Repertoire Antibody Library Constructed from Human Peripheral Blood Lymphocytes with in vitro Immunization with Colorectal Carcinoma-Associated Antigen / 中国肿瘤生物治疗杂志
Chinese Journal of Cancer Biotherapy
; (6)1995.
Article
em Zh
| WPRIM
| ID: wpr-581789
Biblioteca responsável:
WPRO
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.
Texto completo:
1
Índice:
WPRIM
Idioma:
Zh
Revista:
Chinese Journal of Cancer Biotherapy
Ano de publicação:
1995
Tipo de documento:
Article