丹参C4H基因pET32a原核表达载体的构建

Construction of pET32a prokaryotic expression vector of C4H gene in Salvia miltiorrhiza

【目的】克隆丹参中肉桂酸-4-羟化酶基因(SmC4H)的核心区,并构建SmC4H基因的原核表达载体。【方法】设计合成SmC4H基因全长引物,应用PCR克隆SmC4H基因的编码区并添加酶切位点,应用EcoRⅤ、NotⅠ双酶切SmC4H-pGM-T后与原核表达载体pET32a连接,转化到大肠杆菌BL21(DE3)中进行双酶切鉴定,诱导表达重组蛋白并进行SDS-PAGE电泳以及Western Blot分析。【结果】克隆得到了1 200 bp大小的基因片段,经测序鉴定其为SmC4H基因片段;连接表达载体测序结果表明,SmC4H-pET32a构建成功,其诱导表达蛋白经SDS-PAGE检测及Western Blot分析发现,重组蛋白表达效果较好,且主要以包涵体形式存在。【结论】成功构建了SmC4H-pET32a原核表达载体,并成功诱导了SmC4H-pET32a重组蛋白的表达。

【Objective】 The study cloned the core area of cinnamic acid-4-hydroxylase gene（SmC4H） from Salvia miltiorrhiza and constructed prokaryotic expression vector of SmC4H gene.【Method】 We designed and synthesized the primers of full-length about SmC4H gene,then cloned the CDs of SmC4H and added restriction site by PCR.Next we double-digested SmC4H-pGM-T with EcoRⅤ and NotⅠ and ligated with pET32a prokaryotic expression vector,then transformed into E.coli BL21（DE3） and underwent double restriction enzyme digestion.The expression of recombinant protein was analyzed with SDS-PAGE electrophoresis and Western Blot.【Result】 The study cloned 1 200 bp of the gene which was identified as SmC4H gene by sequencing and the SmC4H-pET32a vector was constructed successfully by sequencing and the induced protein was detected by SDS-PAGE and Western Blot analysis,indicating that the expression of recombinant protein which is expressed as inclusion body mostly is better.【Conclusion】 We constructed SmC4H-pET32a prokaryotic expression vector and induced the expression of recombinant protein in SmC4H gene successfully.