人溶血磷脂酸受体2基因的克隆及其在293T细胞中表达

Cloning of Human Lysophosphatidic Acid Receptor 2 and Expression in 293T Cells

在以前的研究中我们发现溶血磷脂酸(Lysophosphatidic acid,LPA)通过溶血磷脂酸受体2(Lysophosphatidic acid receptor 2,LPAR2)介导促进胃癌细胞迁移.为进一步研究LPAR2介导的信号转导机制与功能,本研究试图建立过表达的LPAR2转基因细胞模型.选用人胃癌SGC-7901细胞系的cDNA经RT-PCR法克隆得到LPAR2基因CDS区,并亚克隆到pMD19-T载体,通过测序确认后、经酶切、连接到表达载体pIRES2-EGFP,酶切和测序鉴定获得pIRES2-EGFP-LPAR2重组质粒;通过LipofectamineTM2000介导把表达载体质粒转染进293T细胞.Western Blot法检测结果说明人LPAR2蛋白在293T细胞中得到表达.

In a previous study,it was found that Lysophosphatidic acid receptor 2（LPAR2） me- diating Lysophosphatidic acid induced gastric cancer cell migration. In order to clarify the molecular mechanism of LPAR2 mediated LPA actions,a LPAR2 transgenic cell model was tried to construct further studies. LPAR2 cDNA was cloned by reverse transcription PCR method from human gastric cancer SGC--7901 cells and sequentially cloned into pM19--T vector. After verified by sequencing, LPAR2 cDNA was digested and ligated into eukaryotic expression plasmid plRES2--EGFP. The re- constructed plasmid was identified with enzyme digestion and sequencing; and was then transfected into 293T cells with LipofectamineTM 2000. Western blotting assay showed that LPAR2 proteins were successfully expressed in 293T cells.