小鼠酸性哺乳动物壳多糖酶基因的表达及对真菌细胞壁的水解作用

Expression of mouse acidic mammalian chitinase gene, and hydrolysis of fungal cell wall by the en- zyme

目的克隆和表达酸性哺乳动物壳多糖酶(AMCase),分析其对红色毛癣菌和白念珠菌细胞壁的水解作用。方法从小鼠胃组织中提取总RNA,逆转录成cDNA,扩增AMCase基因,并构建融合表达载体pET28a(+)-AMCase,表达和纯化AMCase蛋白。测定AMCase与红色毛癣菌和白念珠菌细胞壁制备物作用后的N-乙酰氨基葡萄糖浓度。结果成功克隆了小鼠AMCase基因,表达并纯化得到了 AMCase蛋白,纯化后的AMCase可以水解红色毛癣菌和白念珠菌细胞壁。结论在原核系统中成功表达的AMCase基因具有水解红色毛癣菌和白念珠菌细胞壁的活性,提示该蛋白具有潜在的抗真菌功能。

Objective To clone and express the mouse acidic mammalian chitinase （ AMCase ） gene and protein, and to analyze the hydrolysis of the cell walls of Trichophyton rubrum and Candida albicans by this enzyme. Methods Total RNA was isolated from the stomach of BALB/c mouse, and AMCase gene was amplified by RT-PCR. The recombinant fusion expression vector of pET28a （ ＋ ）-AMCase was constructed. AMCase protein was expressed in prokaryotic system and purified. After incubation of AMCase with the cell wall extracts, the level of N- acetylglucosamine was measured. Results AMCase gene was cloned and expressed successfully. Purified AMCase protein can hydrolyse the chitin in the cell walls of T. rubrum and C albicans. Conclusion The AMCase expressed in prokaryotic system can hydrolyse chitin in the cell walls of T. rubrum and C albicans, which implies its antifungal potential.