【作者】 张婷婷；

【导师】 张智英；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2013， 博士

【摘要】 树突状细胞（DCs）是机体免疫细胞中功能最强的抗原递呈细胞，存在于身体各个部位，参与免疫反应的启动和调节过程，在机体免疫监测中发挥着重要作用。酿酒酵母是一种安全、可食用的微生物，因其独特的细胞壁结构，能够特异性地被肠道免疫细胞识别，且在进入免疫细胞后，能够刺激机体产生免疫反应。鉴于酵母被免疫细胞识别这一特性，我们关注于酵母对肠道CD11c⁺DCs的影响机理；并尝试将酵母携带功能基因靶向肠道免疫细胞，通过重组酵母携带的功能基因在免疫细胞中的表达而实现对免疫细胞功能的调控；最后探讨重组酵母携带外源蛋白刺激机体产生抗体的可能性及其应用。本研究首次在活体水平研究口服酵母对肠道CD11c⁺DCs影响的分子机理。通过将酵母口服饲喂小鼠，分离肠道CD11c⁺DCs并进行转录组测序分析后发现，重组酵母进入CD11c⁺DCs后，对CD11c⁺DCs的基因表达进行重编程；其中上调表达基因为1558个，下调表达基因为754个。通过KEGG以及GO分析，发现口服酵母能影响CD11c⁺DCs细胞膜的受体途径，改变细胞膜成分组成；通过信号通路途径影响基因表达；能够影响免疫系统相关基因表达，促进CD4⁺T和CD8⁺T的分化以及Th17细胞的分化，从而调控CD11c⁺DCs的免疫功能。本研究证明通过口服重组酵母能有效介导功能基因在CD11c⁺DCs中表达并促进CD11c⁺DCs成熟。β-catenin基因是Wnt信号通路中一个关键基因，对DCs功能起着重要的调节作用。通过饲喂含有β-catenin（JMY-Pcat）或β-catenin-GFP（JMY-Pcat-GFP）真核表达载体的酵母，均可在肠道CD11c⁺DCs中检测到β-catenin的表达；JMY-Pcat组中CD11c⁺DCs表面共刺激分子CD80、CD83、CD86表达升高，MHCⅡ表达降低；通过免疫组化试验发现在肠道、淋巴结、胸腺和脾脏中均能检测到目的基因β-catenin表达，说明β-catenin能促进DCs成熟；而且JMY-Pcat-GFP组的肠道、淋巴结、胸腺和脾脏中也均可检测到标记基因GFP的表达，说明CD11c⁺DCs成熟后向免疫循环系统迁移。在饲喂酵母5d时，与免疫应答相关的细胞因子IL-12、TNF-α和IFN-γ浓度下降，与免疫耐受相关的IL-6和IL-10浓度升高，机体免疫倾向于免疫耐受。本研究证明重组酵母介导的外源蛋白能够有效诱导机体产生抗体反应。构建适用于生产的整合型酵母JMY1-MSTN，通过口服方式将该酵母饲喂昆白小鼠，能刺激机体产生针对MSTN的特异性抗体，并促进小鼠体重增长。该菌株可在YPD营养培养基中培养，而且无筛选基因，是一种安全、经济、高效酵母生产菌株，且具有提高机体体重的功能，具备作为饲料添加剂的潜能。期间发明一种利用LiAc/NaOH提取酵母总蛋白的方法，该方法对各种酵母菌株具有普遍适用性，提取得到的酵母蛋白质量合格，可满足Western Blot等试验的需求。获得一种转基因小鼠和重组酵母口服免疫介导的无融合单克隆抗体hApoB100的生产方法。利用PiggyBac转座子系统构建转基因载体PB-rtTA-STP-NIT，该载体含有四环素诱导的tet-on系统调控的SV40T和p53基因、与tet-on系统结合的rtTA转录因子以及用于正负向筛选的LoxP-neo-IRES-tk-LoxP基因表达盒，与转座酶载体共注射FVB小鼠受精卵，获得转基因小鼠。分离转基因小鼠的脾细胞，加Dox诱导，可实现脾细胞的永生化。同时构建含有人hApoB100基因的酵母表达载体JMB88-OVA-HA-hApoB100；通过CuSO₄诱导转化JMB88-OVA-HA-hApoB100的酵母菌株JMY1，获得表达hApoB100蛋白的重组酵母菌株JMY1-OVA-HA-hApoB；通过口服免疫转基因小鼠，刺激转基因小鼠产生hApoB100特异性抗体，佐证口服表达外源蛋白的酵母能刺激机体产生抗体的结论；分离和培养具有分泌hApoB100抗体能力的转基因小鼠的脾细胞，经过Dox诱导后可获得能持续培养且保持分泌抗体能力的脾细胞；经过有限稀释法，获得具备持续生长并分泌hApoB100特异性抗体能力的单克隆细胞株；该单克隆细胞株在冻存和复苏操作后，仍能维持细胞生长和分泌hApoB100特异性抗体的能力；单克隆细胞株分泌的单克隆抗体在Western Blot检测时条带单一，特异性好，可满足Western Blot等检测技术对抗体的要求。本研究获得一种高效、安全、快速的无融合单克隆抗体的生产方法。更多还原

【Abstract】 Dendirtic cells （DCs） are the most efficient antigen present cells. As immune sentinels,DCs are ideally positioned throughout the entire body, sample the environment pathogens,transport antigens from the periphery to lymphoid tissues and trigger the immune response.And Saccharomyces cerevisiae, historically used in food precessing, has been demonstratedto enhance human immune system. Based on these observations, we designed series ofexperiments to uncover how the yeast reprogramed intestinal DCs global gene expressionupon uptaken into the DCs. We also engineered the yeast to deliver a functional gene（β-catenin） into DCs and examined the effect of its expression within DCs on immuneresponse. We finally tested the capability of recombinant yeast delivery of protein antigen andfunctional genes into DCs.We demonstrated for the first time how yeast reprogramed intestinal DCs geneexpression.Transcriptome analysis of isolated intestinal CD11c⁺DCs after oral administrationof yeast indicated that yeast up regulated1558genes and down regulated754genes. The GOand KEGG analysis of the DEGs showed that genes involved in the receptor pathway and thecellular compents were significantly influenced by the oral adminstration of the yeast.Thebiological function analysis of these genes indicated that the immune system process wassignificantly changed.We next engineered the yeast to deliver functional gene into intestinal DCs. β-catenin isan essential protein in Wnt/β-catenin pathway and plays an important role in maintaining theimmunity balance in intestine. We constructed a recombinant yeast containing mammaliangene expression cassette β-catenin-HA and β-catenin-HA-GFP driven by CMV promoter, andnamed them as JMY-Pcat and JMY-Pcat-GFP, respectively. Then mouse C57BL/6wasimmunized with recombinant yeast stains JMY-Pcat and JMY-Pcat-GFP by oraladiminstration of the yeast for5days. By analysing the CD11c⁺DCs cells isolated from theintestine with CD11c⁺magnetic activated cell sorting, we confirmed that β-catenin wasexpressed in a higher level in the group JMY-Pcat-GFP. The expression of co-stimulatorymolecules CD80, CD83and CD86of DCs were increased by qRT-PCR detection inJMY-Pcat group, while the expression of MHCⅡ was decreased compared with other groups.We also detected the expression of β-catenin or GFP in intestinal tract, lymph, thymus glandand spleen tissue of JMY-Pcat group or JMY-Pcat-GFP through the immunohistochemical experiment, indicated that the DCs were sitmulated and migrated into the immune tissues. Atthe same time, the concentration of IL-12, TNF-α and IFN-γ which related with immuneresponse were lower in group JMY-Pcat-GFP group, while the concentration of immunetolerance related cytokines IL-6, and IL-10were higher in the JMY-Pcat group orJMY-Pcat-GFP group, suggesting that the over expression of β-catenin in DCs triggeredimmune tolerance in mice.We tested the capability of the recombinant yeast mediated delivery of myostatin proteinantigen into DCs and examined its immune response. During conducting the experiments, wedeveloped a new method for protein extraction from yeast Saccharomyces cerevisiae cellsusing LiAc/NaOH and tested the application of the protein prepared with this new method inWestern Blot. To develop a vaccine grade of recombinant yeast, we constructed a MSTNgene integrated yeast strain. With the oral delivery of this recombinant yeast strain for aperiod of12weeks, the body weight increased significantly compared with the control group.The new MSTN integrated yeast strain can be cultured in YPD medium. Establishment ofsuch yeast strain is a step further toward transformation of yeast cells into edible vaccine togene therapy and feed additives.Finally, we established a hybridoma-free monoclonal antibody production method forhApoB100medicated by SV40T/p53transgenic mice and recombinant yeast oralimmunization technology. The expression plasmid JMB88-OVA-HA-hApoB100wasconstructed by inserting hApoB into JMB88-OVA-MSTN, and used for transformation ofyeast strain JMY1with CuSO₄induction for expression of hApoB100. Then the recombinantyeast JMY1-OVA-HA-hApoB was used to orally immunize SV40T/p53transgenic mice.Then we isolated splenocytes, cultured them in the medium supplemented with Dox forimmortalization, and subsequently subcloned by limited dilution method to obtain the cellclones that could secrete the hApoB100antibody and be immortal. We showed thatsplenocytes derived from transgenic mice harboring a simian virus40large T antigen andmouse p53gene under the control of Tet inducible promoter were conditionally immortalafter doxycycline induction and could produce monoclonal antibodies. This novel approachmay become a method of choice for production of both polyclonal and monoclonalantibodies.更多还原