本文關(guān)鍵詞：人FSHR蛋白的表達及與其受體結(jié)合作用的研究　出處：《新疆大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： FSH33-53肽 FSHR FSHR9-30 FSHR競爭結(jié)合抑制 抗多肽抗體

【摘要】：FSHR(follicle-stimulating hormone receptor)卵泡刺激素受體,是一種七次跨膜受體,屬于G蛋白偶聯(lián)受體家族(GPCRs)成員。GPCRs是迄今發(fā)現(xiàn)的最大的多藥物靶點的受體超家族,其激動劑或拮抗劑常被用于治療各種疾病,在藥物研發(fā)中扮演重要角色。測定配體與受體親和力的結(jié)合實驗是藥物評價、研發(fā)和作用機理研究中不可缺少的部分。激素的結(jié)合從結(jié)構(gòu)上來說,改變受體或配體上的某些肽段就會影響激素的結(jié)合以及下游的信號轉(zhuǎn)導,從功能上來說改變某個結(jié)合位點上的氨基酸,就會影響生理功能的正常發(fā)揮。結(jié)構(gòu)的變化影響正常的機能。通過對其結(jié)構(gòu)的分析,就能更加準確了解生理功能的異常和研究出更有效的治療藥物。FSHR由695個氨基酸組成,分別由細胞外域(ECD)、細胞內(nèi)域(ICD)及跨膜域(TMD)三部分構(gòu)成。氨基端的ECD是由包含349個氨基殘基組成的親水性結(jié)構(gòu)域構(gòu)成,從三維空間上看呈馬蹄形或者U形,存在富含亮氨酸重復(Leucine-rich repeats,LRR)序列,主要參與激素的相互結(jié)合。此前對于受體FSHR的研究中發(fā)現(xiàn),FSHR的細胞外結(jié)構(gòu)域(ECD)被認為是激素選擇性結(jié)合的主要決定區(qū)域。LRR有12個β折疊,每個約有24個氨基酸殘基與FSH產(chǎn)生特異結(jié)合的位點位于LRR5-LRR10之間,LRR的每一個折疊對于激素的結(jié)合都很重要。通過篩查合成肽和FSHR結(jié)構(gòu)表位的預測,發(fā)現(xiàn)在FSHR結(jié)構(gòu)中連接信號肽和胞外區(qū)的9-30這一肽段在激素結(jié)合和信號轉(zhuǎn)導中起關(guān)鍵作用。N端中9-30區(qū)被認為是其獨有的序列,且9-30氨基酸區(qū)域被認為是受體的中和表位。在晶體結(jié)構(gòu)中顯示其一部分氨基酸也參與到激素的結(jié)合中。在先前的研究中,Dattatreyamurty etal發(fā)現(xiàn)FSHR9-30對于激素的結(jié)合有計量依賴性。在此基礎(chǔ)上研究FSHR多肽抗體作為了解結(jié)構(gòu)和功能的探針,這些抗多肽抗體能夠阻斷受體與配體的結(jié)合或是信號轉(zhuǎn)導。當前研究FSH的片段有1-15、33-53、51~(-6)5和81-95,其中33-53和81-95肽參與受體的識別和結(jié)合較好。最近報道FSH33-53肽結(jié)合樹狀大分子對卵巢癌細胞的靶向作用,FSH33-53肽可以使得卵巢癌細胞si RNA沉默。但是與FSH33-53肽所結(jié)合的激素位點還尚未報道,本研究選用FSH33-53肽段,研究其結(jié)合作用。鑒于以上介紹本研究中選用配體FSH33-53多肽與ECD、LRR和9-30作為研究的材料,實驗中初步獲得FSHRN端的ECD、LRR、9-30、29-173和174-331,得到這5個不同肽段的蛋白,為后續(xù)測定與FSH33-53肽的結(jié)合力做準備。免疫新西蘭大白兔制備抗ECD、LRR和9-30多克隆抗體,同時用所制備的多克隆抗體檢測其對結(jié)合力的阻斷作用。從側(cè)面鑒定FSHR的結(jié)合力。從而為今后設(shè)計靶向FSHR的藥物和疫苗提供了參考。研究內(nèi)容包括以下兩方面:1、FSHR蛋白的表達及多克隆抗體的制備首先通過構(gòu)建重組質(zhì)粒制備FSHR胞外區(qū)不同肽段蛋白。以含有FSHR的c DNA為模板,PCR擴增出h FSHR的ECD、LRR、29-173和174-331基因片段,構(gòu)建到p ET22b載體上,經(jīng)酶切和測序結(jié)果顯示構(gòu)建的重組質(zhì)粒正確。轉(zhuǎn)入在BL21大腸桿菌中進行表達,經(jīng)過Ni離子純化柱純化,蛋白大小分別是43kDa、32kDa、22kDa和20kDa,蛋白大小與預期的結(jié)果一致。通過western blot檢測原核蛋白的抗原活性為后續(xù)制備多克隆抗體做準備。其中由于9-30肽氨基酸數(shù)目較少,我們通過多肽合成的方法獲得9-30-KLH蛋白。眾所周知原核蛋白在結(jié)構(gòu)和功能上有一定的缺陷,因此我們也設(shè)計了FSHR的真核蛋白表達,首先構(gòu)建真核表達載體pMT-FSHR,酶切和測序均證明構(gòu)建正確。之后轉(zhuǎn)染果蠅S2細胞,通過熒光顯微鏡顯示轉(zhuǎn)染成功,收集轉(zhuǎn)染5天的細胞培養(yǎng)上清,通過SDS-PAGE檢測FSHR的真核蛋白的表達,但結(jié)果顯示真核蛋白未能獲得。原核蛋白ECD、LRR和9-30肽免疫新西蘭大白兔,收集免疫4次的血清,通過ELISA實驗檢測抗血清效價分別為1:128000、1:64000和1:64000。之后對多克隆抗體進行了功能檢測,首先免疫共沉淀實驗結(jié)果顯示多克隆抗體能與Caov-3細胞表面的FSHR結(jié)合。其次通過多克隆抗體的交叉實驗結(jié)果顯示,抗ECD的多克隆抗體和抗LRR的多克隆抗體之間有交叉反應,而抗9-30多克隆抗體與這兩個多克隆抗體無交叉反應。2、FSHR的結(jié)合阻斷實驗在前期的實驗中我們獲得了h FSHR的ECD、LRR、29-173和174-331蛋白,同時合成短肽蛋白h FSHR9-30-KLH。同時也獲得了抗ECD、LRR和9-30的多克隆抗體。ELISA實驗檢測受體與配體親和力分別為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檢測多克隆抗體競爭結(jié)合FSHR。本研究的結(jié)論,從實驗結(jié)果中可以看出成功獲得人FSHR重組蛋白ECD、LRR、FSHR9-30、29-173和174-331,并且ECD、LRR、9-30蛋白有很好的抗原特異性,在表達人FSHR真核蛋白中,卻未能獲得真核蛋白。在多克隆抗體是制備中,我們獲得了抗ECD、LRR和9-30的多克隆抗體,且抗體效價較高。在抗體交叉實驗中發(fā)現(xiàn)抗ECD的多克隆抗體與抗LRR的多克隆抗體有交叉結(jié)合,抗9-30的多克隆抗體與這兩個多克隆抗體之間無交叉。最后ECD、LRR、9-30、29-173和174-331與FSH 33-53肽的親和力大小中可知9-30的親和力較高,在阻斷效應上可以看出抗ECD的阻斷效應較強�？赡苁请亩伍L,空間結(jié)構(gòu)比較完整,因此阻斷效應較強。本研究通過分析FSHR不同功能片段對于激素的結(jié)合和阻斷作用,初步表明FSH33-53肽與其受體結(jié)合結(jié)合較高的位點位于氨基端9-30區(qū)域,但LRR區(qū)域在FSH結(jié)合過程中也發(fā)揮作用。研究結(jié)果為今后設(shè)計靶向FSHR的藥物和疫苗提供了參考。
[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.

【學位授予單位】：新疆大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R91;Q78

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