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-中国药房

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

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篇名：	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
TITLE：
摘要：	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。
ABSTRACT：	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.
期刊：	2019年第30卷第12期
作者：	罗沈强，田长富
AUTHORS：	LUO Shenqiang，TIAN Changfu
关键字：	重组蛋氨酸酶；诱导条件；单因素试验；GLC细胞；抑制作用
KEYWORDS：	Recombinant methioninase； Induction condition； Single factor test； GLC cells； Inhibitory effect
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