Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.
- Author:
Ye ZHOU
1
;
Zhen-rui CHEN
;
Wei HE
;
Hai-bo LOU
;
Zhe-huan ZHANG
;
Shu-wen LIU
;
Shi-bo JIANG
;
Shu-guang WU
;
Chang-zheng LI
;
Chen ZHOU
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Animals; Antibodies; genetics; immunology; Antibody Specificity; Arthritis, Rheumatoid; immunology; Cell Surface Display Techniques; Cloning, Molecular; Escherichia coli; genetics; metabolism; Genetic Vectors; genetics; HEK293 Cells; Humans; Immunoglobulin G; biosynthesis; genetics; Immunoglobulin Heavy Chains; biosynthesis; genetics; Immunoglobulin kappa-Chains; biosynthesis; genetics; Lymphocytes; immunology; metabolism; Molecular Sequence Data; Peptide Library; Recombinant Proteins; biosynthesis; genetics; immunology; Transfection
- From: Journal of Southern Medical University 2011;31(8):1369-1373
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.
METHODSPeripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.
RESULTSThe libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).
CONCLUSIONThe constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.
