中文摘要：

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Ni^2＋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）12-5、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。

英文摘要：

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-1 sc（Fv）2 was constructed to express the ND-1 sc（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-1 sc（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- 1 sc（Fv）2-5 and ND- 1 sc（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- 1 scFv, and compared with ND- 1 sc（Fv）2-5, ND- 1 sc（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.