Expression And Functional Analysis Of Bmlcad Gene Related To Apoptosis In Silkworm, Bombyx Mori

The ICAD (DFF-45) gene plays an important role in the process of apoptosis (programmed cell death). Cleavage of chromosomal DNA into nucleosomal multimer fragments is a hallmark of apoptosis, which is mediated by the DNA fragmentation factor (DFF) complex. DFF is a heterodimeric protein complex consisting of a 45 kD (DFF45/ICAD) and a 40 kD (DFF40/CPAN/CAD) subunit. ICAD/DFF45 acts as an inhibitor for DFF40 enzymatic activity and chaperone for CAD/DFF-40 during its synthesis. ICAD is a caspase-3 substrate that must be cleaved to be inactivated and allow CAD/DFF40 to execute nuclear internucleosomal DNA fragmentation. BmICAD gene was obtained from the fifth instar larva of Bombyx mori by RT-PCR.The BmICAD cDNA was 844 bp in length,containing a 522 bp open reading frame(ORF),which encoded a protein of 174 amino acids; the molecular weight of this protein was estimated to be 19.6 kD and the isoelectric point was 4.23.The gene was cloned into the pET-28a(+) vector, and over expressed in E.coli BL21(DE3) as His-tagged fusion proteins, induced by IPTG at 25℃. The results of SDS-PAGE showed that the recombinant protein was soluble and was purified by metal-chelating affinity chromatography. rBmICAD was used to generate anti-BmICAD polyclonal antibody.. Anti-BmICAD polyclonal antibody was used to determine the subcellular localization of BmICAD. Immunostaining indicated that BmICAD protein could be found in cytoplasm near the nucleus. Our results indicated that RNAi can inhibit BmICAD gene expression and obviously increase the apoptosis rate of BmN cells. A His pull-down assay was used to further study the possible interaction proteins with BmICAD. Two proteins, DNA supercoiling factor(SCF) and secreted protein acidic and rich in cysteine (SPARC), were found to interact with BmICAD. These results showed that BmICAD can inhibit the apoptosis of BmN cell and SCF and SPARC may play an important role by interacting with BmICAD during this process.