Abstract：Stearoyl-CoA desaturase (SCD), which is also called delta-9 desaturase, is the rate-limiting enzyme of regulating unsaturated fatty acids synthesis in milk, and being as a key enzyme for endogenous synthesis of the conjugated linoleic acid in the meat and milk of ruminants. It has the ability to catalyze saturated fatty acyl coenzyme A to generate monounsaturated fatty acyl coenzyme A. Most importantly, SCD takes an effect on the fatty acids composition and the contents of unsaturated fatty acids in goat (Capra hircus) milk. However, a lack of appropriate tools has hampered further study of SCD. Therefore, goat specific SCD antibody is necessary for the function research of SCD gene fatty acid regulation. The objective of this study was to prepare rabbit (Oryctolagus cuniculus) polyclonal antibodies (pAbs) against SCD through prokaryotically expression and SCD protein purification. The clarified function of the antibody would provide effective tools for the study of SCD functions in fatty acids metabolism. The SCD gene sequence was chosen through bioinformatic analysis mainly using DNAStar software. SCD was amplified by PCR from the cDNA. SCD gene of dairy goat was then sub-cloned into the vector pET32a(+) to construct recombinant plasmid pET32a(+)-SCD which was transformed into Escherichia coli Rosetta (DE3) expression strain. SCD fusion protein was purified by His-tag protein purification kit. Purified fusion protein being mixed with adjuvant was used as antigen to immune 6 months of rabbits to prepare polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to detect the titer of the immune serum. The obtained SCD polyclonal antibodies were used for specificity identification. The sequence with a total of 210 bp length at SCD gene N-terminal was amplified by PCR, and the prokaryotic expression vector pET32a(+)-SCD was successfully constructed. Recombinant protein was successfully expressed in E. coli Rosetta (DE3). Western blotting result showed that SCD fusion protein was correctly expressed which had 6×His tag. Recombinant protein molecular weight was about 30 kD, consistent with the expected protein molecular weight. The best expression condition was identified as follows, 25 ℃, 1 mmol/L of (isopropyl-β-d-thiogalactoside, IPTG), induction for 12 h; SCD fusion protein was mainly expressed in soluble form. Then, the fusion protein from supernatant of lysates was purified using His-tag protein purification beads. The concentration of the obtained protein was 2.5 mg/mL determined by bicinchoninic acid (BCA) Protein Assay Kit. The purified protein was used as antigen to immune rabbits and prepare polyclonal antibody successfully. ELISA result suggested the final titer of the SCD pAbs was 1∶64 000. Western blot analysis showed that the SCD pAbs could identify the specific antigen epitope on the SCD protein expressed in the form of fusion protein and the natural protein. In conclusion, the SCD protein was successfully expressed and purified. The polyclonal antibody with high reactivity and specificity was successfully prepared, which could help researchers explore the biological functions of SCD in future studies.
邱思源 罗军 晏原 朱江江 马功珍. 奶山羊SCD的多克隆抗体制备与鉴定[J]. , 2017, 25(7): 1111-1118.
Jun LUO. Preparation and Identification of Polyclonal Antibody Against SCD of Dairy Goat (Capra hircus). , 2017, 25(7): 1111-1118.
