人胰岛素突变体基因的融合表达

目的　以 pTXB1作为融合表达载体 ,构建人胰岛素突变体 (M insulin)的基因表达克隆 ,表达及纯化。 方法　基因合成胰岛素突变体的cDNA序列 ,酶切后装入融合表达质粒 pTXB1,基因测序正确后转化大肠杆菌BL2 1(DE3) ,诱导表达 ,初步纯化。结果　融合蛋白的分子量约为 330 0 0 ,表达量占菌体总蛋白的 5 0 %以上。融合蛋白主要以包涵体的形式存在 ,洗涤包涵体后融合蛋白的纯度达到 85 %以上。Western blot表明表达蛋白具有胰岛素抗原活性。活性测定显示 ,每升诱导培养后的菌液表达产物中 ,具有的放射免疫活性为 0 .5U。结论　成功地构建了人胰岛素突变体的融合表达体系 ,为进一步的研究奠定了基础。

Expression of human insulin mutant gene in fusion protein form

Objective To construct expression clone of human insulin mutant, M-insulin. Methods cDNA of M-insulin was achieved by gene synthesis, and then prokaryotic expression vector pTXB1-M-insulin was constructed. The expression plasmid was identified with PCR and DNA sequencing. pTXB1-M-insulin was transformed into E.coli BL21(DE3). The expressed protein was identified by SDS-PAGE and Western-blot. Radioimmunoassay was used to test the immune activity of M-insulin fusion protein. Results DNA sequencing demonstrated sequence of M-insulin gene was accurate. SDS-PAGE and densitometric scan analysis showed the molecularweight of M-insulin fusion protein was about 33 000 and its expression level was above 50% of total bacterial proteins. Western-blot demonstrated M-insulin fusion protein could specifically bind insulin antibody. Radioimmunoassay showed the radioimmune activity of M-insulin fusion protein was 0.5 unit per litre bacterial liquid. Conclusion Expression clone of human insulin mutant is successfully constructed.