Expression And Functional Analysis Of Grass Carp Ajuba Protein During GCRV Infection CIK Cells

Ajuba belongs to the Zyxin family and the third group in LIM aomain proteins, which containing double structure of zinc finger domains.The main function of the LIM proteins was participated in the interactions between different proteins. Ajuba is one of the important LIM domain proteins, which involved in assembling the extracellular matrix and taking along with associated proteins to regulate target genes that connect the extracellular matrix and the cytoskeleton. Also, protein Ajuba is associated with different physiological processes as a transcription inhibitory factor, even the occurrence of tumor. And Ajuba involved in the activation of NF-κB pathway, the transcription factor protein family. The NF-κB pathway can control many kinds of molecules that are associated with early immune response and inflammatory response. Hemorrhagic disease of grass carp causing by GCRV has been endangering the development of China’s freshwater aquaculture and causing great losses to the fishermen for decades of years. The research on GCRV molecular structure and gene function are the basis of defining the mechanism of virus infecting the host. However, the research of GCRV proteins interactions with the host is the emphasis and difficulty in prevention of grass carp hemorrhagic disease.Based on this,the following aspects were studied on this study:1. Molecular cloning and expression of Ajuba gene in Grass carpIn the present study, the entire cDNA sequence of the Ajuba gene from grass carp (gcAjuba) according to the yeast two hybrid technology and RACE were characterized. The cDNA of gcAjuba includes 3980 bp and have been submitted to NCBI (GenBank accession number is KC551289.1). It contained an open reading frame (ORF) of 2121 bp encoding a polypeptide of 706 amino acids with an estimated molecular mass of 76.0 kDa and three LIM domains in the C-terminal. Its theoretical pI was 7.87 and the instability index (Ⅱ) was 64.48,indicating that it is an unstable protein. BLAST results showed the amino acid sequence comparison of gcAjuba and its homologues from other species. The gcAjuba coding sequence demonstrated a high degree of amino acid identity with Zebra fish and many other species. Phylogenetic analysis showed that the gcAjuba clustered most closely with the Ajuba protein of other fishspecies (particularly that of Zebra fish) and was furthest phylogenetically from mammalian Ajuba.2. The expression of gcAjuba in response to virus infectionTo examination of the gcAjuba transcriptional level after the virus infection was demonstrated whether GCRV-JX01 regulates the expression of gcAjuba by qRT-PCR. Infected carp kidney (CIK) cells were collected at 0,4,8,12,24 h p.i. The gcAjuba transcriptional level was calculated by 2-ΔΔCT method. The data weresubjected to analysis of Student’s t-test and the P values less than0.05 (P< 0.05) were considered statistically significant.We found that the transcriptional level of gcAjuba was significantly up-regulated at 4 h following the stimulationof virus in vitro.3. The primary research on interaction between gcAjuba and GCRV NS26To investigate the sub-cellular location of the gcAjuba and NS26 proteins, recombinant plasmids pEGFP-gcAjuba and pcDNA3.1-NS26 were co-transfected into CIK cells. His-fused NS26 proteinwas expressed ubiquitously and the EGFP-fused gcAjuba protein was patchily distributed in the cytoplasm. Sub-cellular location of gcAjuba and GCRV-JX01 NS26 proteins did not overlapin the cytoplasm and no direct interaction between gcAjuba and the protein NS26 was detected by CO-IP test in grass CIK cells.Firstly, the full-length cDNA of gcAjuba was isolated and sequenced successfully from the CIK cells. The sequence information and details of the genetic structure of the gcAjuba gene provided critical informationfor further studies investigating the function of this gene in grasscarp. Also, the gcAjuba is determined to be an immediately inducible gene responding to viral infection. This providesa new thinkingfor the prevention and control of GCRV infection mechanism with Ajuba involved in the immune system. But the association of gcAjuba and NS26 could not be confirmedin vivo, which has been suggested that the phosphorylation, methylation or proper folding status of gcAjuba may affect its interaction with NS26 by yeast two-hybrid assay in previous. Presumably, there should be a third partner exist to stimulate the interaction. Thus, it remained to be resolved whether gcAjuba did interact directly with NS26 under certainconditions.