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目的拟从毒力基因ｃｔｘ阴性的Ｏ１３９霍乱弧菌中筛选疫苗候选株。方法用常规鉴定技术、Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ技术和兔肠段结扎试验检测了７株霍乱肠毒素（ＣＴ）基因阴性的Ｏ１３９霍乱弧菌的一般特性、毒性和毒性基因；通过小肠免疫家兔，用ＰＢＬ－ＥＬＩＳＡ、ＥＬＩＳＡ和杀弧菌抗体试验评价抗原性；用家兔主动保护试验和乳鼠被动保护试验研究保护效果。结果７株菌都具有Ｏ１３９霍乱弧菌的一般特性，均不引起肠段积液，毒力基因ｃｔｘ、ｚｏｔ、ａｃｅ和ＲＳ１探针杂交结果均为阴性。Ｂ１３和９４００１免疫后０～２８天血清抗体ＩｇＧ效价一直上升，而９４００２免疫后１４天最高；９４００２的血清杀弧菌抗体峰值最高。Ｂ１３免疫家兔后可保护１０５ＣＦＵ毒株的攻击，９４００１和９４００２免疫后可保护１０５～７ＣＦＵ毒株的攻击，９４００２加强免疫后能保护１０７ＣＦＵ毒株的攻击。Ｂ１３免疫血清１４稀释后对乳鼠的保护率为２０％，９４００１和９４００２的免疫血清１１６稀释后的保护率为５０％。结论７株Ｏ１３９霍乱弧菌属于毒力基因遗传单元缺失，无毒或毒性低，其中９４００２的免疫原性和保护效果较好，且初次免疫后存在免疫记忆
Objective To screen virulent candidate strains of Vibrio cholerae O139 virulent gene ctx. Methods The general characteristics, virulence and virulence genes of seven strains of Vibrio cholerae O139 negative for cholera enterotoxin (CT) gene were detected by routine identification, Southern blotting and rabbit intestine ligation. The rabbits were immunized with PBL-ELISA , ELISA and Vibrio cholerae antibody test to evaluate the antigenicity; the protective effect was studied by active protection test of rabbits and passive protection test of suckling mice. Results All the 7 strains had the general characteristics of Vibrio cholerae O139 and did not cause intestinal effusion. The results of hybridization of virulence genes ctx, zot, ace and RS1 were all negative. Serum IgG titer increased from 0 to 28 days after B13 and 94001 immunization, but was highest at 14 days after 94002 immunization. The peak value of 94002 was the highest. B13 immunized rabbits to protect against 105CFU challenge, 94001 and 94002 immunized to protect against 105-7CFU challenge, and 94002 boosted challenge to protect 107CFU strain. B13 immune serum 14 diluted to protect the rate of 20% of suckling mice, 94001 and 94002 immune serum diluted to 50% protection rate of 50%. Conclusions Seven strains of Vibrio cholerae O139 belong to the deletion of virulence genes, which are non-toxic or toxic. The immunogenicity and protective effect of 94002 are good, and immune memory exists after the first immunization