清道夫受体SR-PSOX基因定点突变的研究  

Gene mutagenesis of scavenger receptor SR-PSOX

作　　者：刘威威[1] 尹岚[1] 张琪敏[1] 陈春霞[1] 戴亚蕾[1] 

机构地区：[1]同济大学医学院免疫学教研室,上海200092

出　　处：《同济大学学报（医学版）》2010年第2期8-13,共6页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金资助项目(30671969)

摘　　要：目的构建清道夫受体SR-PSOX点突变体,揭示该受体与脂质结合的关键位点,为确定SR-PSOX在脂质摄取、泡沫细胞形成中的作用提供模型。方法利用分子克隆及PCR定点突变技术,构建不同突变点的片段并克隆到pEGFP-N3中,形成含有SR-PSOX各联合突变的pEGFP-SR-PSOX突变体质粒。转染293T细胞后采用流式检测受体表达情况。结果从THP-1源性的巨噬细胞中成功克隆SR-PSOX受体并在真核表达载体pEGFP-SR-PSOX基础上获得了SR-PSOX三种突变体(R78、R82H85、R78R82H85)。测序结果表明SR-PSOX序列中发生了预期的突变,使SR-PSOX受体中第78位精氨酸单点突变,第82位精氨酸、第85位组氨酸双点突变,第78、82位精氨酸和第85位组氨酸三点突变,均突变为丙氨酸,并将所有pEGFP-SR-PSOX突变体在293T细胞膜表面成功表达。结论定点突变PCR可以成功构建SR-PSOX受体的3种突变体,使之在293T细胞表面获得表达,为进一步研究SR-PSOX的功能奠定了基础。Objective To study the key points of SR-PSOX receptor for bind and uptake the lipids by constructing SR-PSOX site-directed mutants in order to create and establish a cell expression model. Methods By using molecular cloning and PCR techniques, the fragments of mutants had been cloned into pEGFP-N3 plasmid and formed a series of pEGFP-SR-PSOX mutated recombinant plasmids. The expressions of the mutated SR-PSOX receptor on the surface of cells were detected by flow cytometry with SR-PSOX specific antibody after transfection into 293T cells. Results ORF of SR-PSOX gene fragment was successfully cloned from THP-1 derived macrophage and three site-directed mutants （R78,R82H85,R78R82H85） were created. The sequencing analysis showed that arginine 78,arginine 82 and histidine 85 ,arginine 78,82 and histidine 85 in SR-PSOX were replaced with alanine. All the mutated pEGFP-SR-PSOX receptors were successfully expressed on the surface of 293T cells. Conclusion Site-directed mutated PCR can construct three mutants of SR-PSOX receptor. These receptors expressed effectively with specific antibody on the cell which provides good tools for further SR-PSOX functional studies.

关 键 词：清道夫受体 SR-PSOX 定点突变 转染 

分 类 号：R363.2[医药卫生—病理学]