Objective      To construct a double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL that containing a single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF) receptor KDR

urokinase plasminogen activator（uPA）cleavage site (uPAcs）

tandemly ligated luffin-β and KDEL（Lys-Asp-Glu-Leu）

which is a signal for retention of proteins in the endoplasmic reticulum

and to investigate its cytotoxic effect on non-small cell lung carcinoma (NSCLC) cell line.       Methods      The complete sequence of KDR scFv gene was synthesized. And luffin-β gene was cloned through reverse transcriptase-polymerase chain reaction (RT-PCR). KDR scFv gene was fused together with luffin-β gene by overlaying PCR. The uPAcs sequence was placed the linking position between KDRscFv and luffin-β gene

and the KDEL sequence was fused at the C-terminal of luffin-β gene. In doing so

the fused gene KDRscFv-uPAcs-Luffin-β-KDEL was constructed and cloned into pET-32a(+) vector to form recombinant vector pET-32a(+)/KDRscFv-uPAcs-Luffin-β-KDEL. Subsequently

the recombinant vector pET-32a(+)/KDRscFv-uPAcs-Luffin-β-KDEL was transfected into E.coli BL21

and the fusion protein Trx-EK- KDRscFv-uPAcs-Luffin-β-KDEL（TEKPLK） was expressed under induction

and then purified and digested through enterokinase (EK) to produce double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL（KPLK）. Cell count kit-8 (CCK-8)

RT-PCR and Western blotting were employed to test the cytotoxic effect of KPLK cleavaged by uPA for setting free immunotoxin luffin-β on H460 cells.       Results      After induction

recombinant vector pET-32a(+)/KDRscFv-uPAcs-Luffin-β-KDEL expressed the fusion protein TEKPLK

which contained the Trx tag of vector pET-32a(+)

and its relative molecular mass was about 7.5×10

4

. The immunotoxin protein KPLK

with its relative molecular mass of about 5.8×10

4

was produced by digesting fusion protein TEKPLK with EK. The results of CC

K-8 revealed that immunotoxin KPLK exerted cytotoxic effect on H460 cells in a dose-effect manner. Its IC

50

of anti-tumor was about 35 ng/mL. RT-PCR and Western blotting certified that immunotoxin luffin-β

which could upregulate the expression of caspase-3 gene and its protein in the tumor cells

was released from KPLK protein cleavaged by uPA in vitro.       Conclusion      Fused gene KDRscFv-uPAcs-luffin-β-KDEL and its prokaryotic express vector are successfully constructed. Recombinant fusion immunotoxin KPLK (relative molecular mass about 5.8×10

4

) is successfully prepared. The immunotoxin luffin-β

possessing cytotoxic effect on tumor cells

can be released from KPLK protein cleavaged by uPA in vitro.