| 引用本文： |
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刘威威,尹岚,张琪敏,等.清道夫受体SR—PSOX基因定点突变的研究[J].同济大学学报（医学版）,2010,31(2):8-13. [点击复制]
- LIU Wei-wei,YIN Lan,ZHANG Qi-min,et al.Gene mutagenesis of scavenger receptor SR-PSOX[J].同济大学学报（医学版）,2010,31(2):8-13. [点击复制]
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| 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—PS0x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住组氨酸三点突变，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。 |
| 关键词: 清道夫受体 SR-PSOX 定点突变 转染 |
| DOI：10.3969/j.issnl008-0392.2010.02.003 |
| 投稿时间：2010-01-08 |
| 基金项目:国家自然科学基金资助项目（3067 1969) |
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| Gene mutagenesis of scavenger receptor SR-PSOX |
| LIU Wei-wei,YIN Lan,ZHANG Qi-min,CHEN Chun-xia,DAI Ya-lei |
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| Abstract: |
| 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. |
| Key words: scavenger receptor SR-PSOX site-directed mutagenesis transfection |
