基于pHY300PLK的分泌表达载体的构建

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。

Construction of an secretory expression vector based on pHY300PLK

As quasi public product, watershed resource need to be managed and protected by the government departments, enterprises and the public together. This paper uses the game method to annotate the symbiosis relationship among the participants of the watershed management, and find out that the premise of symbiosis is the cooperation of participants for the common interests. Based on the analysis above, this paper describes the forming process of symbiosis of the participants by using logistic model and analyzes the stability conditions of symbiosis. And then concludes that the behaviors of government will contribute much to the watershed management effect of the enterprises and the public, while the enterprises and the public contribute little to government.