HaSNPV几丁质酶的分离纯化与毒理学研究

Purification and Toxicological Studies of HaSNPV Chitinase

利用重组工程菌GS115-pPIC 9k-ChiA4.0发酵几丁质酶粗液,经(NH4)2SO4盐析、透析、DEAE Sepharose Fast Flow离子交换层析以及Sephadex G-100凝胶过滤层析分离纯化,获得电泳纯的棉铃虫单粒包埋型核型多角体病毒(HaSNPV)几丁质酶,回收率为19.64%,纯化17.74倍;经HaSNPV几丁质酶毒理学研究,通过触杀作用和胃毒作用,几丁质酶使小菜蛾幼虫细胞液化,导致死亡,其作用效果与几丁质酶浓度和作用时间成正比,且胃毒作用大于触杀作用;经小麦根腐、烟草赤星、番茄灰霉和苹果炭疽等植物病原真菌的抑菌实验,表明几丁质酶可以通过酶解真菌细胞壁中的几丁质达到抑菌作用。

Recombinant bacterium GS115-pPIC 9k-ChiA 4.0 fermented chitinase, which was purified by ammonium sulfate precipitation, dialysis , DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography, obtained electrophoretically pure chitinase. Rate of recovery was 19.64%, and purification factor was 17.74. Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus （HaSNPV） chitinase toxicological studies carried out by contact toxicity and stomach toxicity, chitinase liquefied Plutella xylostella larvae cell and caused death. The effect and the concentration of chitinase is proportional to time, and the effect caused by stomach toxicity was more intense than by action of contact toxicity. The result of antibacterial experiments which were performed on Helminthosporium sativum, Tomato botrytis cinerea, Tobacco brown spot, and Colletotrichum gloeosporioides, indicated that chitinase can inhibit fungal by enzymatic hydrolysis of chitin in fungal cell wall.