GM-CSF(12-127)突变体生物学活性的研究

GM－CSF的结构与功能研究表明，N-端的1～13位氨基酸并非生物学活性所必需，且干扰与受体的结合。作者采用PCR突变的方法删除1～11位的氨基酸，保留12位的脯氨酸。突变后的基因经全序列测定证实，之后插入原核高效表达载体pBV220中表达，基因突变后的表达量及表达产物的稳定性均有所提高。大批量表达后经提取包涵体、疏水层析、离子交换层析，最终获得了纯的GM－CSF（12－127）突变体蛋白，其生物学活性比未突变蛋白有明显提高，达3×107U／mg蛋白。受体竞争结合实验显示突变体蛋白受体亲和活性增强。

Study on Biological Activity of GM-CSF (12-127) Mutant

The structure and function of GM-CSF was studied. The result showed that the amino acids 1～13 at the N-terminal of GM-CSF was not essential for its biological activ-ity, and interfered with the receptor binding. By using PCR technique, the amino acids 1～11 were deleted, and the amino acid 12 (Pro) was retained. The mutant GM-CSF cDNA was verified by whole DNA sequencing, then inserted into pBV220(a high experession prokaryot-ic vector) for expression. The expression level and stability of GM-CSF (12-127) mutant were significantly increased. The final GM-CSF (12-127) mutant protein was extracted from E. coli the inclusion body, and purified by hydrophobic and iron exchange chromatographies.The biological activity of the mutant protein reached 3 × 107U/mg protein, that was signifiantly higher than that cf GM-CSF protein. Receptor competitive binding test showed that the affinity of receptor binding of the mutant was significantly improved.