VEGF受体单抗与Luffin双靶融合毒素的构建及其对非小细胞肺癌细胞的抑制作用

Construction of double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL and its cytotoxic effect on non-small-cell lung carcinoma cells

目的构建含血管内皮生长因子(vascular endothelial growth factor,VEGF)受体单链抗体(KDRscFv)、尿激酶纤溶酶原激活剂(urokinase plasminogen activator,uPA)裂解位点(uPAcs)、丝瓜毒素(luffin-β)与KDEL(Lys-Asp-Glu-Leu)内质网驻留信号序列的双靶向融合毒素,并探讨其对非小细胞肺癌(non-small-cell lung carcinoma,NSCLC)细胞的抑制作用。方法全基因合成KDRscFv基因,RT-PCR法克隆luffin-β,重叠PCR法将两基因融合,其连接部位与C端分别引入uPA裂解位点uPAcs与KDEL,形成融合基因KDRscFv-uPAcs-Luffin-β-KDEL。将该融合基因克隆至原核表达载体pET-32a(+)中,转入大肠杆菌后,诱导其表达融合毒素蛋白Trx-EK-KDRscFv-uPAcs-Luffin-β-KDEL(TEKPLK)并纯化TEKPLK。用肠激酶(enterokinase,EK)切割TEKPLK后,纯化与回收靶向融合毒素KDRscFv-uPAcs-Luffin-β-KDEL(KPLK)。采用CCK-8(cell counting kit-8)、RT-PCR、Western blot等方法,体外检测靶向融合毒素KPLK经uPA酶裂解后释放Luffin-β对NSCLC细胞的抑制作用。结果成功诱导重组载体pET-32a(+)/KDRscFv-uPAcs-Luffin-β-KDEL表达相对分子质量约7.5×104含载体表达标签(Trx)的融合毒素蛋白TEKPLK,EK酶切该蛋白获得相对分子质量约5.8×104的靶向融合免疫毒素KPLK。CCK-8法检测表明,KPLK毒素的杀瘤活性成剂量-效应关系,其IC50约为35 ng/mL;RT-PCR和Western blot检测结果显示,KPLK经uPA酶体外裂解后能释放细胞毒素小分子Luffin-β,上调瘤细胞促凋亡基因caspase-3及其蛋白的表达。结论成功构建了KDRscFv-uPAcs-Luffin-β-KDEL融合基因及其原核表达载体,并获得相对分子质量约5.8×104的靶向融合毒素KPLK,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。

Objective To construct a double-targeting fused immunotoxin KDRscFv-uPAcs-Luffin-βKDEL that containing a single-chain variable fragment（scFv） against vascular endothelial growth factor（VEGF） receptor KDR,urokinase plasminogen activator（uPA） cleavage site（uPAcs）,tandemly ligated luffinβ and KDEL（Lys-Asp-Glu-Leu）,which is a signal for retention of proteins in the endoplasmic reticulum,and to investigate its cytotoxic effect on non-small cell lung carcinoma（NSCLC） cell line.Methods The complete sequence of KDR scFv gene was synthesized.And luffin-β gene was cloned through reverse transcriptase-polymerase chain reaction（RT-PCR）.KDR scFv gene was fused together with luffin-β gene by overlaying PCR.The uPAcs sequence was placed the linking position between KDRscFv and luffin-β gene,and the KDEL sequence was fused at the C-terminal of luffin-β gene.In doing so,the fused gene KDRscFv-uPAcs-Luffin-βKDEL was constructed and cloned into pET-32a（＋） vector to form recombinant vector pET-32a（＋） / KDRscFvuPAcs-Luffin-β-KDEL.Subsequently,the recombinant vector pET-32a（＋） / KDRscFv-uPAcs-Luffin-β-KDEL was transfected into E.coli BL21,and the fusion protein Trx-EK-KDRscFv-uPAcs-Luffin-β-KDEL（TEKPLK） was expressed under induction,and then purified and digested through enterokinase（EK） to produce doubletargeting fused immunotoxin KDRscFv-uPAcs-Luffin-β-KDEL（KPLK）.Cell count kit-8（CCK-8）,RT-PCR and Western blotting were employed to test the cytotoxic effect of KPLK cleavaged by uPA for setting free immunotoxin luffin-β on H460 cells.Results After induction,recombinant vector pET-32a（＋）/KDRscFv-uPAcsLuffin-β-KDEL expressed the fusion protein TEKPLK,which contained the Trx tag of vector pET-32a（＋）,and its relative molecular mass was about 7.5 × 104.The immunotoxin protein KPLK,with its relative molecular mass of about 5.8 × 104,was produced by digesting fusion protein TEKPLK with EK.The results of CCK-8 revealed that immunotoxin KPLK exerted cytotoxic effect on H460 cells in a dose-effect manner.Its IC 50 of antitumor was about 35 ng/mL.RT-PCR and Western blotting certified that immunotoxin luffin-β,which could upregulate the expression of caspase-3 gene and its protein in the tumor cells,was released from KPLK protein cleavaged by uPA in vitro.Conclusion Fused gene KDRscFv-uPAcs-luffin-β-KDEL and its prokaryotic express vector are successfully constructed.Recombinant fusion immunotoxin KPLK（relative molecular mass about 5.8 × 104） is successfully prepared.The immunotoxin luffin-β,possessing cytotoxic effect on tumor cells,can be released from KPLK protein cleavaged by uPA in vitro.