HaSNPV几丁质酶基因原核表达、纯化与复性的研究

【作者】 赵健；

【导师】 惠友为；

【作者基本信息】 西北大学 ， 生物化工， 2006， 硕士

【摘要】 棉铃虫核型多角体病毒是我国第一个商品病毒杀虫剂,具有使用安全,害虫不产生抗性等优点,是一种很有发展潜力的可替代生物农药。目前中国棉铃虫单粒包埋型核型多角体病毒（HaSNPV）的基因组序列已经测序完成,在HaSNPV基因组中发现了18家族几丁质酶的序列,该几丁质酶基因是一个晚期非必需基因。由于HaSNPV几丁质酶基因与受染虫体液化和病毒侵染机制密切相关,因而该基因的研究日益引起了人们的重视。 在本研究中我们将删除了N端20个氨基酸的信号肽和C端4个氨基酸的内质网定位序列的HaSNPV几丁质酶基因构建到高效原核表达载体pET28a（+）中,经测序检验后,转化表达宿主菌Rosetta（DE3）,以IPTG为诱导剂诱导表达。最佳IPTG诱导浓度为0.5mM,最佳诱导时间为3h以上,（His）₆-chiA蛋白可占总蛋白的22.6%,1mL菌液约含（His）₆-chiA蛋白65μg,但是（His）₆-chiA蛋白主要以包涵体的形式表达。为了得到可溶性蛋白,我们还将HaSNPV的几丁质酶基因构建到pMAL-c2X载体中,pMAL载体特别有利于外源蛋白的正确折叠,从而形成可溶性的蛋白。MBP-chiA融合蛋白可占总蛋白的14.7%,1mL菌液约含MBP-chiA蛋白47μg,而可溶性的MBP-chiA蛋白占到总MBP-chiA蛋白的30%以上。虽然依旧没有获得有活性的蛋白,但为进一步的研究奠定基础。 在本研究中我们大量培养含有pET28a（+）-chiA质粒的Rosetta（DE3）,诱导表达,收集包涵体蛋白,对其进行初步洗涤纯化,然后用8M尿素溶解,在变性条件下（8M尿素）经镍-次氮基三乙酸（Ni-NTA）柱进行亲和层析纯化,得到的电泳纯的重组几丁质酶蛋白,纯度可达95%以上。然后,我们采用了逐步透析的方法对电泳纯的包涵体蛋白进行复性,于4℃分别对100mL尿素浓度依次为6M-5M-4M-3M-2M-1M-0M的透析液进行透析,每组透析液透析12h,复性效率为28%,1mg复性后蛋白的活性为0.3U。我们也采用了柱上复性的方法对包涵体进行复性,依次以含有6M-5M-4M-3M-2M-1M-0M尿素的refolding buffer的缓冲液洗柱,得到了活性重组几丁质酶蛋白,复性效率约为43%,1mg复性后蛋白的活性为0.5U,此方法可实现蛋白的同时纯化及复性。更多还原

【Abstract】 Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) had been extensively used for the control of H. armigera since the first isolation of this pathogen in Hubei province of China. The genome of this virus has been completely sequenced and a chitinase gene belonging to family 18 chitinases was identified. The chitinase was found to be essential in liquefying infected cadavers.Chitinase gene from HaSNPV was constructed into expression vector pET28a(+) and then transferred into expression host Rosetta(DE3), which was designed to enhance expression of eukaryotic proteins that contained codons rarely used in E. coli. The chitinase gene was successfully expressed as a fusion protein (His)6- chiA with a His-tag at its N terminal. After the recombinant E.coli was induced with 0.5 mM IPTG for 3h, the percentage of the recombinant protein could get to 22.6% of the total E.coli protein, and 1mL culture medium could produce 65μg (His)6- chiA protein. But it was mainly expressed as inclusion body and had no activity, so we constructed the gene into another expression vector pMAL- c2X. About 14.7% of the total E.coli protein was MBP- chiA, and 30% of MBP- chiA protein was soluble. But this fusion protein still had no activity.The inclusion body was expressed in Rosetta(DE3) which contained pET28a(+)-chiA plasmid . (His)6- chiA protein was then purified by Ni-NTA column affinity chromatography under denaturing conditions (8M urea). The (His)6- chiA protein was then refolded by stepwise dialysis with the urea concentration in the base dialysis buffer reduced as follows 6M-5M-4M-3M-2M-1M-0M . After stepwise dialysis, about 28% protein was refolded, and the enzymatic activation of 1 mg refolded (His)6-chiA was 0.3U . The (His)6- chiA protein was also successfully refolded while it was immobilized on Ni-NTA, by washing it using refolding buffer with urea concentration as follows 6M-5M-4M-3M-2M-1M-0M. After that about 43% protein was refolded, and the enzymatic activation of 1 mg refolded protein could get to 0.5U.更多还原