青海血蜱唾液腺重组Hq001蛋白表达及纯化条件的优化
Optimization of expression and purification conditions of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis
王雪薇 1陈开廷 2马静 3刘岳青 1高金亮 2王翠峰4
作者信息
- 1. 内蒙古科技大学包头医学院,内蒙古包头 014040;内蒙古科技大学包头医学院第一附属医院检验科,内蒙古包头 014040;鄂尔多斯市中心医院分子医学实验室,内蒙古鄂尔多斯 017000
- 2. 鄂尔多斯市中心医院分子医学实验室,内蒙古鄂尔多斯 017000
- 3. 内蒙古科技大学包头医学院,内蒙古包头 014040;鄂尔多斯市中心医院分子医学实验室,内蒙古鄂尔多斯 017000
- 4. 内蒙古科技大学包头医学院第一附属医院检验科,内蒙古包头 014040
- 折叠
摘要
目的 对青海血蜱唾液腺重组Hq001蛋白的表达条件(表达菌株及诱导条件)及纯化方式(非变性及变性纯化)进行优化.方法 将重组质粒pET-30a-Hq001分别转化至不同感受态菌株[E.coil BL21(DE3)、E.coil Rosetta(DE3)、E.coil ArcticExpress(DE3)pRARE2],确定最适表达菌株后,对 IPTG 终浓度(0、0.5、1.0 mmol/L)、诱导温度(20、25 ℃)及诱导时间(0、2、4、6、8 h)进行优化.最佳诱导条件表达的菌体经高压均质破碎后,采用非变性(变性-复性-过柱)及变性(变性-过柱-复性)两种方式进行Ni-NTA亲和层析纯化.表达及纯化产物均经12％SDS-PAGE分析.结果 重组Hq001蛋白最适表达菌株为感受态E.coil BL21(DE3);最适诱导条件为:IPTG终浓度0.5 mmol/L,诱导温度25 ℃,诱导时间4 h.非变性纯化方式可获得相对分子质量约为18 800的目的蛋白,大小与预期相符.结论 采用优化的表达条件及纯化方式可获得青海血蜱唾液腺重组rHq001蛋白.
Abstract
Objective To optimize the expression conditions(expression and induction conditions)and purification methods(non-denaturing and denaturing purification)of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis.Methods The recombinant plasmid pET-30a-Hq001 was transformed into competent cells E.coil BL21(DE3),E.coil Rosetta(DE3)and E.coil ArcticExpress(DE3)pRARE2 respectively for the selection of an optimal expression strain.The final con-centration of IPTG(0,0.5,1.0mmol/L),induction temperature(20,25 ℃)and induction time(0,2,4,6,8 h)were optimized.The recombinant bacteria expressed under the ideal induction condition were homogenized by French press and the target protein was purified by passing through a Ni-NTA affinity chromatography column under either native(denaturation-renaturation-column chromatography)or denatured conditions(denaturation-column chromatography-renaturation).The purified products were analyzed by 12％SDS-PAGE.Results E.coil BL21(DE3)was proved to be the most suitable strain for the expression of recombinant Hq001 protein.The optimum induction condition was induction with 0.5 mmol/L IPTG for 4 h at 25 ℃.The target protein with a relative molecular mass of approximately 18 800 was obtained by non-denaturing purification method,and the size was consistent with the expectation.Conclusion The recombinant protein rHq001 in salivary glands of Haemaphysalis qinghaiensis can be obtained by the optimized expression conditions and purification methods.
关键词
青海血蜱/包涵体/变性/复性/纯化/Ni-NTA亲和层析Key words
Haemaphysalis qinghaiensis/Inclusion bodies/Denaturation/Renaturation/Purification/Ni-NTA affinity chromatography引用本文复制引用
基金项目
国家自然科学基金(81760375)
内蒙古自治区自然科学基金(2021MS0803)
内蒙古自治区科技创新引导项目(内财科[2021]381)
出版年
2024
NETL
NSTL
万方数据