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[Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancer].
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Jan; 26(1):16-21.NF

Abstract

OBJECTIVE

To amplify and express the gene encoding murine single-chain fragment variable (scFv) antibody against human cervical cancer and predict the secondary structure and three-dimensional structure of the antibody.

METHODS

The gene fragments coding for the variable region of the heavy and light chains of scFv antibody against human cervical cancer were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, and spliced together using a flexible linker to the antibody. The scFv genes were then cloned into the expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1, respectively. The scFv antibody obtained was assayed using SDS-PAGE, Western blotting, and immunohistochemical analysis, with its secondary structure and three-dimensional structure predicted with Swiss-model and 3dpssm. The physicochemical properties of the antibody were analyzed with Antheprot software.

RESULTS

The expressed scFv antibody was soluble and phage-displayed. The specific binding capacity of the soluble and phage-displayed scFv antibody to the surface-associated antigen of human cervical cancer cell line were further confirmed. The scFv antibody had a relative molecular mass of 32,000 as was consistent to theoretical prediction, with pI of 7.215 and characterized to belong to alpha+beta protein. The antibody contained many protein kinase C phosphorylation sites and casein kinase II phosphorylation sites in both the VH and VL domains. Computer graphic modeling indicated that the linker was isolated from the VH and VL domains, which formed a hydrophobic pocket in the molecule to facilitate antigen binding.

CONCLUSIONS

The soluble and phage displayed scFv antibody expressed in E. coli against human cervical cancer shows high and specific affinity to the cervical cancer cell line surface-associated antigen. The construction and analysis of the molecular model of the antibody may facilitate further studies in engineering of the antibody and exploration of the mechanism of the antigen-antibody interaction.

Authors+Show Affiliations

Medical Laboratory Center, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China. ywang00@163.comNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

chi

PubMed ID

16495168

Citation

Wang, Ying, et al. "[Expression, Structure Prediction and Functional Analysis of Murine Single-chain Fragment Variable Antibody Against Human Cervical Cancer]." Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, vol. 26, no. 1, 2006, pp. 16-21.
Wang Y, Li X, Chen W. [Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancer]. Nan Fang Yi Ke Da Xue Xue Bao. 2006;26(1):16-21.
Wang, Y., Li, X., & Chen, W. (2006). [Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancer]. Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, 26(1), 16-21.
Wang Y, Li X, Chen W. [Expression, Structure Prediction and Functional Analysis of Murine Single-chain Fragment Variable Antibody Against Human Cervical Cancer]. Nan Fang Yi Ke Da Xue Xue Bao. 2006;26(1):16-21. PubMed PMID: 16495168.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Expression, structure prediction and functional analysis of murine single-chain fragment variable antibody against human cervical cancer]. AU - Wang,Ying, AU - Li,Xu, AU - Chen,Wei, PY - 2006/2/24/pubmed PY - 2007/5/2/medline PY - 2006/2/24/entrez SP - 16 EP - 21 JF - Nan fang yi ke da xue xue bao = Journal of Southern Medical University JO - Nan Fang Yi Ke Da Xue Xue Bao VL - 26 IS - 1 N2 - OBJECTIVE: To amplify and express the gene encoding murine single-chain fragment variable (scFv) antibody against human cervical cancer and predict the secondary structure and three-dimensional structure of the antibody. METHODS: The gene fragments coding for the variable region of the heavy and light chains of scFv antibody against human cervical cancer were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, and spliced together using a flexible linker to the antibody. The scFv genes were then cloned into the expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1, respectively. The scFv antibody obtained was assayed using SDS-PAGE, Western blotting, and immunohistochemical analysis, with its secondary structure and three-dimensional structure predicted with Swiss-model and 3dpssm. The physicochemical properties of the antibody were analyzed with Antheprot software. RESULTS: The expressed scFv antibody was soluble and phage-displayed. The specific binding capacity of the soluble and phage-displayed scFv antibody to the surface-associated antigen of human cervical cancer cell line were further confirmed. The scFv antibody had a relative molecular mass of 32,000 as was consistent to theoretical prediction, with pI of 7.215 and characterized to belong to alpha+beta protein. The antibody contained many protein kinase C phosphorylation sites and casein kinase II phosphorylation sites in both the VH and VL domains. Computer graphic modeling indicated that the linker was isolated from the VH and VL domains, which formed a hydrophobic pocket in the molecule to facilitate antigen binding. CONCLUSIONS: The soluble and phage displayed scFv antibody expressed in E. coli against human cervical cancer shows high and specific affinity to the cervical cancer cell line surface-associated antigen. The construction and analysis of the molecular model of the antibody may facilitate further studies in engineering of the antibody and exploration of the mechanism of the antigen-antibody interaction. SN - 1673-4254 UR - https://www.unboundmedicine.com/medline/citation/16495168/[Expression_structure_prediction_and_functional_analysis_of_murine_single_chain_fragment_variable_antibody_against_human_cervical_cancer]_ L2 - http://www.diseaseinfosearch.org/result/7949 DB - PRIME DP - Unbound Medicine ER -