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,去除重组质粒的氨芐抗性基因,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 a-amylase （amy） gene including the promoter, signal peptide and amylase gene was amplified by PCR and then digested and ligated into cloning vector pHY300PLK. The anti-ampcillin gene ofthe 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 thatthe 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-amylasemore 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 secretorycellulase in host strain. These results indicated the promoter and signal peptide of a-amy gene could express not only α-amylase but also foreign protein as well. According to the growth curve of recombinant strains and the plasmid copynumber of pAMY and pAMY1, 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 pAMY 1. Theabove results suggested the cloning vector pHY300PLK was modified into expressing vector and could be used furtherin the expression of other heterogeneous proteins.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第1期52-57,F0003,共7页
Journal of Hunan Agricultural University(Natural Sciences)
基金
现代农业产业技术体系建设专项(CARS 09 06B)
中央级公益性科研院所基本科研专项(2012ZL034)
北京市自然科学基金项目(5083025)
关键词
地衣芽孢杆菌
Α淀粉酶
同源重组
表达质粒
纤维素酶
分泌表达
Bacillus licheniformis
α-amylase
homologous recombination
expression vector
cellulase
secretory expression
作者简介
覃君君（1985-），男，湖南怀化人，硕士，主要从事微生物基因工程的研究，junjunqin@yahoo．cn
通信作者，jiangjunping@yahoo．com．cn
