[Construction and activity analysis of a recombinant immunotoxin composed of PE38 and a disulfide stable single-chain antibody].
OBJECTIVE
To construct the expression vector of a recombinant toxin composed of a disulfide stable single-chain antibody from mAb B3 and PE38 and examine the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells.


METHODS
The V(H) and V(L) fragments of the mAb B3 were ligated by overlaping PCR and the resulting product was cloned to the pET22b expression vector. The PE38 fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by EcoR I and Hind III. The identified expression plasmid was tansformed into E.coli BL21(DE3) followed by IPTG induction. The inclusion body was purified through Q-Sepharose anion exchange column after denaturing and refolding. The binding and cytotoxic ability of the purified products were examined by cell-ELISA and Non-Radioactive Cell Proliferation Assay seperately. The stability assay was performed by incubating the protein sample at 37degrees Celsius.


RESULTS
The expression vector B3dsscFv-PE38-pET was constructed successfully and the expression product existed mainly in the form of inclusion body, accounting for 45% of the totle protein. The refolding product remained the binding ability of the single-chain antibody and exerted cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37degrees Celsius.


CONCLUSIONS
This genetically engineered B3dsscFv-PE38 fusion protein was stable and bifunctional, tumor targeting and tumor cell killing, supporting that it would be a promising candidate for tumor targeted immunotherapy.
Wang
Jian-li
JL
Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
Zheng
Yu-ling
YL
Wang
Bao-li
BL
Guo
Ai-guang
AG
Ma
Ru
R
Jiang
Yong-qiang
YQ
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics*; D019073:HT29 Cells; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins