奶山羊SCD的多克隆抗体制备与鉴定
发布时间：2018-11-17 20:44
【摘要】：硬脂酰辅酶A去饱和酶(stearoyl-Co A desaturase,SCD)既是乳中调控不饱和脂肪酸合成的限速酶,同时又是反刍动物肉及乳中共轭亚油酸内源合成的关键酶。为了制备奶山羊(Capra hircus)SCD的多克隆抗体并进行初步应用。本研究通过生物信息学分析选取SCD基因片段,采用PCR方法扩增SCD基因,连接至p ET32a(+)载体上,原核表达载体p ET32a(+)-SCD转化到大肠杆菌(Escherichia coli)Rosetta(DE3)中诱导表达。使用组氨酸标签蛋白纯化琼脂糖磁珠纯化融合蛋白,纯化的SCD蛋白皮下免疫兔子(Oryctolagus cuniculus)制备多克隆抗体,酶联免疫吸附(enzyme-linked immuno sorbent assay,ELISA)和Western blot检测血清效价和特异性。PCR扩增获得SCD基因N端210 bp的序列,成功诱导原核表达载体p ET32a(+)-SCD在25℃、1 mmol/L异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)、12 h条件下表达,在Rosetta(DE3)上清中获得了可溶的SCD重组蛋白。ELISA检测结果表明,制备的SCD多抗效价达1∶64 000,能特异性检测原核和真核细胞中表达的SCD蛋白。本研究成功表达并纯化了SCD融合蛋白,同时获得了高效价及高特异性的山羊SCD多抗,为进一步研究SCD在羊奶短中链脂肪酸代谢中的调控功能提供了重要的实验工具。
[Abstract]:Stearyl coenzyme A desaturase (stearoyl-Co A desaturase,SCD) is not only a rate-limiting enzyme for regulating the synthesis of unsaturated fatty acids in milk, but also a key enzyme for endogenous synthesis of conjugated linoleic acid in ruminant meat and milk. In order to prepare the polyclonal antibody of dairy goat (Capra hircus) SCD and its preliminary application. In this study, SCD gene fragment was selected by bioinformatics analysis, SCD gene was amplified by PCR method, ligated to p ET32a () vector, and transformed into E. coli (Escherichia coli) Rosetta (DE3 by prokaryotic expression vector p ET32a () -SCD. The fusion protein was purified by using histidine label protein and purified by agarose magnetic beads. The purified SCD protein was subcutaneously immunized with rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody, and enzyme-linked immuno sorbent assay, was obtained by enzyme linked immunosorbent assay (enzyme-linked immuno sorbent assay,). ELISA) and Western blot were used to detect the titer and specificity of serum. The N-terminal 210 bp sequence of SCD gene was amplified by PCR, and the prokaryotic expression vector p ET32a () -SCD was successfully induced at 25 鈩，
本文编号：2338989
[Abstract]:Stearyl coenzyme A desaturase (stearoyl-Co A desaturase,SCD) is not only a rate-limiting enzyme for regulating the synthesis of unsaturated fatty acids in milk, but also a key enzyme for endogenous synthesis of conjugated linoleic acid in ruminant meat and milk. In order to prepare the polyclonal antibody of dairy goat (Capra hircus) SCD and its preliminary application. In this study, SCD gene fragment was selected by bioinformatics analysis, SCD gene was amplified by PCR method, ligated to p ET32a () vector, and transformed into E. coli (Escherichia coli) Rosetta (DE3 by prokaryotic expression vector p ET32a () -SCD. The fusion protein was purified by using histidine label protein and purified by agarose magnetic beads. The purified SCD protein was subcutaneously immunized with rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody, and enzyme-linked immuno sorbent assay, was obtained by enzyme linked immunosorbent assay (enzyme-linked immuno sorbent assay,). ELISA) and Western blot were used to detect the titer and specificity of serum. The N-terminal 210 bp sequence of SCD gene was amplified by PCR, and the prokaryotic expression vector p ET32a () -SCD was successfully induced at 25 鈩，
本文编号：2338989
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