Construction of an HIV-1 Tat-dependent MazF expression vector
10.3760/cma.j.issn.1003-9279.2017.04.019
- VernacularTitle: 依赖HIV-1 Tat的毒素蛋白MazF表达系统的建立
- Author:
Zhenzhou WAN
1
;
Jin YAO
2
;
Yihong HU
2
;
Chiyu ZHANG
2
Author Information
1. Medical Laboratory of Taizhou Fourth People’s Hospital, Taizhou 225300, China
2. Pathogen Diagnostic Center, Institute Pasteur of Shanghai, Chinese Academy of Science, Shanghai 200031, China
- Publication Type:Journal Article
- Keywords:
AIDS;
Tat;
Toxin protein MazF;
Gene therapy
- From:
Chinese Journal of Experimental and Clinical Virology
2017;31(4):362-366
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a Tat-dependent MazF expression vector pcDNA3.1-GFP-HA-MazF-U3TAR.
Methods:HIV-1 U3TAR and MazF gene were amplified from pCR2.1-U3TAR and pET28a-MazF, respectively. Two gene fragments were linked together to form U3TAR-MazF by an overlapping PCR, and then cloned into pMD18T. An N-terminal HA tag was added to MazF to form U3TAR-MazF-HA. After double enzyme digestion using EcoR I and Hind Ⅲ, U3TAR-MazF-HA was reversely inserted into pcDNA 3.1 to form pcDNA3.1-HA-MazF-U3TAR. Then, a fluorescent reporter gene GFP was inserted into the downstream of U3TAR to form pcDNA3.1-GFP-HA-MazF-U3TAR.
Results:The co-transfection experiments with pcDNA3.1-tat-flag showed that pcDNA3.1-GFP-HA-MazF-U3TAR is Tat dependent. MazF was expressed only under the presence of Tat. In addition, compared with the cells transfected with pcDNA3.1-GFP-HA-MazF-U3TAR, less green fluorescent signal was observed in the cells co-transfected with pcDNA3.1-GFP-HA-MazF-U3TAR and pcDNA3.1-tat-flag, indicating that expressed MazF down-regulated the expression of GFP.
Conclusions:The expression vector pcDNA3.1-GFP-HA-MazF-U3TAR will provide an important tool for the development of MazF-based AIDS gene therapy strategies.
