[Construction and expression of anti-human CD3 single chain Fv antibody/human p53 tetramerization domain fusion gene].
OBJECTIVE
To construct anti-human CD3 single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express the fusion protein in Hela cells.


METHODS
The anti-human CD3 scFv was cloned into the previously constructed plasmid pUC18/IgG3/p53 to construct anti-human CD3 scFv /human p53 tetramerization domain fusion gene. After enzyme digestion analysis and sequencing, the fusion gene was subcloned into the expression plasmid pSecTag2-B. Then the plasmid pSecTag2-B containing the fusion gene was transfected into Hela cells. The expressed products were analyzed by SDS-PAGE and Western blot. The binding of the purified fusion protein to human peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry.


RESULTS
DNA sequencing showed the fusion gene was constructed successfully. The expressed product of the fusion gene with a relative molecular mass (M(r)) about 35 000 was confirmed by SDS-PAGE and Western blot. The purified tetrameric anti-human CD3 scFv showed significantly stronger binding to PBMCs than scFv.


CONCLUSIONS
The tetrameric anti-human CD3 scFv which can bind to PBMCs has been successfully expressed and purified for potential use in clinical studies.
Wu
Guo-jun
GJ
Department of Urology Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. wuguojun@fmmu.edu.cn
Bai
Yu-jie
YJ
Wang
Dong
D
Yu
Lei
L
Wang
Zhi
Z
Wang
He
H
Yao
Li-bo
LB
D017252:CD3 Complex / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D017252:CD3 Complex; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C055966:immunoglobulin Fv