利用线性表达框高通量表达人源单克隆抗体

High- Thoughput Expression of IgG by Establishing Gene Expression Cassettes

目的:建立不通过克隆步骤高通量表达来源于人单个B细胞的抗体轻、重链基因的方法。方法和结果:PCR扩增3个末端重叠的DNA片段,即1CMV启动子和编码抗体引导区序列的片段;2抗体Ig G1重链恒定区序列和牛生长激素(BGH)poly(A)信号序列,轻链Igκ恒定区序列和BGH poly(A)信号序列,轻链Igλ恒定区序列和BGH poly(A)信号序列;以及3抗体基因可变区序列V_H、V_κ或V_λ。3个片段通过重叠延伸PCR构建全长线性片段即线性表达框,将此来源于人单个B细胞的配对的抗体轻、重链线性表达框共转染293E细胞,72 h收集上清检测到表达的抗体。结论:构建的抗体基因线性表达框是无须克隆,快速高通量表达抗体基因进行筛选分析的策略。

Objective： To develop an efficient strategy which high-throughput expressed Ig VH and VL genes iso-lated from single B cells as Ig G antibody without a cloning step. Methods Results： Three overlapping DNAfragments were amplified： ①fragment made of the CMV promoter and sequence encoding for an Ig leader; ②ei-ther the H fragment made of the Ig G1 constant region and BGH poly（A） signal sequences, K fragment made ofthe Igκ constant region and BGH poly（A） sequences, or L fragment made of the Igλ constant region and BGHpoly（A） sequences; and ③either the VH, Vκ or Vλ genes amplified from single B cells. Then the linear full-lengthIg heavy- and light-chain gene expression cassettes were assembled by PCR from these fragments. Co-transfectionof paired Ig heavy and light-chain gene in the form of linear gene expression cassettes produced whole Ig G mole-cules with molecular weights of 150 k D. Conclusion： This Ig gene expression cassettes constitute a highly effi-cient strategy for rapid expression of Ig gene for high-throughput screening and analysis without cloning.