摘要： 自然条件下金丝桃素在植物中的含量较低且不稳定，提取工艺受其他物质的影响较大，在一定程度上限制了金丝桃素在医药、食品等领域的应用。为了研究金丝桃素合成途径并实现其体外转化，以贯叶连翘愈伤组织总RNA为模板，经RT-PCR扩增得到了编码金丝桃素合成酶的全长cDNA，即Hyp基因。将该基因克隆到原核表达载体pET30a中，成功构建金丝桃素合成酶表达载体pET30a-Hyp，并转化到大肠杆菌BL21（DE3）中。含有重组质粒的克隆在IPTG诱导下，表达出金丝桃素合成酶融合蛋白，利用Ni柱纯化菌体裂解上清中表达的融合蛋白，对纯化产物进行SDS_PAGE分析，结果Hyp基因得到了高效表达，大小约为20kD。
关键词:
金丝桃素,
贯叶连翘,
金丝桃素合成酶
Abstract: Hypericin has been reported as the active compound found in Hypericum perforatum.Studies have shown that Hypericin has the abilities of anti-depression,anti-Bacteria anti-virus,anti-cancer and so on.But the content of Hypericin of Hypericum perforatum is not enough in natural condition,the extraction of Hypericin was not match the need of applications.To study the synthesis approach and conversion in vitro of Hypericin,the full length cDNA of Hypericin Synthase(Hyp)was amplified from the RNA of Hypericum perforatum by RT-PCR and cloned into the plasmid pET30a to achieve the expression vector pET30-Hyp.Hypericin Synthase(Hyp) could be expressed in E.coli BL21(DE3)after IPTG induction.Hyp was purified from the supernatant of the bacterial lysate by Ni-NTA his bind resin.A protein band with predicted size about 20 KD was detected by SDS_PAGE.
Key words:
Hypericin,
Hypericum perforatum,
pET30a
中图分类号:
王力, 张娜, 张秀清, 孙君社. 金丝桃素合成酶基因的克隆及其表达[J]. 华北农学报, 2005, 20(4): 1-3. doi: 10.3321/j.issn:1000-7091.2005.04.001.
WANG Li, ZHANG Na, ZHANG Xiu-qing, SUN Jun-she. Cloning and Expression of Hypericin Synthase[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2005, 20(4): 1-3. doi: 10.3321/j.issn:1000-7091.2005.04.001.