Disulfide-stabilized single-chain antibody-targeted superantigen: construction of a prokaryotic expression system and its functional analysis.
OBJECTIVE
To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.


METHODS
This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.


RESULTS
The expression vector B3 (scdsFv)-SEA-PET was constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 degrees.


CONCLUSIONS
This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.
Wang
Jian-Li
JL
Department of Life Science, Northwest Sci-Tech University of Agriculture and Forestry, No. 22 Xi-nong Road, Yangling 712100, Shaanxi Province, China.
Zheng
Yu-Ling
YL
Ma
Ru
R
Wang
Bao-Li
BL
Guo
Ai-Guang
AG
Jiang
Yong-Qiang
YQ
D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002470:Cell Survival; D002850:Chromatography, Gel; D003110:Colonic Neoplasms; D004220:Disulfides / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D005818:Genetic Engineering; D006801:Humans; D008113:Liver Neoplasms; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000187:drug effects; D018089:Superantigens / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D018089:Superantigens