The construction and expression of Luffin-β-targeted fusion immunotoxin as well as its inhibitory effect on non-small cell lung cancer cells

Objective: To investigate the inhibitory effect of Luffin-β-targeted fusion immunotoxin, which contains endoplasmic reticulum-resident signal fragment KDEL (Lys-Asp-Glu-Leu-COOH) and the uPAcs (cleavage site of urokinase-type plasminogen activator), on NSCLC (non-small cell lung cancer) cells. Methods: Luffin-β gene was cloned by RT-PCR (reverse transcriptase-polymerase chain reaction). Fused gene Luffin-β-KDEL-uPAcs was constructed by primer extension method, and inserted in pET-32a(+) vector to form the recombinant vector pET-32a(+)/Luffin-β-KDEL-uPAcs. After pET-32a(+)/Luffin-β-KDEL-uPAcs imported into Escherichia coli, the exression of fusion protein TELKP (Trx-EK-Luffin-β-KDEL-uPAcs) was induced, and then the TELKP was purified. EK (enterokinase) was used to digest TELKP to produce the targeting fused immunotoxin LKP (Luffin-β-KDEL-uPAcs). CCK-8 (cell counting kit-8), RT-PCR and Western blotting were employed to test the cytotoxic effect of LKP cleavaged by uPA (urokinase-type plasminogen activator) for setting free immunotoxin Luffin-β on NSCLC cells in vitro. Results: The recombinant vector pET-32a(+)/Luffin-β-KDEL-uPAcs could be induced to express the fusion protein TELKP, which contained Trx tag of vector pET-32a(+) and its relative molecular mass was about 4.88×104. The immunotoxin protein LKP, which contained 290 amine acids and its relative molecular mass was about 3.1 8×1104, could be produced after the fusion protein TELKP was digested by EK. The immunotoxin Luffin-β, which possessed cytotoxic effect on tumor cells, could be released from LKP protein cleavaged by uPA in vitro, and its cytotoxic effect was dose-dependent. Conclusion: Fused gene Luffin-β-KDEL-uPAcs and its prokaryotic express vector are successfully constructed. Recombinant fusion immunotoxin LKP, whose relative molecular mass is about 3.1 8×104, is successfully prepared. The immunotoxin Luffin-β, which possesses cytotoxic effect on tumor cells, can be released from LKP protein cleavaged by uPA in vitro. Copyright © 2013 by TUMOR.