Brown D R, Parker C D
Infect Immun. 1987 Jan;55(1):154-61. doi: 10.1128/iai.55.1.154-161.1987.
Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392. One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B. pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA. FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E. coli, was larger than that expressed in B. pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria. The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases. The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene. Conjugative transfer of pDB441 to B. pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B. pertussis.
百日咳博德特氏菌UT25的DNA被克隆到黏粒pCP13的卡那霉素抗性基因中，以在大肠杆菌LE392中构建一个基因组文库。通过使用兔抗百日咳博德特氏菌抗血清、兔抗丝状血凝素（FHA）抗血清和一种针对FHA的单克隆抗体进行蛋白质免疫印迹鉴定，发现一个含有质粒pDB441的克隆表达了丝状血凝素。FHA是一种220至210千道尔顿的蛋白质，但在大肠杆菌中表达的免疫反应性FHA比在百日咳博德特氏菌中表达的要大，这表明这两种细菌在该蛋白质的加工过程中存在差异。通过使用各种限制性内切酶，将fha基因定位到一个6.5千碱基对的DNA片段上。发现pCP13的卡那霉素抗性基因提供了启动子功能，但可能没有为fha基因提供翻译起始信号。pDB441向百日咳博德特氏菌BP353（一种转座子Tn5诱导的FHA突变体）的接合转移，使FHA的表达比野生型百日咳博德特氏菌更高。
