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根据中华绒螯蟹FAD9基因cDNA序列(Accession Number:JQ693685)设计引物,扩增得到中华绒螯蟹FAD9基因的开放阅读框(ORF),用原核表达载体p Cold-TF DNA成功构建重组表达载体p Cold-fad9,将p Cold-fad9转入大肠杆菌BL21(DE3)p Lys S,在异丙-D-硫代半乳糖苷(IPTG)的诱导下进行表达。SDS-PAGE分析表明,诱导后出现的特异性蛋白条带,大小与预期理论值(95.10 k D)相符。当IPTG浓度为0.3 mmol/L时,在15℃条件下诱导20 h,重组蛋白的表达量最高。目的蛋白主要存在于上清溶液中,为可溶性表达。利用镍离子亲和层析柱对重组蛋白进行了纯化,用Western-blotting方法验证了该重组蛋白可以与anti-His抗体特异性结合。研究结果为中华绒螯蟹FAD9重组蛋白的大量纯化及活性检测奠定基础,也为今后进一步开展脂肪酸去饱和酶功能的研究提供参考。
According to the cDNA sequence of Accession Number (JQ693685) of Eriocheir sinensis, the open reading frame (ORF) of FAD9 gene of Eriocheir sinensis was amplified and the recombinant expression vector p was successfully constructed by using the prokaryotic expression vector p Cold-TF DNA Cold-fad9, p Cold-fad9 was transformed into E. coli BL21 (DE3) p Lys S and expressed under the induction of isopropyl-D-thiogalactoside (IPTG). SDS-PAGE analysis showed that the size of the specific protein bands appearing after induction coincided with the expected theoretical value (95.10 kD). When the concentration of IPTG was 0.3 mmol / L, the recombinant protein was expressed at 15 ℃ for 20 h. The target protein is mainly present in the supernatant solution and is soluble. The recombinant protein was purified by Ni-ion affinity chromatography. The result of Western-blotting confirmed that the recombinant protein could specifically bind anti-His antibody. The results laid the foundation for the large-scale purification and activity determination of FAD9 recombinant protein of Eriocheir sinensis, and provided reference for further research on the function of fatty acid desaturase in the future.