Metase基因高效原核表达体系的建立、表达及活性测定
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摘要
目的:构建L-蛋氨酸γ-裂解酶(Metase)基因的两种高效原核表达载体,建立最佳高效原核表达体系。使来源于恶臭假单胞菌(Pseudomonas Putida)的Metase在大肠杆菌中高效表达,为深入研究Metase抗肿瘤作用奠定基础。
方法:在恶臭假单胞菌来源的Metase基因已经合成(pBSK-Metase)的基础上,通过分子克隆技术,使Metase基因分别与高效原核表达载体pBV220、pGEX-4T-1连接,形成重组表达质粒pBV220-Metase和pGEX-4T-1-Metase。重组子转化感受态大肠杆菌DH5α,涂布于含氨苄抗性的琼脂平板上,挑选白色单克隆菌落。小量提取质粒,PCR鉴定、双酶切鉴定,阳性克隆送往测序。42℃热诱导pBV220-Metase表达,IPTG诱导pGEX-4T-1-Metase表达。优化表达条件。收集菌沉淀,超声波破碎,收集Metase酶液粗品,测定Metase活性,统计分析比较。
结果:成功构建出Metase基因的两种高效原核表达体系pBV220-Metase和pGEX-4T-1-Metase,且经测序证实其中的Metase基因序列都是正确的。重组表达质粒pBV220-Metase和pGEX-4T-1-Metase转化感受态大肠杆菌DH5α后经诱导表达,均能测出Metase活性。
结论:恶臭假单胞菌来源的Metase基因在大肠杆菌中成功地获得高效表达。重组表达质粒pBV220-Metase具有比pGEX-4T-1-Metase更强的活性,可作为Metase的最佳高效原核表达体系。
Objective:To construct two highly efficient prokaryotic expression vectors of L-methionineγ-lyase(Metase) gene,and to establish the best efficient prokaryotic expression system.So that Metase from Pseudomonas putida was highly expressed in E.coli,and lay the foundation for studying anti-tumor effects of Metase.
Methods:On the basis of Metase gene from Pseudomonas putida has been synthesized(pBSK-Metase),Metase gene was connected to the highly efficient prokaryotic expression vector pBV220 and pGEX-4T-1 respectively by molecular cloning technology.Then recombinant expression plasmids pBV220-Metase and pGEX-4T-1-Metase were gengrated.The two recombinant plasmids were transformed into the feeling E.coli DH5α,and then were spreaded on the agar plates which included ampicillin resistance.Next.the white monoclonal colonies were selected. The recombinant plasmids were extracted in small quantities and identified by PCR, two-enzyme digestion.positive clones were sent to sequenc.pBV220-Metase was heat-induced to express by 42℃;pGEX-4T-1-Metase was induced to express by IPTG.Then expressive conditions were optimized.Next.bacterial sediment was collected and broken by ultrasonic wave.Crude Metase was collected and Metase activity was measured.Statistical analvsis and comparison were carried out.
Results:Two highly efficient prokaryotic expression system pBV220-Metase and pGEX-4T-1-Metase of Metase gene were constructed successfully,and the Metase gene sequence was confirmed to be correct by sequencing.After that recombinant expression plasmid pBV220-Metase and pGEX-4T-1-Metase were transformed into E.coli DH5αand induced,both of the Metase activity can be measured.
Conclusion:Metase gene from Pseudomonas putida was highly expressed in Escherichia coli successfully.Recombinant expression plasmid pBV220-Metase had stronger Metase activity than pGEX-4T-1-Metase,and pBV220-Metase could be regarded as the best high-performance prokaryotic expression system of Metase.
方法:在恶臭假单胞菌来源的Metase基因已经合成(pBSK-Metase)的基础上,通过分子克隆技术,使Metase基因分别与高效原核表达载体pBV220、pGEX-4T-1连接,形成重组表达质粒pBV220-Metase和pGEX-4T-1-Metase。重组子转化感受态大肠杆菌DH5α,涂布于含氨苄抗性的琼脂平板上,挑选白色单克隆菌落。小量提取质粒,PCR鉴定、双酶切鉴定,阳性克隆送往测序。42℃热诱导pBV220-Metase表达,IPTG诱导pGEX-4T-1-Metase表达。优化表达条件。收集菌沉淀,超声波破碎,收集Metase酶液粗品,测定Metase活性,统计分析比较。
结果:成功构建出Metase基因的两种高效原核表达体系pBV220-Metase和pGEX-4T-1-Metase,且经测序证实其中的Metase基因序列都是正确的。重组表达质粒pBV220-Metase和pGEX-4T-1-Metase转化感受态大肠杆菌DH5α后经诱导表达,均能测出Metase活性。
结论:恶臭假单胞菌来源的Metase基因在大肠杆菌中成功地获得高效表达。重组表达质粒pBV220-Metase具有比pGEX-4T-1-Metase更强的活性,可作为Metase的最佳高效原核表达体系。
Objective:To construct two highly efficient prokaryotic expression vectors of L-methionineγ-lyase(Metase) gene,and to establish the best efficient prokaryotic expression system.So that Metase from Pseudomonas putida was highly expressed in E.coli,and lay the foundation for studying anti-tumor effects of Metase.
Methods:On the basis of Metase gene from Pseudomonas putida has been synthesized(pBSK-Metase),Metase gene was connected to the highly efficient prokaryotic expression vector pBV220 and pGEX-4T-1 respectively by molecular cloning technology.Then recombinant expression plasmids pBV220-Metase and pGEX-4T-1-Metase were gengrated.The two recombinant plasmids were transformed into the feeling E.coli DH5α,and then were spreaded on the agar plates which included ampicillin resistance.Next.the white monoclonal colonies were selected. The recombinant plasmids were extracted in small quantities and identified by PCR, two-enzyme digestion.positive clones were sent to sequenc.pBV220-Metase was heat-induced to express by 42℃;pGEX-4T-1-Metase was induced to express by IPTG.Then expressive conditions were optimized.Next.bacterial sediment was collected and broken by ultrasonic wave.Crude Metase was collected and Metase activity was measured.Statistical analvsis and comparison were carried out.
Results:Two highly efficient prokaryotic expression system pBV220-Metase and pGEX-4T-1-Metase of Metase gene were constructed successfully,and the Metase gene sequence was confirmed to be correct by sequencing.After that recombinant expression plasmid pBV220-Metase and pGEX-4T-1-Metase were transformed into E.coli DH5αand induced,both of the Metase activity can be measured.
Conclusion:Metase gene from Pseudomonas putida was highly expressed in Escherichia coli successfully.Recombinant expression plasmid pBV220-Metase had stronger Metase activity than pGEX-4T-1-Metase,and pBV220-Metase could be regarded as the best high-performance prokaryotic expression system of Metase.
引文
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[6]KokkinakisDM.Methionine stress:a pleiotropic approach in enhancing the efficacy of chemotherapy[J].CancerLett,2006,233(2):195-207.
[7]Takakura T.Takimoto A,Notsu Y.et al.Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine gamma-lyase conjugated with polyethylene glycol as an antitumor agent[J].Cancer Res.2006,66(5):2807-14.
[8]Gupta A,Miki K,Xu M.et al.Combination efficacy of doxorubicin and adenoviral methioninase gene therapy with prodrug selenomethionine[J].Anticancer Res,2003,23:1181-1185.PMID:12820369.
[9]Zhao R,Domann FE,Zhong W.Apoptosis induced by selenomethionine and methioninase is superoxide mediated and p53-dependent in human prostate cancer cells[J].Mol Cancer Ther,2006,5(12):3275-3284.PMID:17172431.
[10]Yoshido S.Application of methionine free nutrition as an anticancer treatment[J].nutrition,1999,15(5):422-424.
[11]Miki K.XuM An Z,et al.Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model[J].Cancer Gene Ther,2000,7(2):332-338.
[12]Kokkinak DM.Methionine-stress:a pleiotropic approach in enhancing the efficacy of chemotherapy[J].Cancer Lett,2006.233:195-207.
[13]Yuying Tan.Mingxu Xu.Xuezhong Tan.et al.Overexpression and large-scale production of recombinant L-Methionine-a-deamino-γ-gmercaptomethane-lyase for novel anticancer therapy[J].Protain expression and purification.1997,9:233-245.
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