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核盘菌激发子基因SsCut克隆、原核表达及纯化

, PP. 630-636

Keywords: 核盘菌,激发子,载体构建,原核表达,过敏反应

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Abstract:

为开发以病原菌激发子为主要成分的新型生物农药,根据核盘菌的全基因组数据信息,采用逆转录聚合酶链式反应(reversetranscription-PCR,RT-PCR)技术从核盘菌菌株NGA4克隆到编码角质酶基因的全长cDNA序列,在不同条件下进行原核表达,通过Ni2+亲和层析重组蛋白His-SsCut,并进行激发子活性检测.序列分析表明,克隆的基因编码核盘菌角质酶开放阅读框为609bp,预测编码202个氨基酸,并将基因命名为SsCut(Sclerotiniasclerotiorumcutinase).含SsCut/pET32a的表达菌株BL21经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导,于28、30和37℃均能产生44.3kD左右的融合蛋白,其中30℃诱导3h,融合蛋白表达量最高,浸润的烟草出现过敏性坏死反应.经纯化的蛋白His-SsCut和细菌His-Harpin分别浸润烟草叶片出现类似的细胞死亡.表明His-SsCut具有激发子的活性.

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