人胰岛素基因在幼仓鼠肾细胞中的表达及降血糖实验研究

Expression of human insulin gene in baby hamster kidney cells

目的 :构建并筛选人胰岛素基因真核高效表达载体。方法 :常规法构建重组质粒pEF1α ImINS。将重组质粒pEF1α ImINS和胰岛素其他 3个重组质粒分别转染幼仓鼠肾 (BHK )细胞 ,经G418筛选 ,阳性克隆传至 2 0代 ,分别用放射免疫和免疫组化技术分析胰岛素和 (或 )胰岛素原在BHK细胞中表达的情况。并用重组质粒pEF1α ImINS转染BHK细胞的阳性克隆 2 0代的PBS细胞悬浮液 (5× 10 7个 /只 )及其培养上清 (0 .5ml/只 )对昆明小鼠行腹腔注射 ,通过对注射前后小鼠血糖值测定 ,分析转基因产物降血糖的生物学效应。结果 :胰岛素的表达量最高为 7.984μIU/ml ,胰岛素表达水平的灰度值在BHK细胞质中为 177.5± 15.10 ,细胞核中为 150 .3±2 1.43 ;昆明小鼠注射 pEF1α ImINS转染BHK克隆细胞株的悬浮液后 6h与注射前 2 4h血糖值比较 ,差异极显著 (P <0 .0 1)。结论 :BHK细胞中重组质粒 pEF1α ImINS是胰岛素表达量最高的质粒 ;pEF1α ImINS转染的克隆细胞株在小鼠体内有胰岛素表达 。

Objective: To construct and sequence eukar yo tic vector with high effective expression for human insulin. Methods: The recombinant expression plasmid pEF1α-ImINS was constructed accordi ng to the routine method. Four recombinant plasmids of insulin were transfectedinto baby hamster kidney (BHK) cells by using lipofectin reagent. And the expres sion products of insulin gene were detected by using radioimmunoassay (RIA) andimmunohistochemistry when the positive clones were passed to the 20th generation . Then the products of plasmid pEF1α-ImINS were injected into KM mice intraper itoneally to verify whether it has biological effect or not. And the variation o f the concentration of blood sugar was detected 24 hoursbefore injection, 6 an d 72 hours after injection. Results: The highest expression le vel of insulin and/or proinsulin was 7.984μIU/ml detected by RIA. The grey leve l of insulin expressed form pEF1α-ImINS was 177.5±15.10 in cytoplasm, 150.3± 21.43 in nuclear. The statistical analysis showed that the culture medium of BHKcells transfected with pEF1α-ImINS was similar to control group. But the grou p of BHK clone PBS suspension transfected with plasmid pEF1α-ImINS has differe nce between preinjection and postinjection. Conclusion: The ne w recombinant expression plasmid pEF1α-ImINS has the highest expression levelamong the 4 plasmids to express insulin in BHK cells. And the BHK clone cells tr ansfected with pEF1α-ImINS has biological effects.