抗人大肠癌双价单链抗体基因的构建及表达  

Construction and expression of bivalent single-chain antibody against human colorectal carcinoma

作　　者：严丹丹[1] 杨福辉[1] 方瑾[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室卫生部细胞生物学重点实验室,辽宁省沈阳市110001

出　　处：《世界华人消化杂志》2006年第24期2395-2400,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.85-722-18-02;20375047~~

摘　　要：目的:构建抗人大肠癌重组双价单链抗体ND- 1sc(Fv)2的融合基因,并在大肠杆菌中表达．方法:采用基因重组技术借助GGGGS Linker序列,将2个相同的ND-1scFv基因共价连接,构建pET-28a(+)ND-1sc(Fv)2表达载体,并在大肠杆菌中表达双价单链抗体的融合蛋白．表达产物用Ni-NTA resin亲和层析方法纯化,SDS- PAGE和Western blotting检测表达的目的蛋白．间接免疫荧光和ELISA检测抗体的免疫活性．结果:序列测定表明:ND-1sc(Fv)2基因全长1473 bp,包含了2个729 bp的scFv序列和15 bp的GGGGS序列．SDS-PAGE和Western blotting检测显示,融合蛋白的Mr55 000,与预期值一致．ND-1sc(Fv)2表达产物以不溶性包涵体形式存在,经亲和层析纯化后蛋白纯度达90％,间接免疫荧光及ELISA检测表明,ND-1sc(Fv)2保留了亲本抗体的免疫活性,对表达有相应抗原的靶细胞具有特异结合活性,其免疫活性明显高于ND-1scFv．结论:成功地构建了抗人大肠癌双价单链抗体ND-1sc(Fv)2,并在大肠杆菌中高效表达．融合蛋白具有良好的免疫活性．AIM： To construct the fusion gene of recombinant bivalent single-chain antibody ND-1sc（Fv）2 against human colorectal carcinoma and express it in E coil METHODS： The two same ND-1scFv genes against human colorectal carcinoma were covalently linked by a 15-bp linker with sequences encoding GGGGS. The expression vector pET- 28a（＋）ND-1sc（Fv）2 was constructed to express the ND-1sc（Fv）2 fusion protein in E. coli. The product was purified by metal affinity chromatography using Ni-NTA resin and then detected by SDS-PAGE and Western blotting. Its immunity was analyzed by enzyme-linked immunosorbent assay （ELISA） and indirect immunofluorescence assay （IFA）. RESULTS： Sequencing results showed that the ND-1sc（Fv）2 gene was 1473 bp in length, including two scFv of 729 bp and one GGGGS of 15 bp. SDS-PAGE and Western blotting showed that the relative molecular mass of fusion protein was about 55 kDa, in accordance with its predicted Mr value. ND-1sc（Fv）2 was expressed in the form of an inclusion body, and SDS-PAGE analysis of the purified ND-1sc（Fv）2 showed 90% purity. IFA and ELISA revealed that ND-1sc（Fv）2 had specific binding activity to the corresponding antigen-target cells, obviously higher than that of ND-1scFv. CONCLUSION： ND-1sc（Fv）2 gene against human colorectal carcinoma is constructed and expressed successfully in E. coll. The obtained fusion protein exhibits an excellent immunity.

关 键 词：双价单链抗体 ND-1 大肠癌 免疫活性 

分 类 号：R735.34[医药卫生—肿瘤]