Li Xiaojing, Xia Chunli, Li Yijing
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
Wei Sheng Wu Xue Bao. 2009 Aug;49(8):1115-20.
The prepared an oral vaccine by constructing recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens, for preventing poisoning by Clostridium perfringens.
The constructed cell-surface expression plasmid pPG1-alpha/L. 393 and secretion expression plasmid pPG2-alpha/L. 393, both with alpha-toxin gene, were electroporated into L. casei 393, generating recombinant bacteria pPG1-alpha/L. casei 393 and pPG2-alpha/L. casei 393. 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.
Mice immunized with pPG1-alpha/L. casei 393 and pPG2-alpha/L. casei 393 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).
All the results indicated that mice inmmunized by the recombinant L. casei expressing alpha-toxin gene of C. perfringens could elicit regional and systematic immunity response and neutralization ability.
通过构建表达产气荚膜梭菌α毒素基因的重组干酪乳杆菌制备口服疫苗，用于预防产气荚膜梭菌中毒。
将构建的含α毒素基因的细胞表面表达质粒pPG1-α/L. 393和分泌表达质粒pPG2-α/L. 393电穿孔导入干酪乳杆菌393，得到重组菌pPG1-α/干酪乳杆菌393和pPG2-α/干酪乳杆菌393。重组菌株在德氏、罗氏和夏普（MRS）肉汤中用1%乳糖诱导，通过12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质免疫印迹法和间接免疫荧光试验检测目标蛋白。以BALB/C小鼠为动物模型，通过灌胃给予重组菌株进行免疫，分析免疫效果。通过间接酶联免疫吸附测定（ELISA）检测灌胃后小鼠粪便、阴道灌洗液、眼洗液中的特异性抗α毒素蛋白sIgA，通过间接ELISA检测免疫小鼠血清中的特异性IgG。还检测了免疫小鼠对α毒素的抵抗力以及抗体对α毒素的中和能力。
用pPG1-α/干酪乳杆菌393和pPG2-α/干酪乳杆菌393免疫的小鼠可产生显著的抗α毒素抗体，sIgA和循环抗体IgG对α毒素具有完全中和能力。小鼠α毒素攻击试验表明，免疫小鼠可抵抗三倍最小致死剂量（MLD）。
所有结果表明，用表达产气荚膜梭菌α毒素基因的重组干酪乳杆菌免疫小鼠可引发局部和全身免疫反应及中和能力。
