摘要
类枯草杆菌蛋白酶是昆虫致病真菌重要的毒力因子,利用合成的特异性引物通过RT-PCR扩增了球孢白僵菌类枯草杆菌蛋白酶CDEP2基因。将其与质粒pET28a连接,构建重组原核表达载体pET28-CDEP2,转化大肠杆菌BL21(DE3),并用IPTG进行诱导表达。SDS-PAGE检测His-CDEP2融合蛋白以包涵体方式大量表达并对其进行纯化。用SDS-PAGE分离纯化的CDEP2蛋白免疫家兔,制备了多克隆抗血清。Western blotting免疫印迹检测分析表明,该抗血清对CDEP2蛋白有特异性识别能力,可用于对CDEP2蛋白功能的进一步研究。
Subtilisin-like protease was the most important factor for the insecticidal activity of entomopathogenic fungi.Using the specificity primers,a cDNA encoding the subtilisin-like protease CDEP2 gene was amplified from Beauveria bassiana by RT-PCR.The recombination prokaryotic expression plasmid pET28-CDEP2 was constructed by subcloning the maturation CDEP2 protein gene encoding sequence into the vector pET28a.It was transformed into E.coli BL21（DE3） and induced to express by IPTG.SDS-PAGE results showed that the His-CDEP2 fusion protein was high express in the form of inclusion body and purified by affinity chromatography.The rabbits were immuned by the purified CDEP2 protein to produce polyclonal antiserum.Western blotting analysis indicated that the antiserum could react with the corresponding protein,and was suitable to be used for further analysis of CDEP2 protein.
出处
《广东农业科学》
CAS
CSCD
北大核心
2013年第16期131-133,137,共4页
Guangdong Agricultural Sciences
基金
湖南人文科技学院校级青年基金项目(2008QN009)
湖南省大学生研究性学习和创新性实验计划项目(5411009)
关键词
类枯草杆菌蛋白酶
原核表达
融合蛋白
多克隆抗血清
subtilisin-like protease
prokaryotic expression
fusion protein
polyclonal antiserum
