中国麻风皮肤病杂志 ›› 2019, Vol. 35 ›› Issue (5): 257-259.doi: 10.12144/zgmfskin201905257

• 论著 •    下一篇

Rab7表达载体的构建及功能鉴定

李舒康  刘强  李海霞  景海霞  李慎秋   

  1. 湖北省十堰市太和医院(湖北医药学院附属医院)，十堰,442000
  • 出版日期:2019-05-15 发布日期:2019-05-31
  • 通讯作者: 景海霞，E-mail: jinghaixia1210@163.com

Construction of EGFP-Rab7 fusion protein expression vector and identification of its function

LI Shukang, LIU Qiang, LI Haixia, JING Haixia, LI Shenqiu   

  1. Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China
  • Online:2019-05-15 Published:2019-05-31
  • Contact: JING Haixia, E-mail: jinghaixia1210@163.com

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摘要： 目的：构建人Rab7与增强型绿色荧光蛋白（EGFP）融合表达载体，研究其对黑素代谢的影响。方法：分离人外周血淋巴细胞，提取细胞总RNA，逆转录成cDNA，以特异性引物扩增Rab7片段，酶切后连接至EGFP-C1载体并转化至感受态大肠杆菌中，测序分析以确定表达载体构建正确。将构建成功的重组质粒转染A375细胞，检测细胞黑素合成酶的表达水平和黑素含量的变化情况。 结果：EGFP-Rab7重组质粒经测序鉴定构建正确。重组质粒转染A375细胞后，细胞中的酪氨酸酶（TYR）和酪氨酸酶相关蛋白1（TYPR1）表达水平和细胞黑素含量高于空载质粒组。结论：本研究成功构建了EGFP-Rab7融合表达载体，其可增加细胞中的黑素合成酶表达水平和细胞黑素含量。

关键词: Rab7, 融合表达载体, 亚细胞定位, 黑素代谢

Abstract: Objective: To construct EGFP-Rab7 fusion expression vector, and to investigate its effect on melanin synthesis in A375 cells. Methods: Total RNA was extracted from peripheral lymphocytes for reverse transcription of cDNA. Polychain reaction (PCR) was performed with specific primers to amplify Rab7 fragment. PCR products were digested and inserted into EGFP-C1 vector. The recombinant plasmids EGFP-Rab7 were transformed into E. coli DH5a. Positive clones were screened and identified by DNA sequencing. The recombinant plasmid was transfected into A375 cells and the expression level of melanogenic enzymes and melanin content was detected. Results: EGFP-Rab7 expression vectors were verified by DNA sequencing. The expression level of tyrosinase and tyrosinase-related protein 1 and melanin content in A375 cells transfected by EGFP-Rab7 was higher than that in empty transfer plasmid group. Conclusion: EGFP-Rab7 fusion expression vector is successfully constructed which can increase the level of melanin and and melanin synthetase. 

Key words: Rab7, fusion expression vector, subcellular localization, melanin metabolism