[Construction of eukaryotic plasmid of human hCAP gene and the expression and localization of fusion protein].
OBJECTIVE
To construct the expression plasmid of human c-Cbl-associated protein (hCAP) gene and identify the expression and localization of fusion protein.


METHODS
Total mRNA was extracted from CV-1 cells,  and cDNA was formed by reverse transcription. The hCAP coding sequence was amplified by polymerase chain reaction (PCR) and cloned into pEGFP-C1 plasmid. After the hCAP gene was identified by enzyme digestion and sequencing,  the plasmid was transfected into COS-7 cells. The expression of the recombinant plasmid in COS-7 cells was detected by Western blot assay. The localization of pEGFP-hCAP in NIH3T3 cells was observed with laser confocal microscopy.


RESULTS
hCAP was successfully constructed into the pEGFP-C1 expressing vector. The length of the fragment identified by restriction enzyme digestion was 3 879 bp. The expression of pEGFP-hCAP fusion protein with a molecular weight of 169kDa was detected by Western blot. The pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.


CONCLUSIONS
The recombinant plasmid of hCAP gene was successfully cloned into eukaryotic expressing vector,  and the pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.
Department of Orthopedic Surgery, Shengjing Hospital of China Medical University, Shenyang, China.
Tao
T
Shen
Feng
F
Liang
Li-qing
LQ
Yang
Yan
Y
Li
Shuang
S
Liang
Feng
F
Xue
Qin
Q
Fu
D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C119201:ponsin