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

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (5): 670-675.doi:

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

赵莹^1,2，孙哲¹*，刘燕³，胡后银⁴，陈大芳⁵, 娄玉杰²，张宏福¹*

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

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-05-25 发布日期:2009-05-25
通讯作者: 孙哲;张宏福

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

ZHAO Ying^1,2，SUN Zhe¹*， LIU Yan³，HU Hou-ying⁴，CHEN Da-fang⁵，LOU Yu-jie²，ZHANG Hong-fu¹*

1.State Key Lab of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China; 3. College of Animal Science and Technology, Inner Mongolia Agricultural University, Huhhot 010018, China; 4. Institute of Animal Science and Veterinarian of Jinan, Jinan 250002, China; 5. Guizhou Province Livestock and Poultry Seedstock Breeding, Guiyang 550018,China

Received:1900-01-01 Revised:1900-01-01 Online:2009-05-25 Published:2009-05-25

摘要/Abstract

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。

Abstract: Recombinant plasmids pMD18-T-CBHⅡ and pW425t, prokaryotic expression shuttle vector between E.coli and Lactobacillus, were digested with SmaI and SphI enzymes respectively. The purified CBHⅡ gene was subcloned into the expression vector pW425t. Thus,the recombinant pW425t-CBHⅡ 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.

赵莹;孙哲;刘燕;胡后银;陈大芳;娄玉杰;张宏福. 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[J]. 畜牧兽医学报, 2009, 40(5): 670-675.

ZHAO Ying;SUN Zhe;LIU Yan;HU Hou-ying;CHEN Da-fang;LOU Yu-jie;ZHANG Hong-fu. Recombination of CBH Ⅱ Gene with Non-antibiotic Selected Vector and Expression and Activity Detection in Lactobacillus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(5): 670-675.

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