[Expression of CTLA4-EGFP fusion protein in K562 cells and its subcellular localization].
OBJECTIVE
To construct a eukaryotic expression vector for cytotoxic T lymphocyte antigen-4 (CTLA4) gene fused with enhanced green fluorescent protein(EGFP) gene and to analyze the expression and subcellular localization of the fusion protein in K562 cells.


METHODS
The human CTLA4 gene was cloned by RT-PCR and was then inserted into plasmid pEGFP-N1 to construct the expression vector for CTLA4-EGFP fusion gene. The expression and subcellular localization of the fusion protein in transfected K562 cells were analyzed by flow cytometry and confocal microscopy, respectively.


RESULTS
The cDNA of CTLA4 gene was cloned from human peripheral blood cells. The expression vector for CTLA4-EGFP fusion gene was constructed by PCR through introducing a Kozak sequence before the initiation site and deleting the stop codon. Flow cytometry analysis revealed that the proportion of both CTLA4+ and EGFP+ K562 cells was 16% at 24 h after the transfection. Confocal microscopy observation showed that the expressed fusion protein was mainly distributed in the intracellular compartments and had small part on the cell membrane. In contrast, only EGFP expression could be detected in K562 cells transfected with empty vector with diffuse distribution in the cells. Moreover, the expression level of the fusion protein on the cells increased to 29% after stimulation with phorbol ester and ionomycin. In addition, the fusion protein inside the cells tended to move towards and fused with the cell membrane.


CONCLUSIONS
The expression vector for CTLA4-EGFP fusion gene has been constructed successfully and expressed in K562 cells. The expressed fusion protein has similar subcellular localization and transport characteristics to native CTLA4 in activated T cells.
He
Xian-hui
XH
Key Laboratory of Tissue Transplantation and Immunology, Ministry of Education, Guangzhou 510632, China. ozms@jnu.edu.cn
Xu
Li-hui
LH
Liu
Yi
Y
Cai
Xiao-chang
XC
Zeng
Yao-ying
YY
D000069594:Abatacept; D000328:Adult; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D020014:K562 Cells; D018613:Microscopy, Confocal; D010703:Phorbol Esters / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D018796:Immunoconjugates; D007476:Ionophores; D010703:Phorbol Esters; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D000069594:Abatacept