人源化抗体构建过程中假链的产生及应对策略

A Simple and Rapid Protocol for Cloning of the Functional V Kappa Chain from Aberrant-chain-positive Murine Hybridomas

目的在人源化抗体构建过程中,采用方便、快捷的分子克隆手段,消除SP2/0内源性畸形轻链转录本以获得正确的轻链cDNA。方法在mRNA抽提时,不用常规的TRIZOL法抽提总RNA,而采用经腹腔培养,生长状态良好的杂交瘤细胞,用QIAGEN公司Oligotex Direct mRNA Purification Kit,将polyA+的mRNA富集。可有效减少畸变转录本的含量,提高功能型mRNA的丰度。利用polyA+RNA,采用RT-PCR,我们成功地克隆到了轻链可变区序列,序列分析证实读码框完全正确,属于轻链可变区基因。结果NCBI数据库BLAST显示,克隆的基因序列符合小鼠Ig可变区特征,具有正确的CDR和FR功能区及VJ连接区。结论采用polyA+mRNA富集技术,成功克隆获得阻断型抗人CD154 mAb(4F1)单克隆抗体的轻链、重链可变区基因,成功地消除了SP2/0内源性畸形转录本对免疫球蛋白功能性轻链基因的影响。

Objective To eliminate endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by a simple and rapid cloning strategy. Methods In order to eliminate the unfunctional V kappa transcripts which may hinder or prevent the amplification of correct V kappa chain, Oligotex Direct mRNA Purification system was employed to covalently bind polyA^＋ mRNA with higher capacity and accuracy to reduce the endogenous abVk contamination. Results The result of NCBI gene data bank evidenced that the cDNA we got by Oligotex Direct mRNA Purification kit was correct with typical CDRs and FRs domain and functional VJ recombination site. Conclusion This protocol describes the application of a reverse transcriptase PCR strategy to allow the cloning and sequencing of the functional kappa light chain cDNAs from murine hybridomas co-exPressing aberrant endogenous kappa chain mRNAs.