C3SP1-PGL3重组质粒的构建及其在PC12细胞中的表达

Plasmid Construction and Expression in PC12 Cell of Recombinant C3SP1-PGL3

目的:构建含有SP1结合位点的C3启动子的荧光素酶重组质粒。通过瞬时转染证明导入载体的目的基因具有高效的启动子活性。方法:以SF级雄性大鼠的大脑总DNA为模板,根据Genbank中含有SP1结合位点的C3启动子的核苷酸序列设计引物,对大鼠C3基因的启动子进行特异性扩增。以原始质粒p GL3为载体,利用Gibson Assembly连接目的片段和酶切质粒进行分子重组,通过PCR鉴定、酶切鉴定以及测序鉴定后,将该重组质粒通过脂质体介导,转染PC12细胞。活体成像检测和双荧光素酶活性检测得重组质粒的萤火虫荧光素酶活性。结果:PCR鉴定、酶切鉴定表明表达载体p GL-3中插入了含有SP1结合位点的C3启动子基因片断,测序结果表明编码框正确。在PC12细胞中表达出高效的启动子活性。结论:获得能够在PC12细胞内高效表达萤火虫荧光素酶的重组质粒C3SP1-p GL3。

Objective： To construct recombinant plasmid containing the luciferase SP1 binding sites of C3 promoter and to prove the high promoter activity of the target gene obtained by transient transfeetion. Methods： Grade SF male rat brain DNA was used as template, according to the nucleotide sequence of C3 promoter with SP1 binding site in Genbank, the promoter of C3 gene was amplified in the rat. Original plasmid pGL3 as the carrier, making use of Gibson Assembly to link fragment and enzyme plasmid. After using 3 kinds of identification ways that bacteria liquid PCR, double digestion plasmid and genetic sequencing, transfecting recombinant plasmids into PC12 cells ,and then using vivo imaging detection and dual luciferase activity assay to obtain firefly luciferase activity of recombinant plasmid. Results： The study successfully obtains the stable and reliable recombinant plasmid by means of PCR identification, enzyme digestion, and sequencing. What is more ,the recombinant plasmids which were transfected into PC12 cells can transduct with high promoter activity. Conclusion：Recombinant plasmid C3SPI-pGL3 were constructed and efficiently expressed in PC12 cells.