMSB may be involved in a wide variety of biological processes. Moreover, a relatively higher expression level was observed in hemocyte, indicating that ab-TMSB may be involved in the immune response of abalone (H. diversicolor supertexta). In addition, expression levels of ab-TMSB were significantly up-regulated after LPS challenge, which futher suggest that ab-TMSB was involved in ablone (H. diversicolor supertexta) immune response.