N乙酰D葡萄糖胺2差向异构酶基因的克隆、表达及产物纯化

N-乙酰-D-葡萄糖胺-2-差向异构酶基因的克

隆、表达及产物纯化

作者：余云彦，张卫星，闫欣欣，唐琴琴，刘乃华，晏永波，李校，林绍强

【摘要】目的：从猪肾中提取RNA，从中克隆出N-乙酰-D-葡萄糖胺-2-差向异构酶(GNE)基因，并在大肠杆菌E.coli BL21(DE3)中表达。方法：从猪肾中提取总RNA，通过RT-PCR克隆出GNE基因，经测序，感受态大肠杆菌E.coli DH5a转化，质粒抽提，鉴定以及大肠杆菌BL21(DE3)转化表达，通过His-tag亲和纯化(NiSepharose 6 Fast Flow)，G-25柱脱盐，SDS-PAGE电泳检测。结果：RNA提取物经紫外分光光度计检测OD260/OD280=1.77，说明无明显的蛋白质和酚污染。RT-PCR扩增目的片段，测序证明该基因的ORF为1 206 bp，编码402个氨基酸组成的酶蛋白(GNE)。SDS-PAGE显示，纯化蛋白为45 kD 的单一条带，与基因序列推测值一致。结论：GNE基因已被成功地克隆和表达。

【关键词】 N-乙酰-D-萄糖胺-2-差向异构酶;基因;克隆;纯化;猪

Abstract: Objective: To clone the gene of N-acetyl-D-glucosamine-2-epimerase according to RNAextracted

from pig kidney and express it in E.coli BL21 (DE3) and then purify the product. Methods:RNA was extracted from porcine kidney to clone the gene of N-acetyl-D-glucosamine-2-epimerase withRT-PCR followed by sequencing. Recombinant plasmid was converted into E.coli DH5a and to identifyit followed by the conversion of the recombinant plasmid in E.coli BL21 (DE3) and expression. Theprotein was purified by His-tag (Ni Sepharose 6 Fast Flow)，desalted by G-25 column and identifiedby SDS-PAGE. Result: RNA was extracted from porcine kidney and identified with ultravioletspectrophotometer:OD260=0.0346，OD280=0.0195，OD260/OD280=1.77 (between1.7 and 2.2)， it showedthat there was no protein and phenol contamination. Amplification of GNE with RT-PCR，the sequencelength of GNE was 1 206 bp encode for 402 amino acids. The molecular weight of purified proteinwas 45 kD revealed by SDS-PAGE， which was consistent to the analysis based on gene sequence.Conclusion: N-acetyl-D-glucosamine-2-epimerase gene is successfully cloned and expressed in E.coliBL21(DE3).

Key words: N-acetyl-D-glucosamine-2-epimerase;gene;cloning;purification ;pig

N-乙酰-D-神经氨酸(N-Acetylneuraminic acid，Neu5Ac)的酶法合成主要以N-乙酰-D-甘露糖胺(ManNAc)与丙酮酸为底物，在Neu5Ac醛缩酶(Neu5Ac aldolase)的催化下生成Neu5Ac[1]。为克服ManNAc价格昂贵的问题，通过碱性条件[2]或使用N-乙酰-D-葡萄糖胺-2-差向异构酶(GNE)使廉价的N-乙酰-D-葡萄糖胺转化为ManNAc[3]。用Neu5Ac醛缩酶，以ManNAc和丙酮酸为底物进行Neu5Ac酶法合成，已有多篇论文报道[4-6]。酶法合成具有转化率高、提取简单、产品纯度高[7]、环保等优点，鉴于醛缩酶可以从重组微生物中获得[8]，人们倾向于以此方法来生产Neu5Ac。1996年，Maru等人从猪肾中成功克隆出了GNE，该酶的克隆对两步酶法[9]生产Neu5Ac带来了希望，而GNE是酶法合成Neu5Ac最为关键的限速酶[10-11]。本研究从猪肾中克隆出GNE基因，将该基因克隆至表达载体pET28(b)并转入至大肠杆菌E.coli BL21(DE3)中表达，并对其表达产物进行了纯化[12]。

1 材料和方法

1.1 材料

质粒、菌株及工具酶：质粒pBluescri-ptIIKS(+)、pET28(b)(中国科学院上海生命科学研究院杨晟教授惠赠);菌株E.coli DH5a、E.coli BL21(DE3)、限制性内切酶EcoRV、NdeI、EcoR I、T4DNA连接酶和碱性磷酸酶(CIAP)为Takara公司产品，引物由

Takara公司合成。试剂及动物材料：RT-PCR试剂盒、质粒抽提试剂为Takara公司产品;Ni Sepharose 6 Fast Flow购自GE Healthcare;咪唑、尿素为Sigma产品;EDTA、Disodium Salt、0.5 MEDTA(pH .0)、NaCl、Triton X-100、Tris-Hydrochloride、Ammonium persulfate 均购自Promega公司;氯霉素购自Amresco，LB培养基、其他试剂为进口分装或国产;新鲜猪肾购自农贸市场。