全长δ-内毒素cryIA(c)基因在三种原核细胞中的表达  

CLONING AND EXPRESSION OF FULL-LENGTH δ-ENDOTOXIN CrylA(c) GENE IN THREE KINDS OF PROKARYOTIC SYSTEMS USING SHUTTLE VECTOR pHT3101

作　　者：杨远征[1] 齐义鹏[1] 黄永秀[1] 

机构地区：[1]武汉大学病毒研究所,武汉430072

出　　处：《微生物学报》1996年第3期173-180,共8页Acta Microbiologica Sinica

基　　金：国家"八五"攻关项目资助课题

摘　　要：采用随机引物法标记苏芸金芽孢杆菌δ-内毒素cryIA(b)基因:从苏芸金芽孢杆菌HD-73质粒基因文库中筛选到了分别包含δ-内毒素cryIA(c)基因5’端和3’端的6.5kb和4.3kb两个片段,然后分别缺失其非编码区,重连得到全长3.92kb的crylA(c)基因,用穿梭载体pHT3101亚克隆后分别转化大肠杆菌NM522、枯草杆菌AS1176及苏芸金芽孢杆菌晶体缺陷型4D10(H3ab),得到了具有同一质粒的三种工程菌。SDS-PAGE电泳表明此基因在三个宿主系统中均表达了分子量为133300的原毒素,分子量大小与苏芸金芽孢杆菌HD-73晶体蛋白完全相同,免疫检测表明表达蛋白和天然晶体蛋白具有相同的免疫原性。用提取的粗表达产物对二龄小菜蛾幼虫作杀虫试验,证明三种细胞产生的表达蛋白均具有杀虫活性,测得它们的LD_(50)分别为0.311μg/cm^2、0.024μg/cm^2和0.017μg/cm^2。Two fragments, 6.5kb and 4.3kb encoding 5'end and 3'end ofδ-endotoxin cryIA(c) gene, respectively, were selected from the Bacillus thuringiensis kurstaki HD-73 75kb plasmid gene pool using random primer labelling δ-endotoxin cryIA(b) gene probe. The full-length 3.92kb cryIA(c) gene including 5'end 152bp promoter sequence and 3'end 198bp terminater sequence was rebuilt after uncoding sequences were deleted. Three kinds of engineering strains habouring the same recombinant plasmid pHTY1 were obtained after the cryIA( c) gene had been subcloned in shuttle vector pHT3101 and transformed to E. coli NM522, Bacillus subtilis AS1176 and Bacillus thuringiensis crystal-deficient 4D10( H3ab). SDS-PAGE electrophoresis patterns reveal that the crylA(c) gene expressed the same 133 300 protoxin proteins in all three host systems with the same molecular weight to the crystal protein from the Bacillus thuringiensis kurstaki HD-73. Immuno-assays indicate that the expression proteins can react with antiserum of HD-73 paraspore crystal protein in the same pattern as the natural crystalprotein. Bioassays using crude expressed products from three hosts trains reveal that they all showed toxicities to second instar larvae of Plutella xylostella, and their LD50 were calculated to be 0.311μg/ cm2, 0.024μg/ cm2and 0.017 μg / cm2, respectively.

关 键 词：基因表达 δ-内毒素 苏芸金芽孢杆菌 大肠杆菌 

分 类 号：Q939.110.3[生物学—微生物学]