Luffin-β-KDEL-uPAcs融合毒素的构建及其抗胃癌SGC-7901细胞的活性研究

Construction,expression and purification of prokaryotic fusion protein luffin-β-KDEL-uPAcs and its cytotoxic effect on gastric carcinoma cell line SGC-7901

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 a prokaryotic expression vector carrying a fusion gene 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（48.8×103）,and the LKP protein（31.8×103） 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（31.8×103） 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.