D型产气荚膜梭菌Clostripain蛋白的原核表达及其免疫保护效果评价

Prokaryotic expression and evaluation of immune protection effect of Clostripain of Clostridium perfringens type D

摘要:

背景 D型产气荚膜梭菌(Clostridium perfringens)对反刍动物的危害极大，现有的亚单位疫苗多基于已被深入研究的α蛋白、ε蛋白等主要毒力因子，但这些疫苗也存在一定的保护局限性，它们大多仅针对单一毒素诱发的疾病，虽能提供部分保护，却难以抵御其他毒素或多种毒素协同感染所导致的疾病。因此，挖掘新的功能未知且具备良好免疫原性的蛋白，可以为亚单位疫苗的研发提供新思路与候选材料。目的 采用原核表达系统表达并纯化Clostripain蛋白，随后用该蛋白免疫小鼠，以评估其免疫保护效力。方法 通过软件分析出Clostripain蛋白B细胞抗原表位高得分区域，对该蛋白进行原核表达及纯化，用纯化的蛋白免疫小鼠，检测抗体滴度，并检测小鼠血清中各细胞因子的表达水平。随后，用D型产气荚膜梭菌感染小鼠，计算小鼠存活率，观察小鼠组织病理变化，以此评价该蛋白的免疫保护效果。结果 首先，通过预测B细胞线性表位和构象表位，发现Clostripain蛋白具备良好的抗原表位。随后，对该蛋白进行原核表达并纯化。SDS-PAGE结果显示，其条带大小约为79 kDa，Western blotting结果显示出现单一清晰条带。经测定D型产气荚膜梭菌对小鼠LD₅₀为2.90×10⁶ PFU/只。重组蛋白免疫小鼠后多克隆抗体效价达到1:409 600。细胞因子检测结果显示，IL-4和IL-10表达含量较高，与对照组差异显著，说明该蛋白主要诱导Th2型细胞因子表达，介导体液免疫。攻毒试验中免疫组小鼠存活率为80%。病理组织切片分析免疫组小鼠各组织无严重病变。结论 本研究通过pET-32a-Clo重组质粒诱导表达并纯化Clostripain蛋白，重组蛋白免疫的小鼠能产生较高滴度的抗体，具备较好的攻毒保护效果，为后续产气荚膜梭菌致病机制及疫苗的研发奠定了基础。

Abstract:

Background Clostridium perfringens type D poses a severe threat to ruminants. Existing subunit vaccines are primarily based on well-characterized major virulence factors such as α and ε toxins. However, these vaccines exhibit limitations in protective efficacy, as they generally target diseases caused by a single toxin and are often ineffective against infections involving other toxins or multiple toxins. Therefore, identifying novel proteins with unknown functions but strong immunogenicity could offer new avenues and promising candidates for subunit vaccine development.Objective To express and purify the Clostripain via a prokaryotic expression system and evaluate its protective efficacy by immunization of mice.Methods Bioinformatic tools were used to predict high-scoring B-cell epitopes within Clostripain. The gene encoding Clostripain was expressed in a prokaryotic system and the recombinant protein was purified. After mice were immunized with the purified protein, the antibody titers and the expression levels of various cytokines in the mouse serum were measured. Subsequently, the immunized mice were challenged with C. perfringens type D, and the survival rate and histopathological changes were calculated and observed to evaluate the protective effect of the protein.Results Prediction of both linear and conformational B-cell epitopes indicated that Clostripain possessed strong antigenic properties. The protein was successfully expressed and purified. SDS-PAGE results revealed a protein band of approximately 79 kDa, and Western blotting confirmed a single distinct band. The LD₅₀ of C. perfringens type D in mice was 2.90×10⁶ PFU per mouse. Following immunization with the recombinant protein, the polyclonal antibody titer reached 1:409 600. The expression levels of IL-4 and IL-10 in the recombinant protein group were significantly higher than those in the control group, indicating that the protein predominantly induced Th2-type cytokine expression and mediated humoral immunity. In the challenge assay, the survival rate of immunized mice was 80%. Histopathological examination showed no significant lesions in major organs of immunized mice.Conclusion The recombinant Clostripain was successfully expressed and purified in a prokaryotic system via plasmid pET-32a-Clo. Immunization with the recombinant protein elicited high antibody titers in mice and conferred effective protection against pathogenic challenge. These findings lay a foundation for further investigation into the pathogenic mechanisms of C. perfringens and the development of new vaccines.