中国株白喉杆菌白喉毒素全基因编码序列的克隆及其在大肠杆菌中的高效表达  

Cloning, Sequencing and Expression in E. coil of the Structure Gene of Diphtheria Toxin from Chinese Strain of Corynebacterium Diphtheriae

作　　者：张新建[1] 李晶[1] 张宝云[1] 洪涛[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1995年第4期344-348,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：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。By means of polymerase chain reaction(PCR) technology, a 1 .6 kb complete nucleotide sequence that encodes diphtheria toxin(DT) was obtained from a Chinese strain of corynebacterium diphtheria originally isolated from Shenyang in 1990. The resulted restriction enzyme fragments and sequence showed that structural gene from Chinese strain was consistent with other known DT sequences, but some minor variation in base pairing did exist. DT DNA was cloned into BL21(DE3) on the plamind PET-3a and a recombinant DT was expressed successfully wing T7 prmoter. according for 25% of total bacterial protein, Its molecular weight was about 58000. corresponding to the exacted result. Its identity was confirmed by SDS-PAGE. Western-blot and toxicological tests. These results provide an important basis for genetically designing and construction of DT-relaied immuntoxins and vaccines.

关 键 词：白喉杆菌 白喉毒素 克隆 序列分析 大肠杆菌 

分 类 号：R378.71[医药卫生—病原生物学]