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[Construction and expression of single chain Fv antibody against human bladder carcinoma].
Zhonghua Wai Ke Za Zhi. 2001 Oct; 39(10):792-5.ZW

Abstract

OBJECTIVE

To construct and express single chain Fv antibody against human bladder carcinoma, which was expected to have advantages in targeted diagnosis and therapy with the characteristics of lower immunogenicity.

METHODS

Hybridoma BDI-1 cell, which secreted a monoclonal antibody against human bladder carcinoma, was used to isolate total RNA. By reverse transcription, the cDNA was synthesized and used as templates for amplifying the immunoglobulin heavy-and light-chain variable region genes by polymerase chain reaction (PCR). The amplified DNA was ligated into a sequencing vector pUC19 and sequenced with Sanger's method. The VH and VL genes were inserted into expression vector pFUW80. By inducing, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected by ELISA. The 5 x his-tagged ScFv antibodies were purified on IDA-Ni2+ resin by immobilized metal chelate affinity chromatography (IMAC). The purified ScFv antibodies were analyzed by SDS-PAGE.

RESULTS

A full-length of VH and VL genes was 366 and 324 base pairs respectively. Comparing with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from re-arrangement of VH, Dsp2.2 and JH4; the VL gene was VK subgroup IV and from Vk and Jk4. The ScFv antibodies could inhibit 84% of the antigen binding activity of original McAb BDI-1. The purified ScFv antibodies gave a single major band (Mr-29 000) on SDS-PAGE.

CONCLUSION

The single chain Fv antibody against human bladder carcinoma was successfully constructed and expressed.

Authors+Show Affiliations

Institute of Urology, Peking University, Beijing 100034, China.No affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

16201198

Citation

Zhang, M, et al. "[Construction and Expression of Single Chain Fv Antibody Against Human Bladder Carcinoma]." Zhonghua Wai Ke Za Zhi [Chinese Journal of Surgery], vol. 39, no. 10, 2001, pp. 792-5.
Zhang M, Yu L, Huang H. [Construction and expression of single chain Fv antibody against human bladder carcinoma]. Zhonghua Wai Ke Za Zhi. 2001;39(10):792-5.
Zhang, M., Yu, L., & Huang, H. (2001). [Construction and expression of single chain Fv antibody against human bladder carcinoma]. Zhonghua Wai Ke Za Zhi [Chinese Journal of Surgery], 39(10), 792-5.
Zhang M, Yu L, Huang H. [Construction and Expression of Single Chain Fv Antibody Against Human Bladder Carcinoma]. Zhonghua Wai Ke Za Zhi. 2001;39(10):792-5. PubMed PMID: 16201198.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Construction and expression of single chain Fv antibody against human bladder carcinoma]. AU - Zhang,M, AU - Yu,L, AU - Huang,H, PY - 2005/10/6/pubmed PY - 2006/3/15/medline PY - 2005/10/6/entrez SP - 792 EP - 5 JF - Zhonghua wai ke za zhi [Chinese journal of surgery] JO - Zhonghua Wai Ke Za Zhi VL - 39 IS - 10 N2 - OBJECTIVE: To construct and express single chain Fv antibody against human bladder carcinoma, which was expected to have advantages in targeted diagnosis and therapy with the characteristics of lower immunogenicity. METHODS: Hybridoma BDI-1 cell, which secreted a monoclonal antibody against human bladder carcinoma, was used to isolate total RNA. By reverse transcription, the cDNA was synthesized and used as templates for amplifying the immunoglobulin heavy-and light-chain variable region genes by polymerase chain reaction (PCR). The amplified DNA was ligated into a sequencing vector pUC19 and sequenced with Sanger's method. The VH and VL genes were inserted into expression vector pFUW80. By inducing, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected by ELISA. The 5 x his-tagged ScFv antibodies were purified on IDA-Ni2+ resin by immobilized metal chelate affinity chromatography (IMAC). The purified ScFv antibodies were analyzed by SDS-PAGE. RESULTS: A full-length of VH and VL genes was 366 and 324 base pairs respectively. Comparing with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from re-arrangement of VH, Dsp2.2 and JH4; the VL gene was VK subgroup IV and from Vk and Jk4. The ScFv antibodies could inhibit 84% of the antigen binding activity of original McAb BDI-1. The purified ScFv antibodies gave a single major band (Mr-29 000) on SDS-PAGE. CONCLUSION: The single chain Fv antibody against human bladder carcinoma was successfully constructed and expressed. SN - 0529-5815 UR - https://www.unboundmedicine.com/medline/citation/16201198/[Construction_and_expression_of_single_chain_Fv_antibody_against_human_bladder_carcinoma]_ L2 - http://journal.yiigle.com/LinkIn.do?linkin_type=pubmed&issn=0529-5815&year=2001&vol=39&issue=10&fpage=792 DB - PRIME DP - Unbound Medicine ER -