NDPK-A及Cys突变体对A549细胞凋亡和氧化还原对其磷酸转移酶活性的影响

发布时间：2018-12-26 16:18

【摘要】：目的：对人二磷酸核苷激酶A亚基（NDPK-A）及其四种氧化还原异构体表达纯化，在自然及化学氧化还原环境中，研究NDPK-A及四种Cys突变体的磷酸转移酶活性。构建真核重组载体pEGFP-nm23-H1C145S、pEGFP-nm23-H1C109S，瞬时转染人肺腺癌A549细胞中，将重组载体在真核细胞中表达，，研究其诱导肿瘤细胞A549的凋亡活性。方法：在大肠杆菌DH5α中表达NDPK-A基因及其突变体。以DEAE-纤维素弱阴离子交换层析结合Cibacron Blue染料亲和层析技术分别纯化野生型NDPK-A及四种Cys突变体。用HPLC法测定NDPK-A及Cys突变体在自然和化学氧化还原环境下的磷酸基转移酶活性。以pBV220-nm23-H1C145S、pBV220-nm23-H1C109S质粒作为模板，设计合适的引物，进行PCR扩增目的基因，BamH I与EcoR I双酶切后插入pEGFP；将构建成功的重组载体瞬时转染A549细胞后，Western blot方法检测外源目的基因表达水平，流式细胞仪分选绿色荧光表达细胞，测定A549细胞的晚期凋亡率。结果：NDPK-A及突变体在大肠杆菌中高效表达；经纯化分别获得了均一的野生型NDPK-A及突变体蛋白，纯度达到98%；在还原剂（DTT终浓度2.5mM）条件下NDPK-A及突变体的磷酸转移酶活性均高于自然环境下的活性，但在氧化剂H2O2（终浓度0.25mM）条件下的磷酸转移酶活性明显低于自然环境下，且都具有显著性差异。测序鉴定重组载体pEGFP-NDPK-A C145S和pEGFP-NDPK-A C109S成功构建，转染A549细胞后，NDPK-A及其突变体均有表达，并且均能促使A549肿瘤细胞凋亡，凋亡率分别为：41.34±2.55％（NDPK-A），29.77±3.05％（NDPK-AC145S），27.58±4.13％（NDPK-AC109S），突变体NDPK-A C145S，NDPK-A C109S与野生型NDPK-A相比，凋亡率具有显著性差异。结论：氧化还原环境对NDPK-A结构异构及磷酸转移酶活性有一定的影响，这提示氧化还原环境可能调控NDPK-A二硫键的形成,影响蛋白的聚集状态，从而影响蛋白的磷酸转移酶活性。成功构建重组载体pEGFP-NDPK-A C145S、pEGFP-NDPK-A C109S，在A549细胞中均能表达，并且在一定程度上诱导细胞的凋亡。
[Abstract]:Aim: to express and purify human nucleoside diphosphate kinase A subunit (NDPK-A) and its four redox isomers and to study the phosphotransferase activity of NDPK-A and four Cys mutants in natural and chemical redox environments. The eukaryotic recombinant vector pEGFP-nm23-H1C145S,pEGFP-nm23-H1C109S, was constructed for transient transfection of human lung adenocarcinoma cell line A549. The recombinant vector was expressed in eukaryotic cells to study the apoptotic activity of human lung adenocarcinoma cell line A549. Methods: NDPK-A gene and its mutant were expressed in Escherichia coli DH5 伪. Wild type NDPK-A and four Cys mutants were purified by DEAE- cellulose weak anion exchange chromatography and Cibacron Blue dye affinity chromatography. The phosphotransferase activity of NDPK-A and Cys mutants in natural and chemical redox environments was determined by HPLC method. Using pBV220-nm23-H1C145S,pBV220-nm23-H1C109S plasmid as template and designing suitable primers, the target gene, BamH I and EcoR I were digested by PCR and inserted into pEGFP;. After transient transfection of the recombinant vector into A549 cell line, Western blot method was used to detect the expression level of exogenous target gene, flow cytometry was used to select the green fluorescent expression cell, and the late apoptosis rate of A549 cell was determined. Results: NDPK-A and mutants were highly expressed in Escherichia coli, and homogenous wild type NDPK-A and mutant protein were purified, and the purity was 98%. The activity of phosphotransferase in NDPK-A and mutant was higher than that in natural environment under the condition of DTT final concentration (2.5mM). However, the activity of phosphotransferase at the condition of oxidant H2O2 (final concentration 0.25mM) was significantly lower than that in natural environment, and there were significant differences between them. Sequencing confirmed that the recombinant vectors pEGFP-NDPK-A C145S and pEGFP-NDPK-A C109S were successfully constructed. After transfection of A549 cells, NDPK-A and its mutants were expressed, and both of them could induce apoptosis of A549 tumor cells. The apoptotic rates were 41.34 卤2.55% (NDPK-A), 29.77 卤3.05% (NDPK-AC145S), 27.58 卤4.13% (NDPK-AC109S), respectively. The apoptotic rate of the mutant NDPK-A C145SNDPK-AC109S was significantly different from that of wild type NDPK-A. Conclusion: redox environment has a certain effect on NDPK-A structure isomerization and phosphotransferase activity, which suggests that redox environment may regulate the formation of NDPK-A disulfide bond and affect the aggregation of protein. Thus, the phosphoryltransferase activity of the protein was affected. The recombinant vector pEGFP-NDPK-A C145SpEGFP-NDPK-A C109Swas successfully constructed, which was expressed in A549 cells and induced apoptosis to some extent.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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