Locht C, Barstad P A, Coligan J E, Mayer L, Munoz J J, Smith S G, Keith J M
Nucleic Acids Res. 1986 Apr 25;14(8):3251-61. doi: 10.1093/nar/14.8.3251.
We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.
我们从百日咳博德特氏菌中克隆了一个4.5 kb的EcoRI/BamHI DNA片段，该片段包含至少两个负责百日咳毒素表达的基因。通过高压液相色谱法分离出毒素的S4亚基，并确定了其氨基末端氨基酸序列。利用根据蛋白质序列的一部分反向翻译设计的混合合成寡核苷酸探针，鉴定出一个克隆的DNA片段，该片段至少包含毒素S4结构亚基的编码信息。序列分析表明，成熟蛋白是由前体分子经蛋白水解切割产生的。对Tn5诱导的百日咳博德特氏菌毒素缺陷型突变体的Southern印迹分析表明，Tn5 DNA插入到S4亚基基因下游1.3 kb处。这第二个基因可能编码成熟毒素组装所需的另一个亚基或一种非结构转运蛋白，可能存在于同一个多顺反子操纵子中。百日咳毒素基因的分子克隆为开发更安全的重组“新一代”百日咳疫苗奠定了基础。
