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[Generation of anti-rhEPO ScFv by using phage display technology].

Abstract

In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E. coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA. Western blot and Dot-blot. The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD. The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids.

Authors+Show Affiliations

Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

chi

PubMed ID

12515169

Citation

Zhong, L, et al. "[Generation of anti-rhEPO ScFv By Using Phage Display Technology]." Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi = Zhonghua Shiyan He Linchuang Bingduxue Zazhi = Chinese Journal of Experimental and Clinical Virology, vol. 12, no. 1, 1998, pp. 38-42.
Zhong L, Wu S, Yang X. [Generation of anti-rhEPO ScFv by using phage display technology]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 1998;12(1):38-42.
Zhong, L., Wu, S., & Yang, X. (1998). [Generation of anti-rhEPO ScFv by using phage display technology]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi = Zhonghua Shiyan He Linchuang Bingduxue Zazhi = Chinese Journal of Experimental and Clinical Virology, 12(1), 38-42.
Zhong L, Wu S, Yang X. [Generation of anti-rhEPO ScFv By Using Phage Display Technology]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 1998;12(1):38-42. PubMed PMID: 12515169.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Generation of anti-rhEPO ScFv by using phage display technology]. AU - Zhong,L, AU - Wu,S, AU - Yang,X, PY - 2003/1/8/pubmed PY - 2003/2/4/medline PY - 2003/1/8/entrez SP - 38 EP - 42 JF - Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology JO - Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi VL - 12 IS - 1 N2 - In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E. coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA. Western blot and Dot-blot. The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD. The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids. SN - 1003-9279 UR - https://www.unboundmedicine.com/medline/citation/12515169/[Generation_of_anti_rhEPO_ScFv_by_using_phage_display_technology]_ DB - PRIME DP - Unbound Medicine ER -