基因工程重组力达霉素的制备及抗肿瘤活性

目的 制备重组力达霉素rLDM，研究其抗肿瘤活性。方法 构建完整力达霉素辅基蛋白表达载体pET30-sldp，并在大肠埃希菌中诱导表达重组辅基蛋白rLDP，纯化后的rLDP蛋白与力达霉素发色团AE在体外进行组装制备rLDM，MTT法检测rLDM和LDM的体外抗肿瘤活性，利用流式细胞仪检测两者对肿瘤细胞周期的影响。结果 rLDP蛋白以可溶形式在大肠埃希菌中分泌表达，与发色团组装后经HPLC检测在340nm处出现特定吸收峰，表明rLDM制备成功；MTT实验结果显示rLDM和LDM对SKOV3细胞的IC50值相近，无显著性差异；流式细胞仪检测结果证明两者对SKOV3细胞周期的影响趋势相似。结论

Preparation of genetically engineered lidamycin and its antitumor activity

Objective To prepare recombinant lidamycin(rLDM)and study its antitumor activity.Methods The ldp gene was cloned in the experssion vector pET30a(+)and induced in E.coli.The recombinant lidamycin apoprotein rLDP was purified by Ni2+ affinity chromatography.rLDM was prepared by reloading the AE of lidamycin into the rLDP.The cytotoxicity of rLDM and LDM was examined by MTT assay.The analysis of cell cycle was examined by flow cytometry.Results The experssion vector pET30-sldp was constructed,and rLDP was successfully secreted into the culture medium and periplasmic space of E.coli.HPLC showed that rLDP-AE had absorption at 340 nm,meaning rLDM had been reconstituted successfully.The results of MTT assay showed that the IC_(50) value of rLDM for SKOV3 cells was close to that of LDM.FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells.Conclusion Recombinant lidamycin apoprotein rLDP was successfully constructed and expressed in E.coli,rLDM was obtained by molecular reconstitution.As compared with the natural LDM,rLDM shows corresponding activity to cancer cells.