Cloning and expression of Dengue virus nonstructural protein 4A gene and affinity purification of its interacting proteins
10.3760/cma.j.issn.0254-5101.2013.03.005
- VernacularTitle:登革病毒非结构蛋白NS4A的克隆表达及与其相互作用蛋白质的亲和纯化
- Author:
Jun XIA
;
Jiong XIE
;
Peifen ZHANG
;
Yuye LI
;
Chao LIU
;
Xi HUANG
;
Ping ZHANG
- Publication Type:Journal Article
- Keywords:
Dengue virus;
NS4A;
Tandem affinity purification(TAP);
Interaction
- From:
Chinese Journal of Microbiology and Immunology
2013;(3):184-187
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.
