几丁质酶基因表达载体pET-chiB的构建及其在大肠杆菌中的诱导表达

几丁质酶在降解几丁质的过程中起着重要作用,目前人们已从不同微生物体中分离并克隆出了多种几丁质酶基因。实验以pET-22b( )为载体,利用从粘质沙雷氏菌(Serratia marcescens)克隆出的chiB基因,构建出原核生物表达载体pET-chiB。通过表达载体pET-chiB的诱导表达,实验结果显示该基因表达的蛋白为可溶性蛋白,其分子量约为52 kD。利用不同参数包括时间、IPTG浓度和温度诱导表达载体pET-chiB表达并对表达产物进行SDS-PAGE分析,结果显示其诱导表达的最佳参数分别为4 h,0.5 mmol/L和25℃。这些结果为几丁质酶基因的进一步研究和几丁质酶工程菌生产奠定了良好的工作基础。

Construction of the Plasmid pET-chiB Carrying Chitinase Gene and Inducing Expression in E.coli

Chitinase plays important roles in decomposing chitin to GIcNAc. Many chitinase genes have been separated and cloned from different kinds of microbes. In this study, the chitinase chiB gene cloned from Serrat/a marcescens was inserted into the recombinant expression plasmid pET-22b( + ) vectors at the EcoRI site, and the expression vector pET-chiB was transformed into E. coli BL21 ( DE3 ). The expression vector pET-chiB was induced to express and analyzed by SDS-PAGE, and the results indicated that its expression protein was soluble and the protein molecular weight was about 52 kD. The analysis of inducing expression was done by SDS-PAGE. The vectar'pET-chiB was induced to express different time with difference, Isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations and temperatures, and the resuh showed that the best inducing parameters were 4 hours, 0.5 mmol/L and 25℃ respectively. These results provided fundamental data, and materials for the further study on chitinase gene and the producing of chitinase project strains.