SH2B1β基因的克隆表达、单抗制备与鉴定

发布时间：2018-06-02 09:07

本文选题：SH2B1β + 克隆　；参考：《南方医科大学》2009年硕士论文

【摘要】： 目前,肥胖迅速向全球蔓延,已经波及到人们概念中该问题并不严重的东南亚地区。因此,肥胖已成为当今全球医学上越来越严重的问题。当人们的体重大大超过推荐的指标就可以确诊为肥胖症,随着肥胖患病率的增高,与肥胖相关的其他慢性非传染性疾病,如糖尿病、代谢综合征、高血压、心脑血管疾病、睡眠障碍、哮喘、肿瘤等的患病率也呈明显上升的趋势。根据流行病学统计,肥胖发生率的增加与成年人群死亡率的增加呈高度相关。近年来的研究表明,肥胖是由特定的生化因子引起的一系列进食调控和能量代谢紊乱而导致的疾病,与遗传、环境、基因、膳食结构有关,而基因是主要的决定因素。从宏观的水平上看,食物摄入增加、体力活动减少和生热机制的改变导致了脂肪的过多堆积;但从微观水平上分析,成熟脂肪细胞的增多是脂质发生累积的关键因素。肥胖已经不仅仅是影响人体形象美观的问题,还是威胁人类健康的问题。被列为世界上第六位的影响全人类疾病负担的危险因素。 1950年,Ingalls等发现一个基因的阴性突变可以导致肥胖,他将该基因称为肥胖基因(obese gene,ob gene),第一次出现了ob基因的概念。Coleman等用交叉灌流实验证明,某种血源性因子能调节动物摄食、代谢、参与肥胖形成,且这种因子的产生与ob基因有关。1991年Friedman描述了五种可以使小鼠形成肥胖的单基因突变,分别是obese(ob)、diabetes(db)、fat(fa)、tubby(tub)和obese yellow(A~y),为研究肥胖的分子机制奠定了基础。1994年Zhang等利用突变基因的定位克隆技术克隆了小鼠和人的ob基因,同时发现ob基因的突变可以导致肥胖,使肥胖研究真正进入分子时代。瘦素蛋白(Leptin)是ob基因的产物,是由白色脂肪细胞分泌的一种有167个氨基酸残基组成的蛋白质。它除了通过作用于下丘脑特异性受体发挥抑制饮食、减少能量摄入、减轻体重和增加能量消耗的作用外,同时还通过其它组织器官上的受体(胰岛、肝脏、性腺、肾上腺、甲状腺等)在体内共同形成一个复杂的网络作用系统,影响着机体许多生理系统和代谢通路。瘦素蛋白的外周作用还包括调节糖代谢的平衡、促进脂肪分解、抑制脂肪合成、参与免疫调节功能和促进生长发育等。经过长期的研究,人们对瘦素蛋白的生物学作用已经有更进一步的认识:瘦素蛋白在能量代谢调节中并不是一个孤立的激素,而是与许多神经肽、神经递质、激素及其它因子协同发挥作用;在人群中存在有不可忽视的“瘦素抵抗”现象等。这些都是致力把瘦素蛋白单独用作为治疗肥胖症药物的研究者今后需要解决的问题。随着研究的深入,研究者发现SH2B1β蛋白能与瘦素激活的细胞信号蛋白JAK2(非受体酪氨酸激酶)相互作用,调节能量代谢和体重。SH2B1β是SH2B信号接头蛋白家族中的一员,其结构上有SH2结构域,可以结合磷酸化的酪氨酸残基,进而连接不同的信号蛋白,发挥其衔接信号及强化信号通路的作用。SH2B1β广泛分布于下丘脑、肝脏、胰脏、脂肪组织等组织中,参与胰岛素敏感性和血糖平衡的调节以及增强神经生长因子诱导的神经分化。由于SH2B1β是Leptin/JAK2信号通路活化所必需的,如果敲除小鼠的SH2B1β基因,将使小鼠的体重是同窝正常组小鼠的两倍,SH2B1β基因缺失导致严重的瘦素抵抗,食欲过盛和肥胖。SH2B1β通过自身结合JAK2同时募集胰岛素底物蛋白(IRS)结合JAK2复合体,以JAK2/STAT3和IRS2/PI3K两条路径来增强瘦素依赖的信号转导,因而SH2B1β是一种内源的瘦素敏感性增强剂,同时,SH2B1β的过表达还可以抵消蛋白酪氨酸磷酸酶1B(PTP1B)介导的瘦素抑制信号的作用。目前有最新的文献报道指出,脂肪组织与神经组织中的SH2B1β在脂肪细胞的生长分化过程中产生两种相反的效应。虽然神经组织SH2B1β的负调节效应占优,但是脂肪组织SH2B1β是以一种细胞自主的方式来调节脂肪细胞的分化,并在3T3-L1前脂肪细胞的分化过程中呈现表达量进行性增加,同时促进细胞中脂滴的累积以及脂肪分化转录因子PPARγ、C/EBPα的表达。脂肪细胞数量的增多和体积的增大能导致肥胖,而前脂肪细胞分化的增加则是导致成熟脂肪细胞数量增多的主要原因,近年来脂肪细胞的分化调控已成为肥胖及其相关疾病的研究热点。人或动物的脂肪组织成份包括微血管、神经组织、成纤维细胞、脂肪细胞以及前脂肪细胞,脂肪细胞系的发育过程为多能干细胞—间充质干细胞—前脂肪细胞—成熟脂肪细胞,所以有效的调节前脂肪细胞的分化能控制脂肪组织增生、肥大。脂肪细胞分化与胰岛素和胰岛素样生长因子相关,胰岛素和胰岛素样生长因子通过结合其受体激活PI3K和MAPK两条信号转导通路,促进脂肪细胞的分化。SH2B1β作为一种胰岛素受体(IR)和胰岛素样生长因子受体(IGF-IR)活性的正调节蛋白,参与调控脂肪细胞分化的信号通路。目前有关脂肪组织SH2B1β的研究很少,而且研究主要是针对鼠源蛋白。根据文献报道SH2B1β鼠源基因与人源基因的相似性达到85%-87%,本课题着眼于使用人源的SH2B1β基因在体外原核表达重组蛋白后,制备抗SH2B1β的单克隆抗体,通过定性及定量的方法初步探讨该人源蛋白在人源前脂肪细胞分化中的调控作用,为治疗与前脂肪细胞分化关系密切的肥胖及其并发症提供新的药物靶点。由于脂肪干细胞是临界于间充质干细胞与前脂肪细胞时期之间的一种细胞状态,所以本研究通过体外原代培养脂肪干细胞(ADSC)并诱导其成脂分化,收集分化成熟的脂肪细胞后,用TRIZOL一步法提取细胞总RNA。运用RT-PCR方法成功扩增并克隆出SH2B1β基因片段,将所得的目的基因克隆至pET28a(+)表达载体,并在大肠杆菌中进行了大量可溶性表达。用饱和硫酸铵盐析法和DEAE阴离子交换法纯化重组蛋白,重组蛋白通过抗His-tag单抗鉴定后免疫小鼠制备单克隆抗体,分别用ELISA、Western-Blot法检测及鉴定单克隆抗体的特异性。利用RT-PCR技术成功克隆了SH2B1β基因,其大小约为2000bp。经测序并与GenBank中序列进行比对,结果显示插入片段与已报道的SH2B1β基因序列配对一致,无移码突变,大小为2016bp。将编码基因克隆到pET-28a(+)原核表达质粒中,经PCR、限制性酶切分析等鉴定,证实成功构建了SH2B1β-pET28a(+)重组原核表达质粒。经IPTG诱导后,重组质粒SH2B1β-pET28a(+)在大肠杆菌E.coli DE3中表达出His-tag融合重组蛋白,分子量约为80kDa,与理论值基本符合。表达产物主要以可溶性形式存在。经过纯化后,重组蛋白纯度可达80%以上。用抗His-tag单抗对表达产物进行Western-blot分析,可见特异性区带出现,表明该蛋白确实是所要表达的His-tag融合蛋白。进一步用纯化的表达产物免疫小鼠制备免疫血清,ELISA分析表明重组蛋白能与该免疫血清起反应,而与正常小鼠血清不发生交叉反应,说明重组蛋白具有抗原活性。将免疫小鼠脾细胞与小鼠骨髓瘤细胞NS-1融合后筛选到2株单抗分泌细胞。以纯化重组蛋白为抗原,用ELISA法检测单抗细胞株培养上清的效价,OD值分别为1.553～2.210。Western-blot结果显示:抗SH2B1β的2株单抗与人源脂肪组织内提取的天然蛋白发生特异性反应,而阴性对照组则无明显条带出现,说明制备的单抗均为特异性单抗。以上结果表明,我们已经成功地构建了SH2B1β-pET28a(+)原核重组表达质粒,目前国内尚未见关于SH2B1β重组并表达的相关报道。而且在大肠杆菌中大量表达出具有免疫活性的可溶性重组蛋白SH2B1β;筛选并建立了2株分泌抗SH2B1β单克隆抗体的杂交瘤细胞株,为进一步研究该蛋白的生物学功能、在脂肪细胞发育分化过程中所起的作用、对肥胖症潜在治疗价值的验证奠定基础。
[Abstract]:Obesity has spread rapidly around the world and has spread to Southeast Asia, which is not a serious problem in people's concept. Obesity has become a growing problem in global medicine today. The prevalence of chronic non communicable diseases, such as diabetes, metabolic syndrome, hypertension, cardiovascular and cerebrovascular diseases, sleep disorders, asthma, and cancer, has also increased significantly. According to epidemiological statistics, the increase in the incidence of obesity is highly related to the increase in the mortality of adults. Recent studies have shown that obesity is specific. The disease caused by a series of eating regulation and energy metabolism disorder caused by biochemical factors, related to heredity, environment, gene, and dietary structure, and genes are the main determinants. From the macro level, the increase in food intake, the decrease of physical activity and the change of the mechanism of heat generation have resulted in excessive accumulation of fat; but from the micro level. The increase in mature adipocytes is the key factor in the accumulation of lipid. Obesity is not only a problem that affects the beauty of the human body, but also a threat to human health. It is listed as the sixth risk factor affecting the burden of disease in the world.
In 1950, Ingalls and others found that a negative mutation of a gene could lead to obesity. He called the gene obese gene (OB gene), and the first occurrence of the concept.Coleman of the ob gene showed that a certain blood source factor could regulate animal feeding, metabolism, and participate in the formation of obesity, and the production of such factors and ob based factors Five kinds of single gene mutations that could make the mice obese were described in.1991. They were obese (OB), diabetes (DB), fat (FA), tubby (tub) and obese yellow, which laid the foundation for the study of the molecular mechanism of obesity. Now mutations in the ob gene can lead to obesity, making obesity research truly enter the molecular age.
Leptin is a product of the ob gene, a protein composed of 167 amino acid residues secreted by white adipocytes. It is not only able to inhibit diet, reduce energy intake, reduce weight and increase energy consumption by acting on the hypothalamic specific receptors, but also through other tissues and organs. The body (islets, livers, gonads, adrenal glands, thyroid glands, etc.) forms a complex network of networks in the body that affects many physiological and metabolic pathways in the body. The peripheral action of leptin proteins also includes regulating the balance of sugar metabolism, promoting fat decomposition, inhibiting fat synthesis, participating in immunoregulation and promoting growth and development. After a long period of study, there is a further understanding of the biological effects of leptin protein: leptin protein is not an isolated hormone in the regulation of energy metabolism, but a synergistic effect with many neuropeptides, neurotransmitters, hormones and other factors; there is a phenomenon of "leptin resistance" that can not be ignored in the population. All these are the problems to be solved by researchers who are trying to use leptin alone as a treatment for obesity drugs.
With the further research, the researchers found that SH2B1 beta protein can interact with the cell signaling protein JAK2 (non receptor tyrosine kinase) activated by leptin. The regulation of energy metabolism and body weight.SH2B1 beta is a member of the SH2B signal junction protein family. The structure has a SH2 domain, which can be combined with the phosphorylated tyrosine residues and then connect to different types. The role of signal protein, the function of its cohesive signal and intensification signal pathway,.SH2B1 beta is widely distributed in the hypothalamus, liver, pancreas, adipose tissue and other tissues, and participates in the regulation of insulin sensitivity and blood glucose balance and the enhancement of neural differentiation induced by nerve growth factor. Since SH2B1 beta is essential for the activation of Leptin/JAK2 signaling pathway, If the SH2B1 beta gene was knocked out of the mice, the weight of the mice would be two times that of the normal group, SH2B1 beta gene deletion led to severe leptin resistance, excessive appetite and obesity.SH2B1 beta through its own combination of JAK2 and the insulin substrate protein (IRS) combined with the JAK2 complex, with the JAK2/STAT3 and IRS2/PI3K two paths to enhance leptin. Dependent signal transduction, SH2B1 beta is an endogenous leptin sensitifier, and the overexpression of SH2B1 beta can also counteract the role of the protein tyrosine phosphatase 1B (PTP1B) mediated leptin suppression signal.
The latest literature reports that the SH2B1 beta in adipose tissue and nerve tissue produces two opposite effects during the growth and differentiation of adipocytes. Although the negative regulatory effect of SH2B1 beta is dominant, the adipose tissue SH2B1 beta regulates the differentiation of adipocyte by a cellular autonomic formula and before 3T3-L1 In the process of adipocyte differentiation, the expression of the expression is increased, and the accumulation of lipid droplets in the cells and the expression of the fat differentiation transcription factor PPAR gamma, C/EBP a are also promoted.
The increase in the number of adipocytes and the increase in volume can lead to obesity, but the increase in the differentiation of pre adipocytes is the main cause of the increase in the number of mature adipocytes. In recent years, the regulation of adipocyte differentiation has become a hot spot in obesity and related diseases. The composition of human or animal fat tissue includes microvessels and nerve tissue. Fibroblasts, adipocytes, and preadipocytes, the development of the adipocyte system is pluripotent stem cells - mesenchymal stem cells - preadipocytes - mature adipocytes. Therefore, effective regulation of pre adipocyte differentiation can control adipose tissue hyperplasia and hypertrophy. Adipocyte differentiation is associated with insulin and insulin-like growth factors. Insulin and insulin-like growth factors (insulin-like growth factors) promote the differentiation of adipocyte differentiated.SH2B1 beta as a positive regulator of insulin receptor (IR) and insulin like growth factor receptor (IGF-IR) activity by combining its receptor to activate the two signal transduction pathways of PI3K and MAPK, and participate in the regulation of the signal transduction pathway of adipocyte differentiation.
At present, there are few studies on SH2B1 beta in adipose tissue, and the research is mainly aimed at the mouse source protein. According to the literature reports, the similarity of SH2B1 beta mouse source gene and human source gene has reached 85%-87%. This topic focuses on the preparation of monoclonal antibodies against SH2B1 beta by using human SH2B1 beta gene in the expression of recombinant protein of SH2B1 beta in vitro. Quantitative method is a preliminary study of the regulation of the human source protein in the differentiation of prehuman adipocytes, which provides new drug targets for the treatment of obesity and its complications which are closely related to the differentiation of preadipocyte.
Since adipose stem cells are critical to a cell state between mesenchymal stem cells and preadipocytes, this study was used to extract and differentiate mature adipocytes by primary culture of fat stem cells (ADSC) in vitro and to induce adipocyte differentiation. The TRIZOL one step method for extracting total RNA. was successfully amplified by RT-PCR method. The pET28a (+) expression vector was cloned from the SH2B1 beta gene fragment, and a large amount of soluble expression was carried out in Escherichia coli. The recombinant protein was purified by saturated ammonium sulfate salting out and DEAE anion exchange method. The recombinant protein was immunized by anti His-tag monoclonal antibody to prepare monoclonal antibodies, and ELISA and Wes were used respectively. Tern-Blot method was used to detect and identify the specificity of monoclonal antibodies.
The SH2B1 beta gene was cloned successfully by RT-PCR technology. The size of 2000bp. was sequenced and compared with the sequence in GenBank. The results showed that the inserted fragment was matched with the reported SH2B1 beta gene sequence, without the code mutation, the size was 2016bp., and the encoding gene was cloned into the pET-28a (+) prokaryotic expression plasmid, and the restriction enzyme was cut through PCR. It was confirmed that Recombinant Prokaryotic Expression Plasmid of SH2B1 beta -pET28a (+) was successfully constructed.
After IPTG induction, the recombinant plasmid SH2B1 beta -pET28a (+) expressed a His-tag fusion recombinant protein in Escherichia coli E.coli DE3. The molecular weight of the recombinant protein was approximately 80kDa, which was basically in accordance with the theoretical value. The expression product was mainly in the form of soluble form. After purification, the purity of the recombinant protein could reach more than 80%. The expression product was expressed as Western-blo by anti His-tag monoclonal antibody. T analysis showed that the specific zone appeared, indicating that the protein was indeed a His-tag fusion protein to be expressed. Further, the purified expression product was used to immunize mice to prepare the immune sera. ELISA analysis showed that the recombinant protein could react with the immune sera and did not cross reaction with the normal mice serum, indicating that the recombinant protein had antigen activity. Sex.
2 mAb secretory cells were screened by fusion of immunized mouse splenocytes and murine myeloma cell NS-1. The recombinant protein was purified as antigen and ELISA method was used to detect the titer of McAb culture supernatant. The OD value was 1.553 to 2.210.Western-blot, respectively: 2 monoclonal antibodies against SH2B1 beta and natural protein extracted from human adipose tissue Specific reaction was observed, while no obvious bands appeared in the negative control group, indicating that the mAbs prepared were specific mAbs.
The above results show that we have successfully constructed the recombinant expression plasmid of SH2B1 beta -pET28a (+). There is no report on the recombinant and expression of SH2B1 beta, and a large number of soluble recombinant protein SH2B1 beta with immune activity is expressed in Escherichia coli, and 2 monoclonal antibodies secreting anti SH2B1 beta monoclonal antibodies have been screened and established. In order to further study the biological function of the protein, the hybridoma cell line plays a role in the development and differentiation of adipocyte, and lays the foundation for the verification of the potential therapeutic value of obesity.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R589.2;R346

【参考文献】