两种重组蛋氨酸酶表达条件优化及其对人肺腺癌细胞GLC的抑制作用研究

Study on Expression Condition Optimization of Two Recombinant Methioninases and Their Inhibitory Effects on Human Lung Adenocarcinoma Cells GLC

目的:优化两种重组蛋氨酸酶表达的诱导条件,并探讨其对宣威人肺腺癌细胞GLC的抑制作用。方法:将重组蛋氨酸酶表达质粒PGEX-4T1-4A1-MGL和PGEX-4T1-3B8-MGL转染至感受态大肠杆菌DH5α中,用异丙基-β-D-硫代半乳糖苷诱导表达。以目标蛋白表达量为指标,采用单因素试验对诱导前菌液起始光密度(OD600 nm)值、培养温度、诱导时间等诱导条件进行优化。采用亲和层析法对所得重组蛋氨酸酶4A1-MGL、3B8-MGL进行纯化;采用考马斯蓝法检测其质量浓度,十二烷基苯磺酸钠-聚丙烯酰胺凝胶电泳法检测其纯度,分光光度法检测其活性。采用MTT法检测经低、中、高剂量重组蛋氨酸酶(4A1-MGL和3B8-MGL分别均为0.1、0.2、0.4 U/mL)作用24、48、72 h后的细胞增殖情况,并计算细胞抑制率。结果:两种重组蛋氨酸酶表达的最优诱导条件为菌液起始OD600 nm值0.9、培养温度37℃、诱导时间5 h。验证试验结果显示,4A1-MGL、3B8-MGL的蛋白表达量分别为1.52±0.04、1.28±0.03(RSD<3%,n=3)。经纯化后,4A1-MGL的质量浓度为(0.70±0.02)mg/mL,纯度为(96.42±3.15)%,活性为(0.45±0.02)U/mg;3B8-MGL的质量浓度为(0.56±0.02)mg/mL,纯度为(97.43±2.96)%,活性为(0.91±0.03)U/mg。经低、中剂量4A1-MGL和3B8-MGL作用48、72 h,高剂量4A1-MGL和3B8-MGL作用24、48、72 h后,GLC细胞的抑制率均显著升高,且高剂量组作用72 h时显著高于同时间点低、中剂量组(P<0.05)。结论:本研究成功优化了重组蛋氨酸酶表达的诱导条件,所得4A1-MGL和3B8-MGL可剂量依赖性地抑制GLC细胞增殖。

OBJECTIVE:To optimize the expression induction condition of two recombinant methioninases,and to investigate their inhibitory effects on the proliferation of human lung adenocarcinoma cells GLC.METHODS:Recombinant methioninases expression plasmid PGEX-4T1-4A1-MGL and PGEX-4T1-3B8-MGL were transfected into competent Escherichia coli Dh5α,and induced by isopropyl-β-D-thiogalactoside.Using the expression level of target protein as index,the initial OD600 nm value before induction,culture temperature and induction time were optimized by single factor test.The recombinant methioninase 4A1-MGL and 3B8-MGL were purified by affinity chromatography.The concentration of recombinant methioninase was detected by Coomassie blue method.The purity of the product was detected by sodium lauryl benzene sulfonate-polyacrylamide gel electrophoresis;its activity was detected by spectrophotometry.The proliferation of cells was detected by MTT assay after treated with low-dose,medium-dose and high-dose of recombinant methioninases(4A1-MGL or 3B8-MGL was 0.1,0.2,0.4 U/mL)for 24,48,74 h.Inhibitory rate of cells were calculated.RESULTS:The optimal induction condition of two recombinant methioninases included that initial OD600 nm of 0.9,culture temperature of 37℃,induction time of 5 h.The results of validation test showed that protein expression level of 4A1-MGL was 1.52±0.04,that of 3B8-MGL was 1.28±0.03(RSD<3%,n=3).After purification,the concentration,purity and activity of 4A1-MGL were(0.70±0.02)mg/mL,(96.42±3.15)%and(0.45±0.02)U/mg;and those of 3B8-MGL were(0.56±0.02)mg/mL,(97.43±2.96)%and(0.91±0.03)U/mg.After treated with low-dose and medium-dose of 4A1-MGL and 3B8-MGL for 48 and 72 h,treated with high-dose of 4A1-MGL and 3B8-MGL for 24,48 and 72 h,inhibitory rate of GLC cell was increased significantly,and high-dose group for 72 h was significantly higher than low-dose and medium-dose groups at same time point(P<0.05).CONCLUSIONS:The induction conditions of recombinant methioninase expression are successfully optimized in this study.The obtained 4A1-MGL and 3B8-MGL could inhibit the proliferation of GLC cells in a dose-dependent manner.