人溶血磷脂酸受体1基因的克隆、表达载体构建及瞬时转染293T细胞

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 170-175.

人溶血磷脂酸受体1基因的克隆、表达载体构建及瞬时转染293T细胞

李铁威, 赵鹏飞, 马洁, 阿拉坦高勒

（内蒙古大学生命科学学院，呼和浩特 010021）

收稿日期:2013-05-23 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:李铁威，男，研究方向：生物化学与细胞信号转导；E-mail：litieweind@163.com
基金资助:
国家自然科学基金项目（31260210），高等学校博士学科点专项科研基金联合资助课题（20121501110002）

Cloning and Construction of Expression Vector of Human Lysophosphatidic Acid Receptor LPAR1 and Its Transient Transfection into 293T Cell

Li Tiewei, Zhao Pengfei, Ma Jie, Alatan Gaole

(College of Life Sciences, Inner Mongolia University, Huhhot 010021）

Received:2013-05-23 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 从人胃癌细胞中提取RNA然后反转录成cDNA，进而PCR克隆LPAR1（Lysophosphatidic Acid Receptor1）基因，亚克隆到pCR2.1载体，通过测序、酶切、连接到表达载体pIRES2-EGFP，通过酶切、测序鉴定获得pIRES2-EGFP-LPAR1重组质粒；表达载体以Lipofectamine2000介导转染293T细胞。用Real-time PCR法检测外源基因在293T细胞中表达，并通过ELISA检测LPAR1的活性。结果表明，克隆到LPAR1基因并获得了pIRES2-EGFP-LPAR1表达载体，在293T细胞中确认到LPAR1基因的过量表达，并通过LPAR1/Gi信号通路抑制cAMP形成加以验证所克隆LPAR1活性。LPAR1过表达细胞模型的建立，不仅为下一步对该受体功能研究奠定了基础，还使利用LPA剂量依赖性的抑制cAMP的线性关系定量LPA的细胞学方法成为可能。

关键词: LPAR1, 基因克隆, 转基因细胞模型, cAMP应答

Abstract: To detect the over-expression of LPAR1 and the accumulation of cAMP in transfected 293T cells, human LPAR1 gene was cloned and the LPAR1 expressing vector was constructed. LPAR1 CDS sequence was cloned from human gastric cancer cells and sub-cloned into the pCR2.1 vector. By sequencing, digestion and ligation, LPAR1 was directionally cloned into eukaryotic expression plasmid pIRES2-EGFP. The reconstructed plasmid was identified with enzyme digestion and sequencing；the plasmid was transfected into 293T cells with lipofectamine2000. Real-time PCR was performed to assess exogenous LPAR1 expression in 293T cells. ELISA was used to detect the activity of LPAR1. These results indicate that the expression vector containing human LPAR1 gene has been established and LPAR1 gene has also been over-expressed successfully in 293T cells. Through the signaling pathways downstream of LPAR1 mediated under the stimulation of LPA demonstrates the activity of the cloned gene expression of LPAR1 protein. The establishment of LPAR1-overexpressing cell model, not only provides the foundation to research the function of LPAR1 in the next step, but also make it possibly for quantitative the concentration of LPA through the linear relationship between LPA and cAMP.

Key words: LPAR1, Gene cloning, Transgenic cell model, cAMP response

李铁威, 赵鹏飞, 马洁, 阿拉坦高勒. 人溶血磷脂酸受体1基因的克隆、表达载体构建及瞬时转染293T细胞[J]. 生物技术通报, 2013, 0(8): 170-175.

Li Tiewei, Zhao Pengfei, Ma Jie, Alatan Gaole. Cloning and Construction of Expression Vector of Human Lysophosphatidic Acid Receptor LPAR1 and Its Transient Transfection into 293T Cell[J]. Biotechnology Bulletin, 2013, 0(8): 170-175.

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