Fairweather N F, Lyness V A, Pickard D J, Allen G, Thomson R O
J Bacteriol. 1986 Jan;165(1):21-7. doi: 10.1128/jb.165.1.21-27.1986.
The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.
测定了破伤风毒素C片段前30个残基的氨基酸序列，并合成了32种互补寡核苷酸的混合物，每种寡核苷酸长度为17个碱基。通过Southern印迹法鉴定了破伤风梭菌DNA的一个2千碱基（kb）的EcoI片段，并用32P标记的寡核苷酸混合物作为探针将其克隆到大肠杆菌质粒载体pAT153中。鉴定出第二个3.2 kb的Bg/II片段，并用2 kb的EcoRI片段作为探针进行克隆。测定了该DNA 1.8 kb的核苷酸序列，结果表明其编码破伤风毒素的整个C片段和部分B片段。破伤风DNA在大肠杆菌中用pWRL507进行表达，pWRL507是一种含有trp启动子和部分trpE基因的质粒载体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳观察到trpE-破伤风融合蛋白，并显示其与抗C片段抗体发生反应。
