学术
A组乙型溶血性链球菌DNaseB基因亚克隆及pET-28a（＋）-DNaseB载体构建
潘琴[1,2] 王滔[3,4] 陈骏扬[4] 黄祖新[1,2]
[1]福建师范大学生命科学学院,福建福州350108 [2]发育与神经生物学福建省高等学校重点实验室,福建福州350108 [3]福建省立医院儿科,福建福州350001 [4]福建省立医院儿科研究室,福建福州350001
摘 要:
运用PCR扩增技术，以质粒pMD18-T-DNaseB为模板，扩增出0．5kb截短的DNaseB基因，先将该基因片段与pMD19-T载体连接，确定该序列正确并进行大量繁殖后，再将DNaseB基因定向克隆入pET-28a（＋）表迭载体中，构建新的原核表迭载体pET-28a（＋）-DNaseB，转化该重组质粒至受体菌E．coliDH5a中，采用SDS碱裂解法提取该质粒DNA，经EcoRⅠ和HindⅢ双酶切鉴定和核苷酸序列分析，证实插入的基因片段具有正确的DNaseB基因核苷酸序列，将pET-28a（＋）-DNaseB转化至大肠杆菌BL21（DE3）中，经过IPTG诱导其目的蛋白得到了表达．[著者文摘]
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文章出处:
《福建师范大学学报：自然科学版》-2007年23卷6期 -97-101页
分 类 号:
文章编号:
1000-5277（2007）06-0097-05
Subcloning of DNaseB Gene from Group A of Streptococcus and Constructing pET-28a （~）-DNaseB Plasmid
PAN Qin , WANG Tao , CHEN Jun-yang , HUANG Zu-xin （1. College of Life Sciences, Fujian Normal University, Fuzhou 350108, China; 2. State Key Laboratory of Developmental Biology and Neurobiology （Fujian Normal University）, Fuzhou 350108, China; 3. Paediatric Office of Fujian Provincial Hospital, Fuzhou 350001, China; 4. Paediatric Laboratory of Fujian Provincial Hospital, Fuzhou 350001, China）
Abstract:
A 0.5 kb dock-tailed DNaseB gene fragment was amplified by PCR from plasmid pMD18-T- DNaseB . In order to identify its correctness and reproduce this gene abundantly. First , the target gene fragment was cloned into pMD19-T, Then the DNaseB gene was subcloned into pET-28a （＋） resulting pET-28a （＋） -DNaseB. The recombinant plasmid was transformed into E. coli DH5a, The recombinant plasmid DNA was extracted by SDS Alkaline Lysis Method and purified, and was studied in detail by restriction endonuclease and nueleotide sequencing. The result showed that the nucleotide sequence of the inserted DNA fragment was identical to that of DNaseB gene. The recombinant plasmid was transformed into E. coli BL21 （DE3）. The target protein was expressed by IPTG induction .[著者文摘]
Key words:
group A of Streptococcus; DNaseB gene; subcloning
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