生长分化因子5基因原核表达质粒构建和表达及其体内成软骨的诱导活性

Construction and expression of growth differentiation factor-5 gene prokaryotic expression plasmid and its cartilage induction activity in vivo

背景:生长分化因子5在软骨、四肢和关节的发育、形成中起着重要作用,是目前最常应用的研究早期关节发育的标志分子。克隆人生长分化因子5基因可为软骨、关节缺损的修复奠定研究基础。目的:利用基因工程技术在大肠杆菌中表达人生长分化因子5成熟肽,探讨重组蛋白的体内诱导活性。设计、时间及地点:基因水平观察实验,于2006-01/06在南方医科大学分析测试中心完成。材料:人流产胚胎膝关节区软骨组织取自解放军广州军区总医院妇产科,标本获取征得家属同意。10只KM小鼠购自南方医科大学实验动物中心,雌雄各半,体质量18～22g,6～8周龄。方法:通过反转录聚合酶链式反应(RT-PCR)从人胚胎软骨组织克隆生长分化因子5完整成熟肽基因,将所得基因片段插入pET22b(+)载体构建重组原核表达载体pET22b(+)-GDF5,重组质粒转化大肠杆菌BL-21进行IPTG诱导表达。凝胶介质镍离子螯合法纯化目的蛋白,植入小鼠后肢股部肌袋内常规苏木精-伊红染色进行生物学活性鉴定。主要观察指标:琼脂糖凝胶电泳观察目的基因表达条带,测序观察目的基因序列,SDS-PAGE电泳观察目的蛋白表达条带,组织学观察生长分化因子5诱导活性。结果:RT-PCR产物长约350bp,阳性克隆质粒经双酶切可切出约350bp的片段,测序表明与Genbank中的序列完全一致。SDS-PAGE电泳显示重组子转化菌在相对分子质量14100位置处出现特异外源蛋白表达带,和成熟肽相对分子质量13600接近。纯化后体内植入实验组织切片显示,大量的间充质干细胞聚集和增生,并分化成为成软骨细胞,分泌软骨基质并埋入其中变为软骨细胞。结论:通过RT-PCR法从人胚胎软骨组织中成功克隆出人生长分化因子5完整成熟肽基因,可在大肠杆菌中得到高效表达,经纯化后在动物体内具有成软骨诱导活性。

BACKGROUND： Growth differentiation factor-5 （GDF-5） plays an important role in the development and formation of cartilage, extremities, and joints, which is a widely used joint development marker. OBJECTIVE： To express mature pepfide of human GDF-5 in E. coil by the way of genetic engineering, and to explore the inductive activity of recombinant protein in vivo. DESIGN, TIME AND SETTING： The observation experiment based on gene was performed at the Analysis and Testing Center of Southern Medical University from January to June 2006. MATERIALS： Human fetus cartilage tissue was harvested from Department of Gynaecology and Obstetrics, and the consent was obtained from the family. Ten KM mice were purchased from experimental animal center of Southern Medical University, half male and half female, weighing 18--22 g, aged 6-8 weeks. METHODS： The hGDF-5 gene encoding mature peptide was gained by RT-PCR from the total RNA which was extracted from fetus cartilage tissues, and was inserted into the pET22b（＋） vector to construct recombinant prokaryotic expression plasmid pET22b（＋）-GDFS, which was transformed into E. coil BL-21 to be expressed after IPTG induction. Proteins of interest were purified with sepharose chelated with nickel ions （Ni2＋） and then implanted in mouse hindlimb muscle to evaluate the biological activities by routine hematoxylin-eosin staining. MAIN OUTCOME MEASURES： The expression, sequencing of target gene was observed by agarose gel electrophoresis, and the protein expression was detected by SDS-PAGE electrophoresis, meanwhile, the GDF5-inducing activity was evaluate by histological observation. RESULTS： RT-PCR product was about 350 bp in length, which was confirmed by double enzyme digestion of the recombinant plasmid, sequencing result was in agreement with the reported hGDF-5 sequence in Genbank. SDS-PAGE analysis showed a conspicuous band representing a new foreign protein with relative molecular mass of approximately 14 KD after induced expressioin. Cartilage tissues were formed in the mouse muscle where the purified proteins were implanted. CONCLUSION： The integral human GDF-5 mature peptide gene was cloned successfully from human fetus cartilage tissue and a high-yield expression was achieved in E. coli, the purified protein has chondrogenic activities in vivo.