为建立重组基因表达白喉毒素的分离纯化方法,制备出具有生物活性的蛋白质毒素,构建了白喉毒素表达载体pET22b-DT,并转入大肠杆菌BL21(DE3)中诱导表达,以融合表达的His-6作为标签通过亲和层析纯化;表达毒素经蛋白酶酶切后,形成了以二硫键连接的与天然结构相同的带"缺口"的白喉毒素,并测定白喉毒素对豚鼠的急性毒性和细胞毒性。结果表明,该项研究所建立的表达系统有效地表达了DT,表达量为776.26mg/L;高于国内外同类产物的表达量。DT的 N端序列为MGADDVV……,与实验设计一致。重组表达毒素对豚鼠ig染毒的LD50为0.95μg/kg;对CHO-K1、BS-C1、Hela三种细胞的IC50分别为1.709μg/ml、1.424ng/ml、28.947ng/ml。
The principal purpose of this study is to set up the isolation methods for diphtheria toxin expressed by genes, to purify the toxin with complete biological activities, and to make researches on its toxicities. In the experiments, the target gene was inserted into the cloning vectors--pET22b, then the vectors were introduced into host cells -BL21(DE3). The expression of the toxin was induced by addition of IPTG. The expression products were purified by His-6 label affinity chromatography. Then the diphtheria toxin expressed was transferred to PVC membrane using semidry blotter to determine the amino acid sequence of N-terminal. The "nicked" diphtheria toxin linked by disulfide bond was gained by protease hydrolysis. At the same time, acute toxicities of diphtheria toxin in guinea pig, cytotoxicity in three kinds of cells were tested. The results indicated that the expression system established in the study expressed DT effectively. Amino acid sequencing of expressed DT gave the N- terminal sequence- MGADDVV…. This was identical with the experiment project. The LD50 of DT in guinea pig (ig) was 0.64μg/kg; and the IC50 values of DT in CHO-K1, BS-C1 and Hela were 1.709μg/ml, 1.424ng/ml and 28.947ng/ml respectively.
高川,王惠芳,张靖,宋云扬. 基因工程表达白喉毒素的纯化与生物活性评价[J]. 中国生物工程杂志, 2006, 26(0): 1-7.
. purification and biological activities evaluation of diphtheria toxin expressed by gene engineering. China Biotechnology, 2006, 26(0): 1-7.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I0/1
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