中国株白喉杆菌毒素全基因编码序列的克隆

Cloning and sequencing of the structural gene of diphtheria toxin from Chinese strain of Corynebacterium diphtheriae

本实验从我国自己分离的白喉杆菌噬菌体中成功地克隆了白喉毒素的全基因。此基因克隆是以自己设计合成的寡核苷酸为引物，以Ｂ棒状噬菌体ＤＮＡ为模板，用热启动聚合酶链反应（ＰＣＲ）一次扩增出全基因编码序列。该基因全长共１６０５个碱基对。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，核苷酸序列分析并与已知序列比较，结果表明，所获白喉毒素基因的序列与已知序列基本一致，仅有个别碱基位点的差异。该研究为在我国开展白喉毒纱基因工程疫苗及其相关的融合蛋白的研究奠定了必备的基础。

A 1 . 6 kb complete nucleotide sequence that encodes diphtheria toxin ( DT ) was cloned from a Chinese strain of Ctrynebacterium diphtheriae by polymerase chain reaction ( PCR ) . The strain of Ctynebacterium diphtheriae was originally isolated from Shenyang ,Liaoning Province in 1990, and identified by the National Institute for the Control of Pharmaceutial and Biological Products , Beijing. The obtained restriction enzyme fragments and the sequence showed chat DT structural gene from Chinese strain was consistent with other known DT sequences , but some minor variations in base pairing did exist. The open reading frame of the sequence encoding 535 amino acid residues , corresponding to the expected result. This study provided and important basis for the development of novel genetic DT- related immunotoxins or Combined vaccines.