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【目的】构建产气荚膜梭菌(Clostridium perfringens,C. perfringens)α毒素基因的重组干酪乳杆菌口服疫苗,为产气荚膜梭菌毒素中毒的防治提供有效方法。【方法】将构建的重组产气荚膜梭菌α毒素基因细胞表面型载体pPG1及分泌表达载体pPG2电转化乳酸乳杆菌(Lactobacillus casei L.casei),获得阳性重组菌pPG1-α/L.casei393乳酸乳杆菌表面表达系统和pPG2-α/L.casei 393乳酸乳杆菌分泌表达系统。重组菌以1%乳糖为诱导物,在MRS培养基中进行诱导,通过Western-blot和间接免疫荧光方法鉴定,确定目的蛋白的表达。将重组菌口服免疫BALB/c小鼠,收集免疫小鼠粪便及眼冲洗液及外生殖道黏液样本测定小鼠产生抗α毒素的特异性sIgA抗体水平,采集小鼠血液样本测定血清中抗α毒素的特异性IgG抗体水平。并对免疫小鼠进行α毒素的腹腔攻毒试验及对获得的抗血清进行α毒素中和试验测定。【结果】重组干酪乳杆菌pPG1-α/L. casei 393及pPG2-/L. casei 393免疫小鼠能够产生明显的抗α毒素的sIgA和IgG抗体水平,其对α毒素中和试验结果为完全保护。腹腔攻毒实验结果为能抵抗3倍最小致死剂量(minimum lethal dose,MLD100)的α毒素攻击。【结论】表达产气荚膜梭菌α毒素免疫保护性抗原的重组乳酸乳杆菌口服免疫动物能够产生良好的局部和系统体液免疫应答和免疫中和效力。
【Objective】 To construct an oral vaccine of Lactobacillus casei of Clostridium perfringens (C) perfringens gene and provide an effective method for the prevention and treatment of Clostridium perfringens toxin poisoning. 【Method】 The recombinant cell surface vector pPG1 of C. perfringens alpha-toxin gene and the secretion expression vector pPG2 were transformed into Lactobacillus casei L.casei, and the positive recombinant bacteria pPG1-α / L.casei393 Lactobacillus lactis surface expression system and pPG2-α / L.casei 393 Lactobacillus lactis secretion expression system. Recombinant bacteria with 1% lactose as inducer, induced in MRS medium, identified by Western-blot and indirect immunofluorescence to determine the expression of the target protein. BALB / c mice were orally immunized with the recombinant bacterium, and the specific sIgA antibody against alpha toxin in mice was collected from feces and ophthalmic washes of immunized mice and mucus samples from the outer reproductive tract. Blood samples of mice were collected to measure anti-α Toxin-specific IgG antibody levels. The mice inoculated with α-toxin were intraperitoneally challenged and the α-toxin neutralization assay was performed on the obtained antiserum. 【Results】 The results showed that recombinant L. casei pPG1-α / L. Casei 393 and pPG2- / L. Casei 393 immunized mice were able to produce significant levels of sIgA and IgG antibodies to α-toxin, which were completely negative for α toxin neutralization test protection. Intraperitoneal challenge experiments resulted in alpha toxin challenge that was resistant to 3 times the minimum lethal dose (MLD100). [Conclusion] The recombinant Lactobacillus acidophilus orally immunized animals expressing C. perfringens alpha toxin immunoprotective antigens can produce good local and systemic humoral immune response and immune neutralization potency.