Construction of Pertussis-Toxoid-Producing Mutants

百日咳毒素（PT）是百日咳杆菌的典型毒力因子，也是百日咳疫苗所必需的最重要的保护性抗原。作为疫苗的组成部分，需要无毒和免疫保护性的百日咳类毒素。虽然甲醛（FA）处理是一种流行的细菌蛋白毒素的毒素化的方法，PT的解毒，特别是ADP-核糖基转移酶活性的S1的失活，不一定是完全的。由于S1不含赖氨酸残基，因此认为PT的主要B寡聚体部分（S2-S5）被FA处理修饰。此外，某些毒性可通过在高温下孵育类毒素来逆转。我们试图构建B的突变株。通过对百日咳杆菌PT基因、对ADP-核糖基转移酶活性有显著影响的S1基因进行定点突变，构建了百日咳类毒素产生菌。p处的精氨酸 ...更多信息 将第9位转化为赖氨酸，并通过在第129位将谷氨酸另外改变为天冬氨酸、谷氨酰胺或甘氨酸来构建双突变的S1基因。重组百日咳类毒素操纵子并插入到B中。百日咳染色体这些类毒素被组装并表达至近似野生型水平。PT活动，如ADP-核糖基化，CHO-细胞群集，白细胞增多的促进和组胺致敏活动大大减少。特别是，双突变类毒素的毒性在可检测的水平之下。此外，通过脑内或气溶胶激发这些产生类毒素的B。百日咳菌株也减少了。用类毒素或用抗类毒素的小鼠抗体免疫的小鼠分别免受强毒百日咳生物体的脑内或气溶胶攻击。由于突变株产生的类毒素的免疫保护效力与FA处理的百日咳类毒素的免疫保护效力相同，因此突变株将是用于疫苗生产的良好候选物。少

Pertussis toxin (PT) is a typical virulence factor of Bordetella pertussis and the most important protective antigen which must be essential to a pertussis vaccine. As a component of the vaccine, non-toxic and immunoprotective pertussis toxoid is required. Although formaldehyde (FA) treatment is a popular method for toxoiding of bacterial protein toxins, the detoxification of PT, especially inactivation of ADP-ribosyltransferase activity of S1, is not necessarily complete. Since S1 does not contain lysine residue, mainly B oligomer part (S2-S5) of PT is considered to be modified by the FA treatment. Furthermore, some toxicity can be reversed by incubation of the toxoid at high temperature. We have attempted to construct mutant strains of B. pertussis which produce pertussis toxoid by the introduction of site-directed mutations in the PT gene, S1 gene which have profound effects on ADP-ribosyltransferase activity was mutated to construct pertussis toxoid producing strains. Arginine at p … More osition 9 was converted to lysine and doubly mutated S1 genes were constructed by additional changes of glutamic acid to aspartic acid, glutamine or glycine at position 129. The pertussis toxoid operons were reassembled and inserted into the B. pertussis chromosome. These toxoids were assembled and expressed to approximately wild type levels. PT activities such as ADP-ribosylation, CHO-cell clusterization, leukocytosis promotion and histamine sensitization activities were greatly reduced. Particularly, the toxicities of the double mutant toxoids were under detectable level. Furthermore, virulence in mice by intracerebral or aerosol challenge with these toxoid producing B. pertussis strains was also reduced. Mice immunized with the toxoids or with mouse-antibody against the toxoid were protected from the intracerebral or aerosol challenge with virulent pertussis organisms, respectively. Since immunoprotective potency of the toxoid produced by the mutants was the same level to that of FA-treated pertussis toxoid, mutant strains would be good candidates for the vaccine production. Less