CN101638640B - Glucose-6-phosphoric acid dehydrogenase and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof - Google Patents
Glucose-6-phosphoric acid dehydrogenase and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof Download PDFInfo
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- CN101638640B CN101638640B CN2009100923735A CN200910092373A CN101638640B CN 101638640 B CN101638640 B CN 101638640B CN 2009100923735 A CN2009100923735 A CN 2009100923735A CN 200910092373 A CN200910092373 A CN 200910092373A CN 101638640 B CN101638640 B CN 101638640B
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- g6pd
- phosphate dehydrogenase
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- 238000001890 transfection Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 229960004418 trolamine Drugs 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention discloses glucose-6-phosphoric acid dehydrogenase with the amino acid sequence as shown in SEQ ID NO:1. The invention further discloses the nucleotide sequence encoding the glucose-6-phosphoric acid dehydrogenase, comprising the recombinant vector of the nucleotide sequence, the recombinant host cell of the recombinant vector, and a kit which comprises at least one of the glucose-6-phosphoric acid dehydrogenase, the nucleotide sequence, the recombinant vector and the recombinant host cell. The glucose-6-phosphoric acid dehydrogenase in the invention has good heat resistance, high residue enzymatic activity after repeated freeze thawing, and high stability in a wide range of pH value.
Description
Technical field
The present invention relates to a kind of enzyme, the encode nucleotide sequence of this enzyme, the recombinant vectors that comprises described nucleotide sequence comprises the recombinant host cell of described recombinant vectors, and comprises at least a test kit in above-mentioned enzyme, nucleotide sequence, recombinant vectors and the recombinant host cell.More specifically, the present invention relates to a kind of glucose-6-phosphate dehydrogenase (G6PD), the encode nucleotide sequence of this glucose-6-phosphate dehydrogenase (G6PD), the recombinant vectors that comprises described nucleotide sequence, comprise the recombinant host cell of described recombinant vectors, and comprise at least a test kit in above-mentioned glucose-6-phosphate dehydrogenase (G6PD), nucleotide sequence, recombinant vectors and the recombinant host cell.
Background technology
Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase, G6PDH, EC1.1.1.49) be the key regulation and control rate-limiting enzyme of phosphopentose pathway, its major function is synthetic reducing power NADPH or the NADH of providing of biomolecules such as, nitrogen reduction synthetic for lipid acid and gsh, and pentose also is provided for nucleic acid is synthetic.Its concrete catalytic reaction can be shown in following formula one or formula two:
(formula one)
(formula two)
By above reaction formula as can be known, can live by the enzyme of measuring glucose-6-phosphate dehydrogenase (G6PD) at the generating rate of monitoring NADPH of 340nm wavelength place or NADH.
Thereby can be prepared into the corresponding reagent box that comprises glucose-6-phosphate dehydrogenase (G6PD), for example detect the test kit of creatine kinase.Test kit with the detection creatine kinase is an example, and its principle can be under the effect of creatine kinase, and creatine phosphate and ADP reaction generate ATP and creatine.Under hexokinase catalysis, glucose and ATP reaction generate G-6-P and ADP.Under the catalysis of glucose-6-phosphate dehydrogenase (G6PD), G-6-P and NADP
+Reaction generates 6-phosphogluconic acid and NADPH, and NADPH has maximum absorption at the 340nm place, calculates creatine kinase activity by measuring the NADPH generating rate.
But the used glucose-6-phosphate dehydrogenase (G6PD) of present test kit exists poor heat resistance (referring to Fig. 1), transportation, preservation and the use of test kit are had relatively high expectations to envrionment temperature, have influence on the validity and the accuracy of kit measurement easily owing to the sex change of glucose-6-phosphate dehydrogenase (G6PD).For example, disclose the varient of a series of glucose-6-phosphate dehydrogenase (G6PD)s among the JP2001-037480, they are being that remarkable decline (referring to Fig. 2 of this patent) all appears in 60 minutes the enzyme stability of living in 6.7 the imidazoles acetate buffer solution at pH under 52 ℃ the temperature.In addition, the used glucose-6-phosphate dehydrogenase (G6PD) of present test kit can only keep advantages of higher stability (referring to Fig. 3) in narrow pH value scope, the scope of having limited to the tested sample that test kit can measure greatly, higher to the pH conditional request of measuring, thus require also very high to the personnel's that use test kit operative technique level.
Summary of the invention
An object of the present invention is to overcome existing glucose-6-phosphate dehydrogenase (G6PD) poor heat resistance, only can in narrow pH value scope, keep the shortcoming of advantages of higher stability, a kind of good heat resistance is provided, can in wide pH value scope, keeps advantages of higher stability, and the high glucose-6-phosphate dehydrogenase (G6PD) of activity of residual enzyme behind the multigelation.
Second purpose of the present invention provides the nucleotide sequence of the described glucose-6-phosphate dehydrogenase (G6PD) of coding.
The 3rd purpose of the present invention provides the recombinant vectors that comprises described nucleotide sequence.
The 4th purpose of the present invention provides the recombinant host cell that comprises described recombinant vectors.
The 5th purpose of the present invention provides at least a test kit that comprises in above-mentioned glucose-6-phosphate dehydrogenase (G6PD), nucleotide sequence, recombinant vectors and the recombinant host cell.
The present inventor has paid creative work in large quantities, glucose-6-phosphate dehydrogenase (G6PD) sequence to leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides ATCC 12291) is carried out rite-directed mutagenesis at random, obtains the glucose-6-phosphate dehydrogenase (G6PD) that the present invention has the aminoacid sequence shown in the SEQ ID NO:1.And the present inventor is surprised to find that, the gained glucose-6-phosphate dehydrogenase (G6PD), can keep advantages of higher stability in wide pH value scope, and has the high glucose-6-phosphate dehydrogenase (G6PD) of activity of residual enzyme behind the multigelation.
The invention provides a kind of glucose-6-phosphate dehydrogenase (G6PD), wherein, the aminoacid sequence of described glucose-6-phosphate dehydrogenase (G6PD) is the aminoacid sequence shown in the SEQ ID NO:1.
The present invention also provides the nucleotide sequence of the glucose-6-phosphate dehydrogenase (G6PD) of the present invention of encoding.
The present invention also provides the recombinant vectors that comprises nucleotide sequence of the present invention.
The present invention also provides the recombinant host cell that comprises recombinant vectors of the present invention.
The present invention also provides at least a test kit that comprises in glucose-6-phosphate dehydrogenase (G6PD) of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.
Glucose-6-phosphate dehydrogenase (G6PD) provided by the invention has following advantage:
The first, good heat resistance.Referring to Fig. 1 as can be known, glucose-6-phosphate dehydrogenase (G6PD) of the present invention all has relative reactivity more than 90% after 60 minutes 52 ℃ of heating, apparently higher than the relative reactivity of the glucose-6-phosphate dehydrogenase (G6PD) of under the same conditions Comparative Examples 1.
The second, very high at wide pH value scope internal stability.Referring to Fig. 2 and Fig. 3 as can be known, glucose-6-phosphate dehydrogenase (G6PD) of the present invention is handled the relative reactivity that all has under 17 hours the condition more than 78% in 30 ℃ in the multiple damping fluid of pH value 4-7, apparently higher than the relative reactivity of the glucose-6-phosphate dehydrogenase (G6PD) of under the same conditions Comparative Examples 1.
The 3rd, activity of residual enzyme height behind the multigelation.Referring to Fig. 4 as can be known, under the condition of glucose-6-phosphate dehydrogenase (G6PD) of the present invention multigelation 5 times between-20 ℃ and room temperature behind the multigelation activity of residual enzyme still reach 27%, and under the same conditions behind the glucose-6-phosphate dehydrogenase (G6PD) multigelation of Comparative Examples 1 activity of residual enzyme only be 6%.
Description of drawings
Fig. 1 prepares the glucose-6-phosphate dehydrogenase (G6PD) thermostability comparison diagram of embodiment 1 and Comparative Examples 1;
Fig. 2 prepares the glucose-6-phosphate dehydrogenase (G6PD) pH beta stability line figure of embodiment 1;
The glucose-6-phosphate dehydrogenase (G6PD) pH beta stability line figure of Fig. 3 Comparative Examples 1;
Fig. 4 prepares activity of residual enzyme comparison diagram behind the glucose-6-phosphate dehydrogenase (G6PD) multigelation of embodiment 1 and Comparative Examples 1;
Fig. 5 prepares the proteinic SDS-PAGE electrophoresis of embodiment 1 gained photo.
Embodiment
The invention provides a kind of glucose-6-phosphate dehydrogenase (G6PD), wherein, the aminoacid sequence of described glucose-6-phosphate dehydrogenase (G6PD) is the aminoacid sequence shown in the SEQ ID NO:1.
Glucose-6-phosphate dehydrogenase (G6PD) provided by the invention can also be modified, and obtains deutero-protein." deutero-protein " of the present invention refers to have difference on the modified forms that does not influence sequence with the glucose-6-phosphate dehydrogenase (G6PD) with above-mentioned aminoacid sequence.Promptly described " deutero-protein " also comprises the analogue (as D type amino acid) with the amino acid whose residue of natural L type, and has non-natural analogue that exist or synthetic amino acid (as beta-amino acids, gamma-amino acid etc.).
(the not changing primary structure usually) form of modification comprises: the interior or external proteic chemically derived form of body, and as acetylize or carboxylated.Modify and also to comprise glycosylation, as those in proteic synthetic and processing or further carry out glycosylation modified and albumen that produce in the procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
The present invention also provides the nucleotide sequence of the glucose-6-phosphate dehydrogenase (G6PD) of the present invention of encoding.
Known in this field, in 20 kinds of different amino acid of constitutive protein matter, except that Met (ATG) or Trp (TGG) were respectively single password coding, other 18 seed amino acids were respectively by 2-6 codon encode (Sambrook etc., molecular cloning, press of cold spring harbor laboratory, New York, the U.S., second edition, 1989, see 950 pages of appendix D).Promptly because the degeneracy of genetic codon, determine more than one mostly of an amino acid whose codon, the displacement of the 3rd Nucleotide often can not change amino acid whose composition in the triplet codon, and the nucleotide sequence of the gene of the same protein of therefore encoding can be different.Those skilled in the art are according to known password sublist, from aminoacid sequence disclosed by the invention, and the active constant aminoacid sequence of the glucose-6-phosphate dehydrogenase (G6PD) that obtains by described aminoacid sequence, can derive their nucleotide sequence of gene of to encode fully, nucleotide sequence as described in obtaining by biological method (as PCR method, mutation method) or chemical synthesis process, so this partial nucleotide sequence all should be included in the scope of the present invention.On the contrary, utilize dna sequence dna disclosed herein, also can be by means commonly known in the art, for example method (molecular cloning, the press of cold spring harbor laboratory of Sambrook etc., New York, the U.S., second edition, 1989) carry out, by revising nucleotide sequence provided by the invention, obtain and the active consistent aminoacid sequence of glucose-6-phosphate dehydrogenase (G6PD) of the present invention.
Under the preferable case, nucleotides sequence of the present invention is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:2 is carried out that one or several Nucleotide replaces and the nucleotide sequence of the codon same sense mutation that obtains.
Described nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:2 or the SEQ ID NO:3 more preferably.Described nucleotide sequence most preferably is the nucleotide sequence shown in the SEQ ID NO:3.Nucleotide sequence shown in the SEQ ID NO:3 that the present inventor finally determines by a large amount of experiments, owing to may more meet for example codon preference of colibacillary genetic engineering bacterium, thereby more help genetic engineering bacterium expressing protein product.
Nucleotide sequence provided by the invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.For example, those skilled in the art can be easy to obtain template and primer according to nucleotide sequence provided by the present invention and recombinant bacterial strain, utilize PCR to increase and obtain relevant sequence.When sequence is longer, can carry out twice or pcr amplification repeatedly, then the gained fragment be pressed the proper order splicing.In case obtained relevant nucleotide sequence, just can use the relevant aminoacid sequence of the large batch of acquisition of recombination method.Usually the gained nucleotide sequence is cloned into carrier, again in the transgene engineering bacteria, the host cell of the method by routine after the propagation separates and obtains relevant nucleotide sequence then.In addition, also the method for available known artificial chemosynthesis is synthesized relevant nucleotide sequence.
The present invention also provides the recombinant vectors that comprises nucleotide sequence of the present invention.Known in this field, described recombinant vectors generally comprises empty carrier and inserts the goal gene of this empty carrier, and described goal gene is the nucleotide sequence of glucose-6-phosphate dehydrogenase (G6PD) of the present invention.
In the present invention, various carrier known in the art can be selected for use in described " empty carrier " (or title " carrier "), as commercially available various plasmids, clay, phage and retrovirus etc., the preferred pET28b of the present invention (+) plasmid, pMD18-T, pGEMT-Easy or pRSET A.Described empty carrier can comprise multiple certification mark commonly used (for example reporter gene such as fluorescent mark, antibiotic marker) and restriction enzyme site.Construction of recombinant vector can adopt the various endonucleases of the multiple clone site of empty carrier own (as for pUC 18, available Sal I, BamH I, EcoR I etc., for pET28a, available Ndel, XhoI, Nhel, EcoRI, BamH, HindIII etc., can be with EcoR I, Nde I, BamH, HindIII etc. for pET28b) carry out enzyme and cut the acquisition linear plasmid, be connected with the gene fragment that adopts the cutting of identical nucleic acid restriction endonuclease, obtain recombinant plasmid.Preferred described recombinant vectors is pET28b-G6PDH (Y207F+N240S+K312R).The present invention preferably adopts NdeI and XhoI double digestion pET28b and connected PCR product fragment, and linked enzyme connects, and makes up recombinant vectors pET28b-G6PDH of the present invention (Y207F+N240S+K312R).
The present invention also provides the recombinant host cell that comprises recombinant vectors of the present invention.
Method that can be by this area routine in host cell, transforms described recombinant vectors conversion, transduction or transfection as Calcium Chloride Method chemical conversion, high-voltage electric shock, preferred electric shock transforms; Described host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, be preferably intestinal bacteria, Bacillus subtilus, yeast (as pichia spp) or various animal and plant cells, more preferably described host cell is this area genetic engineering bacterium commonly used, as intestinal bacteria, subtilis or pichia spp.Most preferably described host cell is e. coli bl21 (DE3) or e. coli bl21-Rosetta.
Can use this area method commonly used from recombinant host cell, to separate and the purifying glucose-6-phosphate dehydrogenase (G6PD).For example, centrifugation substratum and recombinant host cell, cell debris, affinitive layer purification glucose-6-phosphate dehydrogenase (G6PD) are removed in high-pressure homogenization smudge cells, centrifuging.For the product of the glucose-6-phosphate dehydrogenase (G6PD) of separation and purification gained, can use this area method commonly used to carry out purity and identify.For example, Xylene Brilliant Cyanine G method, Kjeldahl determination, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, enzymic activity, antigen-antibody method, electrophoretic analysis (for example sodium dodecyl sulfate-polyacrylamide gel electrophoresis), analysis by sedimentation, diffusion analysis, permanent solubility method, protein spectrum etc.
The present invention also provides at least a test kit that comprises in glucose-6-phosphate dehydrogenase (G6PD) of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.Under the preferable case, test kit of the present invention comprises glucose-6-phosphate dehydrogenase (G6PD), and do not comprise nucleotide sequence, recombinant vectors and recombinant host cell.Test kit more preferably of the present invention comprises the dry powder of glucose-6-phosphate dehydrogenase (G6PD), and does not comprise nucleotide sequence, recombinant vectors and recombinant host cell.The dry powder that can prepare glucose-6-phosphate dehydrogenase (G6PD) with this area method commonly used, as long as this method can access the dry powder of glucose-6-phosphate dehydrogenase (G6PD), and the activity of not destroying the dry powder of glucose-6-phosphate dehydrogenase (G6PD) gets final product.For example use the vacuum decompression thickener to obtain spissated enzyme liquid, use the dry spissated enzyme liquid of freeze drier then; Perhaps use equipment such as vacuum decompression moisture eliminator.Because glucose-6-phosphate dehydrogenase (G6PD) good heat resistance of the present invention can also use spray-dired technology to obtain the dry powder of this enzyme.Be converted into liquid form in the buffer solution system (for example trolamine damping fluid, phosphate buffered saline buffer, preferred pH is 7.6-8) that the dry powder of glucose-6-phosphate dehydrogenase (G6PD) in use can be by being dissolved in pH7.5-10.
The test kit of detection creatine kinase of the present invention can also comprise that this area is usually used in measuring the composition of the test kit that detects creatine kinase.Corresponding to the glucose-6-phosphate dehydrogenase (G6PD) in the test kit of the present invention can be liquid form and/or solid dry powder form, and other compositions in the test kit of the present invention also can be liquid form and/or solid dry powder form.For example, when test kit of the present invention is liquid form, generally can contain reagent one (R1) and reagent two (R2); Wherein, described reagent one can comprise the hexokinase, the ADP of 1-3mM of G6PDH, 2-3KU/L of NADP, the 1-3KU/L of 15-25mM N-acetylcystein in the 80-120mM imidazole buffer that is dissolved in pH 6.5-7.0,1-3mM ethylenediamine tetraacetic acid (EDTA) (EDTA), 1-3mM; Described reagent two can contain the glucose of the 15-30mM in 40-60mM 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid (CAPSO) damping fluid of pH 9.0-11.0 and the creatine phosphate of 20-40mM.Under the preferable case, described reagent one can comprise the hexokinase, the ADP of 1.5-3mM of G6PDH, 2-3KU/L of NADP, the 1.5-3KU/L of 15-20mM N-acetylcystein in the 80-100mM imidazole buffer that is dissolved in pH 6.8-7.0,1-2mM ethylenediamine tetraacetic acid (EDTA) (EDTA), 1-2mM; Described reagent two can contain the glucose of the 20-30mM in 50-60mM 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid (CAPSO) damping fluid of pH 9.0-10.0 and the creatine phosphate of 25-40mM.
Above-described reagent one and reagent two can pass through Freeze Drying Technique respectively, in conjunction with reduced pressure distillation technique, reverse osmosis technology and ultra-filtration technique etc., are prepared into reagent one dry powder (wherein comprising G6PDH dry powder of the present invention) and reagent two dry powder.Described dry powder can redissolve to original volume of described reagent with the solvent that is selected from deionized water, distilled water and distilled water before detecting sample to be measured.Therefore test kit of the present invention can comprise the reagent one and the reagent two of liquid form, the reagent two that perhaps can comprise reagent one dry powder and liquid form, the reagent one and reagent two dry powder that perhaps can comprise liquid form perhaps can comprise reagent one dry powder and reagent two dry powder of solid form.Test kit of the present invention can comprise the specification sheets that records above-mentioned various component using method and consumption.Therefore, described solution also can be prepared according to prior art according to the record of test kit specification sheets before use by the user and get final product.
Being suitable for using the sample to be measured of test kit of the present invention can be under animal body (the comprising human body) state of health and the various body fluid that produce under the pathological state, for example: tumour hydrops of blood, lymph liquid, celiolymph, gastric juice and various Digestive system, seminal fluid, saliva, tear, sweat, urine, vaginal secretion, milk, amniotic fluid, bile, pleura hydrops (claim not only hydrothorax), segmental bronchus liquid, folliculi liquor, seroperitoneum (but also claiming ascites), pericardium hydrops, hydrarthrosis, each organ or the like.
Further specify the present invention below in conjunction with embodiment, the used reagent of the present invention, substratum are the commercial goods unless stated otherwise.
(1) existing glucose-6-phosphate dehydrogenase (G6PD) G6PDH gene is synthetic
The present inventor has designed primer G1 and G2 (seeing Table 1), utilizes primer G1 and G2 to be utilized the parental gene of pcr amplification G6PDH from Leuconostoc pseudomesenteroides ATCC 12291 bacterial classifications.The pcr amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer G1,400nM primer G2,1.5U Pfu archaeal dna polymerase (Promega, USA), add reaction system with a small amount of leuconostoc pseudomesenteroides of transfering loop picking, transfer to reaction volume with sterilized water and reach 50 μ L.
The pcr amplification reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 45 seconds, 55 ℃ 30 seconds and 72 ℃ 1 minute 30 seconds " then, kept 10 minutes down at 72 ℃ at last.The pcr amplification gene product that obtains is separated with 1% agarose gel electrophoresis, and reclaim the dna fragmentation of the single band of 1.4-1.6kb with QIA rapid extraction gel reagents box (fast and smart company (QIAGEN), Germany).
The amplified production that reclaims is connected to carrier pMD18-T, gets plasmid pMD18-T-parent G6PDH, be converted into host e. coli DH5 α, cultivate by the LB substratum, the LB culture medium prescription is: peptone 1%, yeast extract 0.5%, NaCl 1%, and surplus is a water, and pH 7.0.37 ℃ cultivate 12 hours after, extract plasmid DNA pMD18-T-parent G6PDH, cut and agarose gel electrophoresis separates the back and obtains the dna fragmentation that increases through enzyme.
The gained dna fragmentation is checked order with the full-automatic dna sequencing instrument of 310 types of u.s.a. applied biosystem company, sequencing result shows: containing length in the gained dna fragmentation is the protein coding gene of 1461bp (originate in initiator codon atg and end at terminator codon taa), is the nucleotide sequence shown in the SEQ ID NO:5.
(2) acquisition of G6PDH mutant (Y207F+N240S+K312R) goal gene
With plasmid pMD18-T-parent G6PDH is template, design 207FF and 207FR, 312RF and two pairs of primers of 312RR (seeing Table 1), the Tyr (Y) in the 207th site in the parent G6PDH aminoacid sequence is sported Phe (F), the Lys in the 312nd site (K) sports Arg (R), obtains mutant G6PDH (Y207F+K312R).Concrete steps are as follows:
(A) be template with plasmid pMD18-T-parent G6PDH, utilize primer, amplify fragment G1207FR G1 and 207FR.
(B) be template with plasmid pMD18-T-parent G6PDH, utilize primer, amplify fragment 207FF312RR 207FF and 312RR.
(C) be template with plasmid pMD18-T-parent G6PDH, utilize primer, amplify fragment 312RFG2 312RF and G2.
Amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mMMgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, and 400nM primer G1 and 400nM primer 2 07FR (perhaps are 400nM primer 2 07FF and 400nM primer 312RR, perhaps be 400nM primer 312RF and 400nM primer G2), 1.5U PfuDNA polysaccharase (Promega, USA), 20ng pMD18-T-parent G6PDH, transfer to reaction volume with sterilized water and reach 50 μ L.
The pcr amplification reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 45 seconds, 55 ℃ 30 seconds and 72 ℃ 35 seconds " then, kept 10 minutes down at 72 ℃ at last.
(D) separate through 1% agarose gel electrophoresis, and with QIAquick Extraction Gel Kit (QIAGEN, German) reclaim, with the full-automatic dna sequencing instrument order-checking of 310 types of u.s.a. applied biosystem company, sequencing result shows: obtain three kinds of amplified fragments, i.e. G1207FR, 207FF312RR and 312RFG2.
(E) be template with G1207FR, 207FF312RR and 3,12R,FG2 three bar segment, utilize primer, amplify full-length gene G1 and G2.
Amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mMMgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer G1 and 400nM primer G2,1.5U Pfu archaeal dna polymerase (Promega, USA), 20ng G1207FR, 20ng 207FF312RR and 20ng 312RFG2, transfer to reaction volume with sterilized water and reach 50 μ L.
The pcr amplification reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 45 seconds, 55 ℃ 30 seconds and 72 ℃ 1 minute 30 seconds " then, kept 10 minutes down at 72 ℃ at last.
Separate through 1% agarose gel electrophoresis, and with QIAquick Extraction Gel Kit (QIAGEN, German) reclaim, with the full-automatic dna sequencing instrument order-checking of 310 types of u.s.a. applied biosystem company, sequencing result shows: the gene order that obtains mutant G6PDH (Y207F+K312R).
Full-length gene fragment with mutant G6PDH (Y207F+K312R) is a template then, utilize primer to 240SF and 240SR (seeing Table 1), the Asn (N) in the 240th site in mutant G6PDH (Y207F+K312R) aminoacid sequence is sported Ser (S), obtain the gene order of mutant G6PDH (Y207F+N240S+K312R).Concrete steps are as follows:
(A) be template with mutant G6PDH (Y207F+K312R) fragment that reclaims, utilize primer, amplify fragment G1240SR G1 and 240SR.
(b) be template with mutant G6PDH (Y207F+K312R) fragment that reclaims, utilize primer, amplify fragment 240SFG2 240SF and G2.
Amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mMMgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer G1 and 400nM primer 2 40SR (perhaps 400nM primer 2 40SF and 400nM primer G2), 1.5U Pfu archaeal dna polymerase (Promega, USA), 20ng mutant G6PDH (Y207F+K312R) fragment, transfer to reaction volume with sterilized water and reach 50 μ L.
The pcr amplification reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 45 seconds, 55 ℃ 30 seconds and 72 ℃ 45 seconds " then, kept 10 minutes down at 72 ℃ at last.
Separate through 1% agarose gel electrophoresis, and (QIAGEN German) reclaims, and obtains the full-length gene order of mutant G6PDH (Y207F+N240S+K312R) with QIAquick Extraction Gel Kit.Mutant G6PDH (Y207F+N240S+K312R) is connected to carrier pMD18-T, gets plasmid pMD18-T-mutant G6PDH (Y207F+N240S+K312R), transform host e. coli DH5 α, use the LB culture medium culturing.37 ℃ cultivate 12 hours after, extract plasmid DNA pMD18-T-parent G6PDH, cut and agarose gel electrophoresis separates the back and obtains the dna fragmentation that increases through enzyme.
The gained dna fragmentation is checked order with the full-automatic dna sequencing instrument of 310 types of u.s.a. applied biosystem company, sequencing result shows: containing length in the gained dna fragmentation is the protein coding gene of 1461bp (originate in initiator codon atg and end at terminator codon taa), determine that through dna sequencing the catastrophe point of introducing is errorless, detailed sequence is shown in SEQ ID NO:2.
Table 1
(3) make up recombinant vectors
Cut the protein coding gene that (2) obtain with restriction enzyme NdeI and XhoI enzyme, enzyme is cut the big fragment of gained by using the T4DNA ligase enzyme, is connected to equally on the empty carrier pET28b (+) that cut with restriction enzyme NdeI and XhoI enzyme.To connect product and transform host e. coli DH5 α, use the LB culture medium culturing.37 ℃ cultivate 12 hours after, extract plasmid, cut with restriction enzyme NdeI and XhoI enzyme then, identify two bar segment that obtain to be about respectively 1.5kb and 5kb, prove to have obtained recombinant expression vector pET28b-G6PDH (Y207F+N240S+K312R) through agarose gel electrophoresis.Described recombinant expression vector pET28b-G6PDH (Y207F+N240S+K312R) preserves in pH is the tris damping fluid of 7 50mmol/L.
(4) recombinant vectors is to the conversion of host cell and the cultivation of gained recombinant host cell
Picking is 37 ℃ of single bacterium colonies of cultivating 16 hours e. coli bl21 (DE3) on the LB solid medium, cultivate 16 hours at 37 ℃, the shaking table of 150rpm in liquid LB substratum again.Get 1ml gained liquid culture and transfer in 100ml fresh liquid LB substratum, the rotating speed thermal agitation with 250-300rpm on 37 ℃ of shaking tables was cultivated 2.5 hours.Draw the cultured bacterium liquid of 1.5ml to the 1.5ml centrifuge tube, in cooled on ice after 10 minutes, under 4 ℃, 3000g centrifugal 5 minutes.Supernatant discarded adds the CaCl of 100 μ l at the 0.1mol/L of precooling on ice
2Solution is inhaled moving beating gently up and down with liquid-transfering gun and is spared, and re-suspended cell was placed 20 minutes at ice.Under 4 ℃, 3000g centrifugal 5 minutes then, supernatant discarded added the CaCl of 100 μ l at the 0.1mol/L of precooling on ice
2Solution is inhaled moving beating gently up and down with liquid-transfering gun and is spared, and re-suspended cell obtains competent host cell.
Get the above-mentioned competent e. coli bl21s of 200 μ l (DE3) and place the 1.5ml centrifuge tube, the adding volume is that pET28b-G6PDH (Y207F+N240S+K312R) solution (wherein pET28b-G6PDH (Y207F+N240S+K312R) content is 40ng) of 10 μ l shakes up gently, places on ice 30 minutes.Thermal shock 90 seconds in 42 ℃ of water-baths then placed cooled on ice rapidly 5 minutes then.Add 1ml LB liquid nutrient medium (not containing kantlex) in centrifuge tube, 37 ℃ of shaking culture are 1 hour behind the mixing, make bacterium the restore normal growth state and the kantlex antibiotics resistance gene (Ampr) of expression plasmid coding.Get 100 μ l after above-mentioned bacterium liquid shaken up and coat on the LB screening flat board that contains Kan, face up and place half an hour, treat that bacterium liquid is absorbed the back by substratum fully and is inverted culture dish, cultivated 20 hours for 37 ℃.Colibacillary bacterium colony has appearred on the screening flat board, be that pET28b-G6PDH (Y207F+N240S+K312R) has been converted in e. coli bl21 (DE3) competent cell, obtained the recombinant host cell of present embodiment---e. coli bl21 (DE3)-pET28bG6PDH (Y207F+N240S+K312R).
Preparation 3L LB substratum joins in the 5L fermentor tank, 121 ℃ of sterilizations 15 minutes, adds kantlex after being cooled to room temperature fully, and making its final concentration is 50 μ g/mL.In the gained substratum, add above-mentioned e. coli bl21 (DE3)-pET28bG6PDH (Y207F+N240S+K312R) (10 that 60mL grows to logarithmic phase
6Cells/ml).The cell density that is cultured under 37 ℃ in the fermented liquid sampling reaches 10
7Cells/ml.Being transferred to the TB substratum that 100mL contains 50mg/L with 5% inoculum size then induced 20 hours for 30 ℃.
The TB culture medium prescription is as follows: potassium primary phosphate: 3.4g/L, disodium hydrogen phosphate dodecahydrate: 8.95g/L, ammonium chloride: 2.68g/L, anhydrous sodium sulphate: 0.71g/L, magnesium sulfate heptahydrate: 0.49g/L, yeast powder: 24g/L, peptone: 10g/L, glucose: 0.025%, glycerine: 0.9%, lactose: 0.6%, water is solvent, adjusts pH to 6.8.
(5) separation of protein product is purified
With fermented liquid centrifugal 10min under 5000rpm that (4) obtain, collect the recombinant host cell precipitation.Supernatant discarded is resuspended in the gained recombinant host cell in the Tris-HCl solution that isopyknic pH is 7.5 50mmol/L.With the resuspended cell of high-pressure homogenization crusher machine, centrifugal 20min under 12000rpm collects the supernatant crude enzyme liquid, abandons or adopts the cell debris precipitation.
After above-mentioned gained crude enzyme liquid was 0.22 μ m membrane filtration with the aperture, gained filtrate was carried out affinity chromatography on An Keta purifying person (AKTA Purifier) the nickel affinity chromatography post of AM General electronics corporation (GE company).Wherein, sample-loading buffer is that the pH of 50mmol/L is 7.5 Tris-HCl damping fluid.Behind the sample-loading buffer that flows through two column volumes, use elution buffer, it is that the pH that contains the 50mmol/L of 0.5mol/L imidazoles is 7.5 Tris-HCl damping fluid.When imidazole concentration reaches 0.3mol/L, begin to collect albumen, collect the unimodal end of albumen that real-time monitors.
In the protein solution of collecting, add 40% (W/V) ammonium sulfate precipitation, under the rotating speed of 10000rpm centrifugal 10 minutes then.Collect albumen precipitation, and be 7.5 Tris-HCl damping fluid redissolution with the pH of 0.1mol/L.After gained solution was the membrane filtration of 0.22 μ m with the aperture, gained filtrate was used sephadex G-25 chromatography column (Sephadex G-25) desalination again.The pH that described desalination damping fluid is 0.1mol/L is 7.5 Tris-HCl damping fluid.Sample on using on the good chromatography column of desalination damping fluid balance, wherein the flow velocity of sample solution (being that above-mentioned pH with 0.1mol/L is the protein solution that 7.5 tris-HCl damping fluid redissolves) is 5ml/min, uses desalination damping fluid 3 column volumes of flow velocity wash-out with 10ml/min then.Totally 300 milliliters of liquid under the collection wash-out.
Collected liquid in general refrigerator-10 ℃ freezing 2 hours down, and then-40 ℃ of deep cooling refrigerator pre-freezes 8 hours, in the ALPHA 1-4 of German Ke Ruisite (CHRIST) LSC type freeze drier, with the condition freeze-drying of vacuum tightness 0.04mbar, safe pressure 0.100mbar and condenser temperature-60 ℃ 10 hours.Waiting temperature of charge and Freeze Drying Equipment baffle temperature difference then is zero, and observes 15 seconds internal pressures and indicate when constant, finishes freeze-drying.The proteinic amount of freeze-drying gained is 9.3 grams.Lyophilized protein seals preservation in normal temperature dryer.
Adopt " molecular cloning experiment guide " (Sa nurse Brooker etc., Science Press's (translating), 2002, see 1681 pages of appendix 8) the electrophoretic method of SDS-PAGE of the record proteinic molecular weight that determines present embodiment is about 54.4KD, electrophoresis result is seen Fig. 5, wherein 1 swimming lane is albumen marker, and 2 swimming lanes are the G6PDH of purifying.And according to " biological chemistry " (Wang Jingyan etc., Higher Education Publishing House, 2002, see 168 pages) measuring method (behind the enzymolysis divide peptide section measure) of the prlmary structure of protein of record, the protein that records present embodiment has 486 amino-acid residues (referring to the aminoacid sequence shown in the SEQ ID NO:1), with consistent by the nucleotide sequence coded result shown in the SEQ ID NO:2.Wherein, the difference of aminoacid sequence shown in aminoacid sequence shown in the SEQ ID NO:1 and the SEQ ID NO:4 is: the Tyr on the 207th of the SEQ ID NO:4 is replaced by Phe, and the Asn on the 240th is replaced by Ser, and the Lys on the 312nd is replaced by Arg.
(6) enzyme mensuration alive
Measure enzyme (test result sees the following form 2) alive as follows:
Reagent A: (pH 7.8, contain the MgCl of 3.3mM for 55mM Tris-HCl damping fluid
2)
Reagent B:60mM NAD
+The aqueous solution
Glucose six phosphate aqueous solutions of reagent C: 100mM
Enzyme diluent: 5mM Tris-HCl damping fluid (pH 7.8, contain 0.1% bovine serum albumin)
Concrete steps: in cuvette, add the reagent A of 2.7mL respectively, 0.1mL reagent B, the 0.1mL reagent C after 30 ℃ of temperature were bathed 5 minutes, adds the enzyme liquid of 0.1mL, slight mixing.30 ℃ were reacted the variation of writing down this process numerical value 5 minutes.Promptly measure the variation of the NADH of 340nm place absorbance value.Enzyme unit definition alive is: the amount of reduction generation 1 μ M NADH is an enzyme unit alive in 1 minute.
Formula is calculated in enzyme work
Enzyme (U/mg)=(U/mL) * 1/C alive
Wherein, Vt: cumulative volume (3.0mL)
Vs: enzyme liquid sample volume (0.1mL)
6.22: the molar absorptivity (cm of NADH under condition determination
2/ μ moL)
1.0: optical path length (cm)
Df: extension rate
C: enzyme concn in the solution
To sum up, the present inventor has obtained a kind of new glucose-6-phosphate dehydrogenase (G6PD), and its molecular weight is about 54.4KD, has the aminoacid sequence shown in the SEQ ID NO:1, nucleotide sequence coded by shown in the SEQ ID NO:2.Wherein, those skilled in the art are by reading present embodiment, can know for example SEQ ID NO:2 of nucleotide sequence of the present invention, and can obtain described nucleotide sequence by chemical synthesis process, therefore can be after obtaining having the nucleic acid of this nucleotide sequence, need not to implement present embodiment (1)-(2) step, begin the repetition present embodiment but directly go on foot from present embodiment (3).
According to SEQ ID NO:3 the nucleotide sequence shown in the SEQ ID NO:2 is carried out rite-directed mutagenesis, obtain the same sense mutation nucleotide sequence of the nucleotide sequence shown in the SEQ ID NO:2.Then the nucleotide sequence that obtains is connected on pET28b (+) empty carrier according to the method for preparing embodiment 1, also is transformed in the e. coli bl21 (DE3).Cultivate the gained recombinant host cell under the same conditions, the glucose-6-phosphate dehydrogenase (G6PD) rate ratio of present embodiment prepares embodiment 1 and has improved 20% as a result.
Preparation embodiment 3
Nucleotide sequence shown in the SEQ ID NO:2 is connected on the pRSET A empty carrier according to the method for preparing embodiment 1, also is transformed in the e. coli bl21 (DE3).Cultivate the gained recombinant host cell under the same conditions, glucose-6-phosphate dehydrogenase (G6PD) 9.2 grams that the result obtains.
SEQ ID NO:3 is connected on pET28b (+) empty carrier according to the method for preparing embodiment 1, is transformed among e. coli bl21-Rosetta.Cultivate the gained recombinant host cell under the same conditions, the glucose-6-phosphate dehydrogenase (G6PD) that the result obtains, its rate ratio prepare embodiment 1 and have improved 30%.
Comparative Examples 1
This Comparative Examples is disclosed mutant G6D66M5 (Y207F+N240S) among the JP2001-037480.
Test implementation example 1
The glucose-6-phosphate dehydrogenase (G6PD) activity for preparing embodiment 2-4 and Comparative Examples 1 according to the method test for preparing embodiment 1 record:
Table 2
| Glucose-6-phosphate dehydrogenase (G6PD) | |
|
Preparation embodiment 3 | |
Comparative Examples 1 |
| Enzyme (U/mg) alive | ?425.31 | ?452.60 | ?436.95 | ?450.58 | ?405.09 |
By above result as can be known, the glucose-6-phosphate dehydrogenase (G6PD) enzyme activity of preparation embodiment 1-4 is compared on the same order of magnitude with Comparative Examples 1, can satisfy the requirement to glucose-6-phosphate dehydrogenase (G6PD) of the test kit of measuring creatine kinase concentration in the sample to be measured clinically fully.
Test implementation example 2
Test prepares the thermostability of the glucose-6-phosphate dehydrogenase (G6PD) of embodiment 1-4 and Comparative Examples 1 as follows:
The glucose-6-phosphate dehydrogenase (G6PD) dry powder of 3KU is dissolved in 100mM Tris-HCl, and (pH 6.7, contain 2mMEDTA) in the damping fluid, respectively under 52 ℃, heated respectively 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes, described heating is carried out in the PCR instrument, last residual activity according to G6PDH after the activity test method mensuration heat treated of the G6PDH among the preparation embodiment 1.By the ratio percentage ratio that remaining enzyme is lived and maximum remaining enzyme is lived is that survival rate is an ordinate zou, and be the X-coordinate curve plotting heat-up time.The heat stability testing result of preparation embodiment 1 (aminoacid sequence that obtains of preparation embodiment 2-4 coding with prepare embodiment 1 identical) as shown in Figure 1, Comparative Examples 1 heat stability testing result is as shown in Figure 1.By relatively, prepare the thermostability (not shown) test result of embodiment 2-4 and prepare the basic identical of embodiment 1.
As can be seen from Figure 1, the thermostability of preparation embodiment 1 is significantly greater than Comparative Examples 1, and along with the prolongation of heat-up time, the survival rate of Comparative Examples 1 significantly descends, and preparation embodiment 1 survival rate just slightly descends.Heating is after 60 minutes down at 52 ℃, and preparation embodiment 1 survival rate is at least 92%, and the survival rate of Comparative Examples 1 is less than 25%.
Test implementation example 3
Test prepares the pH stability of the glucose-6-phosphate dehydrogenase (G6PD) of embodiment 1-4 and Comparative Examples 1 as follows:
Prepare the sodium-acetate buffer (pH=4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5) of pH 5.0-7.830mM respectively, the Tris-HCl damping fluid of pH 7.5-8.50.1M (pH=7.8,8.0,8.3,8.5), the glycine buffer of pH 8.5-9.50.1M (pH=8.8,9.0,9.3,9.5).The glucose-6-phosphate dehydrogenase (G6PD) of 3KU is dissolved in respectively in the above-mentioned damping fluid, under 30 ℃, left standstill 17 hours, and measured according to the activity test method of G6PDH among the preparation embodiment 1 (aminoacid sequence that obtains of preparation embodiment 2-4 coding is identical with preparation embodiment 1) then.By the ratio percentage ratio that remaining enzyme is lived and maximum remaining enzyme is lived is that survival rate is an ordinate zou, and the pH value is the X-coordinate curve plotting.The test result of preparation embodiment 1 is seen Fig. 2, and the test result of Comparative Examples 1 is seen Fig. 3.By relatively, prepare the pH stability test result of embodiment 2-4 and the basic identical (not shown) of preparation embodiment 1.
From Fig. 2 and Fig. 3 as can be seen, the pH stability of preparation embodiment 1 significantly is better than Comparative Examples 1, the kind analogy prior art that so comprises the sample to be measured that the test kit of glucose-6-phosphate dehydrogenase (G6PD) of the present invention can be measured is more, the transportation of test kit, the condition of preservation are reduced, the accurate meticulous requirement of operation to the personnel that use test kit reduces, and helps having the popularizing of test kit of glucose-6-phosphate dehydrogenase (G6PD).
Test implementation example 4
The enzyme that will prepare embodiment 1-4 (aminoacid sequence that obtains of preparation embodiment 2-4 coding with prepare embodiment 1 identical) and Comparative Examples 1 is used 50mM Tris-HCl damping fluid respectively, and (pH 7.8, contain the MgCl of 3.3mM
2) be diluted to 1mg/ml and be diluted to 1mg/ml, in-20 ℃ freezing, melt in room temperature again, 5 times repeatedly, measure the residual ratio activity of G6PDH after each freeze thawing respectively according to the activity test method of the G6PDH among the preparation embodiment 1.With the enzyme work before for the first time freezing is 100%, and the percentage ratio that remaining enzyme is lived and freezing preferment is lived after the freeze thawing is that survival rate is an ordinate zou, and number of freezing and thawing is the X-coordinate mapping.By relatively, prepare the multigelation test result of embodiment 2-4 and prepare the basic identical of embodiment 1.
As can be seen from Figure 4, the multigelation stability of preparation embodiment 1 is significantly greater than Comparative Examples 1, and after multigelation 3 times, the survival rate of preparation embodiment 1 is about 50%, and the survival rate of Comparative Examples is about 20%.After the freeze thawing 5 times, the survival rate of preparation embodiment 1 is at least 27%, and Comparative Examples only is 6%.
Preparation embodiment 5-8 creatine kinase test kit
Formulated reagent one and reagent two according to table 3:
Table 3
Comparative Examples 2
Become the test kit of liquid form according to the formulated identical with preparation embodiment 5.Used G6PDH is the G6PDH of Comparative Examples 1 in the Comparative Examples 2.
Test implementation example 5
This test implementation example has been measured two samples to be measured: one is the creatine kinase aqueous standard product of the 200U/L of preparation; Another is 25 years old male sex's being in a good state of health a serum sample to be measured, extracts its 10ml blood, leaves standstill to the hemocyte precipitated and separated, gets upper serum 5ml and is used for the test kit of preparation embodiment 5-8 and Comparative Examples 2 is tested.
The test kit using method is as follows:
Using theoretical factor, is blank calibration with the deionized water.According to following table 4 measuring parameter is set:
Table 4
Method of calculation:
Enzyme (U/mg)=(U/L) * 1/C alive
Wherein, Vt: cumulative volume (0.26mL)
Vs: enzyme liquid sample volume (0.01mL)
6.22: the molar absorptivity (cm of NADH under condition determination
2/ μ moL)
1.0: optical path length (cm)
Df: extension rate
C: enzyme concn in the solution
Measurement result the results are shown in Table 5.
Table 5
As can be seen from Table 5, the test kit test result of preparation embodiment 5-8 than Comparative Examples 2 more near actual value; And the test kit test result standard deviation of preparation embodiment 5-8 is significantly less than Comparative Examples 2, illustrates that test kit performance of the present invention is more stable, and it is more accurate to measure.
Sequence table
＜110〉Beijing Leaderman Biochemistry Co., Ltd
＜120〉glucose-6-phosphate dehydrogenase (G6PD) and nucleotide sequence thereof, recombinant vectors, recombinant host cell and test kit
<130>OICN093788
<160>5
<170>PatentIn?version?3.4
<210>1
<211>486
<212>PRT
<213>Leuconostoc?pseudomesenteroides
<400>1
Met?Val?Ser?Glu?Ile?Lys?Thr?Leu?Val?Thr?Phe?Phe?Gly?Gly?Thr?Gly
1 5 10 15
Asp?Leu?Ala?Lys?Arg?Lys?Leu?Tyr?Pro?Ser?Val?Phe?Asn?Leu?Tyr?Lys
20 25 30
Lys?Gly?Tyr?Leu?Gln?Lys?His?Phe?Ala?Ile?Val?Gly?Thr?Ala?Arg?Gln
35 40 45
Ala?Leu?Asn?Asp?Asp?Glu?Phe?Lys?Gln?Leu?Val?Arg?Asp?Ser?Ile?Lys
50 55 60
Asp?Phe?Thr?Asp?Asp?Gln?Ala?Gln?Ala?Glu?Ala?Phe?Ile?Glu?His?Phe
65 70 75 80
Ser?Tyr?Arg?Ala?His?Asp?Val?Thr?Asp?Ala?Ala?Ser?Tyr?Ala?Val?Leu
85 90 95
Lys?Glu?Ala?Ile?Glu?Glu?Ala?Ala?Asp?Lys?Phe?Asp?Ile?Asp?Gly?Asn
100 105 110
Arg?Ile?Phe?Tyr?Met?Ser?Val?Ala?Pro?Arg?Phe?Phe?Gly?Thr?Ile?Ala
115 120 125
Lys?Tyr?Leu?Lys?Ser?Glu?Gly?Leu?Leu?Ala?Asp?Thr?Gly?Tyr?Asn?Arg
130 135 140
Leu?Met?Ile?Glu?Lys?Pro?Phe?Gly?Thr?Ser?Tyr?Asp?Thr?Ala?Ala?Glu
145 150 155 160
Leu?Gln?Asn?Asp?Leu?Glu?Asn?Ala?Phe?Asp?Asp?Asn?Gln?Leu?Phe?Arg
165 170 175
Ile?Asp?His?Tyr?Leu?Gly?Lys?Glu?Met?Val?Gln?Asn?Ile?Ala?Ala?Leu
180 185 190
Arg?Phe?Gly?Asn?Pro?Ile?Phe?Asp?Ala?Ala?Trp?Asn?Lys?Asp?Phe?Ile
195 200 205
Lys?Asn?Val?Gln?Val?Thr?Leu?Ser?Glu?Val?Leu?Gly?Val?Glu?Glu?Arg
210 215 220
Ala?Gly?Tyr?Tyr?Asp?Thr?Ala?Gly?Ala?Leu?Leu?Asp?Met?Ile?Gln?Ser
225 230 235 240
His?Thr?Met?Gln?Ile?Val?Gly?Trp?Leu?Ala?Met?Glu?Lys?Pro?Glu?Ser
245 250 255
Phe?Thr?Asp?Lys?Asp?Ile?Arg?Ala?Ala?Lys?Asn?Ala?Ala?Phe?Asn?Ala
260 265 270
Leu?Lys?Ile?Tyr?Asp?Glu?Ala?Glu?Val?Asn?Lys?Tyr?Phe?Val?Arg?Ala
275 280 285
Gln?Tyr?Gly?Ala?Gly?Asp?Ser?Ala?Asp?Phe?Lys?Pro?Tyr?Leu?Glu?Glu
290 295 300
Leu?Asp?Val?Pro?Ala?Asp?Ser?Arg?Asn?Asn?Thr?Phe?Ile?Ala?Gly?Glu
305 310 315 320
Leu?Gln?Phe?Asp?Leu?Pro?Arg?Trp?Glu?Gly?Val?Pro?Phe?Tyr?Val Arg
325 330 335
Ser?Gly?Lys?Arg?Leu?Ala?Ala?Lys?Gln?Thr?Arg?Val?Asp?Ile?Val?Phe
340 345 350
Lys?Ala?Gly?Thr?Phe?Asn?Phe?Gly?Ser?Glu?Gln?Glu?Ala?Gln?Glu?Ala
355 360 365
Val?Leu?Ser?Ile?Ile?Ile?Asp?Pro?Lys?Gly?Ala?Ile?Glu?Leu?Lys?Leu
370 375 380
Asn?Ala?Lys?Ser?Val?Glu?Asp?Ala?Phe?Asn?Thr?Arg?Thr?Ile?Asp?Leu
385 390 395 400
Gly?Trp?Thr?Val?Ser?Asp?Glu?Asp?Lys?Lys?Asn?Thr?Pro?Glu?Pro?Tyr
405 410 415
Glu?Arg?Met?Ile?His?Asp?Thr?Met?Asn?Gly?Asp?Gly?Ser?Asn?Phe?Ala
420 425 430
Asp?Trp?Asn?Gly?Val?Ser?Ile?Ala?Trp?Lys?Phe?Val?Asp?Ala?Ile?Ser
435 440 445
Ala?Val?Tyr?Thr?Ala?Asp?Lys?Ala?Pro?Leu?Glu?Thr?Tyr?Lys?Ser?Gly
450 455 460
Ser?Met?Gly?Pro?Glu?Ala?Ser?Asp?Lys?Leu?Leu?Ala?Ala?Asn?Gly?Asp
465 470 475 480
Ala?Trp?Val?Phe?Lys?Gly
485
<210>2
<211>1461
<212>DNA
<213>Leuconostoc?pseudomesenteroides
<400>2
atggtttcag?aaatcaagac?gttagtaact?ttctttggtg?gcactggtga?cttggccaag 60
cgtaagcttt?acccatcagt?tttcaatctt?tataaaaaag?gctacttgca?aaagcatttt 120
gccattgttg?gaacggcccg?tcaagccctc?aatgatgacg?aattcaaaca?attggttcgt 180
gattcaatta?aagatttcac?tgacgatcaa?gcacaagctg?aggcgttcat?cgaacatttc 240
tcataccgtg?cacacgacgt?aacagatgct?gcttcatacg?ctgttttaaa?agaggcgatt 300
gaagaagctg?ccgacaaatt?tgatatcgat?ggcaaccgca?ttttctatat?gtcagttgcg 360
ccacgtttct?ttggtacaat?tgccaaatat?cttaagtcag?aaggcctact?agctgacact 420
ggttacaacc?gtttgatgat?tgaaaagcct?ttcggtacat?catacgacac?agctgccgaa 480
ctccaaaatg?acttggaaaa?cgcatttgat?gataaccaac?tattccgtat?tgaccactac 540
cttggtaagg?aaatggttca?aaacattgct?gcccttcgct?ttggtaaccc?aattttcgat 600
gctgcttgga?acaaggattt?catcaagaac?gttcaagtaa?cattgtcaga?agtcttgggt 660
gtcgaagaac?gtgccggcta?ctatgacaca?gccggtgcat?tgcttgacat?gattcaatcc 720
cacaccatgc?aaattgttgg?ttggttagcc?atggaaaaac?cagaatcatt?cactgacaaa 780
gacattcgtg?ccgctaaaaa?cgcagccttt?aatgctttga?agatctatga?tgaagcagaa 840
gttaacaaat?actttgttcg?tgcacaatat?ggtgccggtg?attcagctga?cttcaagcca 900
taccttgaag?aattagacgt?acctgctgat?tctcgtaaca?ataccttcat?cgccggcgaa 960
ttgcaatttg?atttgccacg?ttgggagggt?gtcccattct?atgtccgttc?aggtaagcgc 1020
ttagctgcta?aacagacacg?ggttgatatc?gtctttaagg?ctggcacgtt?taactttggt 1080
tcagaacaag?aagcacaaga?agctgtcttg?tcaattatca?ttgatccaaa?gggtgctatc 1140
gaattgaagt?tgaacgccaa?gtcagttgaa?gatgctttca?acacacgtac?aattgactta 1200
ggttggactg?tatctgacga?agataagaag?aacacgccag?aaccatacga?acgtatgatt 1260
cacgacacta?tgaatggtga?tggctctaac?ttcgctgact?ggaatggcgt?ttcaatcgct 1320
tggaagttcg?ttgatgctat?ttcagccgtt?tataccgcag?ataaagcacc?acttgaaact 1380
tacaagtcgg?gctcaatggg?tcctgaagca?tccgataaat?tattggctgc?caatggtgat 1440
gcttgggtgt?ttaaaggtta?a 1461
<210>3
<211>1461
<212>DNA
<213>Leuconostoc?pseudomesenteroides
<400>3
atggtgagcg?aaattaaaac?cctggtgacc?ttttttggcg?gcaccggcga?tctggcgaaa 60
cgtaaactgt?atccgagcgt?gtttaacctg?tataaaaaag?gctatctgca?gaaacatttt 120
gcgattgtgg?gcaccgcgcg?tcaggcgctg?aacgatgatg?aatttaaaca?gctggtgcgt 180
gatagcatta?aagattttac?cgatgatcag?gcgcaggcgg?aagcgtttat?tgaacatttt 240
agctatcgtg?cgcatgatgt?gaccgatgcg?gcgagctatg?cggtgctgaa?agaagcgatt 300
gaagaagcgg?cggataaatt?tgatattgat?ggcaaccgta?ttttttatat?gagcgtggcg 360
ccgcgttttt?ttggcaccat?tgcgaaatat?ctgaaaagcg?aaggcctgct?ggcggatacc 420
ggctataacc?gtctgatgat?tgaaaaaccg?tttggcacca?gctatgatac?cgcggcggaa 480
ctgcagaacg?atctggaaaa?cgcgtttgat?gataaccagc?tgtttcgtat?tgatcattat 540
ctgggcaaag?aaatggtgca?gaacattgcg?gcgctgcgtt?ttggcaaccc?gatttttgat 600
gcggcgtgga?acaaagattt?tattaaaaac?gtgcaggtga?ccctgagcga?agtgctgggc 660
gtggaagaac?gtgcgggcta?ttatgatacc?gcgggcgcgc?tgctggatat?gattcagagc 720
cataccatgc?agattgtggg?ctggctggcg?atggaaaaac?cggaaagctt?taccgataaa 780
gatattcgtg?cggcgaaaaa?cgcggcgttt?aacgcgctga?aaatttatga?tgaagcggaa 840
gtgaacaaat?attttgtgcg?tgcgcagtat?ggcgcgggcg?atagcgcgga?ttttaaaccg 900
tatctggaag?aactggatgt?gccggcggat?agccgtaaca?acacctttat?tgcgggcgaa 960
ctgcagtttg?atctgccgcg?ttgggaaggc?gtgccgtttt?atgtgcgtag?cggcaaacgt 1020
ctggcggcga?aacagacccg?tgtggatatt?gtgtttaaag?cgggcacctt?taactttggc 1080
agcgaacagg?aagcgcagga?agcggtgctg?agcattatta?ttgatccgaa?aggcgcgatt 1140
gaactgaaac?tgaacgcgaa?aagcgtggaa?gatgcgttta?acacccgtac?cattgatctg 1200
ggctggaccg?tgagcgatga?agataaaaaa?aacaccccgg?aaccgtatga?acgtatgatt 1260
catgatacca?tgaacggcga?tggcagcaac?tttgcggatt?ggaacggcgt?gagcattgcg 1320
tggaaatttg?tggatgcgat?tagcgcggtg?tataccgcgg?ataaagcgcc?gctggaaacc 1380
tataaaagcg?gcagcatggg?cccggaagcg?agcgataaac?tgctggcggc?gaacggcgat 1440
gcgtgggtgt?ttaaaggcta?a 1461
<210>4
<211>486
<212>PRT
<213>Leuconostoc?pseudomesenteroides
<400>4
Met?Val?Ser?Glu?Ile?Lys?Thr?Leu?Val?Thr?Phe?Phe?Gly?Gly?Thr?Gly
1 5 10 15
Asp?Leu?Ala?Lys?Arg?Lys?Leu?Tyr?Pro?Ser?Val?Phe?Asn?Leu?Tyr?Lys
20 25 30
Lys?Gly?Tyr?Leu?Gln?Lys?His?Phe?Ala?Ile?Val?Gly?Thr?Ala?Arg?Gln
35 40 45
Ala?Leu?Asn?Asp?Asp?Glu?Phe?Lys?Gln?Leu?Val?Arg?Asp?Ser?Ile?Lys
50 55 60
Asp?Phe?Thr?Asp?Asp?Gln?Ala?Gln?Ala?Glu?Ala?Phe?Ile?Glu?His?Phe
65 70 75 80
Ser?Tyr?Arg?Ala?His?Asp?Val?Thr?Asp?Ala?Ala?Ser?Tyr?Ala?Val?Leu
85 90 95
Lys?Glu?Ala?Ile?Glu?Glu?Ala?Ala?Asp?Lys?Phe?Asp?Ile?Asp?Gly?Asn
100 105 110
Arg?Ile?Phe?Tyr?Met?Ser?Val?Ala?Pro?Arg?Phe?Phe?Gly?Thr?Ile?Ala
115 120 125
Lys?Tyr?Leu?Lys?Ser?Glu?Gly?Leu?Leu?Ala?Asp?Thr?Gly?Tyr?Asn?Arg
130 135 140
Leu?Met?Ile?Glu?Lys?Pro?Phe?Gly?Thr?Ser?Tyr?Asp?Thr?Ala?Ala?Glu
145 150 155 160
Leu?Gln?Asn?Asp?Leu?Glu?Asn?Ala?Phe?Asp?Asp?Asn?Gln?Leu?Phe?Arg
165 170 175
Ile?Asp?His?Tyr?Leu?Gly?Lys?Glu?Met?Val?Gln?Asn?Ile?Ala?Ala?Leu
180 185 190
Arg?Phe?Gly?Asn?Pro?Ile?Phe?Asp?Ala?Ala?Trp?Asn?Lys?Asp?Tyr?Ile
195 200 205
Lys?Asn?Val?Gln?Val?Thr?Leu?Ser?Glu?Val?Leu?Gly?Val?Glu?Glu?Arg
210 215 220
Ala?Gly?Tyr?Tyr?Asp?Thr?Ala?Gly?Ala?Leu?Leu?Asp?Met?Ile?Gln?Asn
225 230 235 240
His?Thr?Met?Gln?Ile?Val?Gly?Trp?Leu?Ala?Met?Glu?Lys?Pro?Glu?Ser
245 250 255
Phe?Thr?Asp?Lys?Asp?Ile?Arg?Ala?Ala?Lys?Asn?Ala?Ala?Phe?Asn?Ala
260 265 270
Leu?Lys?Ile?Tyr?Asp?Glu?Ala?Glu?Val?Asn?Lys?Tyr?Phe?Val?Arg?Ala
275 280 285
Gln?Tyr?Gly?Ala?Gly?Asp?Ser?Ala?Asp?Phe?Lys?Pro?Tyr?Leu?Glu?Glu
290 295 300
Leu?Asp?Val?Pro?Ala?Asp?Ser?Lys?Asn?Asn?Thr?Phe?Ile?Ala?Gly?Glu
305 310 315 320
Leu?Gln?Phe?Asp?Leu?Pro?Arg?Trp?Glu?Gly?Val?Pro?Phe?Tyr?Val?Arg
325 330 335
Ser?Gly?Lys?Arg?Leu?Ala?Ala?Lys?Gln?Thr?Arg?Val?Asp?Ile?Val?Phe
340 345 350
Lys?Ala?Gly?Thr?Phe?Asn?Phe?Gly?Ser?Glu?Gln?Glu?Ala?Gln?Glu?Ala
355 360 365
Val?Leu?Ser?Ile?Ile?Ile?Asp?Pro?Lys?Gly?Ala?Ile?Glu?Leu?Lys?Leu
370 375 380
Asn?Ala?Lys?Ser?Val?Glu?Asp?Ala?Phe?Asn?Thr?Arg?Thr?Ile?Asp?Leu
385 390 395 400
Gly?Trp?Thr?Val?Ser?Asp?Glu?Asp?Lys?Lys?Asn?Thr?Pro?Glu?Pro?Tyr
405 410 415
Glu?Arg?Met?Ile?His?Asp?Thr?Met?Asn?Gly?Asp?Gly?Ser?Asn?Phe?Ala
420 425 430
Asp?Trp?Asn?Gly?Val?Ser?Ile?Ala?Trp?Lys?Phe?Val?Asp?Ala?Ile?Ser
435 440 445
Ala?Val?Tyr?Thr?Ala?Asp?Lys?Ala?Pro?Leu?Glu?Thr?Tyr?Lys?Ser?Gly
450 455 460
Ser?Met?Gly?Pro?Glu?Ala?Ser?Asp?Lys?Leu?Leu?Ala?Ala?Asn?Gly?Asp
465 470 475 480
Ala?Trp?Val?Phe?Lys?Gly
485
<210>5
<211>1461
<212>DNA
<213>Leuconostoc?pseudomesenteroides
<400>5
atggtttcag?aaatcaagac?gttagtaact?ttctttggtg?gcactggtga?cttggccaag 60
cgtaagcttt?acccatcagt?tttcaatctt?tataaaaaag?gctacttgca?aaagcatttt 120
gccattgttg?gaacggcccg?tcaagccctc?aatgatgacg?aattcaaaca?attggttcgt 180
gattcaatta?aagatttcac?tgacgatcaa?gcacaagctg?aggcgttcat?cgaacatttc 240
tcataccgtg?cacacgacgt?aacagatgct?gcttcatacg?ctgttttaaa?agaggcgatt 300
gaagaagctg?ccgacaaatt?tgatatcgat?ggcaaccgca?ttttctatat?gtcagttgcg 360
ccacgtttct?ttggtacaat?tgccaaatat?cttaagtcag?aaggcctact?agctgacact 420
ggttacaacc?gtttgatgat?tgaaaagcct?ttcggtacat?catacgacac?agctgccgaa 480
ctccaaaatg?acttggaaaa?cgcatttgat?gataaccaac?tattccgtat?tgaccactac 540
cttggtaagg?aaatggttca?aaacattgct?gcccttcgct?ttggtaaccc?aattttcgat 600
gctgcttgga?acaaggatta?catcaagaac?gttcaagtaa?cattgtcaga?agtcttgggt 660
gtcgaagaac?gtgccggcta?ctatgacaca?gccggtgcat?tgcttgacat?gattcaaaac 720
cacaccatgc?aaattgttgg?ttggttagcc?atggaaaaac?cagaatcatt?cactgacaaa 780
gacattcgtg?ccgctaaaaa?cgcagccttt?aatgctttga?agatctatga?tgaagcagaa 840
gttaacaaat?actttgttcg?tgcacaatat?ggtgccggtg?attcagctga?cttcaagcca 900
taccttgaag?aattagacgt?acctgctgat?tctaaaaaca?ataccttcat?cgccggcgaa 960
ttgcaatttg?atttgccacg?ttgggagggt?gtcccattct?atgtccgttc?aggtaagcgc 1020
ttagctgcta?aacagacacg?ggttgatatc?gtctttaagg?ctggcacgtt?taactttggt 1080
tcagaacaag?aagcacaaga?agctgtcttg?tcaattatca?ttgatccaaa?gggtgctatc 1140
gaattgaagt?tgaacgccaa?gtcagttgaa?gatgctttca?acacacgtac?aattgactta 1200
ggttggactg?tatctgacga?agataagaag?aacacgccag?aaccatacga?acgtatgatt 1260
cacgacacta?tgaatggtga?tggctctaac?ttcgctgact?ggaatggcgt?ttcaatcgct 1320
tggaagttcg?ttgatgctat?ttcagccgtt?tataccgcag?ataaagcacc?acttgaaact 1380
tacaagtcgg?gctcaatggg?tcctgaagca?tccgataaat?tattggctgc?caatggtgat 1440
gcttgggtgt?ttaaaggtta?a 1461
Claims (10)
1. a glucose-6-phosphate dehydrogenase (G6PD) is characterized in that, the aminoacid sequence of described glucose-6-phosphate dehydrogenase (G6PD) is the aminoacid sequence shown in the SEQ ID NO:1.
2. the nucleotide sequence of the glucose-6-phosphate dehydrogenase (G6PD) of encoding is characterized in that, described nucleotides sequence is classified the nucleotide sequence of the described glucose-6-phosphate dehydrogenase (G6PD) of coding claim 1 as.
3. nucleotide sequence according to claim 2, wherein, described nucleotides sequence is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:2 is carried out that one or several Nucleotide replaces and the nucleotide sequence of the codon same sense mutation that obtains.
4. nucleotide sequence according to claim 3, wherein, described nucleotides sequence is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:3.
5. a recombinant vectors is characterized in that, described recombinant vectors is made up of the goal gene of empty carrier and this empty carrier of insertion, and described goal gene is the nucleotide sequence of any described glucose-6-phosphate dehydrogenase (G6PD) among the claim 2-4.
6. recombinant vectors according to claim 5 is characterized in that, described empty carrier is pET28b (+).
7. a recombinant host cell is characterized in that, described recombinant host cell contains claim 5 or 6 described recombinant vectorss.
8. recombinant host cell according to claim 7 is characterized in that, described host cell is e. coli bl21 (DE3) or e. coli bl21-Rosetta.
9. a test kit that detects creatine kinase is characterized in that, described test kit comprises:
The described glucose-6-phosphate dehydrogenase (G6PD) of claim 1.
10. test kit according to claim 9, it is characterized in that described test kit also comprises: the hexokinase, the ADP of 1-3mM of G6PDH, 2-3KU/L that is dissolved in NADP, the 1-3KU/L of 15-25mM N-acetylcystein in the 80-120mM imidazole buffer of pH 6.5-7.0,1-3mM ethylenediamine tetraacetic acid (EDTA), 1-3mM; And be dissolved in the glucose of the 15-30mM in 40-60mM 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid damping fluid of pH 9.0-11.0 and the creatine phosphate of 20-40mM.
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Non-Patent Citations (5)
| Title |
|---|
| JP特开2001-37480A 2001.02.13 |
| JP特开2005-130773A 2005.05.26 |
| JP特开平10-225293A 1998.08.25 |
| 孟泽.葡萄糖-6-磷酸脱氢酶（G6PD）试剂中该酶活力测定方法的改进.《临床检验杂志》.1986,第 4 卷(第 3 期),124-125. * |
| 锺丽民等.聚丙烯酰胺凝胶垂直板电泳检测血清肌酸激酶同工酶.《上海医学检验杂志》.1989,第 4 卷(第 3 期),132-133. * |
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