The Preparation And Identification Of DNA Polymerase Delta Four Subunits And Related Factors Of Zebrafish And The Cloning And Function Analysis Of Epinephelus Akaara IκBα Gene

In this study, the four subunits(Pold1, Pold2, Pold3, Pold4) genes of zebrafish DNA polymerase delta(pol δ) were cloned and expressed in vitro by baculovirus expression system. Zebrafish PCNA gene was cloned, expressed and purified.Zebrafish, as a classic model organism, has 87% genes which are similar to human genes. Zebrafish can not only be used to explore fish pathogenic mechanism, but also be used to explore the pathogenesis of human diseases. Eukaryotic DNA pol δ, the main replicase of chromosomal DNA replication, is involved in various forms of DNA damage repair. As a cofactor of DNA pol δ in the initiation of DNA replication, PCNA is indispensable in the process of DNA replication. In recent years, the purification technology of pol δ has been greatly improved. There are a lot of studies on pathological function of DNA polymerase. However, there are very few DNA polymerase genes characterized in fish so far, and whether the fish has similar function as the mammal DNA pol δ remains unclear. In this study, four subunits’ genes of zebrafish DNA pol δ were cloned and expressed in vitro by baculovirus expression system. Different combinations of those genes were cloned into a baculovirus transfer vector pFBDM. PCR analysis showed that the recombinant expression vectors were constructed successfully. The different subunit combinations of zebrafish DNA pol δ were subcloned into MultiBac baculoviral DNA. The recombinant Bacmids were constructed by transposition. Sf-9 cells were transfection with extracted recombinant Bacmids.Western Blot analysis showed that zebrafish DNA pol δ subunit genes was successfully expressed in Sf-9 cells, and the mass spectrometry(MS) analysis results showed that the recombinant protein were indeed encoded by Pold1, Pold2 and Pold4. PCNA gene of zebrafish was cloned, expressed and purified in prokaryotic expression system. In order to search zebrafish PCNA interacting proteins, the purified PCNA was used to identify the binding protein by affinity chromatography with CNBr-activated SepharoseTM 4B. Totally, 21 proteins was extracted and identified by MS analysis, which involed in many cellular processes, such as DNA replication, DNA repair, cell cycle regulation, etc. It will provide data for the research on functional characterization of zebrafish DNA pol δ, and facilitate our extensive research into understanding how alterations in pol δ function could contribute to the etiology of human cancer.NF-kappa B(NF-κB) inhibitory factor IκBα gene(EaIκBα) from grouper(Epinephelus akaara) was cloned and characterized in this study.The deterioration of aquaculture environment in coastal areas has brought great losses to fish culture industry in China. Grouper is a nutrient rich edible fish. The research of grouper immune system on its related genes and mechanism is conducive to improve the grouper immune response and disease resistance, thus, achieve the goal of health cultivation. NF-κB signal transduction pathway is participated in many physiological activities of immunity and development. EaIκBα gene of E. akaara was cloned and identified by 5’-and 3’-RACE in this study. In order to further analyze the expression pattern and antivirus ability, we analyzed the expression of EaIκBα in prokaryotic expression, and studied its distribution in different tissues. Results showed that the expression of Ea IκBα protein is higher in immune tissues, such as blood cells, head kidney and liver. After the challenge stimulus of LPS, Vibrio alginolyticus and Singapore grouper iridovirus(SGIV), the expression quantity of EaIκBα in the head kidney was tested by fluorescence quantitative PCR analysis. Results showed that its expression is increased in different degrees, indicating that EaIκBα may play a relatively important role in bacterial and viral infections. In addition, our research results showed that the overexpression of EaIκBα protein can inhibit the cytopathic effect(CPE) process of SGIV infection, and reduce the expression of the mainly capsid protein(MCP) and core protein ORF162 in virus. In conclusion, these findings suggested that Ea IκBα may play an important role in immune response of grouper, and provided new theoretical basis for further exploring of the grouper immune mechanism.