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【目的】以抗性棉铃虫Helicoverpa armigera体内发现的一种基因重组导致的嵌合型P450酶CYP337B3作为研究对象,通过基因克隆、体外重组表达、蛋白质结晶等技术手段,获得CYP337B3(△23)的晶体结构,为深入了解棉铃虫P450酶CYP337B3的结构与功能奠定基础。【方法】对CYP337B3编码基因进行了密码子优化,通过基因重组,将其转化至BL21(DE3)感受态细胞中进行原核表达尝试,通过亲和层析及RESOURCETM Q离子交换层析对CYP337B3(△23)进行体外纯化,利用气相悬滴结晶方法对CYP337B3(△23)进行结晶条件的筛选及X射线晶体衍射。【结果】通过密码子优化,实现了CYP337B3(△23)在大肠杆菌原核表达系统内的大量表达,经过体外纯化及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测,CYP337B3(△23)蛋白纯度可达到95%左右,我们对CYP337B3(△23)进行了蛋白结晶条件的筛选,最终获得了CYP337B3(△23)的结晶条件和蛋白晶体。【结论】本文利用气相悬滴结晶方法获得了CYP337B3(△23)的蛋白晶体,为今后CYP337B3的三维结构解析、功能研究以及最终阐述该种新型的棉铃虫抗药机制奠定了良好的基础。
【Objective】 CYP337B3, a chimeric P450 enzyme, was cloned from a resistant Helicoverpa armigera strain. The CYP337B3 (△ 23) gene was cloned by gene cloning, recombinant expression in vitro and protein crystallization. Crystal structure, to lay a foundation for understanding the structure and function of CYP337B3 in cotton bollworm P450. 【Method】 The CYP337B3 gene was codon optimized and transformed into BL21 (DE3) competent cells for genetic prokaryotic expression through genetic recombination. The affinity of CYP337B3 (△) was determined by affinity chromatography and RESOURCETM Q ion exchange chromatography 23) were purified in vitro, the use of gas-phase hanging drop crystallization method CYP337B3 (△ 23) for screening and X-ray crystal diffraction conditions. 【Result】 The results showed that CYP337B3 (△ 23) was highly expressed in E. coli prokaryotic expression system by codon optimization. After in vitro purification and sodium dodecyl sulfate - polyacrylamide gel electrophoresis, CYP337B3 (△ 23) The purity of protein was up to about 95%. We screened the protein crystallization conditions of CYP337B3 (△ 23), finally obtained the crystallization conditions and protein crystals of CYP337B3 (△ 23). 【Conclusion】 In this paper, the crystal structure of CYP337B3 (△ 23) was obtained by the method of gas-phase hanging drop crystallization, which laid a good foundation for the future three-dimensional structural analysis and functional study of CYP337B3 and the ultimate elucidation of the new mechanism of this new type of cotton bollworm.