Molecular cloning of Clostridium perfringens epsilon-toxin gene and its high level expression in E-coli
Molecular cloning of Clostridium perfringens epsilon-toxin gene and its high level expression in E-coli
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DOI：
10.1006/bbrc.1996.1422
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发表时间：
1996-09-24
影响因子：
3.1
通讯作者：
Garg, LC
中科院分区：
文献类型：
--
作者：
Goswami, PP;Rupa, P;Garg, LC
采用PCR方法从D型产气荚膜梭菌（Clostridium perfringens type D）分离株中扩增出ε毒素基因，并将其克隆到氨基末端融合有6个组氨酸标签的T5启动子控制下。携带该构建体的大肠杆菌细胞以包涵体的形式表达高水平的重组蛋白。使用Ni 2 +-次氮基三乙酸（NTA）琼脂糖柱上的单步亲和层析纯化蛋白质。用纯化的重组蛋白免疫家兔后，检测到高滴度的抗体。针对重组蛋白产生的抗体能够识别重组毒素以及天然毒素。抗ε毒素单克隆抗体能在Western blot中检测到重组蛋白。重组蛋白的N端序列与已知的毒素序列相匹配。在摇瓶水平，每升培养物产生高达20 mg的纯ε-原毒素。(C)出版社：Academic Press，Inc.
A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end. Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies. The protein was purified using single step affinity chromatography on a Ni2+-nitrilotriacetic acid (NTA) agarose column. Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected. The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin. Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot. N-terminal sequence of the recombinant protein matched with the known sequence of the toxin. At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture. (C) 1996 Academic Press, Inc.