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,NSC...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。展开更多
1 Introduction In recent years, significant progress has been made in applying immunotoxin (IT)in the therapy of leukemia and marrow transplantation. By 1990, several ITs havebeen put into clinical trials under the pe...1 Introduction In recent years, significant progress has been made in applying immunotoxin (IT)in the therapy of leukemia and marrow transplantation. By 1990, several ITs havebeen put into clinical trials under the permission of FDA (Foodand Drug Administration, USA).展开更多
文摘目的: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。
基金Bio-High Technology Program from the State Commission of Science and Technology, PRC.
文摘1 Introduction In recent years, significant progress has been made in applying immunotoxin (IT)in the therapy of leukemia and marrow transplantation. By 1990, several ITs havebeen put into clinical trials under the permission of FDA (Foodand Drug Administration, USA).
