第一内含子对人胰岛素基因在BHK细胞中表达的影响

The Influence of the First Intron of Human Insulin Gene on Its Expression in BHK Cells

目的 构建人胰胰岛素基因真核表达载体 ,为胰岛素基因研究奠定基础。方法 从人基因组文库中扩增胰岛素基因的第一内含子 ,经测序证实其序列完全正确后 ,与pCMV mINS中的mINS基因融合 ,构建成带有第一内含子的胰岛素基因组基因真核表达质粒pCMV ImINS。分别用pCMV mINS和pCMV ImINS转染BHK细胞 ,经G4 18筛选 ,将阳性克隆传至 2 0代后 ,用放免方法和免疫组化法分析胰胰岛素和 或胰岛素原在BHK细胞中的表达量情况。结果 经放免测定 ,pCMV mINS和pCMV ImINS在BHK细胞中胰岛素和 或胰岛素原的表达量分别为4 0 77μIU ml和 5 0 6 8μIU ml。经免疫组化分析 ,pCMV mINS在BHK细胞质中胰岛素表达水平的灰度值为 190 0±19 5 6 ;pCMV ImINS在BHK细胞质中胰岛素表达水平的灰度值为 186 4± 10 2 4。

Objective To construct eukaryotic higher expression vector which could express human insulin. Methods The first intron of human insulin genomic gene was specifically amplified with the method of PCR and cloned into the expression vector pCMV mINS constructed formerly to form a new recombinant expression plasmid pCMV ImINS. pCMV mINS and pCMV ImINS were transfected into BHK cells by lipofectin reagent respectively. And the expression products of insulin gene were detected by radioimmunoassay (RIA) and immunohistochemistry when the positive clones were passed to 20th generation. Results The expression levels of insulin and/or proinsulin were 4 077μIU/ml and 5 068μIU/ml respectively, detected by RIA. The grey level of insulin expressed from pCMV mINS was 190 0±19 56 in cytoplasm, from pCMV ImINS was 186 4±10 24 in.Conclusion The result showed that the expression level of recombinant plasmids containing the first intron was higher than that of which without the first intron in BHK cells.