Abstract | AIM: 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. CONCLUSION:
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Authors | Kai Chao, Dan He, Hui Yang, Zhong Zhang, Qing Lin, Ai-guang Guo, Hua-liang Huang |
Journal | Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
(Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi)
Vol. 20
Issue 5
Pg. 568-71
(Sep 2004)
ISSN: 1007-8738 [Print] China |
PMID | 15367349
(Publication Type: Journal Article)
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Chemical References |
- Bacterial Toxins
- Escherichia coli Proteins
- Exotoxins
- Immunoglobulin Fragments
- Immunotoxins
- Membrane Proteins
- Recombinant Fusion Proteins
- Virulence Factors
- immunoglobulin Fv
- ADP Ribose Transferases
- Immunophilins
- Peptidylprolyl Isomerase
- FkpA protein, E coli
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Topics |
- ADP Ribose Transferases
(genetics, metabolism)
- Bacterial Toxins
(genetics, metabolism)
- Cell Line, Tumor
- Cell Survival
(drug effects)
- Escherichia coli
(genetics, metabolism)
- Escherichia coli Proteins
- Exotoxins
(genetics, metabolism)
- Genetic Vectors
- Humans
- Immunoglobulin Fragments
(genetics, metabolism)
- Immunophilins
(genetics, metabolism)
- Immunotoxins
(pharmacology)
- Membrane Proteins
(genetics, metabolism)
- Peptidylprolyl Isomerase
(genetics, metabolism)
- Plasmids
- Recombinant Fusion Proteins
(genetics, metabolism, pharmacology)
- Urinary Bladder Neoplasms
(immunology, pathology)
- Virulence Factors
(genetics, metabolism)
- Pseudomonas aeruginosa Exotoxin A
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