人源化抗人肺癌单域抗体基因的构建、表达及活性分析

目的： 构建人源化的抗人肺癌单域抗体hu3D3VH基因, 在大肠杆菌中表达, 对其蛋白活性进行分析.方法： 采用CDR移植技术对mAb3D3的重链可变区进行人源化, 通过重叠PCR获得hu3D3VH的基因.构建pET22（b＋）/hu3D3VH表达载体, 并转化大肠杆菌BL21（DE3）, 在IPTG诱导下表达.表达产物通过Ni亲和层析柱纯化.采用间接ELISA和竞争抑制ELISA法进行活性分析.结果： 通过重叠PCR获得序列正确的目的基因.目的蛋白以包涵体的形式表达, 表达量占菌体总蛋白的30%以上.纯化后, 目的蛋白的纯度达95%以上.hu3D3VH具有与亲本抗体相同的抗原反应性, 并能抑制mAb3D3与L342细胞的结合.结论： 获得的人源化单域抗体hu3D3VH, 保留了与mAb3D3相同的反应性和特异性, 为进一步临床应用奠定了基础.

Gene''''s construction, expression and activity analysis of humanized single-domain antibody against human lung cancer

AIM: To construct the gene of humanized single-domain antibody hu3D3V_H against human lung cancer, to express it in E.coli and analyze its activity. METHODS: The mAb3D3V_H was humanized by using CDRs grafting technique. The hu3D3V_H gene was assembled by overlapping PCR. The expression vector pET22(b )/hu3D3V_H was constructed and transformed into E.coli BL21(DE3) and the hu3D3V_H protein was expressed under IPTG induction. After purification by Ni~ 2 -affinity chromatographic column, the activity of hu3D3VH protein was analyzed by indirect ELISA and competitive inhibition ELISA. RESULTS: The target gene with correct sequence was obtained by overlapping PCR. The hu3D3V_H protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial proteins. After purification, the purity of hu3D3V_H was more than 95%. The reactivity of purified protein was the same as parent antibody, and could inhibit the binding of mAb3D3 to L342 cells. CONCLUSION: The hu3D3V_H still retains the reactivity and specificity of mAb3D3, which lays the foundation for its further clinical application.