Runeberg-Nyman K, Engström O, Löfdahl S, Ylöstalo S, Sarvas M
National Public Health Institute, Helsinki, Finland.
Microb Pathog. 1987 Dec;3(6):461-8. doi: 10.1016/0882-4010(87)90016-7.
Pertussis toxin (PT) is an important virulence determinant of Bordetella pertussis and one of the major protective antigens against whooping cough. The genes coding for PT have recently been cloned, but attempts to express them in Escherichia coli have been unsuccessful. We therefore explored the possibility of expressing these genes in Bacilius subtilis for which efficient vectors are available. The lack of endotoxin in the Gram-positive Bacillus might be an additional advantage for the production of a vaccine component. A DNA fragment coding for S1, one of the subunits of pertussis toxin, was inserted into an alpha-amylase secretion vector and the recombinant plasmid was introduced into B. subtilis. This resulted in high expression of S1, most of which was secreted and therefore found in the culture supernatant. This supernatant had ADP-ribosylating activity similar to that of PT. Western blot with antiserum to B. pertussis holotoxin showed several proteins ranging in size from 28 kDa to 20 kDa reacting in specific manner. About 10% of the protein recognized by the antiserum was of the size expected for native-size S1. The total amount of S1 proteins (full size and truncated) in the culture supernatant was about 100 mg/l. S1 protein made in B. subtilis was partially purified using chromatography with P-cellulose and Blue Sepharose. This preparation was used to immunize rabbits; the immune serum thus obtained recognized subunit S1 of native pertussis toxin.
百日咳毒素（PT）是百日咳博德特氏菌的一种重要毒力决定因素，也是预防百日咳的主要保护性抗原之一。编码PT的基因最近已被克隆，但在大肠杆菌中表达这些基因的尝试均未成功。因此，我们探索了在枯草芽孢杆菌中表达这些基因的可能性，因为有适用于该菌的高效载体。革兰氏阳性菌枯草芽孢杆菌缺乏内毒素，这可能是生产疫苗成分的一个额外优势。将编码百日咳毒素亚基之一S1的DNA片段插入α-淀粉酶分泌载体，并将重组质粒导入枯草芽孢杆菌。这导致了S1的高表达，其中大部分被分泌，因此存在于培养上清液中。该上清液具有与PT相似的ADP-核糖基化活性。用抗百日咳博德特氏菌全毒素血清进行的蛋白质印迹显示，几种大小从28 kDa到20 kDa的蛋白质以特异性方式发生反应。血清识别的蛋白质中约10%是天然大小S1预期的大小。培养上清液中S1蛋白（全长和截短形式）的总量约为100 mg/l。使用P-纤维素和蓝色琼脂糖进行色谱法对枯草芽孢杆菌中产生的S1蛋白进行了部分纯化。该制剂用于免疫兔子；由此获得的免疫血清识别天然百日咳毒素的亚基S1。
