Construction of Pertussis-Toxoid-Producing Mutants

百日咳毒素（Pertussis toxin， PT）是一种典型的百日咳毒力因子，是百日咳疫苗中最重要的保护性抗原。作为疫苗的一个组成部分，需要无毒且具有免疫保护作用的百日咳类毒素。虽然甲醛（FA）处理是一种常用的细菌蛋白毒素的解毒方法，但PT的解毒，特别是S1的adp -核糖基转移酶活性的失活，并不一定是完全的。由于S1不含赖氨酸残基，认为经FA处理后，PT主要是B低聚物部分（S2-S5）被改性。此外，某些毒性可以通过在高温下孵育而逆转。我们试图通过引入PT基因的位点定向突变来构建产生百日咳类毒素的百日咳突变株，通过突变对adp -核糖基转移酶活性有深远影响的S1基因来构建百日咳类毒素产生株。第9位的精氨酸转化为赖氨酸，第129位的谷氨酸进一步转化为天冬氨酸、谷氨酰胺或甘氨酸，构建了双突变的S1基因。百日咳类毒素操纵子重组并插入百日咳双歧杆菌染色体。这些类毒素被组装并表达到大约野生型水平。PT活性如adp核糖基化、cho细胞聚集、白细胞促进和组胺致敏活性大大降低。特别是，双突变类毒素的毒性低于可检测水平。此外，这些产生类毒素的百日咳菌株脑内或气溶胶攻击小鼠的毒力也降低了。用类毒素免疫小鼠或用小鼠抗类毒素抗体免疫小鼠，分别可保护小鼠免受百日咳毒力生物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相同，因此突变株将是疫苗生产的良好候选株。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