HaSNPV几丁质酶重组蛋白的表达、纯化和复性(英文)

Expression of Chitinase Gene of HaSNPV,and Purification,Refolding of Its Recombinant Protein

棉铃虫单核衣壳核型多角体病毒(HaSNPV)是我国第一个商品病毒杀虫剂,具有使用安全、害虫不产生抗药性等优点,是一种很有发展潜力的生物农药。幼虫虫体受病毒感染后,HaSNPV几丁质酶在其液化过程中起了很大的作用,因此可以作为增效剂以显著提高细菌、病毒、真菌等微生物杀虫剂的毒力,并具有更高的安全性。将HaSNPV几丁质酶基因构建到原核表达载体pET28a中,经测序检验后转化至大肠杆菌Rosetta,然后以IPTG作为诱导剂,目标蛋白以包涵体的形式得以成功表达。在变性条件下,包涵体经镍-次氮基三乙酸(Ni-NTA)柱层析纯化,并以两种不同的方法进行复性,均可获得具有活性的HaSNPV几丁质酶。

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus（HaSNPV） has been extensively used for many years to control H.armigera in China.HaSNPV chitinase is considered responsible for the liquefaction process of cadavers,and it may be a safer pesticide to control H.armigera.The HaSNPV chitinase gene was constructed into pET28a and transferred into E.coli strain Rosetta,which contain colons rarely used in E.coli.The chitinase was successfully expressed as（His） 6-chiA.It was expressed as inclusion body and was purified by Ni-NTA Column Affinity Chromatography under denaturing conditions.The inclusion body was then refolded by two different ways,both of which can produce activated enzyme.