促黄体激素释放激素-TAT-p53融合蛋白制备及其对肿瘤细胞增殖作用的影响

Preparation and of luteinizing hormone releasing hormone-TAT-p53 fusion protein and its effect on proliferation of tumor cells

目的制备促黄体激素释放激素(luteinizing hormone releasing hormone,LHRH)-TAT-p53融合蛋白,探讨其抗肿瘤作用。方法以重组质粒pET28a-p53为模板,经PCR扩增目的基因,双酶切,克隆至原核表达载体p ET20b中,构建重组表达质粒p ET20b-LHRH-TAT-p53,并在E. coli中IPTG诱导表达,经金属螯合层析、DEAE弱阴离子交换层析纯化目的蛋白。取对数生长期的HeLa、BGC及MDCK细胞进行细胞活性试验,检测pET28a-p53蛋白活性。结果经双酶切及测序鉴定,重组质粒pET20b-LHRH-TAT-p53构建正确;融合蛋白相对分子质量约54 000,主要以包涵体形式存在,表达量约占菌体总蛋白55%;HeLa及BGC细胞加入纯化复性的重组蛋白后,细胞变圆、色暗、折光性变差,甚至裂解死亡,细胞死亡数增加,而MDCK细胞无变化;一定浓度范围内(25~120μg/m L)蛋白对HeLa及BGC细胞有明显的抑制作用。结论成功制备了LHRH-TAT-p53融合蛋白,该蛋白具有生物活性,对肿瘤细胞有杀伤作用。本研究为下一步制备靶向LHRH的肿瘤治疗药物提供了参考。

Objective To prepare luteinizing hormone releasing hormone(LHRH)-TAT-p53 fusion protein and investigate its antitumor effect.Methods The target gene was amplified by PCR using recombinant plasmid pET28 a-p53 as a template,identified by restriction analysis and cloned into prokaryotic expression vector p ET20 b.The constructed recombinant plasmid pET20 b-LHRH-TAT-p53 was transformed to E.coli and induced with IPTG.The expressed product was purified by metal chelating chromatography and DEAE weak anion exchange chromatography,then refolded,concentrated and subjected to cell activity test in HeLa,BGC and MDCK cells.Results Restriction analysis and sequencing proved that recombinant plasmid pET20 b-LHRH-TAT-p53 was constructed correctly.The expressed fusion protein,with a relative molecular mass of about 54 000,existed in a form of inclusion body and contained 55%of total somatic protein.After addition of purified and re-naturalized recombinant protein,both HeLa and BGC cells were round and dark,with deterioration of refraction even cleavage,and the dead cells increased in quantity.However,MDCK cells showed no significant change.The fusion protein at a certain concentration(25~120μg/m L)showed significantly inhibitory effect on HeLa and BG C cells.Conclusion LHRH-TAT-p53 fusion protein with biological activity was successfully prepared,which showed killing effect on tumor cells.The study provided a reference for further preparation of LHRHtargeting drugs for tumor therapy.