[Soluble expression of recombinant immunotoxin against human bladder carcinoma and its anti-tumor activity].
OBJECTIVE
To construct soluble expression vector of the recombinant immunotoxin against human bladder carcinoma and express and purify the immunotoxin with high biological activity.


METHODS
The gene fragment of the immunotoxin containing the genes encoding the humanized single-chain antibody against human bladder carcinoma and the truncated and modified form of Pseudomonas exotoxin(PE) named PE38/KDEL was digested from the plasmid pABDIT and inserted into the expression vector pTMFK containing the FkpA gene. The recombinant plasmid was used to transform the E.coli strain BL21(DE3)-Star and the immunotoxin was co-expressed with the FkpA which served as the folding assistant factor. The immunotoxin was purified through Ni-NTA agarose and anti-PEAb Sepharose 4B columns. The binding activity of the immunotoxin to the antigen on BIU-87 cells was detected by ELISA. The specific cytotoxic effect of the immunotoxin against BIU-87 cells was assessed by MTT colorimetry.


RESULTS
The immunotoxin was over-expressed in soluble form in E.coli. The immunotoxin showed good binding activity to the membrane antigen on BIU-87 cells. The result of MTT assay demonstrated that purified recombinant immunotoxin could specifically kill BIU-87 cells.


CONCLUSIONS
The immunotoxin was obtained with specific cytotoxicity to human bladder cancer cells, which lays the foundation for the further application of the immunotoxin in the target therapy of human bladder carcinoma.
Chao
Kai
K
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. kchaok@163.com
He
Dan
D
Yang
Hui
H
Zhang
Zhong
Z
Lin
Qing
Q
Guo
Ai-guang
AG
Huang
Hua-liang
HL
D036002:ADP Ribose Transferases / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D020104:Immunophilins / Q000235:genetics* / Q000378:metabolism; D000922:Immunotoxins / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D001749:Urinary Bladder Neoplasms / Q000276:immunology* / Q000473:pathology; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D020104:Immunophilins; D000922:Immunotoxins; D008565:Membrane Proteins; D019696:Peptidylprolyl Isomerase; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv; C068254:FkpA protein, E coli