人GRP94基因慢病毒载体构建及其作用蛋白的筛选

发布时间：2018-03-03 19:53

本文选题：GRP94　切入点：SND1　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:SND1,因为分子量为100kDa,所以又称为p100蛋白或Tudor-SN蛋白。人类SND1最初被发现是作为转录共激活因子能够激活EB病毒细胞核抗原2(Epstein-Barr virus nuclear protein2)所介导的基因表达。SND1蛋白在各物种组织和细胞中分布广泛,在牛、大鼠、小鼠等动物细胞内存在能与人工合成的人SND1抗体相结合的SND1蛋白[1]。SND1在不同生物中具有高度的保守性,参与调控细胞的多种重要生理过程。SND1蛋白具有调节基因转录和剪接加工pre-mRNA的能力,在多种肿瘤的发生发展中起重要作用。但到目前为止,SND1蛋白参与上述肿瘤的发生、发展的具体机制还不清楚,有待于进一步研究。人类主要组织相容性复合体(MHC)被称为人类白细胞抗原(human leukocyte antigen,HLA),该基因位于人染色体的第6号染色体短臂上,编码具有高度多态性的HLA抗原。HLA抗原其本质为一类糖蛋白,由一条α重链和一条β轻链非共价结合而成。HLA在人体生理、病理机制中发挥重要作用,特别是在免疫应答中能够作为递呈分子对抗原进行加工和呈递,形成MHC-抗原肽-TCR复合物启动免疫应答,并对免疫应答起到调节作用。HLA作为某些疾病的遗传标志,对通过对HLA等位基因出现频率的检测,可以作为某些疾病的产前诊断。HLA又被称移植抗原,在器官移植领域开展的针对HLA的研究为移植配型提供可重要的理论依据。机体抗肿瘤的免疫反应机制中,以细胞免疫为主导作用,与体液免疫相互调节,协同杀伤肿瘤细胞。HLA在肿瘤免疫中起到关键作用,研究HLA与肿瘤的关系,有助于了解肿瘤免疫效应中特异性抗原肽,为肿瘤的防治提供依据。葡萄糖调节蛋白94是热休克蛋白90家族中的一员[2],GRP94蛋白本身是一种序列高度保守的蛋白,存在于内质网腔中,作为内质网典型的分子伴侣参与内质网内蛋白的折叠、转运、分泌及降解[3]。同时GRP94能够参与内质网应激,当细胞内出现低糖、缺氧、酸中毒等刺激时,出现错误折叠或未折叠的蛋白质大量聚集,会诱发非折叠蛋白反应,从而导致GRP94表达升高。有文献报道,GRP94能够参与免疫应答过程中MHC类分子的合成,介导肿瘤细胞免疫原性的产生[4]。本课题将FLAG-GRP94目的基因定向导入pLVX-IRES-Puro慢病毒载体,构建人GRP94基因慢病毒载体,进一步筛选稳定表达GRP94蛋白的人宫颈癌HeLa细胞株。随后在用采用FLAG-IP技术钓取HeLa细胞株内的结合蛋白,银染后质谱检测HeLa细胞株内SND1蛋白、HLA-A蛋白及GRP94蛋白的是否表达,并用免疫共沉淀的方法加以验证,从而分析研究SND1蛋白是否能够作为支架蛋白对GRP94所介导的HLA-A蛋白的合成产生影响。研究方法:采用RT-PCR法从HeLa细胞中扩增GRP94基因片段,连接到慢病毒载体p LVX-IRES-Puro中,获得重组载体。瞬时转染293T细胞,采用Western blotting法检测GRP94蛋白表达量。p LVX-FLAG-GRP94重组质粒通过与包装质粒共转染293T细胞,获得重组慢病毒。以慢病毒感染HeLa细胞,筛选并鉴定稳定表达GRP94蛋白的细胞株。利用稳定表达FLAG-GRP94的HeLa细胞株,采用FLAG-IP法,银染后利用质谱筛选结合蛋白SND1、HLA-A、GRP94,并经免疫共沉淀验证。研究结果:(1)PCR法获得了GRP94目的基因片段,将FLAG-GPR94与慢病毒p LVX-IRES-Puro载体双酶切得到相应的片段,并连接获得重组质粒。(2)重组慢病毒载体经双酶切和基因测序比对鉴定正确。(3)He La细胞经慢病毒感染,药物筛选后获得的稳定表达株中GRP94蛋白表达量高于野生型HeLa细胞(P0.01)。(4)利用稳定表达FLAG-GRP94的HeLa细胞株,银染后成功用质谱筛选出GRP94蛋白、SND1蛋白、HLA-A蛋白,通过免疫共沉淀实验GRP94 IP HLA-A、GRP94IP SND1,验证GRP94、HLA-A、SND1三者之间相互结合情况,WB检测。研究结论:本实验成功构建了人GRP94基因慢病毒载体,并通过研究首次发现并提出SND1作为一个衔接蛋白或支架蛋白存在于GRP94与HLA-A的复合物中,推测SND1能够参与MHCⅠ类分子的加工、转运、成熟及降解过程中的某个环节,发挥着对MHC I类分子抗原呈递的重要作用。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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