摘要
【目的】构建玉米大斑病菌StTOXE蛋白的原核表达载体,明确StTOXE融合蛋白的诱导体系和可溶性,为开展该蛋白的功能和结构研究奠定基础。【方法】试验首先克隆了StToxE基因,并通过酶切连接技术将StToxE连接入原核表达载体pET-28a(+)中获得StTOXE融合蛋白表达载体pET28a-StToxE;分别将pET28a(+)和pET28a-StToxE载体分别转入大肠杆菌BL21(DE3)中获得表达菌株BL21-pET28a和BL21-pET28a-StToxE,通过ITPG诱导,SDS-PAGE和Western Blot检测StTOXE融合蛋白的表达及其可溶性。【结果】构建了玉米大斑病菌StToxE基因原核表达载体pET28a-StToxE,并获得转化子BL21-pET28a和BL21-pET28a-St-ToxE;确定了StTOXE融合蛋白的最适ITPG浓度为0.4~0.6 mmol/L,分子量约为72 kD;并确定了StTOXE融合蛋白主要以包含体的形式存在,且为可溶性蛋白。【结论】实验成功构建了StTOXE融合蛋白表达载体pET28a-StToxE;发现ITPG浓度对StTOXE融合蛋白的表达有显著影响;在37℃条件下,StTOXE融合蛋白基本以包涵体形式存在细胞内,且融合蛋白为可溶性蛋白。
【Objective】The present paper aimed to construct the prokaryotic expression vector of Setosphaeria turcia StTOXE protein,and clarify the induction system and solubility of StTOXE fusion protein,which will lay the foundation for the function and structure of the protein.【Method】The StToxE gene was first cloned and ligated into the prokaryotic expression vector pET-28a(+).The StTOXE fusion protein expression vector pET28a-StToxE was obtained by PCR,double enzyme digestion and sequencing.The pET28a(+)and pET28a-St-ToxE vectors were transformed into E.coli BL21(DE3)to obtain expression strains BL21-pET28a and BL21-pET28a-StToxE,respectively.BL21-pET28a and BL21-pET28a-StToxE were induced by ITPG of different concentration,and then the expression and solubility of StTOXE fusion protein were detected by SDS-PAGE electrophoresis and Westen Blot.【Result】The prokaryotic expression vector pET28a-StToxE of StToxE gene was constructed,and the transformants of BL21-pET28a and BL21-pET28a-StToxE were obtained respectively.The optimal ITPG concentration of StTOXE fusion protein was 0.4-0.6 mmol/L,the molecular weight of StTOXE fusion protein was 72 kD,and it was mainly present in the form of inclusion bodies and was a soluble protein.【Conclusion】The StTOXE fusion protein expression vector pET28a-StToxE was successfully constructed in the experiment.It was found that the concentration of ITPG had a significant influence on the expression of StTOXE fusion protein.At 37℃,it was mainly present in the form of inclusion bodies and is a soluble protein.
作者
张运峰
ZHANG Yun-feng(Department of Life Science,Tangshan Normal University,Hebei Tangshan 063000,China)
出处
《西南农业学报》
CSCD
北大核心
2020年第10期2256-2261,共6页
Southwest China Journal of Agricultural Sciences
基金
唐山市应用基础研究计划(19130215g)
河北省自然科学基金(C2019105055)
河北省高等学校科学研究基金(QN2017415)。
