NDPK-A及Cys突變體對(duì)A549細(xì)胞凋亡和氧化還原對(duì)其磷酸轉(zhuǎn)移酶活性的影響
發(fā)布時(shí)間：2018-12-26 16:18
【摘要】：目的：對(duì)人二磷酸核苷激酶A亞基（NDPK-A）及其四種氧化還原異構(gòu)體表達(dá)純化，在自然及化學(xué)氧化還原環(huán)境中，研究NDPK-A及四種Cys突變體的磷酸轉(zhuǎn)移酶活性。構(gòu)建真核重組載體pEGFP-nm23-H1C145S、pEGFP-nm23-H1C109S，瞬時(shí)轉(zhuǎn)染人肺腺癌A549細(xì)胞中，將重組載體在真核細(xì)胞中表達(dá)，，研究其誘導(dǎo)腫瘤細(xì)胞A549的凋亡活性。方法：在大腸桿菌DH5α中表達(dá)NDPK-A基因及其突變體。以DEAE-纖維素弱陰離子交換層析結(jié)合Cibacron Blue染料親和層析技術(shù)分別純化野生型NDPK-A及四種Cys突變體。用HPLC法測(cè)定NDPK-A及Cys突變體在自然和化學(xué)氧化還原環(huán)境下的磷酸基轉(zhuǎn)移酶活性。以pBV220-nm23-H1C145S、pBV220-nm23-H1C109S質(zhì)粒作為模板，設(shè)計(jì)合適的引物，進(jìn)行PCR擴(kuò)增目的基因，BamH I與EcoR I雙酶切后插入pEGFP；將構(gòu)建成功的重組載體瞬時(shí)轉(zhuǎn)染A549細(xì)胞后，Western blot方法檢測(cè)外源目的基因表達(dá)水平，流式細(xì)胞儀分選綠色熒光表達(dá)細(xì)胞，測(cè)定A549細(xì)胞的晚期凋亡率。 結(jié)果：NDPK-A及突變體在大腸桿菌中高效表達(dá)；經(jīng)純化分別獲得了均一的野生型NDPK-A及突變體蛋白，純度達(dá)到98%；在還原劑（DTT終濃度2.5mM）條件下NDPK-A及突變體的磷酸轉(zhuǎn)移酶活性均高于自然環(huán)境下的活性，但在氧化劑H2O2（終濃度0.25mM）條件下的磷酸轉(zhuǎn)移酶活性明顯低于自然環(huán)境下，且都具有顯著性差異。測(cè)序鑒定重組載體pEGFP-NDPK-A C145S和pEGFP-NDPK-A C109S成功構(gòu)建，轉(zhuǎn)染A549細(xì)胞后，NDPK-A及其突變體均有表達(dá)，并且均能促使A549腫瘤細(xì)胞凋亡，凋亡率分別為：41.34±2.55％（NDPK-A），29.77±3.05％（NDPK-AC145S），27.58±4.13％（NDPK-AC109S），突變體NDPK-A C145S，NDPK-A C109S與野生型NDPK-A相比，凋亡率具有顯著性差異。結(jié)論：氧化還原環(huán)境對(duì)NDPK-A結(jié)構(gòu)異構(gòu)及磷酸轉(zhuǎn)移酶活性有一定的影響，這提示氧化還原環(huán)境可能調(diào)控NDPK-A二硫鍵的形成,影響蛋白的聚集狀態(tài)，從而影響蛋白的磷酸轉(zhuǎn)移酶活性。成功構(gòu)建重組載體pEGFP-NDPK-A C145S、pEGFP-NDPK-A C109S，在A549細(xì)胞中均能表達(dá)，并且在一定程度上誘導(dǎo)細(xì)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 偽. 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 鹵2.55% (NDPK-A), 29.77 鹵3.05% (NDPK-AC145S), 27.58 鹵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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
[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 偽. 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 鹵2.55% (NDPK-A), 29.77 鹵3.05% (NDPK-AC145S), 27.58 鹵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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
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