金丝桃素合酶基因的快速克隆及功能鉴定

Rapid cloning and functional characterization of hypericin synthase gene

金丝桃素合酶能催化大黄素生成金丝桃素,根据已发表的金丝桃素合酶的基因序列,设计了6对引物,通过连续重叠PCR快速克隆得到了金丝桃素合酶基因hyp-1。构建含hyp-1基因的原核表达载体pET32ahyp,将该载体导入大肠杆菌进行诱导表达。SDS-PAGE结果表明,hyp-1基因在大肠杆菌中获得表达;Western blot结果表明,重组的Hyp-1蛋白具有特异的免疫活性,表明hyp-1基因在E.coli中得到了表达。酶促反应表明,Hyp-1确实能催化大黄素形成金丝桃素,上述结果表明,本研究克隆的hyp-1基因是金丝桃素合酶基因,具备催化大黄素形成金丝桃素的能力,从而为通过合成生物学技术制备金丝桃素奠定了物质基础。

Hypericin,a red-colored naphtodianthrone,is a natural product synthesized in the medicinal plant Hypericum perforatum,commonly known as St.John＇s wort.Hypericin has attracted a growing attention of the pharmaceutical industry because of its potential application to various therapies,including the treatment of depression and remarkable antiviral and photodynamic activities.hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which,however,has not formed common opinion so far.Six pairs of primers specific to hyp-1 gene were synthesized.The rapid cloning of hyp-1 gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions.All cloned sequences were confirmed by DNA sequencing.A vector named pET32ahyp containing hyp-1 gene was constructed and was transformed into E.coli to induce heterologous expression.SDS-PAGE and Western blot results showed the recombinant Hyp-1 protein was expressed successfully in E.coli.The soluble fraction was used to test the function of the recombinant Hyp-1.Hypericin was detected by LC-MS/MS with emodin as a substrate under in vitro conditions.The above results corroborated the Hyp-1 function,a confusing question,which lay a material foundation for the synthesis of hypericin by synthetic biotechnology.