重组结核分枝杆菌PcaA抗原的诊断潜力及其作为卡介苗启动后亚单位疫苗增强剂的保护功效

IF 1.9 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods Pub Date : 2025-07-26 DOI:10.1016/j.mimet.2025.107202

Yun Xu , Xiaochun Wang , Qiangsen Zhong

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引用次数: 0

摘要

卡介苗（Bacillus calmette - gusamrin， BCG）是目前临床用于预防结核病（TB）的唯一疫苗，然而，它在预防潜伏感染、结核病再激活和提供全面保护方面仍然有限。本研究选择了与持续性感染相关的抗原pcaA基因，成功构建了重组质粒pET28a-PcaA进行蛋白表达和纯化。采用化学发光、酶联免疫吸附试验（ELISA）、流式细胞术和分枝杆菌生长抑制试验（MGIA）进一步验证抗原的特异性。PcaA抗原刺激活动性结核（ATB）和潜伏性结核感染（LTBI）两组结核分枝杆菌（M. tb）患者外周血中IFN-γ、IL-2、IL-8和IgG的表达水平均有显著差异。IL-8联合诊断模型能有效区分ATB和LTBI，而抗pcaa IgG在排除M. tb感染方面表现出色。以二甲基二十二烷基溴化铵(DDA) /胶体锰盐(MnJ)/DM脂质体配制重组PcaA蛋白（rPcaA）免疫小鼠。评估血清特异性抗体水平、脾细胞分泌的细胞因子和脾细胞中多功能T细胞的数量。结果表明，与BCG组相比，BCG + rPcaA-DM疫苗组th1型细胞因子水平、抗体滴度以及IFN-γ+/TNF-α+单阳性和双阳性CD4+和CD8+ T细胞频率显著升高。此外，免疫小鼠的脾细胞和肺细胞显著抑制分枝杆菌的生长。这些研究结果表明，rPcaA-DM疫苗作为卡介苗增强剂，可显著增强Th1极化并提供强大的保护功效，有可能阻止LTBI发展为ATB。

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Diagnostic potential of recombinant Mycobacterium tuberculosis PcaA antigen and its enhancement of protective efficacy as a subunit vaccine booster following BCG priming

Bacillus Calmette-Guérin (BCG) is the only vaccine currently used in clinical practice to prevent tuberculosis (TB), however, it remains limited in preventing latent infection, TB reactivation, and providing comprehensive protection. In this study, the pcaA gene, an antigen associated with persistent infection, was selected, and the recombinant plasmid pET28a-PcaA was successfully constructed for protein expression and purification. The specificity of the antigen was further verified using chemiluminescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and mycobacterial growth inhibition assay (MGIA). Significant differences were observed in the expression levels of IFN-γ, IL-2, IL-8, and IgG in the peripheral blood of patients with Mycobacterium tuberculosis (M. tb) following stimulation with the PcaA antigen in the Active Tuberculosis (ATB) and Latent Tuberculosis Infection (LTBI) groups. An IL-8 combined diagnostic model could effectively distinguish between ATB and LTBI, while anti-PcaA IgG demonstrated strong performance in ruling out M. tb infection. Recombinant PcaA protein (rPcaA) was formulated with liposome dimethyl dioctadecylammonium bromide (DDA) / colloidal manganese salt (MnJ)/DM to immunize mice. Serum-specific antibody levels, cytokines secreted by splenocytes, and the number of multifunctional T cells in splenocytes were assessed. The results indicated that the BCG + rPcaA-DM vaccine group exhibited significantly elevated levels of Th1-type cytokines, antibody titers, and the frequencies of IFN-γ⁺/TNF-α⁺ single and double-positive CD4⁺ and CD8⁺ T cells compared to the BCG group. Furthermore, splenocytes and lung cells from immunized mice significantly inhibited mycobacterial growth. These findings suggest that the rPcaA-DM vaccine, as a BCG booster, significantly enhances Th1 polarization and provides robust protective efficacy, with potential to prevent LTBI progression to ATB.

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来源期刊

Journal of microbiological methods 生物-生化研究方法

CiteScore

4.30

自引率

4.50%

发文量

151

审稿时长

29 days

期刊介绍： The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach. All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.