真核表达载体pEGFP-N1-INSRPTK的构建

Construction of Eukaryotic fluoscent expression vector pEGFP-N-INSRTPK

目的构建含人胰岛素受体酪氨酸蛋白激酶域基因(INSRPTK)的绿色荧光蛋白表达载体pEGFP-N1-IN-SRPTK,为进一步研究胰岛素受体抵抗在多囊卵巢综合症中的作用打下基础。方法应用RT-PCR方法从人胚肾293细胞扩增出两端带有HindIII和EcoRI酶切位点的INSRPTK cDNA编码片段,经回收、纯化、酶切后,依次连接到质粒pMD18-T和pEGFP-N1上。结果酶切及测序表明:重组质粒PEGFP-N1含有INSRPTK片段,方向及序列正确。结论成功构建了人真核表达载体pEGFP-N1-INSRPTK。

Objective To construct Eukaryotic fluoscent expression vector pEGFP-N-INSRPTK for studing the effect of Insulin Resistance on the Polycystic Ovary Syndrome.Methods The cDNA coding sequence of INSRPTK containing the digestion site of Hind III and Sal I was amplified by RT一PCR from the human embryo 293 and then was inserted into pMD18一T plasmid and pEGFP一N1 fluoresccnt expression vector.The recom-bind plasmid was identified by restriction endonuclease digestion、PCR technique and sequencing.Results Restriction erolonuelease digestion and DNA sequencing results showed that the recombinant plasmid pEGFP一N1 contained the fragment of INSRPTK.Conclusion The enkaryotic expression vector pEGFP-N1-INSRPTK was constructed sucessfully.