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[Study on the construction and expression of the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein].
Sheng Wu Gong Cheng Xue Bao. 2008 Mar; 24(3):376-80.SW

Abstract

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 extracellular 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 extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells 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.

Authors+Show Affiliations

Binzhou Medical College, Binzhou 256603, China. jiangwg@gmail.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

18589811

Citation

Jiang, Wenguo, et al. "[Study On the Construction and Expression of the Human 4-1BBL Extracellular domain/anti-CD20 Fab' Fusion Protein]." Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology, vol. 24, no. 3, 2008, pp. 376-80.
Jiang W, Xiong D, Liu F, et al. [Study on the construction and expression of the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein]. Sheng Wu Gong Cheng Xue Bao. 2008;24(3):376-80.
Jiang, W., Xiong, D., Liu, F., Guo, H., Su, Y., Lü, J., & Yang, C. (2008). [Study on the construction and expression of the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein]. Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology, 24(3), 376-80.
Jiang W, et al. [Study On the Construction and Expression of the Human 4-1BBL Extracellular domain/anti-CD20 Fab' Fusion Protein]. Sheng Wu Gong Cheng Xue Bao. 2008;24(3):376-80. PubMed PMID: 18589811.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Study on the construction and expression of the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein]. AU - Jiang,Wenguo, AU - Xiong,Dongsheng, AU - Liu,Fang, AU - Guo,Hongxing, AU - Su,Ye, AU - Lü,Jingli, AU - Yang,Chunzheng, PY - 2008/7/1/pubmed PY - 2009/9/9/medline PY - 2008/7/1/entrez SP - 376 EP - 80 JF - Sheng wu gong cheng xue bao = Chinese journal of biotechnology JO - Sheng Wu Gong Cheng Xue Bao VL - 24 IS - 3 N2 - 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 extracellular 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 extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells 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. SN - 1000-3061 UR - https://www.unboundmedicine.com/medline/citation/18589811/[Study_on_the_construction_and_expression_of_the_human_4_1BBL_extracellular_domain/anti_CD20_Fab'_fusion_protein]_ L2 - https://www.lens.org/lens/search?q=citation_id:18589811 DB - PRIME DP - Unbound Medicine ER -