奶山羊SCD的多克隆抗體制備與鑒定
發(fā)布時(shí)間：2018-11-17 20:44
【摘要】：硬脂酰輔酶A去飽和酶(stearoyl-Co A desaturase,SCD)既是乳中調(diào)控不飽和脂肪酸合成的限速酶,同時(shí)又是反芻動(dòng)物肉及乳中共軛亞油酸內(nèi)源合成的關(guān)鍵酶。為了制備奶山羊(Capra hircus)SCD的多克隆抗體并進(jìn)行初步應(yīng)用。本研究通過(guò)生物信息學(xué)分析選取SCD基因片段,采用PCR方法擴(kuò)增SCD基因,連接至p ET32a(+)載體上,原核表達(dá)載體p ET32a(+)-SCD轉(zhuǎn)化到大腸桿菌(Escherichia coli)Rosetta(DE3)中誘導(dǎo)表達(dá)。使用組氨酸標(biāo)簽蛋白純化瓊脂糖磁珠純化融合蛋白,純化的SCD蛋白皮下免疫兔子(Oryctolagus cuniculus)制備多克隆抗體,酶聯(lián)免疫吸附(enzyme-linked immuno sorbent assay,ELISA)和Western blot檢測(cè)血清效價(jià)和特異性。PCR擴(kuò)增獲得SCD基因N端210 bp的序列,成功誘導(dǎo)原核表達(dá)載體p ET32a(+)-SCD在25℃、1 mmol/L異丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)、12 h條件下表達(dá),在Rosetta(DE3)上清中獲得了可溶的SCD重組蛋白。ELISA檢測(cè)結(jié)果表明,制備的SCD多抗效價(jià)達(dá)1∶64 000,能特異性檢測(cè)原核和真核細(xì)胞中表達(dá)的SCD蛋白。本研究成功表達(dá)并純化了SCD融合蛋白,同時(shí)獲得了高效價(jià)及高特異性的山羊SCD多抗,為進(jìn)一步研究SCD在羊奶短中鏈脂肪酸代謝中的調(diào)控功能提供了重要的實(shí)驗(yàn)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，
本文編號(hào)：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，
本文編號(hào)：2338989
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