Cloning, Bioinformatics Analysis, and Preparation of Polyclonal Antibody for Duck Cellular Inhibitor of Apoptosis...

Cellular inhibitor of apoptosis protein 1 (cIAP1) plays a crucial role in the apoptotic process. This study involved gene cloning, protein expression, bioinformatics analysis, and the preparation of a specific polyclonal antibody. First, the coding sequence of the duck cIAP1 was amplified by PCR and sequenced. Bioinformatics prediction indicated that this protein is a stable hydrophilic protein with no signal peptides and transmembrane domains. It contains characteristic BIR (bacterial IAP repeat) domains. The secondary structure consists of 43% random coils, 28% α-helix, and 8% β-strand, and its 3D structure is highly conserved compared with human and mouse cIAP1. Subsequently, the cIAP1 was cloned into the pET-28a vector. The recombinant His-tagged cIAP1 protein was successfully expressed in Escherichia coli BL21(DE3) upon induction and subsequently purified. Immunization of BALB/c mice with the purified protein emulsified in Freund's adjuvant generated polyclonal antibodies. Indirect ELISA confirmed that the antibody titer exceeded 1:2,048,000. Additionally, Western blotting and immunofluorescence staining demonstrated that the antibody specifically recognized native duck cIAP1 protein without non-specific reactions with negative serum. This study successfully achieved prokaryotic expression of duck cIAP1 and produced a high-titer, highly specific mouse polyclonal antibody. This antibody is suitable for experiments such as Western blotting and immunofluorescence, providing important biological materials and a theoretical basis for further investigation of the anti-apoptotic mechanism of cIAP1 in major diseases of ducks.