产气荚膜梭菌α毒素在干酪乳杆菌中的诱导表达及小鼠口服免疫保护效力的测定

Induced Expression of Alpha-Toxin Gene of Clostridium perfringens in Recombinant Lactobacillus casei and their Immunoprotective in mice

摘要:

摘要: 【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.casei 393 乳酸乳杆菌表面表达系统和pPG2-α/ L.casei393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毒素的腹腔攻毒实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。

Abstract:

Abstract:[Objective] The study was aimed to prepare an oral vaccine by constructing recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens, for preventing poisoning by Clostridium perfringens. [Methods] The constructed cell-surface expression plasmid pPG1-ɑ/L.393 and secretion expression plasmid pPG2-ɑ/L.393, both with alpha-toxin gene, were electroporated into L.casei 393, generating recombinant bacteria pPG1-ɑ/L.casei393 and pPG2-ɑ/L.casei393. The recombinant strains were induced by 1% lactose in De Man, Rogosa and Sharp (MRS) broth, and the target protein was detected by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), Western blot and indirect immunofluorescence assay. BALB/C mouse were used as animal model immunized with recombinant strains by intragastric administration, and the immune efficacy was analyzed. Specific anti- alpha-toxin protein sIgA was detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces, vaginal lavage, eye washing of mice after intragastric administration, and Specific IgG was detected by indirect ELISA in the serum of immunized mice. The resistance of immunized mice to alpha-toxin and the neutralization ability of antibodies to alpha-toxin were also tested. [Results] Mice immunized with pPG1-ɑ/L.casei393 and pPG2-ɑ/L.casei393 could produce remarkable anti-alpha-toxin antibodies, sIgA and circulating antibody IgG had completely neutralization ability against alpha-toxin. The test of alpha-toxin challenge in mice showed that the immunized mice could resist three times’ Minimum Lethal Dose(MLD). [Conclusion] All the results indicated that mice inmmunized by the recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens could elicit regional and systematic immunity response and neutralization ability.