摘要
泽泻鲨烯合酶(Ao SS)催化法呢基焦磷酸[(farnesyl diphosphate,FPP)]合成鲨烯,是碳源流向原萜烷型三萜生物合成的关键调节酶。为深入研究Ao SS基因的功能及表达,课题组将前期克隆获得的建泽泻鲨烯合酶基因(accession No.JX866770)的开放阅读框(ORF)构建到原核表达载体p Czn1上,并在大肠杆菌BL21(Roseta)中进行诱导表达,诱导表达出的融合蛋白主要以包涵体的形式存在,经纯化获得高纯度的目的蛋白;以此目的蛋白进行体外酶促反应验证其功能,其结果显示该目的蛋白具有催化法呢基焦磷酸生成鲨烯的活性;为进一步研究其表达规律,在此基础上,利用该蛋白免疫新西兰兔制备多克隆抗体并纯化,ELISA检测抗体效价大于1∶51 200,Western blotting检测表明其具有较好的特异性;用制备得到的抗体免疫检测建泽泻Ao SS在不同组织中的表达情况,结果显示建泽泻块茎中Ao SS表达最高,其次为叶,根中表达甚微。原核表达载体的成功构建、基因功能的进一步验证及快速免疫检测方法的建立,为进一步开展Ao SS基因功能及其调控的研究奠定基础,为泽泻资源性成分原萜烷型三萜的合成生物学应用提供科学依据。
Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate（ FPP） to form squalene,which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of Ao SS gene,the open reading frame（ ORF） of squalene synthase gene（ accession no. JX866770） from A. orientale was subcloned into a prokaryotic expression vector p Czn1 and induced the expression of Ao SS gene in Escherichia coli BL21（ Roseta）. The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction,the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of Ao SS in A. orientale,the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified,the titer of the antibody was greater than 1∶ 51 200 by ELISA detection,and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of Ao SS in different organs of A. orientale,and the results showed that the Ao SS expression level was the highest in tubers,followed by leaves,and lowest in root. Successful construction of prokaryotic expression vector,validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of Ao SS gene,and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2017年第19期3733-3738,共6页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81673534)
江苏省自然科学基金项目(BK20161576)
江苏省中药优势学科Ⅱ期建设项目(ysxk-2014)
江苏省中药资源产业化过程协同创新中心项目(ZDXM-3-24)
关键词
建泽泻
鲨烯合酶
原核表达
功能验证
免疫检测
Alisma orientale
squalene synthase
prokaryotic expression
functional identification
immunoassay
