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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

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-type plasminogen activator,uPAcs)的Luffin-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,转入大肠埃希菌后,诱导其表达融合蛋白Trx-EK-Luffin-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Luffin-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Luffin-β对NSCLC细胞活性的抑制作用。 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Luffin-β,且后者的体外抗瘤效应呈剂量依赖性。 结论:成功构建了Luffin-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Luffin-β毒素小分子。
标    签   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   Luffin-β   Carcinoma   non-small cell lung   Immunotoxins   Recombinant fusion proteins   Urinary plasminogen activator   Ribosome inactivating proteins   Luffin-β  
 
Abstract
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 pET-32a(+) 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), RTPCR and Western blotting were employed to test the cytotoxic effect of LKP cleavaged by uPA (urokinasetype 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 4.88×104. The immunotoxin protein LKP, which contained 290 amine acids and its relative molecular mass was about 3.18×104, 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 3.18×104, is successfully prepared. The immunotoxin Luffin-β, which possesses cytotoxic effect on tumor cells, can be released from LKP protein cleavaged by uPA in vitro.

中图分类号 R734.2   DOI 10.3781/j.issn.1000-7431.2013.04.004

 
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所属栏目 基础研究

基金项目 重庆市科技攻关计划项目(编号:CSTC,2011AC5188)

收稿日期 2012/12/18

修改稿日期 2013/2/20

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引用该论文: XIANG Ying,LI Qi-ying,WANG Jiang-hong,WANG Li,HUANG De-hong,TANG Xian-jun,ZHANG Man,ZHANG Wen-jun,YANG Tao,XIAO Chun-yan. The construction and expression of Luffin-β-targeted fusion immunotoxin as well as its inhibitory effect on non-small cell lung cancer cells[J]. Tumor, 2013, 33(4): 314~320
项颖,李启英,王江红,王莉,黄德鸿,唐显军,张曼,张文军,杨涛,肖春燕. 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[J]. 肿瘤, 2013, 33(4): 314~320


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