人胰岛素原基因胰外表达重组体的构建及鉴定

Construction and identification of the human proinsulin gene recombinant expressing mature insulin in exopancreas

目的 :构建胰外表达成熟胰岛素的重组体。方法 :将人胰岛素原基因进行二处突变 ,突变后的胰岛素原基因与逆转录病毒载体 pLXSN重组并转染肝癌细胞。提取肝癌细胞基因组DNA ,PCR方法鉴定人胰岛素原基因与载体重组结果及在肝癌细胞基因组中整合情况。同时测定成熟胰岛素的表达。结果 :胰岛素原基因已整合到肝癌细胞基因组。突变的胰岛素原可被广泛存在的furin酶识别 ,在培养基内检测到较高浓度的成熟胰岛素。结论 :成功构建胰外表达成熟胰岛素的突变人胰岛素原基因逆转录病毒重组体。

Objective: To construct a recombinant which expressed and secreted mature insulin in exopancreas.Methods: We performed 2 size-directed mutations on the human proinsulin gene at both B-C and C-A junctions. The mutated proinsulin gene was inserted into the retroviral vector pLXSN and transferred to hepatocarcinoma cells.The genome of hepatocarcinoma cells was extracted.The recombination and the valid integration of human proinsulin gene was testified by PCR technique,meanwhile the mature insulin expressed by the recombinant was estimated. Results: The mutated proinsulin gene had integrated into the genome of hepatocarcinoma cells and become suitable substrates for furin,an ubiquitously expressible endoprotease. High concentration of mature insulin was demonstrated in the culture medium of the hepatocarcinoma cells. Conclusion: The recombinant consisted of mutated human proinsulin gene and retroviral vector expressing mature insulin in exopancreas had been successfully constructed.