建泽泻鲨烯合酶原核表达、功能验证及其免疫检测研究

阅读框（ORF）构建到原核表达载体 pCzn1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AoSS在不同组织中的表达情况，结果显示建泽泻块茎中AoSS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AoSS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。

[关键词] 建泽泻； 鲨烯合酶； 原核表达； 功能验证； 免疫检测

[Abstract] 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 AoSS gene， the open reading frame （ORF） of squalene synthase gene （accession no. JX866770） from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS 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 AoSS 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 AoSS in different organs of A. orientale， and the results showed that the AoSS 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 AoSS gene， and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.

[Key words] Alisma orientale； squalene synthase； prokaryotic expression； functional identification； immunoassay

澤泻Alismatis Rhizoma为泽泻科植物东方泽泻Alisma orientale的干燥块茎，具有利水、渗湿、泄热之功效，道地产区为福建^[1-2]。泽泻的主要药效成分为原萜烷型（protostane）四环三萜类^[2-3]，其结构独特，C-10位和C-14位上有β-CH3，C-8上有α-CH3，C-20为S构型，该类成分具有抗高血脂、降血压、抗HIV1、抗癌等显著活性^[4-7]。近期的研究表明，原萜烷型三萜能促进肝再生和防止ANIT诱导的肝毒性和胆汁淤积^[5-8]，但该类成分在植物体中含量低，分布窄，仅存在于泽泻属等少数植物类群中^[9-11]，限制了其进一步开发利用。生物工程是提高活性成分含量的有效途径之一，而活性物质的生物合成效率与其合成途径中的关键酶密切相关。