大鼠反义转化生长因子βRII/PCDNA3.1(+)真核细胞表达质粒的构建及鉴定

The Construction and Identification of Antisense Transformation Growth Factor β Receptor II/PCDNA3.1(+) Eukaryotic Expression Plasmid with Rat

构建大鼠反义转化生长因子βRⅡ/PCDNA3.1(+)真核细胞表达质粒。查阅文献,采用两对套式引物,运用逆转录巢式PCR技术扩增TGFβRⅡ片段并经测序鉴定,双酶切纯化PCR产物及PCDNA3.1(+),再将TGFβRⅡ反向插入PCDNA3.1(+)线性质粒,即构建成TGFβRⅡ/PCDNA3.1(+)真核细胞表达质粒。将反义质粒转染感受态细胞JM-109,筛选阳性克隆行双酶切鉴定及DNA测序分析证实反义TGFβRⅡ/PCDNA3.1(+)真核细胞表达质粒构建成功。从而为下一步抗肝纤维化基因治疗研究奠定基础。

To construct Antisense Eukaryotic expression Plasmid of Rat transformation growth factor β receptor Ⅱ/ PCDNA3.1 （ ＋ ）. After consulting literature, choosing and synthesizing the proper nested primers of TGFβR Ⅱ, RT-Nest-PCR and gene recombinant techniques were used to construct the fragments of TGFβR Ⅱ . After purification, the PCR products of TGFβR Ⅱ and eukrayotic expression plasmid PCDNA3.1 （ ＋ ） were cut and TGFβR Ⅱ were inserted in revere direction into PCDNA3.1 （ ＋ ）, and then transferred into JM-109 strain, and positive clone was selected. By using enzymecutting and DNA autosequencing, the successful construction of antisense TGFβR Ⅱ eukrayotic expression ptasmid were proved. It may provide a basis for further anti-liver fibrosis gene therapy.