人Leptin基因融合蛋白表达载体构建及对人成骨细胞的作用  

Effect of Leptin fusion protein on growth of human osteoblast cells following constructing Leptin gene expression vector

作　　者：韩国胜[1] 唐天驷[1] 顾勇[1] 董仁斌[1] 陈亮[1] 

机构地区：[1]苏州大学附属第一医院骨科,江苏省苏州市215006

出　　处：《中国组织工程研究与临床康复》2009年第7期1232-1236,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省"科教兴卫"工程项目(RC2007078)~~

摘　　要：背景:瘦素作为一种内分泌激素参与机体能量代谢的调节,近年来瘦素对骨代谢的调节作用也越来越被人们所重视。目的:克隆人Leptin基因,构建重组原核表达载体PEGX-5X-3/Leptin并在大肠杆菌中表达,观察Leptin融合蛋白对人成骨细胞的生长抑制作用。设计、时间及地点:单一样本观察,于2007-07/2008-05在苏州大学附属第一医院骨科实验室完成。材料:新鲜的脂肪组织取自苏州大学附一院(供者知情并同意)、大肠杆菌DH5α、人成骨细胞由苏州大学附属第一医院骨科实验室保种。方法:从人脂肪组织中提取RNA,采用反转录-聚合酶链式反应获得人Leptin全部序列,克隆入带有GST原核细胞表达载体PEGX-5X-3中,实现插入基因的融和表达,用SDS-PAGE和WesternBlot对表达产物进行鉴定,提取并纯化GST-Leptin融合蛋白,以5,10,20,40mg/L不同质量浓度与人成骨细胞共培养,同时设立相应浓度梯度的GST蛋白组,四甲基偶氮唑盐法测两组人成骨细胞的体外增殖情况。主要观察指标:①Leptin基因的克隆与鉴定。②重组质粒的双酶切及聚合酶链反应鉴定。③融合蛋白的表达。④GST-Leptin融合蛋白的纯化。⑤GST-Leptin融合蛋白的鉴定。⑥GST-Leptin融合蛋白对人成骨细胞的生长影响。结果:反转录-聚合酶链反应扩增得到的特异性片段长度约为450bp,以此构建的重组质粒PEGX-5X-3/Leptin,经BamHⅠ和XhoⅠ双酶切后显示5kb和450bp左右的两条片段,测序结果与Genbank中报道的完全一致,证明Leptin基因已成功地克隆到了原核表达载体PEGX-5X-3中。并在大肠杆菌中获得稳定的表达,表达产物的相对分子质量(Mr)同预期值相一致,融合蛋白为Mr45000,GST蛋白为Mr29000,与GST蛋白组相比,20,40mg/L的GST-Leptin融合蛋白可以明显促进细胞的生长(P<0.05),且其促进作用有一定的浓度依赖性。结论:成功构建了PEGX-5X-3/Leptin重组原核表达载体,在E.coliBL21中表达GST-LBACKGROUND： Leptin, an endocrine hormone, regulates energy metabolism of organism. Recently, the effect of leptin on bone metabolism attracts more attention. OBJECTIVE： To construct a recombinant expression vector of PEGX-5X-3/Leptin and express it in E. coli, in addition, to investigate the effect of Leptin-GST fusion protein on the growth of human osteoblasts. DESIGN, TIME AND SETTING： A single sample observation was performed at the Laboratory of Orthopedics in the First Affiliated Hospital of Soochow University. MATERIALS： Fresh fatty tissue obtained from the First Affiliated Hospital of Soochow University, E. coil DH5α, human osteoblasts were preserved by the Laboratory of Orthopedics in the First Affiliated Hospital of Soochow University. METHODS： The Leptin cDNA fragment was amplified from human adipose tissue by reverse transcription polymerase chain reaction （RT-PCR）, then cloned it to the prokaryotic vector PEGX-5X-3 which containing GST, and expressed inductively as a fusion protein in E. coil The expressed GST- Leptin fusion protein was purified via GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot. The human osteoblasts were treated with different concentrations of GST-Leptin fusion protein （5, 10, 20, 40 mg/L） and GST protein for different time, and the proliferation of human osteoblast was measured by MTT assay. MAIN OUTCOME MEASURES： Clone and identification of Leptin gene. The recombinant plasmid was identified by restriction enzyme and PCR. The expression, purification, and identification of GST-Leptin fusion protein, as well as the effects of GST-Leptin fusion protein on human osteoblasts proliferation was observed. RESULTS： The length of specific fragment applied by RT-PCR was 450 bp, and the recombinant plasmid PEGX-SX-3/Leptin presented two bands： 5 kb and 450 bp using respective restriction enzymes BamH I and Xho I. The sequence was identified with that reported in GenBank. It suggested that Leptin had been cloned into PEGX-5X-3 vector Correctly an

关 键 词：瘦素 基因表达 成骨细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]