鼠抗hTNF－α单克隆抗体轻链可变区基因片段的克隆和cDNA序列测定

Cloning and Sequencing of the Light Chain Fragment of Variable region genes of an Anti-hTNF-α Monoclonal Antibody

目的：获得鼠抗人肿瘤坏死因子－α（ｈＴＮＦ－α）单克隆抗体轻链可变区基因序列。方法：采用反转录ＰＣＲ（ＲＴ－ＰＣＲ）方法，以一对针对鼠Ｉｇ轻链可变区（ＶＬ）基因的引物，从分泌鼠抗ｈＴＮＦ－α单克隆抗体的Ｅ６杂交瘤细胞株中扩增和克隆ＶＬ基因片段，用双脱氧链终止法测定其核苷酸序列，并进行计算机分析。结果：此ＶＬ基因长３４２ｂＰ，可编码１１４个氨基酸，为开放读框；有明确的框架区（ＦＲＳ）和抗原互补决定区（ＣＤＲＳ）；含有抗体可变区特征性的两个半胱酸残基。在基因数据库（ＥＭＢＬＧｅｎｅＢａｎｋ１９９５）中，未查见相同序列的基因。结论：此ＶＬ基因系重排的鼠Ｉｇ轻链基因。

Objective: To acquire the light chain fragment of variable region genes of an antihTNF-a monoclonal antibody. Method: Reverse transcriptional-PCR was applied to amplify the light chain variable region gene from total RNA of a cultured hybridoma cell line E6 which secretes the specific anti-hTNF- α McAb. The gene fragment was amplified with a pair of primers designed according to mouse ig VL gene segment. The PCR product was sequenced using the dideoxy chain termination technique. Results: The VL gene consists of 342 base pairs and encodes 114 amino acids. It was an open reading frame. There are four FRs, three CDRs and two characteristic cysteine residues within this VL gene. And we have not found the same VL gene in EMBL Gene Bank 1995. Conclusion: The VL gene was confirmed as a functionally rearranged mouse immunoglobulin linght chain variable region gene.