抗人肿瘤坏死因子-α单链抗体基因的构建及序列测定

Construction and sequencing of the single-chain antibody gene of a human TNF-alpha specific monoclonal antibody

构建鼠抗人肿瘤坏死因子-α（hTNF-α）单链抗体基因．方法：从分泌hTNF-α单抗的小鼠杂交瘤细胞株E6中获得抗体可变区基因的基础上，采用限制性内切酶酶切拼接法，并按VH-Linker-VL的结构将轻、重链可变区基因拼接单链抗体（ScFv）基因，荧光标记测序引物分析其核苷酸序列．结果：构建成长747bp的单链抗体基因，其中连接肽基因长45bP，经序列测定和分析表明为拼接正确、完整的抗hTNF-α单链抗体基因．结论：所构建ScFv为功能性的抗hTNF-α单链抗体基因．

Aim: To construct the single-chain antibody geneof a human tumor necrosis factor (TNF) specific monoclonal antibody. Methods: On the basis of acquiring the heavyand light chain variiable regions from a mouse anti-humanTNF-alpha hybridoma cell line E6, the VH and VL wereconnected into the ScFv by a flexible linker. The ScFv genewere sequenced by 373A auto-sequencing machine made inAmerica. Results: The human TNF-αScFv gene consistedof 747 bp,in which the linker gene was 45 bp. The sequencing analysis showed that the ScFv gene was connected correctly. Conclusion: The data indicate that the constructedgene is the ScFv of the E6 mouse anti-human TNF-αantibody.