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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.
The strategy of constructing human antibody library by hybridoma monoclonal antibody affinity chromatography with purified antigen, antigen in vitro sensitized lymphocytes, and RT-PCR cloning of human antibody genes and phage display technology was developed. The purified colorectal cancer-associated antigen CA was purified by affinity chromatography. -Hb3 was identified by SDS-PAGE and Western blotting. Ten lymphocytes (PBL) of 10 patients with colorectal cancer were sensitized with IL-2 and mitogen in vitro. Lymphoblastization and colony formation were observed. Total RNA was purified and mRNA was purified. Three VH-CHI (γ) genes and five VL-CL (κλ) genes were amplified by RT-PCR. Three kinds of VH (γ) and 8 VL (κλ) genes were cloned by PCR. Through the (Gly_4Ser) _3 the corresponding oligonucleotide link sequence VH and VL gene different combinations of 24 single-chain antibody (ScFv) gene, SfiI enzyme, cloned into fUSE 5RF, using electroporation The expression vector was transformed into MC1061, tetracycline resistance was screened to obtain a library of primary antibodies with 10-6 repertoires. The percentage of insertion of ScFv gene was 85%. This strategy may be universally used for humanization of murine monoclonal antibody.