[1]李宏文,张艳红,邓云华,等.人源性Rab32荧光融合蛋白表达载体的构建及鉴定[J].新乡医学院学报,2016,33(9):745-747.[doi:10.7683/xxyxyxb.2016.09.001]
　LI Hong-wen,ZHANG Yan-hong,DENG Yun-hua,et al.Construction and identification of human pEGFP-Rab32 fusion protein expression vector[J].Journal of Xinxiang Medical University,2016,33(9):745-747.[doi:10.7683/xxyxyxb.2016.09.001]
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人源性Rab32荧光融合蛋白表达载体的构建及鉴定
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
33
期数:
2016年9
页码:
745-747
栏目:
基础研究
出版日期:
2016-08-23

文章信息/Info

Title:
Construction and identification of human pEGFP-Rab32 fusion protein expression vector
作者:
李宏文1; 张艳红1; 邓云华1; 张彩娥2
（1.华中科技大学同济医学院附属同济医院皮肤科，湖北　武汉　430000；2.华中科技大学同济医学院附属同济医院麻醉科，湖北　武汉　430000）
Author(s):
LI Hong-wen1; ZHANG Yan-hong1; DENG Yun-hua1; ZHANG Cai-e2
（1.Department of Dermatology，Tongji Hospital Affiliated to Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430000，Hubei Province，China；2.Department of Anesthesiology，Tongji Hospital Affiliated to Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430000，Hubei Province，China）
关键词:
Rab32; 融合蛋白; 表达载体; 细胞定位; 黑素代谢
Keywords:
Rab32; fusion protein; expression vector; cellular localization; melanin metabolism
分类号:
Q257
DOI:
10.7683/xxyxyxb.2016.09.001
文献标志码:
A
摘要:
目的　构建人源性Rab32荧光融合蛋白表达载体pEGFP-Rab32，并观察Rab32在人黑素细胞瘤细胞系A375中细胞定位情况。方法　收集并提取A375细胞中的总RNA，反转录成cDNA，以特异性引物扩增Rab32片段，经XhoI和KpnI双酶切聚合酶链反应（PCR）产物和pEGFP-C3表达载体，酶切产物经胶回收、连接后转化至感受态大肠杆菌DH5a中，挑取单克隆菌落进行菌液PCR鉴定、酶切鉴定和测序分析，以确定表达载体构建正确。将构建的重组质粒转染A375细胞，Western blot检测细胞中Rab32表达水平，激光共聚焦显微镜观察其细胞定位。结果　pEGFP-Rab32重组质粒经菌落PCR、双酶切和测序鉴定构建正确，转染A375细胞后，Western blot可检测出细胞中pEGFP-Rab32融合蛋白的表达，激光共聚焦显微镜观察到Rab32定位于细胞质中，且大量聚集于细胞核周围。结论　pEGFP-Rab32融合蛋白表达载体成功构建，并观察了Rab32在细胞中的定位，为进一步研究Rab32在黑素代谢中的作用奠定了良好的基础。
Abstract:
Objective　To construct the expression vector of pEGFP-Rab32 of human Rab32 green fluorescent protein fusion protein，and detect its cellular localization in human melanocytoma A375 cells.Methods　Total RNA was extracted from A375 cells for reverse transcription of cDNA.Rab32 fragment was amplified by specific primers，polymerase chain reaction（PCR） product and pEGFP-C3 expression vector was digested by XhoI and KpnI enzyme and then digestion products were transformed into E.coli DH5a after recycling by gel extraction kit and connecting by T4 DNA ligase.Monoclonal bacteria colonies were picked and identified by PCR，enzyme digestion and DNA sequencing to determine the expression vector was constructed correctly.The recombinant plasmid was transfected into A375 cells and the expression levels of Rab32 in cells were detected by Western blot and the laser scanning confocal microscopy was used to observe the cellular localization of Rab32.Results　pEGFP-Rab32 expression vectors were verified by colony PCR，enzyme digestion and DNA sequencing.The pEGFP-Rab32 fusion protein was detected in A375 cells after transfected with the recombinant plasmids by Western blot.Laser scanning confocal microscopy showed that Rab32 localized in cytoplasm and mostly in the peripheral area.Conclusion　pEGFP-Rab32 fusion protein expression vector was constructed successfully and the cellular localization of Rab32 in A375 cells was performed.This research provides a good basis for further study of the role of Rab32 in melanin metabolism.

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更新日期/Last Update: 2016-09-05