大肠癌相关抗原体外致敏法构建人源抗体库

Repertoire Antibody Library Constructed from Human Peripheral Blood Lymphocytes with in vitro Immunization with Colorectal Carcinoma-Associated Antigen

建立杂交瘤单抗亲和层析纯化抗原、抗原体外致敏淋巴细胞和RT-PCR克隆人抗体基因及噬菌体呈现技术构建人源抗体库的策略.将亲和层析纯化的大肠癌相关抗原CA-Hb3经SDS-PAGE和免疫印迹鉴定后,与IL-2和丝裂原于体外致敏10个大肠癌病人各10ml外周血淋巴细胞(PBL),出现淋巴母细胞化和集落形成现象,提取总RNA并纯化mRNA,经RT-PCR扩增3种VH-CHI(γ)基因和5种VL-CL(κλ)基因,再经PCR克隆3种VH(γ)和8种VL(κλ)基因.通过(Gly_4Ser)_3相应的寡核苷酸连接序列将VH和VL基因不同组合连成24种单链抗体(ScFv)基因,经 SfiI酶切,将之克隆入fUSE 5RF,用电穿孔法将此表达载体转化MC1061,四环素抗性筛选得到10~6库容的初级抗体库,ScFv基因插入百分率为85%.该策略将可能普遍用于鼠源单抗人源化.

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(γ) and five VL-CL(κ,λ) genes were amplified from their total RNA and mRNA with RT-PCR. Three VH (γ) and 8 VL (κ,λ) 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.