用简并引物快速克隆苛求芽孢杆菌尿酸酶的编码序列
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摘要
目的:从头克隆苛求芽孢杆菌尿酸酶基因的编码序列。方法:据Edman降解所得N端氨基酸序列(AERTMFYGKGDV)用BLASTp搜索N端相似性>70%细菌尿酸酶;多序列联配比对发现靠近N端和C端的2个保守肽段。据N端已知序列和2个保守肽段设计简并引物进行PCR扩增;将所得片段连接到T载体,转化后筛选阳性克隆测序。结果:据密码子偏好性调整简并引物,获得该尿酸酶从第六位氨基酸到终止密码子之间的编码序列;此尿酸酶共含322个氨基酸残基,原核尿酸酶靠近N端的保守序列ATDSMKNFI从该尿酸酶第70位氨基酸残基左右开始,另一保守序列GKVYTEPR从该尿酸酶第290位氨基酸残基开始但变为GAVFTEPR。结论:用简并引物PCR快速克隆获得了苛求芽孢杆菌尿酸酶的编码序列。
Objective:To clone the whole encoding sequence of the uricase from Bacillus fastidious. Methods:According to the N-terminus amino sequence AERTMFYGKGDV from Edman degradation,bacterial uricases bearing similar N-terminus were sought in NCBI database by BLASTp;through multiple sequence alignment,two short conserved peptide fragments were found,one close to the N-terminus while the other close to the C-terminus. Using the known N-terminus and two conserved peptides,degenerated primers were designed for cloning the encoding sequence of the uricase by PCR;the resulting fragments were inserted into T vector for sequencing. Results:After the adjustment of degeneration of the primers according to the code preference,the encoding sequence of the uricase was obtained from the sixth amino acid residue to the termination code. The translated enzyme contained 322 amino. The conserved peptide fragment ATDSMKNFI started from 70 th amino acid residue while the other conserved peptide fragment GKVYTEPR started from 290 th amino acid residue with the mutated sequence of GAVFTEPR. Conclusion:The encoding sequence of the uricase is cloned rapidly by degenerated primer PCR.
Objective:To clone the whole encoding sequence of the uricase from Bacillus fastidious. Methods:According to the N-terminus amino sequence AERTMFYGKGDV from Edman degradation,bacterial uricases bearing similar N-terminus were sought in NCBI database by BLASTp;through multiple sequence alignment,two short conserved peptide fragments were found,one close to the N-terminus while the other close to the C-terminus. Using the known N-terminus and two conserved peptides,degenerated primers were designed for cloning the encoding sequence of the uricase by PCR;the resulting fragments were inserted into T vector for sequencing. Results:After the adjustment of degeneration of the primers according to the code preference,the encoding sequence of the uricase was obtained from the sixth amino acid residue to the termination code. The translated enzyme contained 322 amino. The conserved peptide fragment ATDSMKNFI started from 70 th amino acid residue while the other conserved peptide fragment GKVYTEPR started from 290 th amino acid residue with the mutated sequence of GAVFTEPR. Conclusion:The encoding sequence of the uricase is cloned rapidly by degenerated primer PCR.
引文
[1]Zhao YS,Yang XY,Lu W,et al.Uricase based methods for determination of uric acid in serum[J].Microchim Acta,2009,164(1-2):1-6.
[2]Sanders GT,Pasman AJ,Hoek FJ.Determination of uric acid with uricase and peroxidase[J].Clin Chim Acta,1980,101(2-3):299-303.
[3]Hande KR,Perini F,Putterman G,et al.Hyperxanthinemia interferes with serum uric acid determinations by the uricase method[J].Clin Chem,1979,25(8):1492-1494.
[4]Duncan PH,Gochman N,Cooper T,et al.A candidate reference method for uric acid in serum.I.Optimization and evaluation[J].Clin Chem,1982,28(2):284-290.
[5]Kroll MH,Elin RJ.Interference with clinical laboratory analysis[J].Clin Chem,1994,40(11 Pt 1):1996-2005.
[6]廖飞.通过预测本底进行酶法分析定量测定体液尿酸含量的方法:中国,ZL 03135649.4[P].2005-08-31.
[7]Liao F,Zhao YS,Zhao LN,et al.Kinetic method for enzymatic analysis by predicting background using uricase as model[J].J Med Coll PLA,2005,20(6):338-344.
[8]Liao F,Zhao YS,Zhao LN,et al.Evaluation of a kinetic uricase method for serum uric acid assay by predicting background absorbance of uricase reaction solution with an integrated method[J].J Zhejiang Univ Sci B,2006,7(6):497-502.
[9]Zhao Y,Zhao L,Yang G,et al.Characterization of an intracellular uricase from Bacillus fastidious A.T.C.C.26904 and its application to serum uric acid assay by a patented kinetic method[J].Biotech Appl Biochem,2006,45(Pt 2):75-80.
[10]赵运胜,赵利娜,杨根庆,等.苛求芽孢杆菌胞外尿酸酶的表征及其在血清尿酸测定中的应用[J].华中科技大学学报(医学版),2007,36(2):239-242.
[11]赵运胜,卜友泉,张宏娟,等.苛求芽孢杆菌基因组DNA提取方法的比较[J].生物技术,2006,16(2):41-42.
[12]Maeda M,Hidaka M,Nakamura A,et al.Cloning,sequencing,and expression of thermophilic Bacillus sp.strain TB-90 urease gene complex in Escherichia coli[J].J Bacteriol,1994,176(2):432-442.
[13]Hibi T,Hayashi Y,Fukada H,et al.Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp.TB-90 urate oxidase[J].Biochemistry,2014,53(24):3879-3888.
[14]Feng J,Liu H,Yang X,et al.Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model[J].Chem Cent J,2013,7(1):69.
[15]Huang Y,Chen Y,Yang X,et al.Optimization of p H values to formulate the bireagent kit for serum uric acid assay[J].Biotechnol Appl Biochem,2015,62(1):137-144.
[16]Zhang C,Yang X,Feng J,et al.Effects of modification of amino groups with poly(ethylene glycol)on a recombinant uricase from Bacillus fastidiosus[J].Biosci Biotechnol Biochem,2010,74(6):1298-1301.
[17]Zhang C,Yang X,Gao A,et al.Comparison of modification of a bacterial uricase with N-hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol)[J].Biotechnol Appl Biochem,2014,61(6):683-690.
[18]Feng J,Li X,Yang X,et al.A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia[J].Arch Pharm Res,2010,33(11):1761-1769.
[19]Feng J,Wang L,Liu H,et al.Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions[J].Appl Microbiol Biotechnol,2015,99(19):7973-7986.
[2]Sanders GT,Pasman AJ,Hoek FJ.Determination of uric acid with uricase and peroxidase[J].Clin Chim Acta,1980,101(2-3):299-303.
[3]Hande KR,Perini F,Putterman G,et al.Hyperxanthinemia interferes with serum uric acid determinations by the uricase method[J].Clin Chem,1979,25(8):1492-1494.
[4]Duncan PH,Gochman N,Cooper T,et al.A candidate reference method for uric acid in serum.I.Optimization and evaluation[J].Clin Chem,1982,28(2):284-290.
[5]Kroll MH,Elin RJ.Interference with clinical laboratory analysis[J].Clin Chem,1994,40(11 Pt 1):1996-2005.
[6]廖飞.通过预测本底进行酶法分析定量测定体液尿酸含量的方法:中国,ZL 03135649.4[P].2005-08-31.
[7]Liao F,Zhao YS,Zhao LN,et al.Kinetic method for enzymatic analysis by predicting background using uricase as model[J].J Med Coll PLA,2005,20(6):338-344.
[8]Liao F,Zhao YS,Zhao LN,et al.Evaluation of a kinetic uricase method for serum uric acid assay by predicting background absorbance of uricase reaction solution with an integrated method[J].J Zhejiang Univ Sci B,2006,7(6):497-502.
[9]Zhao Y,Zhao L,Yang G,et al.Characterization of an intracellular uricase from Bacillus fastidious A.T.C.C.26904 and its application to serum uric acid assay by a patented kinetic method[J].Biotech Appl Biochem,2006,45(Pt 2):75-80.
[10]赵运胜,赵利娜,杨根庆,等.苛求芽孢杆菌胞外尿酸酶的表征及其在血清尿酸测定中的应用[J].华中科技大学学报(医学版),2007,36(2):239-242.
[11]赵运胜,卜友泉,张宏娟,等.苛求芽孢杆菌基因组DNA提取方法的比较[J].生物技术,2006,16(2):41-42.
[12]Maeda M,Hidaka M,Nakamura A,et al.Cloning,sequencing,and expression of thermophilic Bacillus sp.strain TB-90 urease gene complex in Escherichia coli[J].J Bacteriol,1994,176(2):432-442.
[13]Hibi T,Hayashi Y,Fukada H,et al.Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp.TB-90 urate oxidase[J].Biochemistry,2014,53(24):3879-3888.
[14]Feng J,Liu H,Yang X,et al.Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model[J].Chem Cent J,2013,7(1):69.
[15]Huang Y,Chen Y,Yang X,et al.Optimization of p H values to formulate the bireagent kit for serum uric acid assay[J].Biotechnol Appl Biochem,2015,62(1):137-144.
[16]Zhang C,Yang X,Feng J,et al.Effects of modification of amino groups with poly(ethylene glycol)on a recombinant uricase from Bacillus fastidiosus[J].Biosci Biotechnol Biochem,2010,74(6):1298-1301.
[17]Zhang C,Yang X,Gao A,et al.Comparison of modification of a bacterial uricase with N-hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol)[J].Biotechnol Appl Biochem,2014,61(6):683-690.
[18]Feng J,Li X,Yang X,et al.A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia[J].Arch Pharm Res,2010,33(11):1761-1769.
[19]Feng J,Wang L,Liu H,et al.Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions[J].Appl Microbiol Biotechnol,2015,99(19):7973-7986.