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目的 构建能够诱导抗菌抗毒素免疫和抵抗CTXΦ转染的霍乱弧菌生物安全性口服活疫苗候选株。方法 以不产毒的ElTor型霍乱弧菌疫苗候选株IEM10 1为出发菌株 ,以管家基因thyA为选择压力 ,在IEM10 1的thyA基因缺失株IEM10 1 T中 ,通过质粒 染色体致死平衡系统表达霍乱毒素B亚单位基因ctxB和介导CTXΦ噬菌体免疫的rstR基因。在家兔模型中 ,通过检测血清杀弧菌抗体和抗CTB的IgG抗体来评价该新建疫苗候选株的免疫原性 ;以对家兔攻毒试验后肠段积液量来评价其保护力。结果 含ctxB、rstR和大肠杆菌来源的thyA基因的重组质粒在IEM10 1 T中稳定存在 ,GM1 ELISA检测ctxB基因能够很好表达。动物试验表明 ,该新建疫苗候选株能刺激机体产生高滴度的血清杀弧菌抗体和抗CTBIgG抗体 ,能较好抵抗至少 4 μg纯品CT和不同毒株的攻击。结论 应用质粒 染色体致死平衡系统构建了能抵抗CTXΦ转染和稳定表达ctxB的生物安全性抗菌抗毒口服活疫苗候选株 ,其在家兔模型中有较好的免疫原性和保护力。
Objective To construct a live vaccine candidate strain of Vibrio cholerae that can induce antibacterial and antitoxin immunity and resist CTXΦ transfection. Methods The virulent ElTor Vibrio cholerae vaccine candidate strain IEM10 1 was used as a starting strain and the housekeeping gene thyA was used as a selective strain to express cholera toxin in IEM10 1 T of IEM10 1, a lethal balance system of plasmid chromosome. B subunit gene ctxB and the rstR gene that mediates CTXΦ phage immunization. In the rabbit model, the immunogenicity of the new vaccine candidate strain was evaluated by detecting the serum Vibrio cholerae antibody and the anti-CTB IgG antibody. The protective capacity of the vaccine was evaluated based on the amount of effusion in the intestinal tract after the challenge test in rabbits. Results The recombinant plasmid containing ctxB, rstR and E. coli-derived thyA gene was stable in IEM10 1 T, and ctxB gene was detected by GM1 ELISA. Animal experiments show that the new vaccine candidate can stimulate the body to produce high titer of serum Vibrio cholerae anti-CTBIgG antibodies, and can be better resistant to at least 4 g of pure CT and different strains of the attack. Conclusion The bioassay antibacterial and anti-virus oral live vaccine candidate strain resistant to CTXΦ transfection and stable expression of ctxB was constructed by plasmid lethal balance system. It has good immunogenicity and protective ability in rabbit model.