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 SI00A4 is required to explore the regulatory mechanism of SI00A4 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 SI00A4. We digested the fragment and PGEX-6p-I vector with restriction enzymes,and linked them together. The products of linking was transformed into competent cells (Topl0F'). We selected positive colonies which were verified with PCR and gel electropboresis. We transformed the recombinant vector into competent cells (BL21 (DE3) pLys S), and expression of protein was induced by the lactose analog isopropyl β -D thiogalaetoside (IPTG). Successful Expression of S100A4 protein was demonstrated by SDS-PAGE and western blot. In the end we purified the fusion protein. This experiment successfully produced S100A4 molecule belonging to sika through prokaryotic expression system.%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.
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