MHCI类分子HLA-A30人鼠嵌合人源化小鼠流感模型

HLA是定位在人第6号染色体短臂上一组紧密连锁的基因群，编码主要组织相容性复合物(MHC)。这个区域全长约3600kb，包括有239个基因座位，其中约40%的表达基因与人体的免疫功能密切相关。从染色体端粒到着丝点方向，依次排列着MHC I类、MHC II类和MHCIII类基因。MHC I类基因包括HLA-A、B、C三个经典基因，分别编码I类抗原A、B、C的α链，而其β链则位于第15号染色体上的β2m基因编码。MHCI 类区域还编码一些非经典分子，包括HLA-E、F、G等，此区域还包括一些假基因，例如HLA-H、J、K等。和MHC I类抗原肽加工与递呈有关的蛋白LMP2（PMSB8）、LMP7（PSMB9）、TAP1和TAP2则被位于MHC II类基因区域的基因所编码。I类分子将抗原肽递呈给CD8+ I T细胞，促进其活化为CTL效应细胞，从而发挥特异性杀伤靶细胞的功能。

在过去的几十年，研究学者尝试且已制备了多种MHC I类人源化小鼠模型，例如A1，A2，A3，A11，A24，B7，B27 或Cw3等。这些MHC类分子人源化转基因小鼠已被广泛用于传染性疾病、自身免疫疾病的研究，例如强直性脊柱炎（AS）、类风湿性关节炎（RA）、牛皮癣、胰岛素依赖性糖尿病（IDDM）、重症肌无力（MG）、多发性硬化症（MS）等。此外，这些人源化转基因小鼠在疫苗的研发和测试、评价治疗药物的安全性和免疫原性方面也发挥重要作用。MHC I类人源化小鼠的研制经历了三个阶段。第一个阶段，研究人员分离出HLA-A基因片段，直接显微注射入小鼠受精卵，制备第一代MHCI类分子人源化小鼠，例如HLA-A*0201、HLA-A*2401、HLA-B27等。研究初步显示，HLA-A分子可以表达在小鼠细胞膜上，并产生有限的CTL免疫反应。为了提高小鼠CTL免疫反应，研究人员将人HLA-A分子第二、三外显子和小鼠第四、五、六、七、八外显子连接在一起，构成人鼠嵌合分子，研制了第二代人源化小鼠（HHM小鼠）模型。随后将HHM小鼠和mβ2m/H2-Db等基因敲除小鼠结合，产生了第三代HLA-A类分子人源化小鼠模型（HHD小鼠），消除了小鼠内源性I类、II类分子的影响。但是由于抗原肽的加工及递呈过程存在缺陷，人源化小鼠模型仍然产生了有限的CTL免疫反应。

MHC I类人源化小鼠模型有多种用途。第一，MHC I类分子HLA-B27人源化小鼠作为动物模型被用于脊柱关节炎（spondyloarthritis）、一种自体免疫性疾病的研究。第二，MHC I类分子人源化小鼠，尤其是第二代HHM小鼠、第三代HHD小鼠，已经被广泛用于抗原表位筛选的研究。第三，MHC I类分子人源化小鼠，例如HLA-A*0201，可以用于来自多种病毒的抗原表位筛选，包括vacciniavirus ,severe acute respiratory syndrome-associated coronavirus,cytomegalovirus(CMV) 和 hepatitis delta virus。此外，这些人源化小鼠也可用于病毒免疫逃逸机制研究。第四、MHC I类分子人源化小鼠也可以用于肿瘤抗原表位筛选，例如MAGE蛋白、Mucin-1、HuD、HSP70、MDM2等。第五、基因治疗方法相关的抗原表位筛选及鉴定。基因治疗成功与否某种程度上决定于宿主对治疗药物的免疫反应。人源化小鼠可以用于基因治疗抗原表位的筛选及治疗过程评价，例如杜氏肌营养不良症DMD。第六、MHC I类分子人源化小鼠可以用于研究肿瘤免疫治疗方法的效率、副作用，以及治疗方法的改进，尤其是肽类疫苗相关的肿瘤免疫治疗。不同的MHCI类人源化小鼠，例如HLA-A2、HLA-A11、HLA-A24等也可用于评价免疫治疗中疫苗的疗效以及触发的CTL免疫反应，进而对疫苗抗原表位进行定点修饰从而达到精准肿瘤免疫治疗的目的。MHC I类分子人源化小鼠也可用于不同疫苗免疫方法的评价，例如纳米颗粒DNA疫苗、RNA疫苗、腺相关病毒疫苗、重组蛋白等。。

小鼠H-2 I类分子和人MHC I类分子主要存在以下几种差异。第一、I类分子重链基因序列存在一定差异，且A2-A3链的糖基化模式也不一样，这就决定了小鼠和人MHC I类分子抗原结合特性存在物种的差异性。第二、I类分子轻链β2m不同。人类hβ2m只有一种基因型，而小鼠存在三种基因型，五种亚型。人I类分子重链更倾向和人类hβ2m结合。人hβ2m不但具有稳定I类分子二聚体复合物结构的功能，而且可以修饰人I类分子重链结构而触发CTL免疫反应。第三、抗原肽加工和递呈过程涉及的分子不同。首先，蛋白酶体具有蛋白酶活性，可以裂解外源蛋白产生C末端修饰的、能够被I类分子复合物识别的肽段。尽管小鼠和人蛋白酶体进化上存在很大的保守性，但是早期使用HLA-B27分子体外研究发现，人MHC I类分子复合物更倾向于识别人蛋白酶体PSMB8/PSMB9产生的肽段。其次，TAP泵不同。早期使用缺失和拯救试验研究已显示TAP1/TAP2在抗原肽加工和递呈过程具有重要作用。后期使用HLA-A3和HLA-A11分子进行的体外试验进一步研究显示抗原肽加工和递呈过程存在TAP1/TAP2限制性，人MHC I类分子更倾向结合人TAP1/TAP2加工运输的抗原肽。最后，MHC I类抗原加工过程的分子伴侣在人和小鼠之间也存在物种差异性。第四、基因表达调控不同。HLA基因的表达调控是免疫应答中极其关键的环节，目前有关MHC I类分子基因表达调控研究较少。HLA基因的表达调控主要在转录水平上，由反式作用的转录因子和基因编码区5’上游的順式作用元件控制。启动子区的差异可以影响组蛋白的乙酰化及转录复合物的组装。基因上游长达几Kb的座位控制区（locus control region, LCR）也可影响到组蛋白的乙酰化及转录复合物的组装。在小鼠H2E基因已经发现位于基因上游20Kb以外的LCR调控元件，在人HLA-DR、HLA-DQ基因上游5-50kb或更远区域也发现了类似的LCR调控元件。HLA基因上游调控区的等位多态性也可引起MHC II类分子α和β链产物量的不平衡，导致α和β链的错配而引起疾病的发生。病毒的感染，例如CMV、HBV、Ad12等，也可影响蛋白质组装、运输或者分子伴侣的合成，进而减少HLA基因在细胞膜上的表达。其中Ad12的感染可以显著减少TAP蛋白的转录水平，从而影响I类分子从细胞质向粗面内质网的运输，进而有益于病毒发生免疫逃逸。HLA类分子也存在转录后水平的调节。5’-UTR和3’-UTR在转录后水平的基因表达调控发挥着重要作用，控制着mRNA稳定性和降解速率，决定着翻译速率和利用效率，而且还可对特定mRNA的时空性表达起重要作用。HLA-DRA、HLA-DQA1、HLA-DP基因均存在转录后水平的调控。不同等位基因型在抗原递呈细胞(APC)表面表达强度不同，从而改变了免疫反应的信号强度，促进炎症反应生成。此外，MHC类分子的表达能够决定免疫系统发育过程中T细胞群的形成，因此其表达水平对T细胞的发育命运至关重要。

尽管目前已经制备了大量MHC I类分子人源化小鼠，例如HLA-A02、HLA-A11、HLA-A24等，但是这些人源化小鼠使用的转基因构建均是较小的基因片段（<30kb），缺乏I类分子基因座相关调控元件。建立外源转基因的表达水平接近或达到原有的生理水平的动物模型，有利于从动物整体水平研究MHC I类HLA-A基因的功能。为此，我们建立两种人源化小鼠模型，一种整合了HLA-A基因座位点基因组大片段，调控中国人种特异性I类分子基因型HLA-A30的表达。另一种是整合HLA-PSMB8/PSMB9/TAP1/TAP2基因座位点基因组大片段，调控I类分子抗原肽加工递呈分子的表达。在整个HLA-A和HLA-PSMB8/PSMB9/TAP1/TAP2基因调控序列调控下，研究这些分子在临床药物筛选、疫苗筛选、病毒等诱发的机体免疫应答反应中发挥的协同免疫调节作用。

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1 PR8流感接种方法滴鼻；采样时间点D3 D7

小鼠按体重随机分成 C57，HLA-A30-7#， HLA-A30-9#, HLA-A30-12#, HLA-A30-18#或19# 备注：HLA-A30 7#和19#可以选择5只小鼠单独进行感染，取D7天肺组织进行QPCR检测病毒载量、HLA-A30基因表达水平及细胞因子检测，Western blot定量检测脾脏中HLA-A30的表达水平，（脾脏组织和/或肺组织流式检测HLA-A30基因表达水平和NK细胞）

2 测定指标：

（1）小鼠日常观察

观察模型组和空白组小鼠的活动状况、精神状态、饮水进食等情况。

（2）小鼠体重指标

在每个时间点，对模型组和空白组小鼠称量体重。

时间点：感染当天即为0天，感染后第一天，感染后第三天，感染后第七天为采样点。

（3）组织病理学观察

感染后3D, 7D三个时间点，每组取两只小鼠，处死后，取肺组织、分β别称重，组织固定过夜后，30%蔗糖脱水48H，OCT包埋后制作冰冻切片，做H&E染色组织病理学检测。

（4）肺部病毒载量定量PCR

3D, 7D三个时间点M1基因

（5）流式细胞术检测 7D肺部和脾脏、

脾脏 HLA-A/B/C表达水平、NK细胞、CD19+B细胞、CD4+/CD3+/CD8+/CD3+

肺部HLA-A/B/C表达水平、NK细胞、CD4+/CD3+、CD8+/CD3+、 、CD19+B细胞

（6）定量PCR检测 （3D, 7D） 肺脏、脾脏、胸腺

检测HLA-A30基因表达水平 HLA-A30E3-H2Db QPCR，hβ2M, mMHCI+mβ2m（流感病毒对鼠MHCI内源性和外源性人MHCI基因调控是否一致）

（7）ELISA检测细胞因子表达水平IFNy, TNFa，IL-6

（8）Western blot: 肺脏、脾脏、胸腺中蛋白hβ2M和HLA-A表达水平，订购hβ2M抗体

CRP基因敲除小鼠和人CRP转基因小鼠在赛业（苏州）生物科技有限公司模式动物研究中心完成，该中心具有符合国际标准的动物模型制备相关完备监督管理制度。动物的繁殖及感染模型评估在中国疾控中心动物中心完成，所有操作在SFP级别下遵守二级生物安全管理规定进行，实验动物尸体实行无害化处理，对生态环境无影响。

建立了整合HLA-A基因座位点的人鼠嵌合MHCI类分子HLA- hβ2m /A30人源化转基因小鼠，HLA-A0101基因调控序列来自BAC克隆CH501-67J3，包含完整的HLA-A0101基因座位点、48kb的5’—UTR和52kb的3’—UTR。

HLA-A30基因型在中国人群存在特异性分布。HLA基因的不同表达水平对免疫应答反应进行调控。抗原加工递呈分子在MHC I类分子基因表达调控方式存在差异，HLA-A基因表达调控元件尚不清楚。将较完整HLA-A基因组区域导入野生型小鼠体内，可获得接近HLA-A生理水平表达的人源化转基因小鼠模型。因此筛选整合不同HLA-A30基因组区域的转基因小鼠，可在体内水平研究HLA-A30基因的表达调控机制。模型也可有效筛选能引起人HLA-A30限制性免疫反应的物质。

我们又利用不同HLA-A30转基因小鼠建立流感病毒感染模型。 整合不同HLA-A30基因组片段的人源化小鼠，HLA-A30基因表达水平在各个组织器官中不同人源化小鼠品系之间是不同的。流感病毒感染后，HLA-A30基因表达是否在感染后期表达下调（病毒的免疫逃逸）；对鼠源内源性I类分子和人源嵌合分子调控是否一致？检测不同品系人源化小鼠（三个代表性的品系 9#,12#,18#）的病毒载量、细胞因子分泌水平、肺组织病理变化、免疫细胞群变化（流式细胞分析），期望回答问题：在各个组织器官HLA-A30基因表达水平不同，流感病毒感染后免疫反应是否一致。