单环刺螠纤溶酶Ⅲ基因克隆及原核表达
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摘要
单环刺螠(Urechis unicinctus)纤溶酶(UFE)是由本实验室分离纯化得到的一组有抗凝和纤溶活性及较好生物安全性的纤溶酶。本研究为获得重组表达的UFE,根据该纤溶酶Ⅲ的(UFEⅢ)N端测序结果,设计简并引物,扩增出该蛋白的部分基因编码序列,利用5′-RACE PCR获得(UFEⅢ)的cDNA全长序列863bp,其中开放阅读框编码261个氨基酸。通过生物信息学分析其为丝氨酸蛋白酶家族成员,并与之前实验室提取的UFEⅢ进行同源性分析,推测其为一组同工酶。以PMD18-T-UFE为模板,扩增单环刺螠纤溶酶的成熟肽片段,构建重组表达载体pET32a-UFE和pET28a-UFE,转入Rosetta2(DE3)菌中诱导表达,UFEⅢ分别以可溶蛋白和包涵体的形式得到了表达。
Urechis unicinctus Fibrinolytic enzymes(UFE)were isolated and purified by our laboratory. Each of them had great anticoagulant activity,plasmin activity and histocompatibility.According to the N-terminal amino acid sequence of the UFEⅢ,degenerate primers were designed to amplify part of the UFEⅢ.Then a 863bp length complete cDNA sequence which encoding the UFEⅢ was amplyfied on the basis of the result of 5′-RACE PCR.The ORF coded 258animo acids.The result of this cDNA sequence analyzed by bioinformatics software demonstrated that UFEⅢ was one of the tryptase family members.Homology analysis of UFEⅢ and UFEⅡ suggested that they were a group of isoenzymes. Mature peptide fragment was obtained by employing PMD18-T-UFE as the temeplate.The recombinant plasmids pET32a-UFE/pET28a-UFE were constructed and then transformed into E.coli Rosetta2(DE3) strain,UFEⅢ was expressed as soluble proteins and inclusion bodies.
Urechis unicinctus Fibrinolytic enzymes(UFE)were isolated and purified by our laboratory. Each of them had great anticoagulant activity,plasmin activity and histocompatibility.According to the N-terminal amino acid sequence of the UFEⅢ,degenerate primers were designed to amplify part of the UFEⅢ.Then a 863bp length complete cDNA sequence which encoding the UFEⅢ was amplyfied on the basis of the result of 5′-RACE PCR.The ORF coded 258animo acids.The result of this cDNA sequence analyzed by bioinformatics software demonstrated that UFEⅢ was one of the tryptase family members.Homology analysis of UFEⅢ and UFEⅡ suggested that they were a group of isoenzymes. Mature peptide fragment was obtained by employing PMD18-T-UFE as the temeplate.The recombinant plasmids pET32a-UFE/pET28a-UFE were constructed and then transformed into E.coli Rosetta2(DE3) strain,UFEⅢ was expressed as soluble proteins and inclusion bodies.
引文
[1]王菲,刘静波,王二雷,等.动植物蛋白中抗凝溶栓活性物质研究进展[J].食品研究与开发,2010,31(9):194-196.
[2]王乐,王君高.纳豆激酶的研究进展[J].中国酿造,2008(10):3.
[3]葛涛,梁国栋.蚓激酶研究进展[J].中国生物工程杂志,2003,23(4):51.
[4]李莹,陈斌,敬俊锋,等.纳豆激酶基因的克隆及其在大肠杆菌中的表达[J].重庆师范大学学报,2012,29(1):78-80.
[5]Xu Z R,Yang Y M,Gui Q F.Expression,purification,and characterization of recombinant lumbrokinase PI239in Escherichia coli[J].Protein Expression and Purification,2010,69(2):198-203.
[6]初金鑫,蔡文娣,韩宝芹,等.单环刺螠纤溶酶Ⅰ的性质和溶栓活性[J].天然产物研究与开发,2010,22:661-664.
[7]Wang Dianliang,Liu Wanshun,Han Baoqin.Biochemical and enzymatic properties of UFE,a novel marine fibrinolytic enzyme[J].Applied Biochemistry and Biotechnology,2007,26(2):84-93.
[8]蒋仲青,刘万顺,韩宝芹,等.单环刺螠纤溶酶的分离纯化及溶栓活性的初步研究[J].中国海洋大学学报:自然科学版,2009,39:138-142.
[9]Wang Dianliang,Jiang Hezuo,Wang Ruiling.Purification and characterization of a novel fibrinolytic enzyme from a marine animal,Urechis unicinctus[J].China Biotechnology,2010,30(8):42-51.
[10]J.萨姆布鲁克.分子克隆实验指南[M].金冬雁.第2版.北京:科学出版社,1998:55.
[11]蒋仲青.单环刺螠纤溶酶的分离纯化及其药效学研究[D].中国海洋大学,2009.
[12]韩宝芹,冯伊琳,毕庆庆,等.单环刺螠纤溶酶Ⅱ的基因克隆[J].中国海洋大学学报:自然科学版,2011,41(7/8):91-96.
[13]Anthony L Fink.Protein aggregation,folding aggregates,inclusion bodies and amyloid[J].Folding&Design,1998,3(1):9-23.
[14]Wetlaufer D B,Ristow S.Acquistion of three-dimensional structure of proteins[J].Annu Rev Biochem,1973,42:135-158.
[15]龙英娜,刘焕奇,王明志.重组包涵体的纯化与复性[J].兽医临床,2008(4):70-71.
[2]王乐,王君高.纳豆激酶的研究进展[J].中国酿造,2008(10):3.
[3]葛涛,梁国栋.蚓激酶研究进展[J].中国生物工程杂志,2003,23(4):51.
[4]李莹,陈斌,敬俊锋,等.纳豆激酶基因的克隆及其在大肠杆菌中的表达[J].重庆师范大学学报,2012,29(1):78-80.
[5]Xu Z R,Yang Y M,Gui Q F.Expression,purification,and characterization of recombinant lumbrokinase PI239in Escherichia coli[J].Protein Expression and Purification,2010,69(2):198-203.
[6]初金鑫,蔡文娣,韩宝芹,等.单环刺螠纤溶酶Ⅰ的性质和溶栓活性[J].天然产物研究与开发,2010,22:661-664.
[7]Wang Dianliang,Liu Wanshun,Han Baoqin.Biochemical and enzymatic properties of UFE,a novel marine fibrinolytic enzyme[J].Applied Biochemistry and Biotechnology,2007,26(2):84-93.
[8]蒋仲青,刘万顺,韩宝芹,等.单环刺螠纤溶酶的分离纯化及溶栓活性的初步研究[J].中国海洋大学学报:自然科学版,2009,39:138-142.
[9]Wang Dianliang,Jiang Hezuo,Wang Ruiling.Purification and characterization of a novel fibrinolytic enzyme from a marine animal,Urechis unicinctus[J].China Biotechnology,2010,30(8):42-51.
[10]J.萨姆布鲁克.分子克隆实验指南[M].金冬雁.第2版.北京:科学出版社,1998:55.
[11]蒋仲青.单环刺螠纤溶酶的分离纯化及其药效学研究[D].中国海洋大学,2009.
[12]韩宝芹,冯伊琳,毕庆庆,等.单环刺螠纤溶酶Ⅱ的基因克隆[J].中国海洋大学学报:自然科学版,2011,41(7/8):91-96.
[13]Anthony L Fink.Protein aggregation,folding aggregates,inclusion bodies and amyloid[J].Folding&Design,1998,3(1):9-23.
[14]Wetlaufer D B,Ristow S.Acquistion of three-dimensional structure of proteins[J].Annu Rev Biochem,1973,42:135-158.
[15]龙英娜,刘焕奇,王明志.重组包涵体的纯化与复性[J].兽医临床,2008(4):70-71.