抗人TNF-α单抗基因的克隆及鉴定

Cloning and identification of variable region genes of anti TNF α McAb

目的克隆抗人ＴＮＦ－α鼠单抗得可变区基因以构建人－鼠嵌合抗体表达载体。方法采用ＲＴ－ＰＣＲ技术，以前导肽序列的引物从１个分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆抗体轻链、重链可变区基因（Ｖκ，ＶＨ），在大肠杆菌中表达Ｆａｂ段核实其功能活性。结果分别得到了２个Ｖκ和２个ＶＨ基因。ＤＮＡ序列测定表明，其中１个轻链可变区基因为骨髓瘤细胞系中固有的无功能基因。１个重链可变区基因经原核系统表达测活表明无抗体活性。另一个轻链和重链可变区基因的成熟蛋白编码部分与从第一骨架区引物所克隆的、可在大肠杆菌表达出抗体活性的Ｖκ、ＶＨ序列相符。将该轻链、重链基因分别克隆到了人－鼠嵌合轻链、重链表达载体中。结论通过原核表达系统核实。

Objective To clone and identy the genes of anti human TNF α McAb. Methods The entire V H and V L genes from a mouse hybridoma secreting anti TNF α monoclonal antibody were cloned by RT PCR, by using 5' primers for leader sequences. Results Two V H and V L genes were obtained. DNA sequence analysis showed that one of the Vκ gene was the aberrant Vκ transcript derived from the myeloma fusion partner. One of the V H was tested by using E.coli secretary expression system and no function was found. The other V H and Vκ had the same sequences as those of the bacterially expressed functional V genes which were cloned by using primers of the first framework region. The V H and V L genes were inserted into human mouse chimeric antibody light and heavy chain expression vectors respectively. Conclusions The variable genes of anti TNF α McAb are obtained the functiou of which was verified through E.coli expression system.