不同连接肽的双价单链抗体基因的构建及表达

采用基因重组技术分别借助不同长度的连接肽G4S和(G4S)3],将两个相同的抗人大肠癌单链抗体基因ND-1scFv共价连接,构建表达载体pET-28a( )ND-1sc(Fv)2,并在大肠杆菌BL21中表达ND-1sc(Fv)2的融合蛋白。应用Ni2 亲和层析方法对表达产物进行纯化,SDS-PAGE、免疫荧光法(IFA)和ELISA对纯化后的蛋白质进行纯度和免疫活性分析。结果表明成功构建了表达载体pET-28a( )ND-1sc(Fv)2,并在大肠杆菌中获得高效表达,其表达产物以不溶性包涵体形式存在。纯化后ND-1sc(Fv)2-5、ND-1sc(Fv)2-15的蛋白质纯度分别为90%和86%。IFA及ELISA结果表明,二者均保留了亲本抗体的免疫活性,对表达在人大肠癌细胞上的肿瘤相关抗原LEA具有特异结合活性,其免疫活性均明显高于ND-1scFv,其中ND-1sc(Fv)2-15的免疫活性更接近于亲本单抗ND-1,该抗体有望成为大肠癌临床导向诊断和治疗的理想载体。

Construction and Expression of Bivalent Single-chain Antibodies with Different Linker Sequence against Human Colorectal Carcinoma

To engineer and express the bivalent covalent scFv of ND-1mAb against human colorectal carcinoma, the two same ND-1scFv gene were linked by different length linker with sequences encoding G4S and (G4S)3. The expression vector pET-28a( )ND-1sc(Fv)2 was constructed to express the ND-1sc(Fv)2 fusion protein in E.coli BL21. The expressed product was purified by metal affinity chromatography using Ni-NTA resin. The purity and immunoreactivity were analyzed by SDS-PAGE, Immunofluorescence assays (IFA) and ELISA. The results demonstrated that pET-28a( )ND-1sc(Fv)2 gene was constructed and expressed successfully in E.coli BL2 in the form of an inclusion body. The purity of ND-1sc(Fv)2 proteins with 5 amino acid linker and 15 amino acid linker were 90% and 86% respectively. IFA and ELISA revealed that both of ND-1sc(Fv)2-5 and ND-1sc(Fv)2-15 had specific binding activity to the tumor-associated antigen LEA expressed in human colorectal carcinoma cells and obviously higher than that of ND-1scFv, and compared with ND-1sc(Fv)2-5, ND-1sc(Fv)2-15 showed the activity closer to ND-1mAb. They may become potentially useful in clinical diagnosis and therapy as a carrier for human colorectal carcinoma.