CAJ | 학술논문

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IC_(50)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.结论大肠埃希菌表达的力达霉素辅基蛋白和发色团体外组装得到的重组力达霉素,具有和天然力达霉素相当的体外抗肿瘤活性.
목적 제비중조력체매소rLDM,연구기항종류활성.방법 구건완정력체매소보기단백표체재체pET30-sldp,병재대장애희균중유도표체중조보기단백rLDP,순화후적rLDP단백여력체매소발색단AE재체외진행조장제비rLDM,MTT법검측rLDM화LDM적체외항종류활성,이용류식세포의검측량자대종류세포주기적영향.결과 rLDP단백이가용형식재대장애희균중분비표체,여발색단조장후경HPLC검측재340nm처출현특정흡수봉,표명rLDM제비성공;MTT실험결과현시rLDM화LDM대SKOV3세포적IC_(50)치상근,무현저성차이;류식세포의검측결과증명량자대SKOV3세포주기적영향추세상사.결론 대장애희균표체적력체매소보기단백화발색단체외조장득도적중조력체매소,구유화천연력체매소상당적체외항종류활성.
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.