人源性Rab38荧光融合蛋白表达载体的构建及功能鉴定

Construction of human EGFP-Rab38 fusion protein expression vector and identification of its function

目的构建人源性Rab38与EGFP融合蛋白表达载体pEGFP-Rab38,观察Rab38在人黑素细胞瘤细胞系A375中的细胞定位情况,为进一步研究Rab38在黑素代谢中的作用提供实验依据。方法收集A375细胞,提取总RNA并逆转录成cDNA。特异性引物PCR扩增Rab38片段并插入pEGFP-C3载体,随后转化至感受态大肠杆菌DH5α中,挑取单克隆菌落进行鉴定。将鉴定正确的重组表达载体转染A375细胞,检测细胞中Rab38和Tyrp1(酪氨酸酶相关蛋白1)的表达并观察Rab38的亚细胞定位。结果 pEGFP-Rab38重组质粒经菌液PCR、双酶切和测序鉴定正确;转染A375细胞后,激光共聚焦显微镜观察到Rab38定位于胞浆中,且大量聚集于细胞核周围;实时定量PCR和Western blot可检测出细胞中EGFP-Rab38融合蛋白的表达,且细胞中Tyrp1的表达明显升高。结论融合蛋白表达载体pEGFP-Rab38成功构建,观察了Rab38在细胞中的定位并初步研究了其对黑素合成关键蛋白Tyrp1的影响,为进一步研究Rab38在黑素代谢中的作用奠定了良好的基础。

Objective To construct the expression vector for EGFP- Rab38 fusion protein,and detect its subcellular localization in A375 cells,thus providing basis for further study of its role in melanin metabolism.Methods cDNA was obtained by reverse transcription of total RNA of A375 cells.Specific primers were designed and Rab38 was amplified by polymerase chain reaction（PCR）.After digested by Xhol and Kpnl,PCR products were inserted into pEGFP- C3 vector by T4 DNA ligase.Then the recombinant plasmids were transformed into competent E.coli DH5α.The pEGFP- Rab38 recombinant plasmid was transfected into A375 cells for the detection of subcellular localization of Rab38 and expression of Rab38 and Tyrpl（Tyrosinase related protein 1）.Results pEGFP- Rab38 recombinant plasmid was verified by colony PCR,enzyme digestion and DNA sequencing.A 51 kD EGFP- Rab38 was detected by western blot.Overexpression of Rab38 increased the expression of Tyrpl.Confocal microscope showed that Rab38 localized in the cytoplasm and mostly in the peripheral area.Conclusion The expression vector for EGFP- Rab38 fusion protein was successfully constructed and an investigation of the subcellular localization of Rab38 in A375 cells was performed.Our research provides a good basis for future study of the role of Rab38 in melanin metabolism.