草鱼重组瘦蛋白的制备及其对JAK2-STAT3信号通路的作用研究

Preparation of Recombinant Leptin from Grass Carp(Ctenopharyngodon idellus) and Its Effect on JAK2-STAT3 Signaling Pathway

瘦素(leptin)是主要由脂肪细胞分泌的蛋白质,具有调节机体食物摄入和能量代谢的功能。为了获得具有生物活性的瘦蛋白,本研究采用PCR技术从草鱼脑垂体中扩增出瘦素的cDNA开放阅读框(open reading frame,ORF)序列,用于构建pGEX4T-1-CiLEP重组质粒(带GST标签),并将重组质粒在大肠杆菌BL21中进行原核表达。经SDS-PAGE和Western-blot检测证实,在47 kD处有与预期相对分子质量相符的融合重组瘦蛋白(即含GST标签的草鱼重组瘦蛋白)。体内注射试验结果表明,与注射GST标签蛋白相比,纯化后的融合重组瘦蛋白的注射对草鱼脑垂体和肠道的瘦素受体基因及JAK2-STAT3信号通路相关基因的表达有上调作用。以上结果为进一步研究草鱼重组瘦蛋白对JAK2-STAT3信号通路相关基因表达的影响和深入了解草鱼瘦素基因的功能及其调控机制奠定了基础。

Leptin is a protein which is secreted by adipocyte and plays roles in regulating food intake and energy metabolism.In order to obtain the leptin protein with biological activity,the PCR technology was used to amplify the open reading frame(ORF)sequence of leptin from the pituitary of grass carp.The recombinant plasmid(pGEX4T-1-CiLEP)(containing GST marker)was constructed and transformed into the E.coli BL21 competent cells for leptin expression in grass carp.SDS-PAGE and Western-blot analyses showed that the recombinant fusion leptin(GST marker plus recombinant leptin)was expressed with relative molecular mass of 47 kD in grass carp.In vitro injection experiments showed that compared with injection of GST marker,injection of purified recombinant fusion leptin up-regulated the leptin receptor(Lep-R)gene and the JAK2-STAT3 signaling pathway in grass carp pituitary and intestine.These results enrich the theoretical foundation in understanding the function and regulation mechanism of grass carp leptin gene.