發(fā)布時間：2018-03-03 19:53

  本文選題：GRP94　切入點：SND1　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的:SND1,因為分子量為100kDa,所以又稱為p100蛋白或Tudor-SN蛋白。人類SND1最初被發(fā)現(xiàn)是作為轉(zhuǎn)錄共激活因子能夠激活EB病毒細(xì)胞核抗原2(Epstein-Barr virus nuclear protein2)所介導(dǎo)的基因表達(dá)。SND1蛋白在各物種組織和細(xì)胞中分布廣泛,在牛、大鼠、小鼠等動物細(xì)胞內(nèi)存在能與人工合成的人SND1抗體相結(jié)合的SND1蛋白[1]。SND1在不同生物中具有高度的保守性,參與調(diào)控細(xì)胞的多種重要生理過程。SND1蛋白具有調(diào)節(jié)基因轉(zhuǎn)錄和剪接加工pre-mRNA的能力,在多種腫瘤的發(fā)生發(fā)展中起重要作用。但到目前為止,SND1蛋白參與上述腫瘤的發(fā)生、發(fā)展的具體機制還不清楚,有待于進(jìn)一步研究。人類主要組織相容性復(fù)合體(MHC)被稱為人類白細(xì)胞抗原(human leukocyte antigen,HLA),該基因位于人染色體的第6號染色體短臂上,編碼具有高度多態(tài)性的HLA抗原。HLA抗原其本質(zhì)為一類糖蛋白,由一條α重鏈和一條β輕鏈非共價結(jié)合而成。HLA在人體生理、病理機制中發(fā)揮重要作用,特別是在免疫應(yīng)答中能夠作為遞呈分子對抗原進(jìn)行加工和呈遞,形成MHC-抗原肽-TCR復(fù)合物啟動免疫應(yīng)答,并對免疫應(yīng)答起到調(diào)節(jié)作用。HLA作為某些疾病的遺傳標(biāo)志,對通過對HLA等位基因出現(xiàn)頻率的檢測,可以作為某些疾病的產(chǎn)前診斷。HLA又被稱移植抗原,在器官移植領(lǐng)域開展的針對HLA的研究為移植配型提供可重要的理論依據(jù)。機體抗腫瘤的免疫反應(yīng)機制中,以細(xì)胞免疫為主導(dǎo)作用,與體液免疫相互調(diào)節(jié),協(xié)同殺傷腫瘤細(xì)胞。HLA在腫瘤免疫中起到關(guān)鍵作用,研究HLA與腫瘤的關(guān)系,有助于了解腫瘤免疫效應(yīng)中特異性抗原肽,為腫瘤的防治提供依據(jù)。葡萄糖調(diào)節(jié)蛋白94是熱休克蛋白90家族中的一員[2],GRP94蛋白本身是一種序列高度保守的蛋白,存在于內(nèi)質(zhì)網(wǎng)腔中,作為內(nèi)質(zhì)網(wǎng)典型的分子伴侶參與內(nèi)質(zhì)網(wǎng)內(nèi)蛋白的折疊、轉(zhuǎn)運、分泌及降解[3]。同時GRP94能夠參與內(nèi)質(zhì)網(wǎng)應(yīng)激,當(dāng)細(xì)胞內(nèi)出現(xiàn)低糖、缺氧、酸中毒等刺激時,出現(xiàn)錯誤折疊或未折疊的蛋白質(zhì)大量聚集,會誘發(fā)非折疊蛋白反應(yīng),從而導(dǎo)致GRP94表達(dá)升高。有文獻(xiàn)報道,GRP94能夠參與免疫應(yīng)答過程中MHC類分子的合成,介導(dǎo)腫瘤細(xì)胞免疫原性的產(chǎn)生[4]。本課題將FLAG-GRP94目的基因定向?qū)�入pLVX-IRES-Puro慢病毒載體,構(gòu)建人GRP94基因慢病毒載體,進(jìn)一步篩選穩(wěn)定表達(dá)GRP94蛋白的人宮頸癌HeLa細(xì)胞株。隨后在用采用FLAG-IP技術(shù)釣取HeLa細(xì)胞株內(nèi)的結(jié)合蛋白,銀染后質(zhì)譜檢測HeLa細(xì)胞株內(nèi)SND1蛋白、HLA-A蛋白及GRP94蛋白的是否表達(dá),并用免疫共沉淀的方法加以驗證,從而分析研究SND1蛋白是否能夠作為支架蛋白對GRP94所介導(dǎo)的HLA-A蛋白的合成產(chǎn)生影響。研究方法:采用RT-PCR法從HeLa細(xì)胞中擴(kuò)增GRP94基因片段,連接到慢病毒載體p LVX-IRES-Puro中,獲得重組載體。瞬時轉(zhuǎn)染293T細(xì)胞,采用Western blotting法檢測GRP94蛋白表達(dá)量。p LVX-FLAG-GRP94重組質(zhì)粒通過與包裝質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,獲得重組慢病毒。以慢病毒感染HeLa細(xì)胞,篩選并鑒定穩(wěn)定表達(dá)GRP94蛋白的細(xì)胞株。利用穩(wěn)定表達(dá)FLAG-GRP94的HeLa細(xì)胞株,采用FLAG-IP法,銀染后利用質(zhì)譜篩選結(jié)合蛋白SND1、HLA-A、GRP94,并經(jīng)免疫共沉淀驗證。研究結(jié)果:(1)PCR法獲得了GRP94目的基因片段,將FLAG-GPR94與慢病毒p LVX-IRES-Puro載體雙酶切得到相應(yīng)的片段,并連接獲得重組質(zhì)粒。(2)重組慢病毒載體經(jīng)雙酶切和基因測序比對鑒定正確。(3)He La細(xì)胞經(jīng)慢病毒感染,藥物篩選后獲得的穩(wěn)定表達(dá)株中GRP94蛋白表達(dá)量高于野生型HeLa細(xì)胞(P0.01)。(4)利用穩(wěn)定表達(dá)FLAG-GRP94的HeLa細(xì)胞株,銀染后成功用質(zhì)譜篩選出GRP94蛋白、SND1蛋白、HLA-A蛋白,通過免疫共沉淀實驗GRP94 IP HLA-A、GRP94IP SND1,驗證GRP94、HLA-A、SND1三者之間相互結(jié)合情況,WB檢測。研究結(jié)論:本實驗成功構(gòu)建了人GRP94基因慢病毒載體,并通過研究首次發(fā)現(xiàn)并提出SND1作為一個銜接蛋白或支架蛋白存在于GRP94與HLA-A的復(fù)合物中,推測SND1能夠參與MHCⅠ類分子的加工、轉(zhuǎn)運、成熟及降解過程中的某個環(huán)節(jié),發(fā)揮著對MHC I類分子抗原呈遞的重要作用。
[Abstract]:Objective: To study the SND1, because the molecular weight of 100kDa, also known as P100 protein or Tudor-SN protein. The human SND1 was first discovered as a transcriptional co activator can activate EB virus nuclear antigen 2 (Epstein-Barr virus nuclear protein2) mediated gene expression of.SND1 protein is widely distributed in various species, tissues and cells in the bovine, rat, animal cells in mice can be combined with synthetic human SND1 antibody of SND1 [1].SND1 protein is highly conserved in different organisms, are involved in the regulation of cell in many important physiological processes of.SND1 protein has the ability to regulate gene transcription and splicing of pre-mRNA, play an important role in the occurrence and development of tumors. But so far, the SND1 protein is involved in tumorigenesis and development of specific mechanisms is unclear and needs further study. The human major histocompatibility Complex (MHC) is called the human leukocyte antigen (human leukocyte, antigen, HLA), the short arm of chromosome sixth. The gene is located on human chromosome HLA, encoding.HLA antigen with high polymorphism and its essence is a kind of glycoprotein, by an alpha heavy chain and a light chain of non covalent binding of beta and.HLA play an important role in human physiology, pathological mechanism, especially as presenting molecules of antigen processing and presentation in the immune response, the formation of MHC- peptide -TCR complex to activate the immune response, and the immune response plays a regulatory role of.HLA as a genetic marker of some diseases, the frequency of detection of HLA alleles,.HLA can be used as prenatal diagnosis of certain diseases also known transplantation antigens, the study on HLA in the field of organ transplantation can provide important theoretical basis for the transplantation. The anti tumor machine body The immune response mechanism, leading role in cellular immunity, humoral immunity and mutual regulation, coordination of killing tumor cells.HLA plays a key role in tumor immunity, to study the relationship between HLA and tumor specific antigen, contribute to the understanding of tumor immunity peptide, provide the basis for prevention and treatment of cancer. Glucose regulated protein 94 is heat shock protein 90 in the family of a member of [2], GRP94 protein is a highly conserved protein in the endoplasmic reticulum lumen, as molecular chaperones of endoplasmic reticulum in typical endoplasmic reticulum in protein folding, translocation, secretion and degradation of [3]. and GRP94 could be involved in endoplasmic reticulum stress, when the low sugar. Cell hypoxia, acidosis, stimulation, emergence of misfolded or unfolded proteins accumulate, can induce the unfolded protein response, resulting in increased expression of GRP94. There are reports in the literature, GRP94 can participate in immune Synthesis of MHC molecular response in the process of tumor cells mediated by the immunogenicity of [4]. will be the subject of the FLAG-GRP94 gene directed into pLVX-IRES-Puro lentiviral vector, to construct a lentiviral vector containing human GRP94 gene, further screening of stable expression of GRP94 protein in human cervical cancer cell line HeLa by using FLAG-IP technology. Then in the fishing binding protein HeLa cells, mass spectrometry after silver staining detection of SND1 protein in HeLa cells, HLA-A protein and GRP94 protein expression and immunoprecipitation verified, so as to analyze the influence of HLA-A protein synthesis of SND1 protein can be used as a scaffold protein of GRP94 is mediated by the method of amplification. The GRP94 gene fragment from HeLa cells by RT-PCR method and connected to the lentiviral vector of P LVX-IRES-Puro, the recombinant plasmid was transfected to 293T cells. Using Western blotting method. Detection of GRP94 protein expression of.P by recombinant plasmid LVX-FLAG-GRP94 and packaging plasmids were transfected into 293T cells to obtain recombinant lentivirus. The infection of HeLa cells with lentivirus, screening and identification of stable expression of GRP94 cells. HeLa cells FLAG-GRP94 expression by stable, using the method of FLAG-IP, after silver staining by mass spectrometry screening binding protein SND1 HLA-A, GRP94, and by immunoprecipitation test. Results: (1) PCR obtained by GRP94 gene fragments, FLAG-GPR94 and P LVX-IRES-Puro lentivirus vector digested the corresponding fragment, and connected with the recombinant plasmid. (2) the recombinant lentiviral vector by restriction enzyme digestion and gene sequencing identification right. (3) He La cells by lentivirus infection was higher than that of wild type HeLa cells expressing GRP94 protein drug screened stable expression strains (P0.01). (4) using HeLa cell line with stable expression of FLAG-GRP94 After silver staining, successfully screened by mass spectrometry and GRP94 protein, SND1 protein, HLA-A protein, GRP94 and IP by CO immunoprecipitation assay of HLA-A, GRP94IP SND1, GRP94 HLA-A, SND1 verification, combining three, WB detection. Conclusion: This study successfully constructed GRP94 gene lentiviral vector, and through study on the complex was first discovered and proposed SND1 as an adaptor protein or scaffold proteins exist in GRP94 and HLA-A, that SND1 can participate in the processing of MHC class I molecules transport, a link in the process of maturation and degradation, plays an important role in MHC class I molecules in antigen presentation.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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本文編號：1562456