Ub-HBcAg-CTP融合蛋白诱导特异性细胞毒性T淋巴细胞反应的实验研究

发布时间：2018-08-25 10:15

【摘要】：慢性乙型肝炎仍是严重危害人类健康的疾病,现今全球大约有3.5亿人感染乙肝病毒(hepatitis B virus,HBV)。目前采取的治疗方法,均不能彻底清除病毒而使病毒持续存在于肝细胞内,造成感染慢性化,并可继发肝硬化、肝细胞癌。机体清除HBV感染起关键作用的是HBV特异性细胞毒T淋巴细胞(cytotoxic T lymphocyte,CTL)。泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)是一种高度选择性的蛋白降解系统,它广泛存在于真核细胞内,并依靠ATP酶而起作用,对维持细胞乃至整个生物体的正常生理功能具有重要的作用。泛素化的抗原蛋白被蛋白酶体复合物识别后被降解为若干小肽段,与主要组织相容性复合体-I(major histocompatibility complex,MHC)类分子结合后被抗原提呈细胞识别,诱导特异性CTL反应。目前有许多研究表明,泛素化的抗原进入细胞后可被UPS系统有效降解及提呈,从而增强抗原诱导的免疫应答。正常情况下,由于膜屏障的存在,蛋白、多肽等很难进入细胞内,某些蛋白具有很强的不依赖于受体的跨膜活性,这类短肽被称为细胞穿透肽(cell penetrating peptide,CPP)。目前应用最为广泛的细胞穿透肽是HIV-Tat(human immunodeficiency virus transactivator oftranscription,HIV-Tat)蛋白。PTD(protein transduction domain)是Tat蛋白行使跨膜功能的核心片段,包含该区段的蛋白具有穿透细胞膜的功能。胞浆转导肽(cytoplasmic transduction peptide,CTP)为PTD的衍生体,是一种新型的转导肽系统,它去除了核定位信号而能携带蛋白类大分子穿越细胞膜并专一性定位于胞浆中的。本研究通过构建Ub-HBc Ag-CTP融合基因表达质粒,并进行蛋白的表达及纯化,利用Ub-HBc Ag-CTP融合蛋白体外刺激树突状细胞(dendritic cell,DC)成熟并诱导特异性CTL,体内免疫BALB/c小鼠,对Ub-HBc Ag-CTP融合蛋白诱导特异性CTL的免疫功能进行了探讨。实验共分三部分:(1)Ub-HBcAg-CTP融合蛋白的表达、纯化及其体外诱导小鼠髓源性树突状细胞成熟的研究;(2)Ub-HBc Ag-CTP融合蛋白促进T淋巴细胞增殖,诱导特异性ctl反应的体外研究;(3)ub-hbcag-ctp融合蛋白免疫balb/c小鼠诱导特异性ctl反应的体内研究。第一部分Ub-HBcAg-CTP融合蛋白的表达、纯化及其体外诱导小鼠髓源性树突状细胞成熟的研究目的:构建Ub-HBcAg-CTP融合基因的原核表达载体,并进行蛋白的表达及其纯化,体外观察其对诱导小鼠髓源性树突状细胞成熟的作用。方法:以质粒pcDNA3.1(-)-Ub-HBcAg中的Ub-HBc Ag融合基因为模板设计引物,上游引物带有CTP基因序列,进行PCR扩增,PCR产物克隆到原核表达质粒pMAL-c2X中,菌落PCR阳性克隆测序,鉴定正确的质粒转化大肠杆菌BL21(DE3),并进行蛋白纯化及Western blotting鉴定。同时构建并表达对照组蛋白HBcAg-CTP及Ub-HBcAg。体外分离培养近交系BALB/c小鼠髓源性树突状细胞,加重组白细胞介素-4(interleukin,IL-4)及粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)。树突状细胞培养至第5天时加入Ub-HBc Ag-CTP、HBc Ag-CTP、Ub-HBcAg及HBc Ag诱导DC成熟。激光共聚焦显微镜观察免疫荧光在细胞中的分布及定位,并对荧光强度进行定量分析,流式细胞术检测树突状细胞表面分子的表达,并以酶联免疫吸附法(ELISA)检测树突状细胞的上清液中细胞因子IL-12p70的含量。结果:PCR扩增分别得到820bp、590bp及780bp大小的条带,分别为Ub-HBcAg-CTP、HBc Ag-CTP及Ub-HBcAg融合基因。将其克隆至表达质粒pMAL-c2X中,阳性克隆菌落测序正确,并在DE3中获得诱导表达。Western blotting鉴定分别为Ub-HBcAg-CTP、HBc Ag-CTP及Ub-HBc Ag融合蛋白。体外成功诱导培养并鉴定小鼠骨髓源性DC,免疫荧光法证实Ub-HBcAg-CTP能够穿透DC膜进入细胞质,而不能进入细胞核;Ub-HBcAg-CTP能明显上调DC表面分子CD80、CD83、CD86及MHC-I类分子的表达;Ub-HBc Ag-CTP组诱导DC分泌的IL-12p70水平明显高于对照组。结论:成功构建了Ub-HBcAg-CTP及对照融合表达质粒并诱导表达,Ub-HBcAg-CTP具有穿透树突状细胞膜并定位于胞浆的能力,明显增强DC表面共刺激分子的表达及促进DC的成熟及分化,明显增强DC分泌IL-12p70等细胞因子的能力。第二部分Ub-HBc Ag-CTP融合蛋白促进T淋巴细胞增殖,诱导特异性CTL反应的体外研究目的:探讨经Ub-HBc Ag-CTP融合蛋白致敏的DC体外增强T淋巴细胞增殖能力及诱导特异性细胞毒T淋巴细胞(CTLs)的作用。方法:体外分离培养小鼠髓源性DC,不同组融合蛋白加入树突状细胞中诱导其成熟及分化后,与分离培养的小鼠T淋巴细胞共同培养,ELISA法检测T细胞上清液中干扰素(interferon,IFN)-γ、IL-2、IL-4及IL-10的分泌水平,流式细胞仪检测胞内细胞因子水平,细胞计数试剂盒-8(CCK-8)试剂盒检测T淋巴细胞增殖反应,乳酸脱氢酶(LDH)释放试验检测特异性CTL活性。结果:Ub-HBc Ag-CTP融合蛋白可以明显上调细胞因子IFN-γ和IL-2的水平;流式细胞仪检测的由Ub-HBc Ag-CTP融合蛋白所诱导的CTL水平明显高于对照组及空白组;Ub-HBc Ag-CTP诱导树突状细胞刺激T细胞增殖能力明显高于对照组;Ub-HBc Ag-CTP融合蛋白组诱导的CTL与对照组相比,具有明显的特异性杀伤作用。结论:经Ub-HBc Ag-CTP融合蛋白诱导成熟的DC能明显刺激Th1型细胞因子的分泌,增强T淋巴细胞增殖能力,明显增加CTLs的表达并增强特异性CTL活性。第三部分Ub-HBc Ag-CTP融合蛋白免疫BALB/c小鼠诱导特异性CTL反应的体内研究目的:探讨Ub-HBc Ag-CTP融合蛋白在BALB/c小鼠体内有效诱导HBV特异性CTL反应,并初步探讨Ub-HBc Ag-CTP融合蛋白诱导CTL的机制,研究Ub-HBc Ag-CTP融合蛋白诱导CTL与JAK/STAT信号通路的关系。方法:BALB/c小鼠随机分为实验组Ub-HBc Ag-CTP(50μg),对照组HBc Ag-CTP(50μg)、Ub-HBc Ag(50μg)、HBc Ag(50μg)及空白组(100μl生理盐水),经肌肉免疫小鼠,每周一次,共3次。最后一次免疫后1周,收集小鼠T淋巴细胞,ELISA法检测T淋巴细胞分泌细胞因子;流式细胞仪检测T淋巴细胞内的细胞因子;酶联免疫斑点(ELISPOT)法检测特异性T淋巴细胞;CCK-8试剂盒检测T淋巴细胞增殖活性;LDH释放试验检测特异性CTL活性;Western blotting法检测小鼠T淋巴细胞JAK/STAT信号通路中各信号分子的表达水平。结果:Ub-HBc Ag-CTP融合蛋白能有效刺激小鼠T淋巴细胞分泌Th1型细胞因子;流式细胞仪及ELISPOT法检测该融合蛋白诱导的CTL水平明显高于其他组;且该融合蛋白诱导的T淋巴细胞增殖活性和CTL活性明显高于对照组及空白组;Ub-HBc Ag-CTP融合蛋白可以明显上调T淋巴细胞内Jak2,Tyk2,STAT1及STAT4的表达水平,而对Jak1,Jak3及STAT6的表达无统计学意义。结论:Ub-HBc Ag-CTP融合蛋白免疫BALB/c小鼠后,能有效刺激T淋巴细胞分泌Th1型细胞因子及增加CTLs的表达,并能提高T淋巴细胞增殖活性及CTL活性,以上免疫效应可能与JAK/STAT信号通路的活化有关。
[Abstract]:Chronic hepatitis B is still a serious disease endangering human health. There are about 350 million people infected with hepatitis B virus (HBV) in the world today. Current treatment methods can not completely eliminate the virus and make it persist in the liver cells, causing chronic infection, and secondary cirrhosis, hepatocellular carcinoma. Infection plays a key role in HBV-specific cytotoxic T lymphocyte (CTL). Ubiquitin-proteasome system (UPS) is a highly selective protein degradation system, which exists widely in eukaryotic cells, and depends on ATPase to maintain the integrity of cells and even the whole organism. Ubiquitinized antigen proteins are recognized by proteasome complexes and degraded into small peptides. They are recognized by antigen presenting cells after binding to major histocompatibility complex (MHC) molecules and induce specific CTL reactions. Normally, because of the existence of membrane barrier, proteins, peptides, etc. are difficult to enter the cell. Some proteins have strong transmembrane activity independent of receptors. These short peptides are called cell penetrating peptides. The most widely used cell penetrating peptide is HIV-Tat protein. PTD (protein transduction domain) is the core fragment of the transmembrane function of Tat protein. The protein containing this fragment has the function of penetrating cell membrane. Smc transduction peptide (CTP), a derivative of PTD, is a new type of peptide transduction system. It removes nuclear localization signal and carries protein macromolecules across cell membrane and specifically locates in cytoplasm. In this study, we constructed Ub-HBc Ag-CTP fusion gene expression plasmid, expressed and purified the protein, using Ub-HBc Ag-C. TP fusion protein stimulated dendritic cell (DC) maturation in vitro and induced specific CTL. BALB / c mice were immunized in vivo. The immune function of specific CTL induced by Ub-HBcAg-CTP fusion protein was investigated. The experiment was divided into three parts: (1) Expression, purification and induction of myeloid dendritic cells in vitro by Ub-HBcAg-CTP fusion protein. Studies on cell maturation; (2) Ub-HBcAg-CTP fusion protein promotes T lymphocyte proliferation and induces specific CTL reaction in vitro; (3) in vivo study on the induction of specific CTL reaction in balb/c mice immunized with ub-hbcag-ctp fusion protein. Part I Expression, purification and in vitro induction of mouse myeloid dendritic cells by Ub-HBcAg-CTP fusion protein Objective: To construct the prokaryotic expression vector of Ub-HBcAg-CTP fusion gene, express and purify the protein, and observe its effect on inducing maturation of murine myeloid dendritic cells in vitro. Colony PCR positive cloning and sequencing were carried out to identify the correct plasmid transformed into Escherichia coli BL21 (DE3) and to purify the protein and identify it by Western blotting. The control group proteins HBcAg-CTP and Ub-HBcAg were constructed and expressed in vitro. Dendritic cells, interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in aggravating group. DC maturation was induced by adding Ub-HBc Ag-CTP, HBc Ag-CTP, Ub-HBcAg and HBc Ag on the 5th day of culture. The distribution and localization of light in the cells were analyzed quantitatively. The expression of surface molecules of dendritic cells was detected by flow cytometry. The content of cytokine IL-12p70 in the supernatant of dendritic cells was detected by enzyme-linked immunosorbent assay (ELISA). Results: A band of 820 bp, 590 BP and 780 BP was amplified by PCR, respectively. Ub-HBcAg-CTP, HBc Ag-CTP and Ub-HBcAg fusion genes were cloned into the expression plasmid pMAL-c2X. The positive clones were sequenced correctly and expressed in DE3. The fusion proteins of Ub-HBcAg-CTP, HBc Ag-CTP and Ub-HBc Ag were identified by Western blotting. It was confirmed that Ub-HBcAg-CTP could penetrate DC membrane into cytoplasm but not into nucleus; Ub-HBcAg-CTP could significantly up-regulate the expression of CD80, CD83, CD86 and MHC-I molecules on DC surface; and the level of IL-12p70 secreted by Ub-HBcAg-CTP group was significantly higher than that of control group. Ub-HBcAg-CTP has the ability to penetrate the dendritic cell membrane and localize in the cytoplasm. It can significantly enhance the expression of costimulatory molecules on the surface of DC, promote the maturation and differentiation of DC, and enhance the ability of DC to secrete IL-12p70 and other cytokines. Part II Ub-HBcAg-CTP fusion protein promotes the proliferation of T lymphocytes and induces specific CTL reaction. Objective: To investigate the effect of DC sensitized by Ub-HBc Ag-CTP fusion protein on enhancing T lymphocyte proliferation and inducing specific cytotoxic T lymphocyte (CTLs) in vitro. Methods: Mice myelogenous DC was isolated and cultured in vitro. Different groups of fusion proteins were added to dendritic cells to induce its maturation and differentiation, then T lymphocyte was isolated and cultured. The secretion levels of interferon-gamma, IL-2, IL-4 and IL-10 in the supernatant of T cells were detected by ELISA, cytokine levels were detected by flow cytometry, T lymphocyte proliferation was detected by CCK-8 kit, and specific CTL activity was detected by lactate dehydrogenase (LDH) release assay. B-HBc Ag-CTP fusion protein can significantly up-regulate the levels of cytokines IFN-gamma and IL-2; flow cytometry showed that the level of CTL induced by Ub-HBc Ag-CTP fusion protein was significantly higher than that of control group and blank group; Ub-HBc Ag-CTP induced dendritic cells proliferation was significantly higher than that of control group; Ub-HBc Ag-CTP induced dendritic cells proliferation was significantly higher than that of Ub-HBc Ag-CTP fusion protein group; Conclusion: Mature DC induced by Ub-HBc Ag-CTP fusion protein can significantly stimulate the secretion of Th1 cytokines, enhance the proliferation of T lymphocytes, significantly increase the expression of CTLs and enhance the specific CTL activity. Part III Ub-HBc Ag-CTP fusion protein immunized BALB/c mice. Objective: To investigate the effect of Ub-HBc Ag-CTP fusion protein on inducing specific CTL reaction in BALB/c mice and the mechanism of inducing CTL by Ub-HBc Ag-CTP fusion protein. Methods: BALB/c mice were randomly divided into two groups. Ub-HBc Ag-CTP (50 ug), HBc Ag-CTP (50 ug), Ub-HBc Ag (50 ug), HBc Ag (50 ug), HBc Ag (50 ug) and blank group (100 UG normal saline) were immunized to mice once a week for three times. Cytokines; Enzyme-linked immunodot assay (ELISPOT) to detect specific T lymphocytes; CCK-8 kit to detect T lymphocyte proliferation activity; LDH release assay to detect specific CTL activity; Western blotting to detect the expression of signal molecules in mouse T lymphocyte JAK/STAT signaling pathway. Results: Ub-HBc Ag-CTP fusion protein was effective. The CTL level induced by the fusion protein was significantly higher than that of other groups, and the proliferation and CTL activity of T lymphocytes induced by the fusion protein were significantly higher than those of control group and blank group; Ub-HBc Ag-CTP fusion protein could significantly up-regulate Jak in T lymphocytes. Conclusion: Ub-HBc Ag-CTP fusion protein can effectively stimulate T lymphocytes to secrete Th1 cytokines and increase the expression of CTLs, and can increase the proliferation and CTL activity of T lymphocytes, which may be related to JAK/ST. Activation of AT signaling pathway is involved.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R512.62

【相似文献】