人铁氧还蛋白表达载体构建
|
摘要
目的为了将人铁氧还蛋白(adrenodoxin,FDX)在293T细胞中表达,扩增FDX的ORF,将FDX的ORF插入到哺乳动物细胞表达载体pcDNA的NotI/KpnI,构建表达载体。方法以GeneBank报道的FDX的mRNA序列(NM_004109.5)为模板设计引物,在5'和3'分别引入限制性酶切位点NotI和KpnI,从HepG2的cDNA中扩增FDX的ORF,将FDX的PCR产物和空载体pcDNA3.1用NotI和KpnI双酶切4 h,酶切产物用DNA回收试剂盒回收。将FDX的PCR酶切产物分别连接到空载体NotI/KpnI,连接产物转化DH5α,培养过夜。挑单克隆,用PCR鉴定阳性克隆并测序。结果经1%琼脂糖凝胶电泳和EB染色,观察到一500 bp条带,阳性克隆的测序结果与GeneBank报道的核酸序列一致。结论应用常规分子克隆方法已成功构建了FDX的真核表达载体pcDNA-FDX。
Objective: To express human ferredoxin(FDX)in 293 T cells, the open read frame of FDX was amplified and inserted into the NotI/KpnI sites of pcDNA3.1/myc-His(-) A. Methods: Specific primers were designed on the basis of the mRNA sequence of FDX( NM_004109.5) reported by GeneBank. The restriction enzymes NotI and KpnI were respectively introduced into the site of 5'end and 3'end. The ORF fractions of FDX were amplified from the cDNA of HepG2 cells. The PCR products and the empty vector were individually digested for 4 h with NotI and KpnI restriction enzymes. The digestion products were purified by gel extraction kit. The digestion fractions of FDX were ligated into the NotI/KpnI site of pcDNA3.1/myc-His(-) A, and then directly transformed into Escherichia coli DH5α cultured over night. Single colon was picked and characterized with PCR amplification and sequenced.Results: PCR products of FDX were separated by a 1% agarose gel with ethidium bromide staining. One band of500 bp was observed. The amino acid sequence of positive clone characterized by sequencing is consistent with the sequence reported by GenBank. Conclusion:.The eukaryotic expression vector of human ferredoxin gene was successfully constructed by conventional molecular cloning method.
Objective: To express human ferredoxin(FDX)in 293 T cells, the open read frame of FDX was amplified and inserted into the NotI/KpnI sites of pcDNA3.1/myc-His(-) A. Methods: Specific primers were designed on the basis of the mRNA sequence of FDX( NM_004109.5) reported by GeneBank. The restriction enzymes NotI and KpnI were respectively introduced into the site of 5'end and 3'end. The ORF fractions of FDX were amplified from the cDNA of HepG2 cells. The PCR products and the empty vector were individually digested for 4 h with NotI and KpnI restriction enzymes. The digestion products were purified by gel extraction kit. The digestion fractions of FDX were ligated into the NotI/KpnI site of pcDNA3.1/myc-His(-) A, and then directly transformed into Escherichia coli DH5α cultured over night. Single colon was picked and characterized with PCR amplification and sequenced.Results: PCR products of FDX were separated by a 1% agarose gel with ethidium bromide staining. One band of500 bp was observed. The amino acid sequence of positive clone characterized by sequencing is consistent with the sequence reported by GenBank. Conclusion:.The eukaryotic expression vector of human ferredoxin gene was successfully constructed by conventional molecular cloning method.
引文
[1]柯霞,丁冠军,孙骏,等.VD3羟化酶及其电子传递链的体外构建及活性研究[J].中国生物工程杂志,2016,36(5):89-96.
[2]杨超,胡红涛,吴平,等.高等植物铁氧还蛋白-NADP-氧化还原酶研究进展[J].植物生理学报,2014,52(9):1353-1366.
[3]陈倩.嗜酸氧化亚铁硫杆菌中个铁氧还蛋白铁硫簇组装机理研究[D].湖南:中南大学,2011.
[4]Kerstin M E,Michael R,Rita B.Adrenodoxin-AVersatile Ferredoxin[J].IUBMB Life,2012,64(6):506-512.
[5]Guengerich F P.Human cytochrome P450 enzymes[M].4thed.New York:Springer,2015.
[6]Girvan H M and Munro A W.Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology[J].Current Opinion in Chemical Biology,2016(31):136-145.
[7]邱星辉.冷欣夫.细胞色素P450酶系的结构功能与应用前景[M].北京:科学出版社,2001.
[8]Ewen K M,Kleser M,Bernhardt R.Adrenodoxin:the archetype of vertebrate[2Fe-2S]cluster ferredoxins[J].Biochim Biophys Acta,2011,1814(1):111-125.
[9]Uchida E E,Kagawa N,Sakaki T,et al.Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES[J].Biochemical and biophysica research communications,2004,323(2):505-11.
[10]Shinkyo R,Sakaki T,Kamakura M,et al.Metabolism of vitamin D by human microsomal CYP2R1[J]Biochemical and biophysical research communications2004,324(1):451-7.
[11]Tang E K,Tieu E W,Tuckey R C.Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles[J].FEBS J,2012,279(19):3749.
[12]Sawada N,Sakaki T,Kitanaka S,et al.Enzymatic properties of human 25-hydroxyvitamin D3 1a-hydroxylase Coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli[J]European Journal of Biochemistry,1999,265(3):950-956.
[13]LaSala D,Shibanaka Y,Jeng A Y.Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase Inhibitors[J]Analytical Biochemistry,2009,394(1):56-61.
[2]杨超,胡红涛,吴平,等.高等植物铁氧还蛋白-NADP-氧化还原酶研究进展[J].植物生理学报,2014,52(9):1353-1366.
[3]陈倩.嗜酸氧化亚铁硫杆菌中个铁氧还蛋白铁硫簇组装机理研究[D].湖南:中南大学,2011.
[4]Kerstin M E,Michael R,Rita B.Adrenodoxin-AVersatile Ferredoxin[J].IUBMB Life,2012,64(6):506-512.
[5]Guengerich F P.Human cytochrome P450 enzymes[M].4thed.New York:Springer,2015.
[6]Girvan H M and Munro A W.Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology[J].Current Opinion in Chemical Biology,2016(31):136-145.
[7]邱星辉.冷欣夫.细胞色素P450酶系的结构功能与应用前景[M].北京:科学出版社,2001.
[8]Ewen K M,Kleser M,Bernhardt R.Adrenodoxin:the archetype of vertebrate[2Fe-2S]cluster ferredoxins[J].Biochim Biophys Acta,2011,1814(1):111-125.
[9]Uchida E E,Kagawa N,Sakaki T,et al.Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES[J].Biochemical and biophysica research communications,2004,323(2):505-11.
[10]Shinkyo R,Sakaki T,Kamakura M,et al.Metabolism of vitamin D by human microsomal CYP2R1[J]Biochemical and biophysical research communications2004,324(1):451-7.
[11]Tang E K,Tieu E W,Tuckey R C.Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles[J].FEBS J,2012,279(19):3749.
[12]Sawada N,Sakaki T,Kitanaka S,et al.Enzymatic properties of human 25-hydroxyvitamin D3 1a-hydroxylase Coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli[J]European Journal of Biochemistry,1999,265(3):950-956.
[13]LaSala D,Shibanaka Y,Jeng A Y.Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase Inhibitors[J]Analytical Biochemistry,2009,394(1):56-61.