三种鸭防御素真核表达载体的构建及其表达分析

Construction of Eukaryotic Expression Vectors of Three Duck Defensins and Their Expression Analysis

为研究鸭防御素的真核表达,人工合成了鸭防御素AvBD2、AvBD10和AvBD12基因,并将其插入到真核表达质粒pDoubleEx-EGFP-flag-N中构建重组表达质粒;重组表达质粒经酶切鉴定正确后,分别转染HEK293细胞进行表达,用RT-PCR和Westem-blot鉴定目的基因的表达情况。结果表明,酶切鉴定证实目的基因成功插入到真核表达质粒中;真核表达质粒转染HEK293细胞24 h后,在荧光显微镜下可见绿荧光蛋白表达;RT-PCR能检测到目的基因mRNA,但Western-blot没有检测到目的蛋白的表达条带。

To study the eukaryotic expression of duck defense, duck defensins genes AvBD2, AvBD10 and AvBD12 were artificially synthesized and inserted into the eukaryotic expression plasmid pDoubleEx-EGFP-flag-N to construct recombinant expression plasmids. After identification by enzyme digestion, recombinant expression plasmids were transfected into HEK293 cells respectively. And the expression of target genes were analyzed by using RT-PCR and Westem-blot. The results showed that target genes were inserted into the eukaryotic expression plasmid successful; 24 hours after the transfection of recombinant expression plasmids into HEK293 cells, the expression of green fluorescent protein were visible under the fluorescence microscope; RT-PCR could detected the target genes＇ mRNA, but western blot did not detect the expression of target proteins. This study constructed eukaryotic expression vectors of three duck defenses, and try to express the defensins in HEK293 cells. It provided reference for further research of eukaryotic expression of duck defensins.