[Construction of pNTAP-MK2 eukaryotic expression plasmid and establishment of a cell line for its stable expression].
OBJECTIVE
To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2.


METHODS
The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay.


RESULTS
The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei.


CONCLUSIONS
The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.
Department of Pathophysiology and Key Laboratory for Proteomics of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Da-an
DA
Wang
Xiao-wei
XW
Gong
Ai-hua
AH
Liu
Zhu-zhong
ZZ
Mei
Dan
D
Wang
Xiao-yan
XY
Ming
Xu
X
Wang
Jie
J
Wei
Peng
P
Deng
Yong
Y
Jiang
D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000099190:MAP-Kinase-Activated Kinase 2
D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2