背景：鲍曼不动杆菌是一种广泛存在于环境中的革兰氏阴性菌，尤其常见于医院环境，特别是在重症监护病房中。随着耐药性逐渐增加，鲍曼不动杆菌已成为临床治疗中的一大挑战。为了应对这一问题，开发非抗生素疗法尤为重要，疫苗作为预防措施可以有效缓解这一问题。外膜蛋白因其良好的免疫原性和高度保守性，成为了疫苗开发中的重要靶点。病毒样颗粒（VLPs）作为疫苗递送系统的核心组件，通过高效展示抗原，显著增强免疫系统对抗原的识别与反应。SpyTag/SpyCatcher系统是一种创新的分子连接技术，能够通过共价键牢固地将目标外膜蛋白成分结合到VLPs上，从而提高疫苗的免疫效果。细菌仿生囊泡（BBV）作为一种新型的疫苗递送系统，能够有效包装并稳定外膜蛋白，模拟细菌感染的过程，有助于提高抗原的免疫原性，从而促进更强的免疫反应。通过以上创新策略，我们可以期待未来能开发出更为有效的预防措施，以应对鲍曼不动杆菌耐药性带来的挑战。

目的：由于外膜蛋白的复性制备较为复杂，本研究的第一部分旨在优化OmpW的表达、复性和纯化工艺，以提高复性蛋白的纯化效率，为其在疫苗开发或功能研究中的应用奠定坚实基础。本研究的第二部分将聚焦于使用AP205病毒样纳米颗粒并结合SpyCatcher与Spy技术递送OmpW；使用细菌仿生囊泡BBV递送外膜蛋白22、W、A，为临床耐药菌的治疗提供新的治疗方案。

方法：在本文第一部分，我们将外膜蛋白W（OmpW）的基因克隆于不同表达载体（pET-N-His-PreScission与pThioHisA），并转入大肠杆菌BL21（DE3）中，分别置于不同诱导温度（25℃、30℃、37℃）与诱导时间（0h、2h、4h、6h、8h）表达，收集菌液破碎离心以获得可溶性上清与包涵体沉淀，SDS-PAGE电泳分析重组蛋白可溶性；使用5种洗涤剂(Triton X-100、Triton X-114、尿素、吐温80、吐温20)分别或交叉洗涤包涵体沉淀，SDS-PAGE电泳分析洗涤效果；包涵体变性蛋白柱上复性或透析复性，利用Ni-NTA纯化OmpW目的蛋白，SDS-PAGE电泳并BCA法测量蛋白浓度。在第二部分的研究中，我们利用IEDB与AlphaFold3预测OmpW的B细胞抗原表位短肽序列，并与OmpW全长蛋白通过SpyCatCher与SpyTag系统分别与AP205共价偶联，同时，我们还利用细菌仿生囊泡（BBV）技术，将外膜蛋白OmpA、OmpW和Omp22进行包裹递送。采用上述基于外膜蛋白的多策略免疫方法，对6-8周龄的ICR雌性小鼠进行免疫（第0天、第14天与第28天），并在每次免疫一周后采血，评估疫苗是否诱导了显著的体液免疫反应。在最后免疫后三周后，腹腔注射鲍曼不动杆菌临床分离株，以评估疫苗对于小鼠感染后生存率、心肝脾肺肾等主要脏器细菌载量、血清生化、炎症水平的影响。

结果：在第一部分的研究中，我们成功将OmpW基因克隆整合至（pET-N-His-PreScission与pThioHisA）两个质粒，并构建了原核表达载体，重组蛋白在25℃、30℃、37℃诱导0h、2h、4h、6h、8h均表达于包涵体沉淀中，且在37°诱导8h达到了表达高峰；鉴定得到了包涵体沉淀的最佳洗涤剂组合：0.5％（吐温80、吐温20、TritonX-100）与3M尿素交叉洗涤1次；250ml的菌液样品经柱上复性得到目的蛋白47mg，并且纯度高达为85％，透析复性后镍柱纯化仅得到6mg，柱上复性蛋白的获取效率为透析复性的7.8倍。在第二部分的研究中，我们成功纯化了外膜蛋白OmpW、Omp22、OmpA与AP205纳米颗粒，并制备了BBV，并将OmpW与其B细胞抗原表位短肽成功共价连接至AP205纳米颗粒，与呈递OmpW、Omp22和OmpA的BBV组分别免疫6-8周龄的ICR雌性小鼠后，发现相比于蛋白的单独免疫组，AP205+OmpW组或BBV+22/W/A的联合免疫组在体液免疫水平上显著提高，后续在攻击保护实验上也与之对应。AP205+OmpW或BBV+22/W/A联合免疫组显著提高了小鼠的生存率，降低了心肝脾肺肾等主要脏器的细菌载量，血清生化实验的AST与CK水平进一步验证了AP205+OmpW或BBV+22/W/A联合免疫组对心脏与肝脏的保护效率的优势，并且减轻了由细菌攻击引发的炎症反应。

结论：成功建立了优化的OmpW包涵体表达条件、洗涤方案及基于柱上复性的纯化技术，为基于OmpW的耐药性鲍曼不动杆菌外膜蛋白疫苗应用以及生物学功能的研究提供了重要基础。并且成功构建了基于外膜蛋白的BBV+22/W/A、AP205+OmpW联合免疫疫苗新策略，展现了其在抗菌免疫领域的巨大潜力，针对传统的细菌疫苗提供了更为先进的开发平台，为多重耐药菌感染这一全球公共卫生挑战开辟了新的防治路径。

Background: Acinetobacter baumannii is a Gram-negative bacterium commonly found in the environment, particularly in hospital settings, especially in intensive care units. As antibiotic resistance continues to rise, A. baumannii has become a significant challenge in clinical treatment. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has greatly reduced treatment options, putting immense pressure on traditional antibiotic therapies. To address this issue, developing non-antibiotic therapies is crucial, and vaccines can effectively reduce antibiotic use, thereby minimizing the development of antibiotic resistance and reducing the risk of severe infections. Outer membrane proteins (OMPs) are important vaccine targets due to their strong immunogenicity and high conservancy. Virus-like particles (VLPs), as core components of vaccine delivery systems, significantly enhance the immune response by efficiently presenting antigens to the immune system. The SpyTag/SpyCatcher system is an innovative molecular linkage technology that covalently attaches target outer membrane protein components to VLPs, thereby improving vaccine efficacy. Bacterial membrane vesicles (BBVs), as a new type of vaccine delivery system, can effectively package and stabilize outer membrane proteins, simulating the process of bacterial infection. The unique structure of BBVs enhances the immunogenicity of the antigens, promoting a stronger immune response. With these innovative strategies, we can expect to develop more effective preventive measures to tackle the challenges posed by A. baumannii antibiotic resistance.

Objective: Due to the complexity of refolding outer membrane proteins, the first part of this study aims to optimize the expression, refolding, and purification processes of OmpW to improve the purification efficiency of refolded proteins, providing a solid foundation for its application in vaccine development or functional studies. The second part of the study will focus on using AP205 virus-like nanoparticles combined with the SpyCatcher/SpyTag system and BBV technology to deliver outer membrane proteins, evaluating the protective efficacy in a sepsis mouse model.

Methods: In the first part of this study, the OmpW gene was cloned into different expression vectors (pET-N-His-PreScission and pThioHisA) and transformed into E. coli BL21 (DE3). The protein was expressed at different induction temperatures (25°C, 30°C, 37°C) and induction times (0h, 2h, 4h, 6h, 8h), with cell lysates centrifuged to obtain soluble supernatants and inclusion body precipitates. SDS-PAGE was used to analyze the solubility of the recombinant protein. Five detergents (Triton X-100, Triton X-114, urea, Tween 80, and Tween 20) were used separately or in combinations to wash the inclusion body precipitates, and SDS-PAGE was employed to evaluate the washing efficiency. Inclusion bodies were refolded either on-column or by dialysis, and Ni-NTA purification was used to purify the OmpW protein. SDS-PAGE and BCA assays were used to determine protein concentration. In the second part of the study, B-cell epitope short peptide sequences of OmpW were predicted using IEDB and AlphaFold3, and these peptides were covalently coupled to AP205 nanoparticles through the SpyCatcher/SpyTag system. Additionally, BBV technology was employed to package and deliver OmpA, OmpW, and Omp22. Mice (6-8 weeks old, ICR females) were immunized on days 0, 14, and 28, and blood was collected one week after each immunization. The humoral immune response was evaluated, and three weeks after the final immunization, mice were intraperitoneally challenged with A. baumannii strain Ab.1 to assess vaccine effects on survival rate, bacterial load in major organs (heart, liver, spleen, lungs, kidneys), serum biochemistry, and inflammation levels.

Results: In the first part of the study, we successfully cloned the OmpW gene into the pET-N-His-PreScission and pThioHisA plasmids and constructed the prokaryotic expression vectors. Recombinant proteins were expressed in inclusion bodies at 25°C, 30°C, and 37°C after induction for 0h, 2h, 4h, 6h, and 8h, with the highest expression observed at 37°C after 8 hours. The optimal detergent combination for washing inclusion bodies was 0.5% (Tween 80, Tween 20, Triton X-100) combined with 3M urea, washing once. A total of 47 mg of target protein was obtained by column refolding from 250 ml of bacterial culture with a purity of 85%. After dialysis refolding, only 6 mg was obtained through Ni-column purification, with column refolding showing a 7.8-fold higher efficiency than dialysis. In the second part of the study, we successfully purified the outer membrane proteins OmpW, Omp22, and OmpA, and AP205 nanoparticles, and prepared BBVs. OmpW and its B-cell epitope peptides were covalently linked to AP205 nanoparticles. When immunized in combination with BBV or AP205, the immune response was significantly enhanced compared to the protein-only groups. In the subsequent challenge experiment, the AP205 or BBV combined immune groups showed improved survival rates, reduced bacterial load in major organs, and better serum biochemistry results. These groups also exhibited superior protection of the heart and liver, improved immune modulation, and reduced inflammation induced by bacterial infection.

Conclusion: We successfully established optimized conditions for OmpW inclusion body expression, washing protocols, and on-column refolding purification techniques, providing an important foundation for the application of A. baumannii outer membrane protein-based vaccines and functional studies. Furthermore, we developed a novel multi-strategy immunization approach combining BBV and AP205 for outer membrane proteins, demonstrating significant potential in antibacterial immunity. This approach not only provides an advanced platform for traditional bacterial vaccine development but also offers new solutions for combating novel antibiotic-resistant pathogens and challenging diseases.