牛胰腺DNaseⅠ基因的合成、表达及功能验证
|
摘要
从NCBI下载牛胰腺DNaseⅠ蛋白序列,利用密码子优化软件(Codon Optimization)进行序列优化,设计并合成24条首尾重叠的引物合成786bp的DNaseⅠ基因,构建重组载体pET30a-DNaseⅠ,转入BL21(DE3),ArcticExpress(DE3),BL21(DE3)pLysS 3种感受态中诱导表达后,纯化目的蛋白并验证其活性.结果表明:BL21(DE3),ArcticExpress(DE3)两种感受态菌落生长异常,且未纯化到目的蛋白,BL21(DE3)pLysS感受态菌落生长正常,但未经诱导和葡萄糖诱导表达的菌液中也未纯化到目的蛋白,IPTG诱导扩大培养的菌液中纯化到0.15 mg/mL的目的蛋白.经验证,酶原液能彻底消化λ-DNA和不同大小的质粒DNA,酶稀释液消化产物电泳检测显示条带拖尾弥散,表明酶稀释液只能消化部分λ-DNA.
Deoxyribonulease I(DNaseⅠ)sequence from bovine pancreas,786 bp in size,acquired from NCBI and optimized by Codon Optimization,was synthesized,using SOE PCR(gene splicing by overlap extension PCR)with 24 overlapped primers.The recombinant expression vector pET30 a-DNaseⅠ was successfully constituted,and was transformed into the competent cells of E.coli,BL21(DE3),ArcticExpress(DE3)and BL21(DE3)pLysS.Then,DNaseⅠwas purified and its activity was detected.The results showed that the colonies in BL21(DE3)and ArcticExpress(DE3)did not grow properly and no target protein was gained.The colony in BL21(DE3)pLysS induced by glucose or without induction grew normally,but no purified target protein was obtained.In amplification culture of BL21(DE3)pLysS induced by IPTG,0.15 mg/mL DNaseⅠwas purified.DNaseⅠactivity detection indicated that the raw liquid of the enzyme could completely dissolveλ-DNA and plasmid DNA of various sizes,while its diluted liquid partially dissolvedλ-DNA only.
Deoxyribonulease I(DNaseⅠ)sequence from bovine pancreas,786 bp in size,acquired from NCBI and optimized by Codon Optimization,was synthesized,using SOE PCR(gene splicing by overlap extension PCR)with 24 overlapped primers.The recombinant expression vector pET30 a-DNaseⅠ was successfully constituted,and was transformed into the competent cells of E.coli,BL21(DE3),ArcticExpress(DE3)and BL21(DE3)pLysS.Then,DNaseⅠwas purified and its activity was detected.The results showed that the colonies in BL21(DE3)and ArcticExpress(DE3)did not grow properly and no target protein was gained.The colony in BL21(DE3)pLysS induced by glucose or without induction grew normally,but no purified target protein was obtained.In amplification culture of BL21(DE3)pLysS induced by IPTG,0.15 mg/mL DNaseⅠwas purified.DNaseⅠactivity detection indicated that the raw liquid of the enzyme could completely dissolveλ-DNA and plasmid DNA of various sizes,while its diluted liquid partially dissolvedλ-DNA only.
引文
[1]倪玉华,张建军,孙宝贵.DNA酶Ⅰ的研究进展[J].国际病理科学与临床,2006,26(6):531-535.
[2]MANNHERZ H G,PEITSCH M C,ZANOTTI S,et al.A New Function for an Old Enzyme:The Role of DNaseⅠin Apoptosis[M].Curr Top Microbiol Immunal,1995,198:161-174.
[3]OLIVERI M,DAGA A,LUNARDI C,et al.DNaseⅠBehaves as a Transcription Factor Which Modulates Fas Expression in Human Cells[J].European Journal of Immunology,2004,34(1):273-279.
[4]NAPIREI M,WULF S,MANNHERZ H G.Chromatin Breakdown During Necrosis by Serum Dnase1and the Plasminogen System[J].Arthritis&Rheumatology,2004,50(6):1873-1883.
[5]BARANOVSKII A G,BUNEVA V N,NEVINSKY G A.Human Deoxyribonucleases[J].Biochemistry Mosc,2004,69(6):587-601.
[6]萨姆布鲁克J,拉塞尔D W.分子克隆实验指南[M].北京:化学工业出版社,2008.
[7]张杏.牛胰腺脱氧核糖核酸酶I在毕赤酵母中的表达及其酶学性质的研究[D].武汉:湖北大学,2012.
[8]WORRALL A F,CONNOLLY B A.The Chemical Synthesis of a Gene Coding for Bovine Pancreatic DNaseⅠand Its Cloning and Expression in Escherichia coli[J].Journal of Biological Chemistry,1991,265(35):21889-21895.
[9]CHEN C Y,LU S C,LIAO T H.Cloning,Sequencing and Expression of a cDNA Encoding Bovine Pancreatic Deoxyribonuclease I in Escherichia coli:Purification and Characterization of the Recombinant Enzyme[J].Gene,1998,206(2):181-184.
[10]赵鹃,王霞,李炳志,等.合成生物学中的DNA组装技术[J].生命科学,2013,25(10):983-991.
[11]XIONG AS1,YAO Q H,PENG R H,et al.PCR-Base Accurate Synthesis of Long DNA Sequences[J].Nature Protocols,2006,1(2):791-797.
[12]HECKMAN K L.PEASE L R.Gene Splicing and Mutagenesis by PCR-Driven Overlap Extension[J].Nature Protocols,2007,2(4):924-932.
[13]柴冉,孙强,邱立友.降落-重叠PCR法四重融合构建平菇同源重组片段[J].中国生物化学与分子生物学报,2012,28(4):375-379.
[14]丁红雷,潘竞,张卫军,等.幽门螺杆菌cagT蛋白的表达、纯化及免疫原性研究[J].西南大学学报(自然科学版),2010,32(4):46-50.
[15]黄兰香,李建新,周林,等.鸡胚液中蛋白质动态电泳分析[J].西南大学学报(自然科学版),2005,27(6):885-887.
[16]SALNIKOW J,LIAO T H,MOORE S,et al.Bovine Pancreatic Deoxyribonuclease A.Isolation,Composition,and Amino Acid Sequences of the Tryptic and Chymotryptic Peptides[J].Journal of Biological Chemistry,1973,248(4):1480-1488.
[17]孙晶晶,刘亚娟,秦琴,等.蜡梅α-半乳糖苷酶基因的克隆和表达分析[J].西南大学学报(自然科学版),2015,37(1):25-32.
[18]杨震,杨可,侯曼美,等.人组织型纤溶酶原激活剂原核表达载体的构建及在大肠杆菌中的初步表达[J].西南大学学报(自然科学版),2012,34(4):77-83.
[19]董战旗,张军,胡楠,等.家蚕核型多角体病毒IE1的多克隆抗体制备及鉴定[J].西南大学学报自然科学版,2014,36(10):76-81.
[20]ZHOU B Y,ZHOU L,LIU M C,et al.Expression and Purification of a Fusion Gene TSO45W-4B-TSOL18of Taenia Solium in Escherichia coli Arctic Express(DE3)and Preparation of Rabbit antiserum[J].Chinese Journal of Endemiology,2013,32(6):619-624.
[21]GAO C E,HONG M,LIU W Q,et al.Construction of a Prokaryotic Expression Vector for Breast Cancer Transductive Peptide and Expression in E.coli BL21(DE3)pLysS[J].Chinese Medicinal Biotechnology,2010,5(3):177-180.
[22]HAO D C,ZHU P H,YANG S L,et al.Medium Optimization for the Production of Human Cytochrome P4502C9by Escherichia coli BL21(DE3)pLysS Using Plackett Burman Design and Gray Relational Analysis[J].Drug Metabolism Reviews,2006,38(57):101-102.
[23]KOSTYUKOVA A S,HITCHCOCK-DEGREGORI S E.Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function[J].Journal of Biological Chemistry,2004,279(7):5066-5071.
[2]MANNHERZ H G,PEITSCH M C,ZANOTTI S,et al.A New Function for an Old Enzyme:The Role of DNaseⅠin Apoptosis[M].Curr Top Microbiol Immunal,1995,198:161-174.
[3]OLIVERI M,DAGA A,LUNARDI C,et al.DNaseⅠBehaves as a Transcription Factor Which Modulates Fas Expression in Human Cells[J].European Journal of Immunology,2004,34(1):273-279.
[4]NAPIREI M,WULF S,MANNHERZ H G.Chromatin Breakdown During Necrosis by Serum Dnase1and the Plasminogen System[J].Arthritis&Rheumatology,2004,50(6):1873-1883.
[5]BARANOVSKII A G,BUNEVA V N,NEVINSKY G A.Human Deoxyribonucleases[J].Biochemistry Mosc,2004,69(6):587-601.
[6]萨姆布鲁克J,拉塞尔D W.分子克隆实验指南[M].北京:化学工业出版社,2008.
[7]张杏.牛胰腺脱氧核糖核酸酶I在毕赤酵母中的表达及其酶学性质的研究[D].武汉:湖北大学,2012.
[8]WORRALL A F,CONNOLLY B A.The Chemical Synthesis of a Gene Coding for Bovine Pancreatic DNaseⅠand Its Cloning and Expression in Escherichia coli[J].Journal of Biological Chemistry,1991,265(35):21889-21895.
[9]CHEN C Y,LU S C,LIAO T H.Cloning,Sequencing and Expression of a cDNA Encoding Bovine Pancreatic Deoxyribonuclease I in Escherichia coli:Purification and Characterization of the Recombinant Enzyme[J].Gene,1998,206(2):181-184.
[10]赵鹃,王霞,李炳志,等.合成生物学中的DNA组装技术[J].生命科学,2013,25(10):983-991.
[11]XIONG AS1,YAO Q H,PENG R H,et al.PCR-Base Accurate Synthesis of Long DNA Sequences[J].Nature Protocols,2006,1(2):791-797.
[12]HECKMAN K L.PEASE L R.Gene Splicing and Mutagenesis by PCR-Driven Overlap Extension[J].Nature Protocols,2007,2(4):924-932.
[13]柴冉,孙强,邱立友.降落-重叠PCR法四重融合构建平菇同源重组片段[J].中国生物化学与分子生物学报,2012,28(4):375-379.
[14]丁红雷,潘竞,张卫军,等.幽门螺杆菌cagT蛋白的表达、纯化及免疫原性研究[J].西南大学学报(自然科学版),2010,32(4):46-50.
[15]黄兰香,李建新,周林,等.鸡胚液中蛋白质动态电泳分析[J].西南大学学报(自然科学版),2005,27(6):885-887.
[16]SALNIKOW J,LIAO T H,MOORE S,et al.Bovine Pancreatic Deoxyribonuclease A.Isolation,Composition,and Amino Acid Sequences of the Tryptic and Chymotryptic Peptides[J].Journal of Biological Chemistry,1973,248(4):1480-1488.
[17]孙晶晶,刘亚娟,秦琴,等.蜡梅α-半乳糖苷酶基因的克隆和表达分析[J].西南大学学报(自然科学版),2015,37(1):25-32.
[18]杨震,杨可,侯曼美,等.人组织型纤溶酶原激活剂原核表达载体的构建及在大肠杆菌中的初步表达[J].西南大学学报(自然科学版),2012,34(4):77-83.
[19]董战旗,张军,胡楠,等.家蚕核型多角体病毒IE1的多克隆抗体制备及鉴定[J].西南大学学报自然科学版,2014,36(10):76-81.
[20]ZHOU B Y,ZHOU L,LIU M C,et al.Expression and Purification of a Fusion Gene TSO45W-4B-TSOL18of Taenia Solium in Escherichia coli Arctic Express(DE3)and Preparation of Rabbit antiserum[J].Chinese Journal of Endemiology,2013,32(6):619-624.
[21]GAO C E,HONG M,LIU W Q,et al.Construction of a Prokaryotic Expression Vector for Breast Cancer Transductive Peptide and Expression in E.coli BL21(DE3)pLysS[J].Chinese Medicinal Biotechnology,2010,5(3):177-180.
[22]HAO D C,ZHU P H,YANG S L,et al.Medium Optimization for the Production of Human Cytochrome P4502C9by Escherichia coli BL21(DE3)pLysS Using Plackett Burman Design and Gray Relational Analysis[J].Drug Metabolism Reviews,2006,38(57):101-102.
[23]KOSTYUKOVA A S,HITCHCOCK-DEGREGORI S E.Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function[J].Journal of Biological Chemistry,2004,279(7):5066-5071.