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通过聚合酶链式反应（ＰＣＲ）从一中国株中型白喉产毒杆菌的β噬菌体基因组中克隆出１０６５碱基对的白喉毒素全基因编码序列，将ＰＣＲ产物直接克隆到ｐＧＥＭ－Ｔ／载体系统，经有关限制性内切酶消化，核苷酸序列分析表明，成功的克隆出白喉毒素全基因编码序列。利用上下游引物中导人的ＮｄｅＩ和ＢａｍＨＩ位点，将白喉毒素基因插入原核表达载体ＰＥＴ－３ａ，从而构建出白喉毒素表达载体ＰＥＴ／ＤＴ。以ＢＬ２１（ＤＥ３）为工程菌，用ＩＰＴＧ诱导Ｔ７启动子进行表达，经ＳＤＳ－ＰＡＧＥ分析表明，在５８０００处可见一产物条带，表达量可达菌体蛋白的２５％，经豚鼠毒性实验和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明，表达产物与白喉杆菌分泌的毒素有相同的免疫学及生物学活性。
The full-length coding sequence of 1065 base pairs of diphtheria toxin was cloned from the β-phage genome of a Chinese strain of T. diphtheriae by polymerase chain reaction (PCR). The PCR product was directly cloned into pGEM-T / vector system, Nucleotide sequence analysis showed that the full-length coding sequence of diphtheria toxin was cloned successfully by restriction endonuclease digestion. The diphtheria toxin gene was inserted into the prokaryotic expression vector PET-3a using the NdeI and BamHI sites of the upstream and downstream primers to construct the diphtheria toxin expression vector PET / DT. The T7 promoter was induced by IPTG using BL21 (DE3) as the engineering strain. SDS-PAGE analysis showed that a product band was found at 58000 with an expression level of 25% of that of the bacterial protein. Western-blot experiments showed that the expressed product had the same immunological and biological activities as the toxin secreted by diphtheria.