酸性纤维素酶CBHⅡ基因与非抗性穿梭表达载体的重组及在乳酸杆菌的表达及其活性检测  

Recombination of CBH Ⅱ Gene with Non-antibiotic Selected Vector and Expression and Activity Detection in Lactobacillus

作　　者：赵莹[1,2] 孙哲[1] 刘燕[3] 胡后银 陈大芳[5] 娄玉杰[2] 张宏福[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室,北京100193 [2]吉林农业大学动物科技学院,长春130118 [3]内蒙古农业大学动物科技学院,呼和浩特010018 [4]济南市畜牧兽医研究所,济南250002 [5]贵州省畜禽良种场,贵阳550018

出　　处：《畜牧兽医学报》2009年第5期670-675,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30500364);中国博士后基金项目(20060390897)

摘　　要：以SmaI和SphI双酶切pMD18-T-CBHⅡ和以胸苷酸合成酶基因(thymidylate synthase,thyA)为选择压力的非抗生素抗性的穿梭表达载体pW425t,胶回收酸性纤维素酶CBHⅡ基因和载体大片段,并将纯化的CBHⅡ基因和表达载体pW425t大片段进行连接,构建出可以在乳酸菌与大肠杆菌之间穿梭表达的原核表达重组质粒pW425t-CBHⅡ。将pW425t-CBHⅡ转化至thyA基因缺陷型的乳酸杆菌感受态细胞中,通过质粒提取、酶切鉴定、PCR鉴定、测序分析和生长功能弥补筛选阳性克隆。SDS-PAGE分析,可见约49.6 ku的蛋白,并且刚果红染色显示重组乳酸杆菌可产生明显的水解圈。Recombinant plasmids pMD18-T-CBH Ⅱ and pW425t, prokaryotic expression shuttle vector between E. coli and Lactobacillus, were digested with Sinai and SphI enzymes respectively. The purified CBH Ⅱ gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-CBH Ⅱ was constructed, and then was transformed into the competence thyA gene-mutant Lactobacillus DOMLaS107. Treated lysates of bacterium were loaded directly on SDS-PAGE, and approximately 49.6 kD protein was observed, and recombinant Lactobacillus can produced clear hydrolysis halos on the Congo-Red-CMC plate.

关 键 词：纤维素酶 CBHⅡ基因 乳酸杆菌 表达 

分 类 号：Q784[生物学—分子生物学] Q786