Repertoire Antibody Library Constructed from Human Peripheral Blood Lymphocytes with in vitro Immunization with Colorectal Carcinoma-Associated Antigen
- VernacularTitle:大肠癌相关抗原体外致敏法构建人源抗体库
- Author:
Zhiwei HU
;
Qubing SUN
- Publication Type:Journal Article
- Keywords:
antigen;
neoplasm;
human immunoglobulin gene;
reverse transcription;
polymerase chain reaction;
combinatorial antibody library
- From:
Chinese Journal of Cancer Biotherapy
1995;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
A Strategy was established for construction of repertoire antibody library with affinity chromatography purifying antigen, antigen immunizing human lymphocytes, RT-PCR and phage display technology. The colorectal carcinoma-associated antigen CA-Hb3 was purified with affinity column and analysed with SDS-PAGE and Western-blot, then applied for immunizing peripheral blood lymphocytes (PBL) in vitro. The PBL were isolated from ten patients with colorectal carcinoma and cultured with interleukin 2 and pokeweed mitogen, lymphoblast-like cells occurred and colonies formed after immunization. Three VH-CHl(?) and five VL-CL(?,?) genes were amplified from their total RNA and mRNA with RT-PCR. Three VH (?) and 8 VL (?,?) genes were reamplified and randomly combined to construct 24 single-chain variable fragments (ScFv) genes through (Gly_(4)Ser)_(3) linker. ScFv genes digested with Sfi I were cloned into fUSE 5 vector and transformed into MC1061 with electroporation. Repertoire antibody library was obtained with 10~(6) tetracycline-resistant colonies, in which the percentage of ScFv inserts was 85 % . This strategy might be used for humanizing mouse-original monoclonal antibody.
