SARS-3CL蛋白酶基因在杆状病毒载体中的构建及其转染

摘要

Objective: To construct severe acute respiratory syndrome (SARS)coronavirus 3CL protease gene into transforming vector prepare recombinant baculovirus and transduct it into infect insect cells to express SARS-3CL protease. Mothods: The 3cl-Teasy and pFastBac HTb bacmida were amplified. The 3cl gene was cloned into baculovirus transforming vector pFastBac HTb by enzyme-digest- and-ligase method,which was named recombinant pFB HTb-3cl. The pFB HTB-3cl was transformed into E.coli DH10Bac competent cells.The positive colonies were screened by three antibiotics and blue-white patch method. The bacmid-HTb-3cl recombinant baculovirus bacmid was obtained and purified to transfect St9 insect cells.The protease expressed in Sf9 insect cells were identified by SDS-PAGE. Results: Recombinant expression vector was obtained successfully. The 3CL protease expressed in insect cells were identified by SDS-PAGE. Conclusion: The expression of 3CL protease in insect cells provided foundation for detecting protein activities and screening inhibitor against SARS-3CL protease.%目的:构建SARS冠状病毒3CL蛋白酶基因的杆状病毒重组供体质粒,包装重组3CL蛋白酶的杆状病毒,感染昆虫细胞进行表达.方法:首先扩增含3cl-Teagy和pFagtBac HTh的转化菌,用酶切连接法构建重组转座质粒pFB HTb-3cl.将该质粒转化E.coli DH10Bac感受态菌,在菌体内进行重组,并经三重抗性和蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HTb-3cl,对重组质粒Bacmid-HTB-3cl进行纯化并转染St9昆虫细胞包装杆状病毒,利用病毒感染St9昆虫细胞并进行蛋白表达.利用SDS-PAGE检测蛋白表达情况.结果:成功构建了重组表达载体并得到了重组杆状病毒,SDS-PAGE检测到有3CL蛋白酶表达.结论:3CL蛋白酶在昆虫细胞中的表达为蛋白活性的检测及抑制剂的筛选奠定了基础.