The Preliminary Study On Cloning,Expression And Functi On Of Grass Carp(Ctenopharyngodon Idellus) TRIM32

Grass cotta(Ctenopharyngodon idellus)is China's important freshwater fish,it was large-scale farmed in many areas of China's.However,with the vigorous development of grass carp aquaculture,many kinds of diseases frequently broke out.there is no healthy and effective treatment.this situation seriously hindered the healthy development of grass carp aquaculture.Because fish-specific immune system is not completely development,natural immune system is more important for resiste various pathogens invasion.The TRIM(tripartite-motif protein)family is an important member in natural immune system,which involved in cell proliferation,differentiation,apoptosis,tumor suppression,antiviral activity and other processes.TRIM family also called as RBCC family,from the N-terminal to the C-terminal is the RING domain,B-box domain,Coiled-coil domain.In mammals,it has been confirmed that TRIM32(tripartite-motif protein 32)play the role of E3 ubiquitin ligase and regulate the signal pathway in immune response.The main subject of the present study was grass carp TRIM32,the aim of this study is to providing a further theoretical basis for prevention and treatment of grass carp disease.The main research contents include the following aspects:1.The TRIM32 full-length sequence of grass carp was cloned using PCR method.It was 1980 bp in length encoding 660 amino acids.2.The TRIM32 protein has three typical structural features of the TRIM family.Sequence analysis indicated that TRIM32 of grass carp shared the highest identity with zebrafish,which belongs to the same branch on the phylogenetic trees.3.Immunofluorescence and Western blotting showed that TRIM32 successfully expressed in cytoplasmic speckles of EPC cells.4.Tissue distribution analysis showed that TRIM32 was widely expressed,especially in the renal tissue.TRIM32 was significantly higher in zygote period,cleavage and blastula stage than other stages during different stages of embryonic development,and then decreased until hatching in which it was significantly increased.5.Further studies have shown that grass carp TRIM32 can activate the nuclear factor NF-?B based on dual luciferase assay and induce inflammation.6.In addition,Construction of grass carp TRIM32 prokaryotic expression vector and preparation of polyclonal antibodies.