第一部分：

背景：单纯疱疹病毒1型（Herpes simplex virus type 1，HSV-1）是一种人类普遍感染的病原体，其与口唇疱疹、新生儿脑炎、阿尔兹海默症等疾病的发生发展密切相关。目前抗病毒治疗均无法根除HSV-1感染，只能对症治疗，并且新的HSV-1耐药毒株不断出现，因此新的抗病毒药物研发就显得格外迫切。HSV-1正在持续地威胁公共卫生安全，然而，HSV-1的裂解性感染、潜伏和再激活等过程如何转换尚未被完全阐明。因此，研究调控HSV-1感染的关键信号/代谢通路和宿主因子，可以为进一步研究及开发针对HSV-1感染的治疗药物提供新线索。

方法：我们首先基于课题组前期研究发现，HSV-1感染可显著重塑细胞的代谢过程并诱导胆汁酸的分泌，本研究系统探究了HSV-1感染对胆汁酸代谢通路的调控作用及分子机制。首先，我们通过qRT-PCR、ELISA等技术进一步验证了HSV-1感染对胆汁酸代谢通路的影响，鉴于胆汁酸作为核受体法尼醇受体（Farnesoid X Receptor，FXR）的天然配体可通过与其结合促进其核转位，我们进一步采用核质分离实验，免疫荧光等实验观察HSV-1感染后核受体FXR的入核情况。同时，通过蛋白质免疫印迹实验（Western blot）分析HSV-1感染后FXR蛋白表达水平的变化。另外，为阐明FXR在HSV-1复制中的功能，我们也在体外使用FXR特异性的抑制剂及激动剂处理后观察，FXR对HSV-1复制增殖的影响，同时通过构建FXR基因敲除及过表达细胞模型进行同步验证。最后，我们通过HSV-1病毒的立即早期基因ICP0、ICP4、ICP27启动子转录活性分析、染色质免疫共沉淀（ChIP）、荧光共定位，蛋白质免疫共沉淀（CoIP）等方法深入解析了胆汁酸代谢下游分子FXR影响HSV-1复制增殖的分子机制。

结果：1、HSV-1感染调控胆汁酸代谢通路：qRT-PCR及ELISA结果显示HSV-1感染可显著增强宿主细胞的胆汁酸代谢相关基因表达及促进胆汁酸累积。2、核质分离实验及免疫荧光实验结果显示，HSV-1感染后累积的胆汁酸可作为FXR配体，与其结合并促进FXR发生转位至细胞核，增强其转录调控功能。Western blot分析表明，HSV-1感染可降低FXR的蛋白表达水平。3、通过FXR激动剂、抑制剂处理及敲除/过表达FXR实验证实，FXR的激活可以促HSV-1的转录与复制，而抑制FXR则显著降低病毒复制效率。4、染色质免疫共沉淀（ChIP），荧光共定位和蛋白质免疫共沉淀（Co-IP）实验揭示，核内FXR可与RNA聚合酶Ⅱ（RNA poly Ⅱ），细胞周期蛋白T1（Cyclin T1）以及病毒转录激活因子VP16形成转录复合物直接结合到HSV-1的立即早期基因（ICP0、ICP27、ICP4）的启动子区域，增强其转录活性，从而促进病毒复制。

结论：法尼醇受体FXR通过促进病毒立即早期基因转录而促进病毒复制，细胞感染HSV-1后通过下调FXR表达而抵抗病毒复制，是细胞的一种抗病毒机制。因此，FXR是一种潜在的抗HSV-1裂解感染的干预靶点。

第二部分：

背景：社区获得性肺炎通常源于肺炎支原体的耐大环内酯类菌株，但目前还没有针对这种菌株的有效疫苗。

目的：本研究旨在设计一种带有CTLA-4胞外结构域的新型多表位肺炎支原体疫苗，为肺炎支原体的防治提供新策略。并提出了针对肺炎支原体和其他病原微生物的疫苗免疫信息学策略。

方法：利用免疫信息学方法确定肺炎支原体P30粘附蛋白的优势B细胞和T细胞表位，然后通过与抗原递呈细胞靶向的“导航分子”CTLA-4细胞胞外结构区结合，构建了疫苗序列。随后验证了疫苗的理化性质、抗原性和致敏性。通过分子动力学模型来确认与TLR-2、TLR-4、B7-1和B7-2的相互作用。最后对疫苗的表达进行了电子克隆。

结果：所设计的疫苗表现出抗原性和非过敏性。与TLR-2、TLR-4、B7-1和B7-2对接后的分子动力学模拟实验表明，所构建的疫苗与这些分子之间存在稳定的相互作用。电子克隆证实了疫苗基因在昆虫杆状病毒载体中的有效表达。

结论：本研究提出了一种针对肺炎支原体和其他致病性非病毒和非细菌病原微生物疫苗分子设计的策略，这种疫苗免疫信息学方法为新型疫苗开发带来了新的策略和途径。

Part one:

Background: Herpes simplex virus type 1 (HSV-1) is a pathogen that is commonly infected in humans and is closely associated with the development of diseases such as herpes orofacialis, neonatal encephalitis, and Alzheimer's disease. Current antiviral therapies are unable to eradicate HSV-1 infection and can only treat symptoms, and new drug-resistant strains of HSV-1 continue to emerge, making the development of new antiviral drugs particularly urgent. HSV-1 is a persistent threat to public health and safety, yet how the processes of cleavage infection, latency, and reactivation of HSV-1 are transformed has not been fully elucidated. Therefore, studying the key signaling/metabolic pathways and host factors that regulate HSV-1 infection could provide new clues for further research and development of therapeutic agents against HSV-1 infection.

Methods: We firstly based on our group's previous research finding that HSV-1 infection can significantly remodel cellular metabolic processes and induce bile acid secretion, and in this study, we systematically explored the regulatory effects and molecular mechanisms of HSV-1 infection on the bile acid metabolic pathway. First, we further verified the effect of HSV-1 infection on the bile acid metabolic pathway by qRT-PCR, ELISA, etc. Given that bile acid, as a natural ligand of Farnesoid X Receptor (FXR), can promote its nuclear translocation by binding to FXR, we further used nucleoplasmic separation assay, immunofluorescence and other experiments to observe the nuclear translocation of FXR after HSV-1 infection. At the same time, we analyzed the changes in the protein expression level of FXR after HSV-1 infection by Western blot. In addition, to elucidate the function of FXR in HSV-1 replication, we also observed the effect of FXR on HSV-1 replication and proliferation after treatment with FXR-specific inhibitors and agonists in vitro, which was simultaneously verified by constructing FXR knockdown and overexpression cell models. Finally, we deeply analyzed the molecular mechanism of the downstream molecule of bile acid metabolism, FXR, affecting the replication and proliferation of HSV-1 by analyzing the transcriptional activity of ICP0, ICP4, and ICP27 promoters of HSV-1 virus in the immediate early stage, Chromatin Immunoprecipitation (ChIP), fluorescence co-localization, and Co-Immunoprecipitation (Co-IP).

Results: 1. HSV-1 infection regulates the bile acid metabolic pathway: qRT-PCR and ELISA results showed that HSV-1 infection significantly enhanced the expression of bile acid metabolism-related genes and promoted bile acid accumulation in host cells. 2. Nucleoplasmic isolation and immunofluorescence experiments showed that bile acid accumulated after HSV-1 infection could act as a ligand for FXR, binding to it and promoting its translocation to the nucleus. Western blot analysis showed that HSV-1 infection could reduce the protein expression level of FXR. 3. FXR agonist, inhibitor treatment and knockdown/overexpression of FXR experiments demonstrated that activation of FXR could promote the transcription and replication of HSV-1, whereas inhibition of FXR significantly reduced the efficiency of viral replication. 4. Chromatin Immunoprecipitation (ChIP), fluorescence co-localization and Co-Immunoprecipitation (Co-IP) experiments revealed that intranuclear FXR could form transcriptional complexes with RNA polymerase II (RNA poly Ⅱ), Cyclin T1, and viral transcriptional activator VP16 to directly bind to the immediate-early genes (ICP0, ICP27, ICP4) of HSV-1, to enhance their transcriptional activity, thereby promoting viral replication.

Conclusion: The farnesol receptor FXR promotes viral replication by facilitating viral immediate early gene transcription, and cells resist viral replication by down-regulating FXR expression after HSV-1 infection, an antiviral mechanism in cells. Thus, FXR is a potential target for intervention against HSV-1 lysis infection.

Part two:

Background: Community-acquired pneumonia often stems from the macrolide-resistant strain of Mycoplasma pneumoniae, yet no effective vaccine exists against it.

Objective: The aim of this study was to design a novel multisite Mycoplasma pneumoniae vaccine with CTLA-4 to provide new insights into the control of Mycoplasma pneumoniae.

Methods: This study proposes a vaccine-immunoinformatics strategy for Mycoplasma pneumoniae and other pathogenic microbes. Specifically, dominant B and T cell epitopes of the Mycoplasma pneumoniae P30 adhesion protein were identified through immunoinformatics method. The vaccine sequence was then constructed by coupling with CTLA-4 extracellular region, a novel molecular adjuvant for antigen-presenting cells. Subsequently, the vaccine's physicochemical properties, antigenicity, and allergenicity were verified. Molecular dynamics modeling was employed to confirm interaction with TLR-2, TLR-4, B7-1, and B7-2. Finally, the vaccine underwent in silico cloning for expression.

Results: The vaccine exhibited both antigenicity and non-allergenicity. Molecular dynamics simulation, post-docking with TLR-2, TLR-4, B7-1, and B7-2, demonstrated stable interaction between the vaccine and these molecules. In silico cloning confirmed effective expression of the vaccine gene in insect baculovirus vectors.

Conclusions: This vaccine-immunoinformatics approach holds promise for the development of vaccines against Mycoplasma pneumoniae and other pathogenic non-viral and non-bacterial microbes.