| Cardiovascular diseases seriously threat human beings. Occurrence of cardiovascular diseases is a complex process, which was exactly regulated by a series of transcription factors. Thus, understanding the normal regulation network and mechanisms of cardiovascular development can help understanding the pathogenetic mechanism of cardiovascular diseases.The CG8419 gene is the homolog of Trim45 previously cloned by our lab, which is highly conserved on evolution. Previous study by Drosophila model shows that CG8419 may be involved in Drosophila hematopoietic development. CG9090 and mrj are two candidate proteins interacted with CG8419 previously identified by using a yeast two-hybrid system.First, the full-length ORF of CG8419, CG9090 and mrj were constructed into pMD-18T plasmids, and after confurmed by enzyming and sequencing identified, the eukaryotic expression plasmids pCMV-tag2B-CG8419, pCMV-myc-CG9090ć'pCDNA3.1B-mrj were constructed. Three genes were transfected to HEK293 cells to study the relationship by using co-immunoprecipitation (CO-IP) assay. The results showed that CG8419 and CG9090 or CG8419 and mrj showed a interaction between the two genes. In order to study the exact interaction sites, CG8419 was subcloned to the plasmids pCMV-tag2B-Ring, pCMV-tag2B-RB2, pCMV-tag2B-RB 1, pCMV-tag2B-B2F, pCMV-tag2B-B1F and pCMV-tag2B-FLMN. The CO-IP assays were performed between CG8419 subclones with CG9090 and mrj and the results showed that CG8419 interacted with CG9090 and mrj, seperately, mainly by B-BOX domain, however, ring domain also gave an effect. The analyses by CO-IP assay showed that the Pfam:Mito_carr dormain of CG8419 interacted with CG9090, and The DnaJ dormain of CG8419 interacted with mrj.To sum up, our results confurmed that CG8419 interacted with CG9090 and mrj by its B-BOX dormain, CG9090 interacted with CG8419 by its Pfam:Mito_carr dormain, and mrj interacted with CG8419 by its DnaJ dormain, which establish an bases for further study on the CG8419 signaling pathway. |
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