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

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.