目的构建含尿激酶型纤溶酶原激活剂(urokinase plasminogen activator,uPA)裂解位点(uPAcs)和KDEL(Lys-Asp-Glu-Leu)驻留信号序列的丝瓜毒素(Luffin-β)基因的原核载体,表达并纯化其融合毒素蛋白Luffin-β-KDEL-uPAcs(LKP),并探讨融合毒素蛋白LKP抗胃癌SGC-7901细胞的活性。方法 RT-PCR两步法克隆Luffin-β基因,引物延伸法构建Luffin-β-KDEL-uPAcs融合基因并亚克隆至原核表达载体pET-32a(+)中,诱导其表达融合蛋白Trx-EK-Luffin-β-KDEL-uPAcs(TELKP)并纯化TELKP,肠激酶(enterokinase,EK)切割TELKP后,纯化与回收目的毒素蛋白LKP,SDS-PAGE对LKP蛋白予以检测鉴定,高效液相色谱法(HPLC)对其进行纯度检测。采用cell counting kit-8(CCK-8)、RT-PCR、West-ern blot等方法,体外检测毒素蛋白LKP经uPA酶裂解后释放Luffin-β的抗胃癌SGC-7901细胞的活性。结果成功诱导重组载体pET-32a(+)/Luffin-β-KDEL-uPAcs表达相对分子质量约48.8×103含载体表达标签(Trx)的融合免疫毒素TELKP,EK酶切该蛋白获含290个氨基酸,相对分子质量约31.8×103的目的蛋白LKP。SDS-PAGE检测鉴定表明,LKP蛋白与预期大小一致,其纯度达98.8%。CCK-8、RT-PCR、Western blot等法检测显示,LKP经uPA酶体外裂解可释放具杀瘤活性的Luffin-β小分子毒素。结论成功克隆到Luffin-β-KDEL-uPAcs融合基因,并将其构建于原核表达载体pET-32a(+)中,且诱导该载体表达了相对分子质量约31.8×103的融合毒素LKP。LKP毒素经uPA酶体外裂解能释放具杀瘤活性的Luffin-β小分子毒素。 Objective To construct the Luffin-β gene containing urokinase plasminogen activator cleavage site (uPAcs) and KDEL (Lys-Asp-Glu-Leu) resident signal sequence Prokaryotic vector to express and purify its fusion toxin protein Luffin-β-KDEL-uPAcs (LKP), and to investigate the activity of fusion toxin LKP against gastric cancer SGC-7901 cells. Methods The Luffin-β gene was cloned by RT-PCR and the Luffin-β-KDEL-uPAcs fusion gene was constructed by primer extension and subcloned into prokaryotic expression vector pET-32a (+). After cleavage of TELKP by Luffin-β-KDEL-uPAcs (TELKP) and purification of TELKP, the target toxin protein LKP was purified and recovered. The LKP protein was detected by SDS-PAGE and identified by high performance liquid chromatography ) For purity testing. The anti-gastric cancer SGC-7901 cells, which release Luffin-β, after the cleavage of toxin LKP by uPA, were detected by cell counting kit-8 (CCK-8), RT-PCR and West-ern blot. RESULTS: The recombinant vector pET-32a (+) / Luffin-β-KDEL-uPAcs was successfully expressed with a molecular weight of about 48.8 × 103. The fusion immunotoxin TELKP containing the vector expressing tag (Trx) was digested with EK. The protein contained 290 amino acids , The relative molecular mass of about 31.8 × 103 of the target protein LKP. SDS-PAGE detection and identification showed that the LKP protein with the expected size, the purity of 98.8%. CCK-8, RT-PCR, Western blot and other tests showed that LKP could release Luffin-β small molecule toxin with cytotoxicity after in vitro cleavage by uPA. Conclusion The fusion gene Luffin-β-KDEL-uPAcs was cloned successfully and constructed into the prokaryotic expression vector pET-32a (+). The recombinant fusion protein LKP was induced to express the fusion protein LKP with a relative molecular mass of about 31.8 × 103. LKP toxin is released by in vitro cleavage by uPA to release the tumorigenic Luffin-beta small molecule toxin.