中华绒螯蟹Δ9脂肪酸去饱和酶基因克隆与原核表达

Prokaryotic expression of fatty acyl-CoA Δ9 desaturase in Eriocheir sinensis

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。

Fatty acyl-Co A Δ9 desaturase（FAD9） is a membrane-bound enzyme anchored in the endoplasmic reticulum that plays an important role regulating cell membrane fluidity and fatty acid metabolism. The FAD9 sequence has two transmembrane domains and three amino acid motifs. Purifying the FAD9 protein can be difficult because of its membrane structural characteristics. Fish species frequently express FAD9 heterologously. The Chinese mitten crab, Eriocheir sinensis, is an important aquatic species. We designed primers based on the FAD9 cDNA sequence from E. sinensis（Accession Number： JQ693685） to obtain the opening reading frame（ORF）. The ORF was subcloned into the p Cold-TF DNA prokaryotic expression vector to generate the p Cold TF-fad9 recombinant expression vector, which was transformed into E. coli BL21（DE3）p Lys S, and FAD9 was expressed successfully in E. coli BL21（DE3）p Lys S using IPTG induction. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that the recombinant protein had an approximate molecular weight of 95.10 k D, which was consistent with the theoretical molecular weight, and that it was mainly detected in the supernatant. We tested different IPTG concentrations（0.1 mmol/L, 0.3 mmol/L, 0.5 mmol/L, 0.8 mmol/L, 1.0 mmol/L, and 1.2 mmol/L）, induction temperatures（35℃, 25℃, 18℃, and 15℃）, and induction times（2 h, 5 h, 10 h, 15 h, and 20 h）. The results showed that when the IPTG concentration was 0.1 mmol/L, expression of the recombinant protein was significantly lower than that at the other IPTG concentrations. Temperatures of 15–18℃ and an induction time of 20 h were optimal. The highest recombinant protein expression was detected when the IPTG concentration was 0.3 mmol/L, with a 20-h incubation at 15℃. The fusion protein was identified by purification and western blotting. The protein contained a 6×His-tag, so we used His-tag nickel ion affinity chromatography to purify the protein and an anti-6×His-tag antibody for western blotting. The results showed that the p Cold TF-fad9 recombinant protein was successfully expressed in E. coli, and western blotting revealed that the p Cold TF-fad9 recombinant protein was specifically recognized by the 6×His antibody, indicating that the recombinant protein had antigen activity. Our study provides basic methods to purify and detect the activity of E. sinensis FAD9 and will promote further study on the functions of FADs.