[The construction of anti-EGFR single chain variable fragment fused with gelonin toxin prokaryotic expressing plasmid and the study of its anti-tumor effects].

OBJECTIVE

To construct a prokaryotic expressing plasmid for recombinant immunotoxin which fused anti-EGFR scFv together with gelonin toxin, express and verify its function.

METHODS

The gene fragments coding anti-EGFR single chain fragment were amplified with PCR and cloned into pET32a vector which contains gelonin toxin. The new plasmid was transformed into BL21 (DE3) cells. The induced inclusion bodies were denatured, refolded and purified through SP Sepharose Fast Flow Column. The purified immunotoxin rEG was identified by western blot analysis, and the bioactivity was identified using cell immnuohistochemistry and MTT assay.

RESULTS

The expressing vector pET32a-rEG has been constructed correctly, confirmed by restriction endonuclease digestion and sequencing. The recombinant immunotoxin rEG was purified after denaturing the inclusion bodies, refolding and cationic exchange chromatograph. The purified protein rEG had the right immunology specificity, rEG could efficiently target to EGFR positive cells identified by cell immnuohistochemistry. And the result of MTT assay showed rEG could specifically kill EGFR positive cells.

CONCLUSION

The recombinant immunotoxin rEG with high purity and biologic activity was prepared in this study, which would become the basic for the further study of the biologic function of rEG.