A组乙型溶血性链球菌DNaseB基因亚克隆及pET-28a(+)-DNaseB载体构建

运用PCR扩增技术,以质粒pMD18-T-DNaseB为模板,扩增出0.5 kb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( )表达载体中,构建新的原核表达载体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.

Subcloning of DNaseB Gene from Group A of Streptococcus and Constructing pET-28a (+)-DNaseB Plasmid

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.