枯草芽孢杆菌C-36内切葡聚糖酶基因的克隆及其在大肠杆菌中融合表达

以自行分离筛选出的天然枯草芽孢杆菌（Bacillus subtilis）C-36的染色体DNA为模板，PCR扩增得到含有内切葡聚糖酶基因的DNA片段，将其克隆到pMD-18T载体中，序列分析表明，克隆得到的DNA片段全长1602bp，编码一个含有499个氨基酸的多肽。与其他芽孢杆菌内切葡聚糖酶基因序列比对，其核苷酸同源率为90％～93％，其编码的氨基酸序列的同源性在90％～98％，已将此基因注册GenBank（DQ782954）。将含内切葡聚糖酶基因的重组克隆质粒进行亚克隆，用Kpn I和EcoR I双酶切后，与相同酶切的表达载体pET-32a相连接，并导入大肠杆菌BL21中表达。蛋白质电泳实验结果表明在6．47×10^4处有表达蛋白带。经测定表达蛋白比酶活力达99．02U／mL，为出发菌C-36（63．78U／mL）的1．55倍。

Cloning,sequencing,and expression of endoglucanase gene from Bacillus subtilis C-36 in Escherichia coil

A gene encoding endoglucanase from Bacillus subtilis C-36 was cloned into E. coil cloning plasmid pMD-18T by PCR amplification. The whole length of the gene was 1602 bp, encoding 499 amino aeids. The nueleotide sequence of the gene shared high degree of sequence similarity to that of other typical bacillus in GenBank. The similarities were between 90% and 93%. The gene has been registered in GenBank （Accession number DQ782954）. The gene from recombinant cloning plasmid was subcloned into Kpn I and EcoR I site of expression plasmid pET-32a in E. coil. The recombinant expression plasmid was transformed into E. coli strain BL21. The result of SDS-PAGE showed that about 5.3 × 10^4 protein of the endoglucanase was expressed in E. coli strain BL21. The enzymatic activity of E. coli expressed strain was 99. 02 U/mL,it was about 1.55-fold higher than that of the original strain.