梅花鹿鹿茸S100A4融合蛋白在大肠杆菌中的表达纯化  

Expression and Purification of S100A4 Fusion Protein of Deer Antler in E.coli BL21

作　　者：刘东[1,2] 邢秀梅[2] 赵海平[2] 褚文辉[2] 鲁晓萍[2] 王大涛[2] 李春义[2] 

机构地区：[1]江苏科技大学,江苏镇江212018 [2]中国农业科学院特产研究所,吉林吉林132109

出　　处：《特产研究》2011年第2期7-10,共4页Special Wild Economic Animal and Plant Research

基　　金：国家自然科学基金(31070878)

摘　　要：我们的前期研究表明,鹿茸发生和再生都是依赖干细胞的过程。鹿茸的干细胞存在于鹿未来鹿茸发生区的骨膜中,即生茸骨膜。AP细胞表达特有的分子S100A4,推测为鹿茸发生的关键调节分子。进一步从分子水平阐述S100A4在鹿茸发生中的调节机制需要高质量S100A4的纯品。为了解决这一问题,我们针对已从梅花鹿AP细胞反转录出的S100A4基因序列,设计了含有EcoR I和BamH I酶切位点的上下游引物,并扩增出了目的片段。其后将S100A4基因片段和PGEX-6P-1载体酶切并进行了连接,转入Top10F’感受态细胞中,涂板筛选出了阳性克隆,进行了菌液PCR及双酶切鉴定,再将重组质粒转入了BL21(DE3)pLys S表达菌株进行了IPTG诱导表达。用聚丙烯酰胺凝胶电泳及western blot鉴定表明融合蛋白成功表达,随后对融合蛋白进行了纯化。本研究成功地实现了鹿本身特有的S100A4基因的体外表达。Our early research demonstrated that antler generation and regeneration both depend on stem cells.Stem cells of antlers exist in the periosteum that will develop into an antler.The periosteum of this area is called antlerogenic periosteum（AP）.AP expresses S100A4 molecule,which is thought to be critical for regulating antler generation.High quality of pure S100A4 is required to explore the regulatory mechanism of S100A4 in antler regeneration.In order to meet this demand,we designed the primers which include the cutting sites of EcoR I and BamH I to clone the gene of S100A4.We digested the fragment and PGEX-6p-1 vector with restriction enzymesa,nd linked them together.The products of linking was transformed into competent cells（Top10F）’.We selected positive colonies which were verified with PCR and gel electrophoresis.We transformed the recombinant vector into competent cells（BL21（DE3） pLys S）,and expression of protein was induced by the lactose analog isopropylβ-D thiogalactoside（IPTG）.Successful Expression of S100A4 protein was demon-strated by SDS-PAGE and western blot.In the end we purified the fusion protein.This experiment successfully produced S100A4molecule belonging to sika through prokaryotic expression system.

关 键 词：S100A4 PGEX-6P-1 BL21(DE3)pLysS 蛋白表达 

分 类 号：Q786[生物学—分子生物学]