芽孢杆菌分泌型表达载体的构建

Construction of a Secretion-expression Vector of Bacillus

以地衣芽孢杆菌(B.licheniformis)α-淀粉酶信号肽编码区为基础构建了具有Pst Ⅰ、Nhe Ⅰ、Sma Ⅰ、BamH Ⅰ、Sal Ⅰ多酶切点的芽孢杆菌分泌型载体pAMY403,其分泌效率与质粒pAMY 413相同,而表达水平提高了50%,并能使外源β-内酰胺酶基因正常表达和分泌。质粒pAMY403的多酶切点可以满足外源基因插入产生非融合蛋白质,也可满足以三种不同读码框架插入外源基因产生融合蛋白质,因而能适应构建不同分泌型工程菌株的要求。

A Bacillus protein secretion-expression vector pAMY403 has been constructed based on the promoter and signal sequence coding region of α-amylase gene from Bacillus licheniformis. It possesses a polylinker with the restriction sites of Pst I -Nhe I -Sma I -BamH I -Sal I after signal peptide coding region. The α-amylase gene carried by plasmid pAMY403 in B. subtilis can be expressed and secreted as its parental plasmid pAMY413. When β-Iactomase gene from B. licheniformis is cloned at SmaI sites of pAMY403, it can express and secret normally. Polilinkers of plasmid pAMY403 provide cloning sites both for inserting heterogenes to produce non-fusion protein (e. g. insert at NheI or PstI site)and for protein fusion in different reading frames at other restriction sites. Therefore plasmid pAMY403 can adapt to any project of constructing chimeric secretion plasmid in B. subtilis.