[Construction of a eukaryotic expression vector for alpha-1-antitrypsin and the localization of the expression product in NIH 3T3 cells].
OBJECTIVE
To construct a eukaryotic expression vector for alpha-1-antitrypsin (AAT) and detect its expression and localization in NIH 3T3 cells.


METHODS
The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences for mouse AAT (GenBank accession No. NM_009243) were amplified by RT-PCR and cloned into hemagglutinin (HA)-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, which were observed under fluorescence microscope.


RESULTS
The recombinant plasmid was verified by PCR, enzyme digestion and sequence analysis, and the fusion protein was highly expressed in NIH 3T3 cells. Under fluorescence microscope, the fusion protein was found to distribute mainly in the cytoplasm.


CONCLUSIONS
The expression vector for AAT-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells to allow future functional study of AAT in mammalian cells.
Liu
Cheng-Wu
CW
Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province, Southern Medical University, Guangzhou, China. gxmulchw@126.com
Hu
Shui-Wang
SW
Chen
Deng-Yu
DY
Feng
Guo-Kai
GK
Deng
Peng
P
Jiang
Yong
Y
D000818:Animals; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D006388:Hemagglutinins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000235:genetics
D006388:Hemagglutinins; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin