【作者】 李凯；

【导师】 季明春；

【作者基本信息】 扬州大学 ， 免疫学， 2017， 硕士

【摘要】 Ⅰ型糖尿病(Type 1 diabetes,T1D)是一种自身免疫性疾病,体内的细胞毒性T淋巴淋巴细胞(cytotoxicTlymphocyte,CTL)识别并杀伤胰岛β细胞,诱发胰岛炎,造成体内由胰岛β细胞分泌的胰岛素水平降低,机体血糖指数升高,最终引起糖尿病的发生。正常机体生理情况下,体内针对胰岛β细胞的CTL本应被阴性选择后清除或者选择性沉默,但由于遗传变异、病毒感染等因素,出现异常增殖,并能在膜表面表达可与主要组织相容性复合物一类分子(Major histocompatibility complex,MHC-I)提呈的胰岛p细胞相关的抗原肽相结合的特异性T细胞抗原受体T细胞(抗原)受体T cell receptor,TCR,这些抗原肽包括葡萄糖-6-磷酸酶催化亚基相关蛋白(Glucose-6-phosphatase catalyzes acyl-related proteins,IGRP)、胰岛素(Ins)、谷氨酸脱羟酶(Glutamatedecarboxylase,GAD)等。成熟的效应T淋巴细胞通过全身血液循环选择性迁移趋化募集到胰岛中,分泌穿孔素、颗粒酶等多种细胞因子来损伤胰岛β细胞。锚合到MHC-Ⅰ类分子的抗原结合槽中的IGRP206-214九肽(VYLKYNVFC)可以通过与TCR的结合将抗原信息传递给特异性CTL,而可溶性的抗原肽-MHC-I类分子也能达到相同的效果。课题组前期通过引入单链三聚体(single chain trimer,SCT)以及二硫键捕获(disulfide trapping,dt)技术,设计了 IGRP206-214-H-2Kd-dtSCT 的编码基因并通过原核表达系统制备了相关蛋白,验证了蛋白活性。本课题改用真核系统来表达IGRP206-214-H-2Kd-dtSCT(Eukaryotic-IGRP206-214dtSCT,eIGRP206-214-dtSCT),研究真核表达系统是否对目的蛋白具有糖基化修饰以及初步验证糖基化修饰的蛋白是否具有相同的生物活性。目的:将IGRP206-214-H-2Kd-dtSCT的编码基因利用质粒重组到巴斯德酵母的染色体中,从而获得稳定的转录翻译;通过甲醇发酵诱导获得目的蛋白;确认巴斯德酵母对目的蛋白进行了糖基化修饰;制备IGRP206-214-H-2Kd-dtSCT-四聚体(IGRP-Tetramer)以及四聚体偶联毒素(IGRP-SAP),体外流式检测真核蛋白的生物学活性。方法:(1)取本实验室保存的DH5α-pet22b-IGRP20-214-H-2Kd-dtSCT菌株,设计上下游引物,PCR获得IGRP206-214-H-2Kd-dtSCT的编码基因,设计基因序列,以引物补足5’端信号肽α-因子、带入HRV 3C识别位点、Avi-Tag、6×his-tag以及双酶切位点,插入到pPICZαA质粒中,并通过中间宿主菌大肠杆菌DH5α得到稳定转录。小提质粒,并单酶切线性化后电转到巴斯德酵母GS115中,博来霉素抗性筛选阳性克隆,并筛选高表达菌株。甲醇诱导培养基培养重组GS115,诱导目的蛋白的分泌表达,48-72h后过滤、浓缩培养基上清,上蛋白纯化系统。纯化后的目的蛋白切除C端多余氨基酸片段,生物素化后,再次利用蛋白纯化系统去除游离的生物素以及HRV 3C protease。纯化的目的蛋白制备四聚体,验证蛋白生物素化效率。并偶联毒蛋白皂草素。(2)rIGRP206-214-H-2Kd-dtSCT-Tetramer(原核表达的 IGRP206-214 单链三聚体:Recombinant-IGRP206-214dtSCT,rIGRP206-214dtSCT)偶联 PE 制备用于特异性流式检测 IGRP抗原特异性 CTL 的荧光蛋白(PE-IGRP-dtSCT);Dot-blot、western blot 鉴定eIGRP206-214-H-2Kd-dtSCT蛋白的构象正确性;PNGase F切除真核系统表达的目的蛋白上的糖链,飞行时间质谱比对切除前后分子量变化,确认糖基化修饰的存在;可溶性eIGRP206-214-Tetramer联合IL-2添加到12周龄NOD雌鼠脾脏淋巴细胞培养液中共培养72h,流式检测IGRP抗原特异性CTL细胞频率变化;可溶性eIGRP206214-Tetramer偶联SAP添加到12周龄NOD雌鼠脾脏淋巴细胞培养液中共培养7天,流式检测IGRP抗原特异性CTL细胞频率变化。结果:(1)构建的重组质粒pPICZaA-IGRP206-214-H-2Kd-dtSCT成功导入GS115菌株中,使菌株获得Zeocin抗性,PCR验证与对照组双酶切的DH5a中的pPICZaA-IGRP206-214-H-2Kd-dtSCT有相同大小的条带,并且条带大小符合前期设计要求,基因测序结果说明构建的基因片段符合设计要求。甲醇诱导后培养基中与对照转化空质粒组相比51kDa处出现明显的条带。纯化后目的蛋白纯度达到95%以上。生物素化结果显示去除了大部分游离生物素,生物素化效率检测,以蛋白:Streptavidin质量比不同浓度的情况,目的蛋白依然完全形成多聚体,目的蛋白完全生物素化。(2)成功制备了 PE-IGRP-dtSCT,并且排除了荧光蛋白的非特异性;采用H-2Kd构象单抗,蛋白印迹结果显示真核系统表达的蛋白具有天然的正确空间构象;eIGRP206-214-dtSCT体外实验表明能够随着体系中蛋白浓度的提升,促进IGRP抗原肽特异性CTL的细胞频率增加,并且有显著差异;eIGRP206214-dtSCT偶联SAP后能够显著降低IGRP抗原肽特异性CTL的细胞频率,并且较rIGRP206-214-dtSCT偶联SAP具有更好的杀伤作用。结论:巴斯德酵母真核表达系统能够用来表达IGRP抗原肽-MHC Ⅰ抗原肽,表达的重组蛋白具有糖基化修饰以及正确的空间构象,能够与表面的TCR结合刺激细胞增殖,偶联毒素后促进IGRP抗原特异性CTL细胞频率下降。本课题为此后真核系统表达抗原肽-MHC Ⅰ类分子复合物提供了参考,并且有助于靶向毒蛋白的体内研究。更多还原

【Abstract】 Type 1 diabetes is a kind of autoimmune disease.Cytotoxic T lymphocyte identify and kill islet beta cells in vivo that cause insulitis and drive the level of insulin down,secreted by islet beta cells in vivo.This leads to the occurrence of diabetes along with the rise of glycemic index.Although CTL,which aimed at islet beta cells,should be removed by negative or selected silence,it exprss specific TCR on the membrane surface to identify antigen peptide about islet beta cells presented by MHC class I.The antigen peptide include IGRP、GAD、insulin.Mature effect T cells choose to move to islet by the whole body blood circulation system.It secrets perforin,granular enzyme and other cytokines to injure in pancreatic β-cell.IGRP206-214(VYLKYNVFC)anchor close to the antigen binding cleft of MHC class I which may give antigen information to specific CTL.Also,soluble antigen peptide-MHC class I molecules can achieve the same effect.Early,my project designed coding genes about the IGRP206-214-H-2Kd-dtSCT and prepared protein by prokaryotic expression system along with technology of single chain trimer disulfide trapping.This project use eukaryotic expression system to express protein for studying whether glycosylation modification of the target protein exist in eukaryotic expression system and making sure whether glycosylation modified protein has the same biological activity.[Objective]The coding gene of IGRP206-214-H-2Kd-dtSCT in recombinant plasmid is integrated into the chromosome of the pasteur yeast and gets a stable transcription.The target protein is induced in methanol medium,of which the galactosylated modification from pichia pastoris is confirmed.The tetramer is prepared with IGRP206-214-H-2Kd-dtSCT and linked with the saporin,of which biological activity is detected by FACS.[Methods](1)For getting encoding genes of the targting protein from DH5a plasmid,we designed Upstream and downstream primers.The design process including addition of HRV 3C-Tag、6×his-tag、Avi-tag、alpha factor of signal peptide and double enzyme locis,is inserted into the pPICZaA plasmid.This encoding genes gets a stable transcription in DH5α escherichia coli.The plasmid is extracted and transformed into saccharomyces pastorianus of GS115 by electroporation after single enzyme linearization.The positive clones are screened by a resistanta of ampicillin,which high expression of strain be screeninged.Target protein is innduced in methanol medium.Culture supernatant is filtered and enriched after 48-72h.The excess c-terminal amino acid fragment is removed from the purified target protein,of which the free fraction part is removed by Bio-Rad purifier.The target protein is grouped into tetramer and linked with saporin.The efficiency of biotin protein is verified.(2)rIGRP206-214-H-2Kd-dtSCT-Tetramer linked with PE is prepared as fluorescent protein.It is used in FACS.Conformation correctness of eIGRP206-214-H-2Kd-dtSCT is confirmed by Dot-blot and western-blot.The change of molecular weight about the protein digested by PNGase enzyme,is confirmed by ESI-TOF-MS for Confirming the presence of glycosylation modification.Splenic lymphocytes of 12W female NOD are trained in culture added with soluble SAP-eIGRP206-214-Tetramer after 7 days.The frequency of specific CTL is tested by FACS.[Results](1)The recombinant plasmid constructed with IGRP206-214-H-2Kd-dtSCT is insected in GS115 train to which gives a ampicillin resistant.The same size bands of PCR consequence is observed in double Digests consequence of recombinant plasmid,of which molecular weight is confirmed to the preliminary design.Gene sequencing results are consistent.Obvious stripe of 51kDa is confirmed in methanol induction medium compared with the control group.The fineness of the purified target protein is above 95%.The most free biotin is removed by Bio-rad purification system.Multimerization of the target protein is formed in1:1 mass ratio about the target protein to streptavidin.(2)The nonspecific of fluorescent protein is shut off after the pereparetion of PE-IGRP-dtSCT.The correct space conformation of protein expressed in eukaryotic system is confirmed by the consequence of dot-blot and western blot.The protein of eIGRP206-214-dtSCT promotes the increasing of cell frequency as the concentration of protein in system in vitro and the results have significant differences.The protein of sap-eIGRP206-214-dtSCT reduces cell frequency of the IGRP antigen peptide specific CTL significantly which has better killing effect compared with the protein of sap-rIGRP206-214-dtSCT.[Conclusion]The recombinant proteins of IGRP antigen peptide with MHC class I expressed in pasteur yeast eukaryotic system.The expressed recombinant proteins has the glycosylation modification as well as the correct space conformation which can stimulate cell proliferation combined to TCR in cell membrane.It can reduce the cell frequency of IGRP antigen peptide specific CTL coupled with saporin.This topic provides reference for the recombinant proteins of antigen peptide-MHC class I expressed in eukaryotic expression system and helps studies about targeting toxic proteins in vivo.更多还原