Luffin-β靶向融合免疫毒素的构建、表达及对非小细胞肺癌的抑制作用  

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

作　　者：项颖[1] 李启英[1] 王江红[1] 王莉[1] 黄德鸿[1] 唐显军[1] 张曼[1] 张文军[1] 杨涛[1] 肖春燕[1] 

机构地区：[1]重庆市肿瘤研究所生物治疗及血液肿瘤科,重庆400030

出　　处：《肿瘤》2013年第4期314-320,共7页Tumor

基　　金：重庆市科技攻关计划项目(编号:CSTC;2011AC5188)

摘　　要：目的:探讨含内质网驻留信号序列KDEL(Lys-Asp-Glu-Leu-COOH)及尿激酶型纤溶酶原激活物裂解位点(cleavage site of urokinase-typeplasminogen activator,uPAcs)的Lun-β靶向融合免疫毒素对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞的抑制活性。方法:RT-PCR法克隆Lun-β基因,引物延伸法构建Lun-β-KDEL-uPAcs融合基因,并亚克隆至原核表达载体pET-32a(+)中,转入大肠埃希菌后,诱导其表达融合蛋白Trx-EK-Lun-β-KDEL-uPAcs(简称TELKP),并纯化TELKP蛋白。用肠激酶(enterokinase,EK)切割TELKP后,纯化与回收靶向融合免疫毒素Lun-β-KDEL-uPAcs(简称LKP)。采用CCK-8、RT-PCR和蛋白质印迹等方法,体外检测毒素LKP经uPA酶裂解后释放的Lun-β对NSCLC细胞活性的抑制作用。结果:成功诱导重组载体pET-32a(+)/Lun-β-KDEL-uPAcs表达相对分子质量约4.88×104的含载体表达标签Trx的融合免疫毒素蛋白TELKP,EK酶切该蛋白获得含290个氨基酸、相对分子质量约3.18×104的靶向融合免疫毒素LKP。LKP经uPA酶体外裂解后能释放具肿瘤杀伤活性的细胞毒素小分子Lun-β,且后者的体外抗瘤效应呈剂量依赖性。结论:成功构建了Lun-β-KDEL-uPAcs融合基因及其原核表达载体,并获得相对分子质量约3.18×104的靶向融合免疫毒素LKP,该毒素经uPA酶体外裂解后能释放具杀瘤活性的Lun-β毒素小分子。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 pET32a（＋） 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 4.88×10^4. The immunotoxin protein LKP, which contained 290 amine acids and its relative molecular mass was about 3.18×10^4, 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 3.18×10^4, is successfully prepared. The immunotoxin Luffin-β, which possesses cytotoxic effect on tumor cells, c

关 键 词：癌 非小细胞肺 免疫毒素类 重组融合蛋白质类 尿纤溶酶原激活物 核糖体失活蛋白质类 Lun-β 

分 类 号：R734.2[医药卫生—肿瘤]