摘要： 为使苎麻生物脱胶果胶裂解酶PelG403高效表达。将pelG403从重组质粒pEASY-Blunt E1-G403更换至质粒拷贝数更高的pET28a载体中，导入Escherichia coli BL21（DE3）后进行诱导表达。采用DNS法进行酶活力测定，通过SDS-PAGE和Western Blot分析检验PelG403表达效果，并对其序列进行分析鉴定。结果表明：pET28a-G403/BL21的果胶裂解酶活力为335.8 U/mL，较pEASY-Blunt E1-G403/BL21提高了3.05倍；工程菌pET28a-G403/BL21表达产物分子量比预测带His标签的融合蛋白分子量（43.6 ku）略小；PelG403含387个氨基酸，N末端前35个氨基酸为信号肽；理论pI值为7.64，有4个半胱氨酸，不稳定系数为27.42；其蛋白结构域含有典型的右手β-螺旋结构，属于Pec lyase C家族。因此，将pelG403从重组质粒pEASY-Blunt E1-G403更换至pET28a载体中，并导入E.coli BL21（DE3）进行诱导表达，是苎麻脱胶果胶裂解酶基因pelG403高效表达的方法和途径，同时对其序列进行分析鉴定，为果胶裂解酶的纯化、关键位点分析、分子改造及其脱胶功能特性研究奠定基础。

关键词: 苎麻, 生物脱胶, 果胶裂解酶, 原核表达, 生物信息学

Abstract: In order to efficiently express the pectate lyase PelG403 from ramie biodegumming. This study replaced pel G403 from the recombinant plasmid pEASY-Blunt E1-G403 to the pET28a vector with a higher plasmid copy number, and then introduced Escherichia coli BL21(DE3) to induce expression. The enzyme activity was determined by DNS method, and the expression effect of PelG403 was tested by SDS-PAGE and Western Blot analysis, and the sequence was analyzed and identified. The results showed that the pectate lyase activity of pET28a-G403/BL21 was 335.8 U/mL, which was 3.05 times higher than that of pEASY-Blunt E1-G403/BL21;the molecular weight of the expressed product of pET28a-G403/BL21 was slightly smaller than that of the predicted fusion protein with His tag(43.6 ku);PelG403 contained 387 amino acids, and the first 35 amino acids at the N-terminal were signal peptides;the theoretical pI value was 7.64, 4 cysteines, and the instability coefficient was 27.42;its protein domain contained a typical right-handed β-helix structure and belonged to the Pec lyase C family. Therefore, replacing pelG403 from the recombinant plasmid pEASY-Blunt E1-G403 into the pET28a vector and introducing E.coli BL21(DE3) for inducible expression was a method and approach for high-efficiency expression of the ramie degummed pectate lyase gene pelG403, and its sequence was analyzed and identified. It laid a foundation for the purification of pectate lyase, key site analysis, molecular modification and the study of its degumming functional characteristics.

Key words: Ramie, Biological degumming, Pectate lyase, Prokaryotic expression, Bioinformatics

中图分类号: 

• Q78