人4-1BBL胞外区／抗CD20 Fab＇融合蛋白的构建和表达  

Study on the Construction and Expression of the Human 4-1BBL Extracellular Domain/anti-CD20 Fab' Fusion Protein

作　　者：姜文国[1] 熊冬生 刘芳[2] 郭红星 苏晔 吕晶丽 杨纯正 

机构地区：[1]滨州医学院,滨州256603 [2]中国协和医学科大学,中国医学科学院血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《生物工程学报》2008年第3期376-380,共5页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863)资助(No.2003AA215080);山东省自然科学基金资助(No.Q2006C02)~~

摘　　要：研究共刺激分子4-1BBL在肿瘤靶向治疗方面的作用,用PCR和overlap PCR方法构建人4-1BBL胞外区/抗CD20 Fab’融合蛋白表达载体,并用双脱氧终止法测定DNA序列;采用亲和层析法纯化该产物,并用SDS-PAGE和HPLC鉴定纯化产物;采用玫瑰花环试验鉴定纯化产物与靶细胞的结合活性。DNA序列测定结果表明:人4-1BBL胞外区/抗CD20Fab’融合蛋白已构建成功。表达可溶性产物的产量达200μg/L以上,纯度较高,具有与激活的Jurkat(4-1BBL+)和Raji细胞(CD20+)结合的活性。这将为非何杰金氏淋巴瘤免疫治疗、靶向治疗提供新的思路。Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extraceUular domain/anti-CD20 Fab＇ fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab＇ expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extraceIlular domain / anti-CD20 Fab＇ fusion protein was corrected. The fusion protein was recovered in high yield （up to 200 μg/mL） after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat ceils and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab＇ fusion protein was induced to express in E. coli 16C9, The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.

关 键 词：4-1BBL CD20 靶向治疗 免疫治疗 

分 类 号：Q78[生物学—分子生物学] S512.1[农业科学—作物学]