Fernando G J, Murray B, Zhou J, Frazer I H
University of Queensland, Department of Medicine, Princess Alexandra Hospital, Brisbane, Australia.
Clin Exp Immunol. 1999 Mar;115(3):397-403. doi: 10.1046/j.1365-2249.1999.00813.x.
E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity.
E7是与宫颈癌相关的16型人乳头瘤病毒（HPV16）产生的主要致癌蛋白。该蛋白在大肠杆菌中作为谷胱甘肽-S-转移酶（GST）融合蛋白表达。从细菌裂解物中收集富含E7的包涵体，将其溶解在10 M尿素中，然后使用阴离子交换柱色谱法纯化该蛋白。通过连续的Triton X-114萃取去除内毒素后，获得了高纯度（约90%）的材料，适用于人体临床试验。该材料具有免疫原性，用作疫苗时可保护小鼠免受HPV16 E7 DNA转染的肿瘤细胞系的攻击。基于这一观察结果，E7GST融合蛋白目前正在用于一项针对HPV16诱导的宫颈癌疫苗的人体临床试验。该融合蛋白可用凝血酶切割以去除GST融合部分，并通过制备性SDS凝胶电泳进一步纯化，以获得纯度>98%的游离E7。
