-
外体(exosomes)是由细胞产生的直径在30~100 nm的小囊泡状结构[1]。外体表面和内部可以携带多种物质,在细胞与细胞间以及各种蛋白质、脂类和核酸的细胞间扮演运输工的角色[2]。目前,普遍用于标记外体结构的蛋白标记物包括CD63、CD81 和 TSG101等[2-3]。几乎所有的细胞均可分泌外体并且广泛存在于各类体液中[4],随体循环在体内流动作用于邻近的或者远距离的受体细胞。研究发现包括活化的B淋巴细胞、树突状细胞以及巨噬细胞在内的抗原呈递细胞所分泌的外体都具有活化和调节免疫应答的功能[5]。这些抗原呈递细胞分泌的外体携带 MHC-I、MHC-II 和CD86 等诱导性共刺激分子(ICOS),并具有激活T细胞增殖的能力[6]。据此,有望利用抗原呈递细胞分泌的外体作为多肽抗原的天然载体,制备成外体疫苗,激活机体产生针对该多肽的特异性免疫反应。
疫苗接种是预防与控制家禽疫病的重要手段。禽冠状病毒—鸡传染性支气管炎病毒(Infectious bronchins virus, IBV) ,是全球鸡呼吸道疾病的主要病原体之一,给养鸡业带来严重的经济损失[7-9]。由于IBV血清型复杂多样、抗体交叉保护力低,所以弱毒疫苗的使用无法有效控制感染并加剧病毒变异[10-11]。在防控此类传染性疾病时,动物群体免疫亚单位疫苗等非复制性、无致病力的疫苗能够在一定程度上提高疫苗接种的安全性[12],但是亚单位疫苗免疫原性差,难以激活细胞免疫,因此不适用于初次免疫,其免疫效果还受免疫佐剂、免疫次数的明显影响[13]。如果利用抗原呈递细胞源外体激活细胞免疫的优势,在鸡APC细胞的外体中装载IBV病毒亚单位抗原,则可能有效提高多肽抗原的免疫原性。有研究报道了LPS刺激鸡巨噬细胞系HD11细胞分泌的外体可作为佐剂增强细胞免疫反应[14]。此外,由于树突状细胞难以在体外稳定传代,而鸡巨噬细胞已有可稳定传代的细胞系,并且B淋巴细胞的体外活化复杂于巨噬细胞。利用巨噬细胞的永生化细胞系开展外体疫苗的研发可能具有更好的应用性。
基于上述背景,本研究以鸡巨噬细胞系HD11源外体作为研究对象,以IBV病毒中可诱导机体产生中和抗体的S1蛋白为模式抗原,构建装载抗原的重组外体,并进一步对外体中S1蛋白的表达进行鉴定,以明确是否成功构建装载抗原的重组外体。携带抗原的重组HD11源外体将作为重要的生物学材料,用于后续巨噬细胞源外体载体疫苗研发以及相应免疫应答机制的研究。
Construction of exosomes of recombinant HD 11 cells harboring the IBV S1 protein
-
摘要: 为了给禽类巨噬细胞源外体在免疫应答的功能研究和家禽外体载体疫苗的研发奠定基础,本研究以鸡传染性支气管炎病毒(Infectious bronchins virus, IBV)S1蛋白作为模式抗原,通过慢病毒表达系统技术实现在HD11细胞中稳定表达S1蛋白,再利用超速离心从细胞培养物上清中分离纯化得到外体。蛋白免疫印分析、电镜与粒径分析检测等结果表明,在该重组HD11细胞分泌的外体中可检测到稳定的S1蛋白表达,显示成功构建了装载IBV S1蛋白的重组外体。Abstract: The exosomes of antigen presenting cells (Antigen presenting cell, APC) participate in the immune response process, and can activate the adaptive immune response after carrying the antigen. As a kind of antigen-presenting cells, the related functions of poultry macrophage-derived exosomes are still unknown. The S1 protein of chicken infectious bronchitis virus (IBV) was used as the model antigen, the S1 protein was stably expressed in HD11 cells through the lentiviral expression system, and then the exosomes were separated and purified from the cell culture supernatant by ultracentrifugation. The protein immunoblot analysis, electron microscopy and particle size analysis detection showed that stable S1 protein expression can be detected in the exosomes secreted by the recombinant HD11 cells. Recombinant exosomes harboring IBV S1 protein were successfully constructed, which can provide a reference for the functional study of avian macrophage-derived exosomes in immune response and the development of poultry exosome vector vaccines.
-
Key words:
- exosome /
- HD11 cell /
- IBV /
- S1 protein
-
-
[1] ZABOROWSKI M P, BALAJ L, BREAKEFIELD X O, et al. Extracellular vesicles: composition, biological relevance, and methods of study [J]. Bioscience, 2015, 65(8): 783 − 797. doi: 10.1093/biosci/biv084 [2] RAPOSO G, STOORVOGEL W. Extracellular vesicles: exosomes, microvesicles, and friends [J]. The Journal of Cell Biology, 2013, 200(4): 373 − 83. doi: 10.1083/jcb.201211138 [3] COLOMBO M, RAPOSO G, THÉRY C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles [J]. Annual Review of Cell and Developmental Biology, 2014, 30: 255 − 89. doi: 10.1146/annurev-cellbio-101512-122326 [4] BORGES FT, REIS LA, SCHOR N. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases [J]. Braz J Med Biol Res, 2013, 46(10): 824 − 830. doi: 10.1590/1414-431X20132964 [5] BOBRIE A, COLOMBO M, RAPOSO G, et al. Exosome secretion: molecular mechanisms and roles in immune responses [J]. Traffic (Copenhagen, Denmark), 2011, 12(12): 1659 − 1668. doi: 10.1111/j.1600-0854.2011.01225.x [6] RAMACHANDRA L, QU Y, WANG Y, et al. Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation [J]. Infection and Immunity, 2010, 78(12): 5116 − 5125. doi: 10.1128/IAI.01089-09 [7] FERNANDO F S, DA SILVA MONTASSIER MDF, SILVA K R, et al. Nephritis associated with a S1 variant brazilian isolate of infectious bronchitis virus and vaccine protection test in experimentally infected chickens [J]. International Journal of Poultry Science, 2013, 12(11): 639. doi: 10.3923/ijps.2013.639.646 [8] NII T, ISOBE N, YOSHIMURA Y. Effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct [J]. Theriogenology, 2014, 81(8): 1129 − 1138. doi: 10.1016/j.theriogenology.2014.02.002 [9] YU L, JIANG Y, LOW S, et al. Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens [J]. Avian Diseases, 2001, Apr,45(2): 416 − 424. [10] FARSANG A, ROS C, RENSTRöM L H, et al. Molecular epizootiology of infectious bronchitis virus in Sweden indicating the involvement of a vaccine strain [J]. Avian Pathology, 2002, 31(3): 229 − 236. doi: 10.1080/03079450220136530 [11] MCKINLEY E T, HILT D A, JACKWOOD M W. Avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination [J]. Vaccine, 2008, 26(10): 1274 − 1284. doi: 10.1016/j.vaccine.2008.01.006 [12] CAVANAGH D. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus [J]. Avian Pathology, 2003, 32(6): 567 − 582. doi: 10.1080/03079450310001621198 [13] CAVANAGH D, CASAIS R, ARMESTO M, et al. Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins [J]. Vaccine, 2007, 25(30): 5558 − 5562. doi: 10.1016/j.vaccine.2007.02.046 [14] HONG Y, LEE J, VU T H, et al. Exosomes of lipopolysaccharide-stimulated chicken macrophages modulate immune response through the MyD88/NF-κB signaling pathway [J]. Developmental and Comparative Immunology, 2021, Feb,115: 103908. [15] THÉRY C, AMIGORENA S, RAPOSO G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids[J]. Current Protocols in Cell Biology, 2006 Apr, chapter 3: unit 3.22. [16] LINDENBERGH M F S, STOORVOGEL W. Antigen presentation by extracellular vesicles from professional antigen-presenting cells [J]. Annual Review of Immunology, 2018, Apr,36: 435 − 459. [17] WANG G, WANG Z, ZHUANG P, et al. Exosomes carring gag/env of ALV-J possess negative effect on immunocytes [J]. Microbial Pathogenesis, 2017, Nov,112: 142 − 147. [18] SU Q, ZHANG Y, CUI Z, et al. Semen-derived exosomes mediate immune escape and transmission of reticuloendotheliosis virus [J]. Frontiers in Immunology, 2021, 12: 735280. doi: 10.3389/fimmu.2021.735280 [19] BANSAL S, PERINCHERI S, FLEMING T, et al. Cutting edge: circulating exosomes with COVID spike protein are induced by BNT162B2 (Pfizer-biontech) vaccination prior to development of antibodies: a novel mechanism for immune activation by mrna vaccines [J]. Immunol, 2021, 207(10): 2405 − 2410. doi: 10.4049/jimmunol.2100637 [20] QURESHI N, VOGEL S, WAY C, et al. The proteasome [J]. Immunologic research, 2007, 31(3): 243 − 260. [21] 宋延华, 刘福安. 鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探[J]. 中国病毒学., 1999, 14(3): 72 − 75. [22] 戴亚斌, 陈德胜, 丁铲, 等. 表达鸡传染性支气管炎病毒JS/95/03株S1蛋白的重组杆状病毒构建[J]. 病毒学报., 2003, 19(1): 86 − 90. [23] 廖明, 辛朝安, 王林川. 鸡传染性支气管炎病毒S1基因在昆虫细胞中的表达[J]. 中国兽医学报., 1997, 17(6): 22 − 26. [24] 李中华, 肖运才, 胡思顺, 等. 传染性支气管炎病毒S1蛋白基因的克隆与原核表达[J]. 中国兽医杂志., 2015, 51(4): 35 − 38.