摘要
Objective: To clone the variable region genes (VH and VL) of murine McAb against γ-seminoprotein, and to construct a single chain Fv (ScFv) gene. Methods: Total RNA was extracted from the hybridoma cell line E4B7, which secretes McAb against γ-seminoprotein, and subjected to reverse transcription. The VH and VL genes were amplified by PCR, cloned into pUC19 vector and their sequences were analysed. The VH and VL genes were ligated into a ScFv gene by a peptide linker. The ScFv was inserted into the prokaryotic fusion protein expression vector pGEX-4T-1 and efficiently expressed in Escherichia coli. Results: The E,B, ScFv gene consisted of 741 bp,encoding 247 amino acid residues. A 45 bp linker sequence was found between VH and VL and the predicted amino acid sequence of the linker was (Gly4Ser)3. The expression of GST-ScFv fusion protein was induced by IPTG. SDS-PAGE showed a novel protein band with an apparent molecular mass of 52 000, which was about 40% of total bacterial protein. After primary purification and renaturation, the fusion protein was further purified by GST affinity chromatography. The fusion protein was digested with thrombin and the ScFv was separated. The binding activity of the ScFv was confirmed by competitive binding inhibition assay in vitro. Conclusion: Anti-human γ-seminoprotein ScFv has been successful1y constructed for the use in clinical studies.
Objective: To clone the variable region genes (VH and VL) of murine McAb against γ-seminoprotein, and to construct a single chain Fv (ScFv) gene. Methods: Total RNA was extracted from the hybridoma cell line E4B7, which secretes McAb against γ-seminoprotein, and subjected to reverse transcription. The VH and VL genes were amplified by PCR, cloned into pUC19 vector and their sequences were analysed. The VH and VL genes were ligated into a ScFv gene by a peptide linker. The ScFv was inserted into the prokaryotic fusion protein expression vector pGEX-4T-1 and efficiently expressed in Escherichia coli. Results: The E,B, ScFv gene consisted of 741 bp,encoding 247 amino acid residues. A 45 bp linker sequence was found between VH and VL and the predicted amino acid sequence of the linker was (Gly4Ser)3. The expression of GST-ScFv fusion protein was induced by IPTG. SDS-PAGE showed a novel protein band with an apparent molecular mass of 52 000, which was about 40% of total bacterial protein. After primary purification and renaturation, the fusion protein was further purified by GST affinity chromatography. The fusion protein was digested with thrombin and the ScFv was separated. The binding activity of the ScFv was confirmed by competitive binding inhibition assay in vitro. Conclusion: Anti-human γ-seminoprotein ScFv has been successful1y constructed for the use in clinical studies.
