Expression of scFv SA3 against hepatoma fused with enhanced green fluorecsent protein and its targeted ability in vivo
10.3969/j.issn.1672-7347.2011.10.008
- VernacularTitle:人源抗肝癌单链抗体SA3与增强型绿色荧光蛋白的融合表达及其体内靶向性研究
- Author:
Jian HUANG
;
Yuehui LI
;
Fengjie GUO
;
Yongqing TONG
;
Jiajia WANG
;
Jinyue HU
;
Guancheng LI
- Publication Type:Journal Article
- Keywords:
hepatoma;
scFv;
EGFP;
fusion expression;
targeted therapy;
prokaryotic expression
- From:
Journal of Central South University(Medical Sciences)
2011;36(10):979-986
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.
