Calderwood S B, Acheson D W, Goldberg M B, Boyko S A, Donohue-Rolfe A
Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
Infect Immun. 1990 Sep;58(9):2977-82. doi: 10.1128/iai.58.9.2977-2982.1990.
We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter. The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography. The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit. B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells. This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.
我们构建了一种质粒表达载体（pSBC32），该载体在可诱导的trc启动子控制下编码志贺毒素/志贺样毒素I的B亚基。所编码的B亚基被转运至周质空间，通过使用受体类似物亲和色谱法，可从周质提取物中一步纯化出毫克量的该蛋白。纯化后的B亚基与针对志贺毒素的多克隆抗血清以及对B亚基具有特异性的单克隆抗体均能正常相互作用。在该系统中纯化得到的B亚基为五聚体（如同天然全毒素），并且在阻断志贺全毒素与HeLa细胞结合方面具有生物学活性。该表达系统或许能够快速纯化出足量的志贺毒素B亚基，以尝试进行结晶，或者研究其作为疫苗单独使用或与合适的多糖抗原偶联使用时的功效。
