靶向抗肿瘤蛋白iRGD-CDD的原核表达及生物活性鉴定

Prokaryotic Expression,Purification and Antitumor Activity Identification of Tumor Targeting iRGD-CDD Fusion Protein

目的:原核表达并纯化具有特异性成纤维细胞激活蛋白(FAPα)酶切位点的靶向抗肿瘤GP-CDD-iRGD融合蛋白,利用FAPα的酶切功能切除融合标签,检测其对FAPα阳性肿瘤细胞株的毒性。方法:设计并合成GP-CDD-iRGD基因,插入p GEX-4T3载体,构建重组表达质粒,转化至BL21大肠杆菌感受态细胞中,IPTG诱导表达,SDS-PAGE分析重组融合蛋白的表达,Western blot检测融合蛋白的表达,经GST亲和柱纯化融合蛋白后,通过体外细胞毒性试验(MTT法)和细胞凋亡实验评价该融合蛋白的靶向抗肿瘤活性。结果:成功构建重组原核表达质粒p GEX-GPCDD-iRGD,可溶性表达相对分子量约为36 k Da的融合蛋白GST-GP-CDD-iRGD,纯化后蛋白纯度约为90%,经MTT实验测定其对FAPα阳性4T1细胞株的ED50约为18.5μmol/L,流式细胞术检测到其对FAPα阳性4T1细胞株具有选择性毒性作用,早期凋亡比例达到约28%。结论:原核表达的重组融合蛋白GP-CDD-iRGD对FAPα阴性4T1细胞株未显示毒性,而对FAPα阳性4T1细胞株具有显著的促凋亡作用,为进一步研究其在体内的靶向抗肿瘤活性提供了依据。

Objective： To express tumor targeting GP-CDD-iRGD fusion protein with specific fibroblast activation protein（ FAPα） restricting sites in prokaryotic cells,purify the expressed product and determine its toxicity to FAPα positive and negative tumor cell lines,evaluate the feasibility to remove the fusion tag in vivo.Methods： Design and synthesis the gene of GP-CDD-iRGD,insert it into p GEX-4T3 vector. The recombinant plasmid was transformed into E. coli BL21 cells for expression under induction of IPTG. The expressed recombinant fusion protein was analyzed for expression level and form by SDS-PAGE,then purified by GST affinity tag protein purification kit,evaluate the anti-tumor activity and FAPα targeted cleavage effect through in vitro toxicity tests. Results： Restriction analysis and sequencing proved that recombinant plasmid p GEX-GPCDD-iRGD was constructed correctly. Recombinant GP-CDD-iRGD fusion protein,with a relative molecular mass of about 36000,was solublely expressed and reached a purity of about 90% after purification. ED50 of the protein is about 18. 5μmol / L. Conclusion： It showed significant toxicity to FAPα-positive 4T1 cells while FAPα-negative cell lines with no effect which established a foundation of further reasearch of its antitumor activity in vivo.