Objective　　　To construct a prokaryotic expression vector carrying a fusion g

ene containing tandemly ligated luffin-β

KDEL (Lys-Asp-Glu-Leu) and urokinase plasminogen activator cleavage site (uPAcs)

to express and purify the fusion protein

and to investigate the cytotoxic effect of the fusion protein on gastric carcinoma cell line SGC-7901.　　　 Methods　　　Luffin-β gene was cloned by two-step reverse transcriptase-polymerase chain reaction (RT-PCR). The fusion gene of luffin-β-KDEL-uPAcs was obtained by primer extension

and was inserted into pET-32a(+) vector to construct a recombinant vector pET-32a(+)/luffin-β-KDEL-uPAcs. The fusion protein Trx-EK-luffin-β-KDEL-uPAcs (TELKP) was expressed under induction

purified

and digested by enterokinase (EK) to obtain luffin-β-KDEL-uPAcs (LKP). The LKP protein was purified and its purity was determined by high performance liquid chromatography (HPLC). Cell counting kit-8 (CCK-8)

RT-PCR and Western blotting were employed to detect the cytotoxic effect of LKP on gastric carcinoma cell line SGC-7901 after uPA cleavage.　　　 Results　　　The recombinant vector pET-32a(+)/luffin-β-KDEL-uPAcs could successfully express the fusion protein TELKP (48.8×10

3

)

and the LKP protein (31.8×10

3

) could be obtained by digesting the fusion protein TELKP with EK. The purity of the LKP protein was about 98.8%. The results of CCK-8

RT-PCR and Western blotting revealed that immunotoxin luffin-β

which had cytotoxic effect on tumor cells

could be released from the LKP protein cleaved by uPA in vitro.　　　 Conclusion　　　The fusion gene luffin-β-KDEL-uPAcs and its prokaryotic expression vector are successfully constructed. Recombinant fusion protein LKP (31.8×10

3

) is successfully prepared. The immunotoxin luffin-β

which possesses cytotoxic effect on tumor cells

could be released from the LKP protein cleaved by uPA in vitro.