Cloning and molecular characterization of an ovine ABCD4 cDNA sequence, recombinant protein expression, and polyclonal antibody generation in Ujimqin sheep

Introduction ATP-binding cassette subfamily D member 4 (ABCD4) is an ATP-binding cassette transporter involved in intracellular cobalamin trafficking and has also been identified as a candidate gene associated with vertebral-number variation in livestock. However, molecular information and experimental resources for ovine ABCD4 remain limited. This study aimed to characterize a RACE-derived ABCD4 cDNA sequence from Ujimqin sheep, analyze its sequence and predicted molecular features, establish a recombinant protein expression system, and generate a polyclonal antibody resource for subsequent studies. Methods Total RNA isolated from Ujimqin sheep liver was subjected to rapid amplification of cDNA ends (RACE), followed by sequence analysis, phylogenetic comparison, physicochemical characterization, recombinant expression in Escherichia coli, protein purification, and antibody production. Results A 1,874-bp ABCD4 cDNA sequence was obtained, containing a predicted 1,209-bp open reading frame encoding a 402-amino-acid polypeptide. Sequence and bioinformatic analyses showed that the RACE-derived sequence retained conserved features of the ABC transporter family, although its predicted protein length was shorter than that of canonical mammalian ABCD4 proteins. For recombinant antigen production, the ABCD4 expression construct was introduced into E. coli BL21 (DE3), and higher recombinant protein expression was observed after induction with 0.5 mM IPTG at 37 °C for 4 h. The recombinant protein was predominantly recovered from the insoluble fraction and purified by Ni-NTA affinity chromatography. Purified recombinant protein was then used to immunize New Zealand White rabbits solely for polyclonal antiserum production, and the resulting antisera showed strong immunoreactivity against the recombinant antigen. Discussion Overall, this study provides a RACE-derived ovine ABCD4 cDNA sequence together with recombinant protein and antibody resources that may support future investigations of ABCD4 expression, localization, and biological function in sheep.