扁豆几丁质酶基因的克隆及表达

Cloning and expression of a chitinase gene from dolichos lablab

以扁豆总RNA为模板,通过RT-PCR技术扩增到长度为885 bp的扁豆几丁质酶基因cDNA,其编码294个氨基酸,目的蛋白的分子量为28.429 kDa.以该基因构建pET-21a-chi表达载体并在E.coli BL21(DE3)中表达.30℃下,用0.1 mmol.L-1IPTG,诱导5 h,表达产物的酶活力为36.25 U.mL-1.通过常压Sephadex G-50凝胶过滤色谱,DEAE-650C常压与高效离子交换色谱对表达产物进行纯化.目标蛋白达到电泳纯(SDS电泳一条带),几丁质酶的比活力108.42 U.mg-1.表达的蛋白质产物主要以可溶性形式存在.

The chitinase gene cDNA was amplified by RT - PCR with total RNA extracted from dolichos lablab as the template. The length of cDNA was 885 bp, which encoded 294 amino acids with the predicted molecular weight of 28.429 kDa. The gene was used to construct the plasmid of pET-21a-chi and expressed in E. coil BI21 （DE3） strain. At 30 ℃, after induction for 5 h with 0. 1 mmol · L^-1 IPTG, the intracellular chitinase activity of 36.25 U · mL^-1 was reached in the recombinant strain. The expressed product was purified by gel filtration chromatography on Sephadex G -50 and high performance ion exchange chromatography on DEAE -650C. The purified targeted protein appeared as a single band on SDSPAGE, and the specific enzyme activity was 108.2 U · mg^-1 The plant- derived chitinase gene can be expressed stably in E. coli BL21 （DE3） in the soluble form.