scTRAIL在E.coli中的重组表达及抗肿瘤活性的研究

目的:重组表达人单链TRAIL（single-chain TRAIL，scTRAIL）及研究其在抗肿瘤中的作用。方法:通过重组PCR获得编码scTRAIL的基因序列，构建含编码scTRAIL基因的重组质粒，将重组表达质粒转化到大肠埃希菌(E.coli)BL21（DE3），通过IPTG诱导，破碎细菌，上清行Amylose Resin亲和层析初步纯化融合蛋白。通过MTT法检测融合蛋白对人胰腺胆管上皮细胞SW1990、人大细胞肺癌NCI-H460和人脐静脉内皮细胞(HUVECs)的增殖抑制作用。结果:成功构建并克隆编码scTRAIL的基因序列，经IPTG诱导表达，表达产物的表达量约占菌体蛋白的20%左右，获得了相对分子质量约为110 kD的可溶性重组蛋白MBP-scTRAIL。MBP-scTRAIL分别作用于NCI-H460和SW1990，其增殖抑制作用IC50分别约为111 ng/ml和250 ng/ml，且MBP-scTRAIL增殖抑制率明显高于TRAIL114-281（P<0.01）。结论:scTRAIL的抗肿瘤效果优于单体TRAIL114-281，是一种安全、有效的抗肿瘤生物制品。

Recombinant expression and anti-tumor effects of single-chain TRAIL in E.coli

Objective:To investigate the expression of a single-chain TRAIL (scTRAIL) and study its anti-tumor effects.Methods:The genetic sequence of coded scTRAIL was obtained by means of PCR.One recombinant plasmid containing scTRAIL gene was structured，and it was transformed in E.coli BL21 and then expressed by isopropyl-β-d-thiogalactoside induction.Amylose resin affinity purification column was used to purify the obtained protein.The fusion protein inhibiting the growth of pancreatic cancer cells (SW1990)，lung cancer cells (NIC-H460) and human umbilical vein endothelial cells (HUVECs) were detected by means of MTT.Results:The genetic sequence of coded scTRAIL was successfully structured and cloned.The expression level of fusion protein was about 20% of total somatic protein by IPTG，while the relative molecular mass was about 110 kD.MBP-scTRAIL was obtained.The MBP-scTRAIL was capable of significantly inhibiting the proliferation of cancer cells (NCI-H460 and SW1990).There was positive correlation between inhibitory effects and the density，and the inhibitory rate was obviously higher than that of TRAIL114-281(P<0.01).The levels of IC50 exerted on NCI-H460 and SW1990 are around 111 ng/ml and 250 ng/ml respectively.Conclusion:Proven to be a safe and effective anti-tumor biological medicine，scTRAIL has better effects than those of TRAIL114-281.