人脂蛋白脂酶基因重组质粒的构建及其表达

目的研究野生型人脂蛋白脂酶(LPL)cDNA重组表达质粒的构建了及其在COS-1细胞中的表达。方法采用RT-PCR法从人大网膜脂肪组织获取LPL cDNA基因,以pcDNA3.1Zeo( )质粒作为载体,运用基因克隆技术构建野生型人LPLcDNA重组质粒,限制性内切酶酶切、PCR以及双向测序鉴定重组质粒;运用L ipofectam ine 2000TM将重组pcDNA3.1Zeo( )-LPLcDNA质粒导入COS-1细胞,RT-PCR法检测LPL mRNA,ELISA法和比色法分别检测细胞裂解液及细胞培养基中LPL浓度及活性。结果经限制性内切酶酶切及PCR鉴定,证实LPL基因已被连接到pcDNA3.1Zeo( )质粒中;DNA双向测序证实其产物与基因库中LPL基因序列完全一致。LPL mRNA和蛋白表达及其活性测定结果表明,pcDNA3.1Zeo( )-LPL cDNA重组质粒已转染入COS-1细胞中。结论实验成功构建野生型人LPL cDNA重组表达质粒,并证实其能在COS-1细胞中有效表达。

Construction and Expression of Recombinant Wild Lipoprotein Lipase Gene Plasmid

Objective To study the construction of recombinant wild lipoprotein lipase(LPL) gene plasmid and its expression in COS-1 cells. Methods The LPL cDNA was isolated from the human epiploon adipose tissue by means of RT-PCR.The LPL cDNA was ligated into the pcDNA3.1Zeo( ).The recombinant pcDNA3.1Zeo( )-LPL cDNA was identified by endonucleases,PCR and DNA sequencing.COS-1 cells were transfected with the recombinant LPL gene plasmid using Lipofectamine 2000~(TM).The LPL mass in cells and the culture medium were determined by a Markit-M LPL Kit.Spectrophotometry was used to measure the LPL activity. Results The LPL gene was ligated into the pcDNA3.1Zeo( ) plasmid identified by endonucleases and PCR.The sequence of the LPL gene was the same as the sequence of the Gene Bank identified by DNA sequencing.Wild pcDNA3.1Zeo( )-LPL(cDNA) plasmids was transformed into the COS-1 cells. Conclusion The recombinant plasmid pcDNA3.1Zeo( )-LPL cDNA could be constructed and successfully transformed into the COS-1 cells.