Fisher K S, Maxwell I H, Murphy J R, Collier J, Glode L M
Department of Medicine, University of Colorado Cancer Center, Denver 80262.
Infect Immun. 1991 Oct;59(10):3562-5. doi: 10.1128/iai.59.10.3562-3565.1991.
We previously demonstrated that cells can be killed through transfection of an expression plasmid that encodes the diphtheria toxin A-chain fragment (DT-A). This report describes the construction of expression plasmids containing three mutant DT-A-coding sequences substituting glutamic acid 148 with aspartic acid, serine, or glutamine which are known to have 100- to 300-fold-reduced ADP-ribosylation activity measured in vitro. The toxicity of these constructs was determined in cotransfection experiments using HeLa and 293 cells with a luciferase expression plasmid as the reporter. Dose responses were compared for the three new DT-A mutant plasmids and for the corresponding plasmids containing wild-type DT-A and the previously characterized tox 176 mutant. The dose required to produce 50% inhibition of control luciferase expression in 293 embryonic kidney cells for the five plasmids ranged from 0.01 micrograms for wild-type DT-A to 1.2 micrograms for the least toxic plasmid, which replaces glutamic acid 148 with glutamine. In conclusion, a wide range of DT-A toxicity can be achieved by using plasmid expression vectors that encode different DT-A mutations.
我们之前证明，通过转染编码白喉毒素A链片段（DT-A）的表达质粒可杀死细胞。本报告描述了含有三个突变DT-A编码序列的表达质粒的构建，这些序列将谷氨酸148分别替换为天冬氨酸、丝氨酸或谷氨酰胺，已知在体外测定时其ADP-核糖基化活性降低了100至300倍。在共转染实验中，使用HeLa细胞和293细胞以及荧光素酶表达质粒作为报告基因，测定了这些构建体的毒性。比较了三种新的DT-A突变体质粒以及含有野生型DT-A和先前表征的tox 176突变体的相应质粒的剂量反应。在293胚胎肾细胞中，使对照荧光素酶表达产生50%抑制所需的剂量，对于这五种质粒，范围从野生型DT-A的0.01微克到毒性最低的质粒（将谷氨酸148替换为谷氨酰胺）的1.2微克。总之，通过使用编码不同DT-A突变的质粒表达载体，可以实现广泛的DT-A毒性范围。
