摘要
目的构建在肝细胞内增强表达成熟人胰岛素的生理调控性人胰岛素原基因重组质粒。方法利用PCR定点突变技术在B-C及C-A连接处已两点突变的人胰岛素原基因上进行B10位突变,并在其上游连接肝细胞特异的3倍体葡萄糖反映元件(GLRE3)和胰岛素样生长因子结合蛋白-1启动子(IGFBP-1P)。经酶切后将含有调控元件的3点突变人胰岛素原基因(GLRE3-IGFBP-1P-3mINS)插入逆转录病毒载体(pLXSN),脂质体介导转染大鼠肝癌细胞CBRH7919后检测成熟胰岛素表达情况及与含有调控元件的2点突变人胰岛素原基因(GLRE3-IGFBP-1P-3mINS)表达体的表达差异。结果人胰岛素原基因的B10位组氨酸编码序列CAC突变为门冬氨酸编码序列GAC。构建了含调控元件的B10位门冬氨酸人胰岛素原基因逆转录病毒载体质粒(pLXSN-GLRE3-IGFBP-1P-3mINS),酶切、PCR及测序鉴定各段基因碱基序列及连接方向正确。pLXSN-GLRE3-IGFBP-1P-3mINS经脂质体包裹转染大鼠肝癌细胞,细胞外液含5.0、25.0mmol/L的葡萄糖浓度下胰岛素含量分别为5.03±0.72、43.90±2.30mU/L。未进行B10位突变的重组体转染组在同样葡萄糖浓度下胰岛素含量分别为<2.00、2.10±0.23mU/L。结论成功构建了含调控元件的B10位门冬氨酸人胰岛素原基因逆转录病毒载体重组质粒,该重组体已整合入鼠肝癌细胞基因组,其表达效率显著提高并受葡萄糖生理性调控。
Objective： To construct the glucose-activated human proinsulin gene recombinants intensively expressing mature insulin in hepatocytes. Methods： To perform B10 site-directed mutagenesis in the B-C and C- A site-mutated human proinsulin gene. Then the glucose-responsive elements （GLRE3） specifically expressing in hepatocytes and insulin-like growth factor binding protein-1 promotors（IGFBP-1P） were ligated to the upstream of the mutated human proinsulin gene. The three site-mutated human proinsulin gene containing regulatory elements （GLRE3-IGFBP-1P-3mINS） was inserted into the retroviral vector （pLXSN） after restrictive endonuclease digestion, and transfered into rat＇s hepatoeareinoma cells CBRH7919 mediated by liposomes. The mature insulin level was detected in the culture media and the expressive difference between GLRE3-IGFBP-1P-3mINS and two site-mutated human proinsulin gene recombinants （GLRE3-IGFBP-1P-2mINS） was compared. Results The sequence of CAC coding for Histidine at B10 site of human proinsulin gene was mutated to the sequence of GAC coding for AsparticAcid. The retroviral vector plasmids containing three site mutated human proinsulin gene and regulatory elements（pLXSN- GLRE3- IGFBP-1P-3mINS） was constructed, and the correctness of base sequence and colligated direction was identified via restriction endonuclease, PCR, and sequencing. The recombined plasmids was encapsuled by liposomes and transfered to the hepatocarcinoma cells. The insulin level measured in the culture media containing 5.0mmol/L and 25mmol/L of glucose was 5.03 ± 0. 72,43.90 ± 2.30mU/L respectively. While the insulin level under same hepatocarcinoma cells transferred with two site-mutated glucose concentretion in the culture media of the human proinsulin gene recombinants was 〈 2.00,2.10 ± 0.23mU/L correspondingly. Conclusions ： The recombined plasmids of the mutated human proinsulin gene and retroviral vector weve constructed successfully, the mutated human proinsulin gene was colligated with regulatory elements at the upstream and the coding sequence of the B10 site was modified to the coding sequence of aspartic acid. The recombinants was integrated into the genome of the hepatocarcinoma cells, insulin regulated physiologically by glucose and the expressive efficiency was enhanced which may secret mature significantly.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2005年第9期44-49,共6页
China Biotechnology
基金
辽宁省自然科学基金资助项目(962293)
