人FSHR蛋白的表达及与其受体结合作用的研究

发布时间：2018-01-17 18:13

本文关键词：人FSHR蛋白的表达及与其受体结合作用的研究　出处：《新疆大学》2017年硕士论文　论文类型：学位论文

【摘要】：FSHR(follicle-stimulating hormone receptor)卵泡刺激素受体,是一种七次跨膜受体,属于G蛋白偶联受体家族(GPCRs)成员。GPCRs是迄今发现的最大的多药物靶点的受体超家族,其激动剂或拮抗剂常被用于治疗各种疾病,在药物研发中扮演重要角色。测定配体与受体亲和力的结合实验是药物评价、研发和作用机理研究中不可缺少的部分。激素的结合从结构上来说,改变受体或配体上的某些肽段就会影响激素的结合以及下游的信号转导,从功能上来说改变某个结合位点上的氨基酸,就会影响生理功能的正常发挥。结构的变化影响正常的机能。通过对其结构的分析,就能更加准确了解生理功能的异常和研究出更有效的治疗药物。FSHR由695个氨基酸组成,分别由细胞外域(ECD)、细胞内域(ICD)及跨膜域(TMD)三部分构成。氨基端的ECD是由包含349个氨基残基组成的亲水性结构域构成,从三维空间上看呈马蹄形或者U形,存在富含亮氨酸重复(Leucine-rich repeats,LRR)序列,主要参与激素的相互结合。此前对于受体FSHR的研究中发现,FSHR的细胞外结构域(ECD)被认为是激素选择性结合的主要决定区域。LRR有12个β折叠,每个约有24个氨基酸残基与FSH产生特异结合的位点位于LRR5-LRR10之间,LRR的每一个折叠对于激素的结合都很重要。通过筛查合成肽和FSHR结构表位的预测,发现在FSHR结构中连接信号肽和胞外区的9-30这一肽段在激素结合和信号转导中起关键作用。N端中9-30区被认为是其独有的序列,且9-30氨基酸区域被认为是受体的中和表位。在晶体结构中显示其一部分氨基酸也参与到激素的结合中。在先前的研究中,Dattatreyamurty etal发现FSHR9-30对于激素的结合有计量依赖性。在此基础上研究FSHR多肽抗体作为了解结构和功能的探针,这些抗多肽抗体能够阻断受体与配体的结合或是信号转导。当前研究FSH的片段有1-15、33-53、51~(-6)5和81-95,其中33-53和81-95肽参与受体的识别和结合较好。最近报道FSH33-53肽结合树状大分子对卵巢癌细胞的靶向作用,FSH33-53肽可以使得卵巢癌细胞si RNA沉默。但是与FSH33-53肽所结合的激素位点还尚未报道,本研究选用FSH33-53肽段,研究其结合作用。鉴于以上介绍本研究中选用配体FSH33-53多肽与ECD、LRR和9-30作为研究的材料,实验中初步获得FSHRN端的ECD、LRR、9-30、29-173和174-331,得到这5个不同肽段的蛋白,为后续测定与FSH33-53肽的结合力做准备。免疫新西兰大白兔制备抗ECD、LRR和9-30多克隆抗体,同时用所制备的多克隆抗体检测其对结合力的阻断作用。从侧面鉴定FSHR的结合力。从而为今后设计靶向FSHR的药物和疫苗提供了参考。研究内容包括以下两方面:1、FSHR蛋白的表达及多克隆抗体的制备首先通过构建重组质粒制备FSHR胞外区不同肽段蛋白。以含有FSHR的c DNA为模板,PCR扩增出h FSHR的ECD、LRR、29-173和174-331基因片段,构建到p ET22b载体上,经酶切和测序结果显示构建的重组质粒正确。转入在BL21大肠杆菌中进行表达,经过Ni离子纯化柱纯化,蛋白大小分别是43kDa、32kDa、22kDa和20kDa,蛋白大小与预期的结果一致。通过western blot检测原核蛋白的抗原活性为后续制备多克隆抗体做准备。其中由于9-30肽氨基酸数目较少,我们通过多肽合成的方法获得9-30-KLH蛋白。众所周知原核蛋白在结构和功能上有一定的缺陷,因此我们也设计了FSHR的真核蛋白表达,首先构建真核表达载体pMT-FSHR,酶切和测序均证明构建正确。之后转染果蝇S2细胞,通过荧光显微镜显示转染成功,收集转染5天的细胞培养上清,通过SDS-PAGE检测FSHR的真核蛋白的表达,但结果显示真核蛋白未能获得。原核蛋白ECD、LRR和9-30肽免疫新西兰大白兔,收集免疫4次的血清,通过ELISA实验检测抗血清效价分别为1:128000、1:64000和1:64000。之后对多克隆抗体进行了功能检测,首先免疫共沉淀实验结果显示多克隆抗体能与Caov-3细胞表面的FSHR结合。其次通过多克隆抗体的交叉实验结果显示,抗ECD的多克隆抗体和抗LRR的多克隆抗体之间有交叉反应,而抗9-30多克隆抗体与这两个多克隆抗体无交叉反应。2、FSHR的结合阻断实验在前期的实验中我们获得了h FSHR的ECD、LRR、29-173和174-331蛋白,同时合成短肽蛋白h FSHR9-30-KLH。同时也获得了抗ECD、LRR和9-30的多克隆抗体。ELISA实验检测受体与配体亲和力分别为0.21×10~(-6)、0.45×10~(-6)和0.056×10~(-6) mol/L、0.43×10~(-6)和1.1×10~(-6),ELISA检测多克隆抗体竞争结合FSHR。本研究的结论,从实验结果中可以看出成功获得人FSHR重组蛋白ECD、LRR、FSHR9-30、29-173和174-331,并且ECD、LRR、9-30蛋白有很好的抗原特异性,在表达人FSHR真核蛋白中,却未能获得真核蛋白。在多克隆抗体是制备中,我们获得了抗ECD、LRR和9-30的多克隆抗体,且抗体效价较高。在抗体交叉实验中发现抗ECD的多克隆抗体与抗LRR的多克隆抗体有交叉结合,抗9-30的多克隆抗体与这两个多克隆抗体之间无交叉。最后ECD、LRR、9-30、29-173和174-331与FSH 33-53肽的亲和力大小中可知9-30的亲和力较高,在阻断效应上可以看出抗ECD的阻断效应较强。可能是肽段长,空间结构比较完整,因此阻断效应较强。本研究通过分析FSHR不同功能片段对于激素的结合和阻断作用,初步表明FSH33-53肽与其受体结合结合较高的位点位于氨基端9-30区域,但LRR区域在FSH结合过程中也发挥作用。研究结果为今后设计靶向FSHR的药物和疫苗提供了参考。
[Abstract]:FSHR (follicle-stimulating hormone receptor) follicle stimulating hormone receptor, a seven transmembrane receptor, which belongs to the family of G protein coupled receptors (GPCRs).GPCRs member is found so far the largest number of drug target receptor superfamily and its agonists or antagonists are used in the treatment of various diseases, play an important role in drug in research and development. According to the experimental determination of ligand and receptor affinity is indispensable for drug evaluation, study on the mechanism of development and effect of hormone binding part. From the structure, change of receptor or ligand on some peptides can affect hormone binding and signal transduction, from the function, change a combination of amino acid sites on that will affect the physiological function. The normal functioning of the changes in the structure. Through the analysis of the structure, can more accurately understand the physiological function of the abnormal and A more effective treatment of.FSHR consists of 695 amino acids in research, respectively by the extracellular domain (ECD), intracellular domain (ICD) and transmembrane domain (TMD) is composed of three parts. The ECD is hydrophilic amino terminal domain consists of 349 amino residues, see horseshoe shaped or the U shape from the three-dimensional space, there is a leucine rich repeat (Leucine-rich repeats LRR) sequence, combined with each other mainly involved in hormone receptor FSHR. Prior to the study found that the extracellular domain of FSHR (ECD) is considered to be the main hormone determining region.LRR selective binding of 12 beta folding, each about 24 amino acid residues with FSH specific binding sites located between LRR5-LRR10, LRR of each hormone combination for folding are very important. By predicting the screening of synthetic peptides and FSHR epitopes, found in the FSHR structure connecting the signal peptide and the extracellular domain of 9 The -30 peptide in the hormone binding and signal transduction plays a key role in the.N end of the 9-30 area is considered to be its unique sequence, and the amino acid 9-30 area is considered as a receptor neutralizing epitope. The display part of the amino acid involved in hormone binding in the crystal structure. In the previous study, Dattatreyamurty etal FSHR9-30 has found that measurement depends on the hormone combination. The probe based on FSHR polypeptide antibody as the understanding of the structure and function of these anti peptide antibody can block the binding of receptor and ligand or signal transduction. The current research on the FSH fragment of 1-15,33-53,51~ (-6) 5 and 81-95, the recognition of 33-53 and 81-95 peptide participate in the receptor and binding better. Recently reported FSH33-53 peptide binding dendrimers on ovarian cancer cell targeting, FSH33-53 peptide can make ovarian cancer cells Si RNA and FSH33-53 but silent. The combination of the peptide hormone site has not yet been reported, this study used FSH33-53 peptide on the binding. In view of the above introduced in this study using ligand FSH33-53 peptide and ECD, LRR and 9-30 as research materials, the initial FSHRN end of the ECD, LRR, 9-30,29-173 and 174-331, which are 5 different peptides the protein, preparation and adhesion of FSH33-53 peptide for subsequent determination. New Zealand white rabbits were immunized to prepare anti ECD polyclonal antibody, LRR and 9-30, at the same time using polyclonal antibodies to detect the inhibition of binding force. The binding force from the side of the identification of FSHR. Thus for the future design target to FSHR drugs and vaccines to provide a reference. The research contents include the following two aspects: 1, the expression of FSHR protein and preparation of polyclonal antibody by recombinant plasmid preparation of FSHR extracellular domain protein. Different peptides containing FSHR C DNA to die In PCR, amplified h FSHR ECD, LRR, 29-173 and 174-331 gene were constructed into P ET22b vector and confirmed by enzyme digestion and sequencing showed that the recombinant plasmid was transferred to the right. BL21 expression in Escherichia coli, purification by Ni ion chromatography, protein size were 43kDa, 32kDa, and 22kDa 20kDa, protein size and expected results. The activity of Western blot antigen detection prokaryotic protein for the subsequent preparation of polyclonal antibody preparation. Due to the small number of 9-30 peptide, we obtained 9-30-KLH protein by peptide synthesis. As everyone knows there are some defects in the structure and function of prokaryotic protein, therefore we also designed the eukaryotic protein expression of FSHR, we construct the eukaryotic expression vector pMT-FSHR, enzyme digestion and sequencing were proved correctly constructed. After transfection of Drosophila S2 cells by fluorescence microscopy showed close successful transfection. In 5 days of transfection cell culture supernatant, the expression of eukaryotic protein SDS-PAGE detection of FSHR, but the results showed that the eukaryotic protein failed to obtain prokaryotic protein. ECD, LRR and 9-30 peptide in New Zealand white rabbits were immunized 4 times, collecting immune serum, the titer of the antiserum by ELISA assay respectively for 1:128000,1:64000 and 1:64000. to polyclonal antibodies were the first function test, CO immunoprecipitation experiments showed that the polyclonal antibody could bind to Caov-3 FSHR on the cell surface. Secondly, through cross experiment of polyclonal antibody showed that the cross reaction between anti ECD polyclonal antibody and anti LRR polyclonal antibody and anti 9-30 polyclonal antibody and the two polyclonal antibodies had no cross reaction with.2, FSHR combined with blocking experiments in previous experiments we obtained the H FSHR ECD, LRR, 29-173 and 174-331 protein, H protein and peptide FSHR9-30 -KLH. also received anti ECD polyclonal antibody affinity,.ELISA assay of receptor and ligand LRR and 9-30 were 0.21 (-6) * 10~, 0.45 * 10~ (-6) and 10~ (-6) 0.056 * mol/L, 0.43 * 10~ (-6) and 1.1 * 10~ (-6), ELISA polyclonal detection competitive antibody binding FSHR. the conclusions of this study, can be seen from the experimental results obtain the recombinant FSHR protein ECD, LRR, FSHR9-30,29-173 and 174-331, and ECD, LRR, 9-30 protein antigen has good specificity, the expression of human FSHR eukaryotic proteins, but failed to obtain eukaryotic protein. Polyclonal antibody is in preparation, we obtained anti ECD polyclonal antibody, LRR and 9-30, and the antibody titers were higher in antibody cross experiments showed that the polyclonal anti ECD antibody and anti LRR polyclonal antibody has no cross intersection between anti 9-30 polyclonal antibody and two polyclonal antibody. Finally, ECD, LRR, 9-30,29- The 9-30 of the 173 high affinity and 174-331 with FSH 33-53 peptide affinity, the blocking effect can be seen on the blocking effects of anti ECD peptide may be strong. Long, space structure is complete, so the blocking effect is strong. Through the analysis of FSHR of different functional fragments for hormone binding and inhibition, preliminary results show FSH33-53 peptide binding to its receptor located in the N-terminal region 9-30 higher binding sites, but also with the LRR region plays a role in the process of FSH. The results for the future design of FSHR targeting drugs and vaccines to provide reference.

【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91;Q78

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