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[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it]
Daihui Weng1, Yingfeng Lei2, Yangchao Dong2
1Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi' an 710032; Department of Clinical Laboratory, Second Artillery General Hospital, Beijing 100088, China.
Objective:
To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay.
Methods:
Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay.
Results:
The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system.
Conclusion:
TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.
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