摘要：
用PCR、酶切、连接方法将地衣芽孢杆菌耐高温α-淀粉酶基因(amy)表达单元(包括启动子、信号肽及淀粉酶基因)克隆到pHY300PLK中,并用反向PCR、同源重组方法,去除重组质粒的氨苄抗性基因,以利用α-淀粉酶基因的启动子和信号肽高效表达自身蛋白及外源蛋白.结果表明:连入了α-淀粉酶表达单元的pHY300PLK,即pAMY质粒能够分泌表达α-淀粉酶,且去除了氨苄抗性基因的pAMY质粒,即pAMY1质粒能更有效的表达α-淀粉酶；将纤维素酶基因连接到pAMY1质粒中淀粉酶信号肽的下游,得到的重组质粒pCEL能分泌表达纤维素酶,表明α-淀粉酶基因的启动子和信号肽不仅能启动自身蛋白的表达,也能启动外源蛋白的表达;重组菌的生长情况与质粒拷贝数的比较表明,与PAMY质粒相比去除了氨苄抗性基因的pAMY1质粒对重组菌的生长没有影响,且pAMY1质粒在重组菌的复制效率更高,能更高效的表达蛋白,以上结果证明pHY300PLK克隆质粒已成功改造成表达质粒,下一步可用于更多外源基因的分泌表达.%The expression unit of heat-stable α-amylase (amy) gene including the promoter, signal peptide and amylase gene was amplified by PCR and then digested and ligated into cloning vector pHY300PLIC The anti-ampcillin gene of the recombinant plasmid was deleted by inverse PCR and homologous recombination for high expression of auto-protein and heterogeneous protein using the promotor and signal peptide of α-amy gene.The result indicated that the pHY300PLK with anti-ampcillin gene, namely, plasmid pAMY, could express secretory α-amylase in host strain while the plasmid pAMY without anti-ampcillin gene, namely, plasmid pAMY1, could express the secretory α-amylase more efficiently.Then the cellulase gene was inserted into the vector pAMY1 at the position immediately after the promoter and signal peptide of α-amy gene, the resulting recombinant plasmid, named pCEL could express secretory cellulase in host strain.These results indicated the promoter and signal peptide of α-amy gene could express not only a-amylase but also foreign protein as well.According to the growth curve of recombinant strains and the plasmid copy number of pAMY and pAMYl, deletion of the anti-ampcillin showed no influence on the growth of the recombinant strain while improved the replication efficiency of the plasmid, leading to higher expression of protein by pAMY1.The above results suggested the cloning vector pHY300PLK was modified into expressing vector and could be used further in the expression of other heterogeneous proteins.
