CN1379100A - High-temp. resistant dihydroorotate synthase gene and its coded polypeptide and preparing method - Google Patents
High-temp. resistant dihydroorotate synthase gene and its coded polypeptide and preparing method Download PDFInfo
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Abstract
The invention discloses a high-temperature resistant dihydroorotate synthase gene, a polypeptide coded by the gene and a preparation method of the gene. It relates to a separated DNA coding with high-temperature resistant dihydroorotate synthase activity or its functional equivalent variant and the polypeptide with high-temperature resistant dihydroorotate synthase activity or its functional equivalent variant produced by using said separated DNA by means of recombinant DNA technology. The invention clones and separates the high temperature resistant dihydroorotate synthase gene based on sequencing and analyzing Tengchong thermophilic anaerobe whole genome. The present invention relates to transgenic microbe or transgenic animal and plant for producing high temperature dihydroorotate synthase and recovering the enzyme coded by said gene. In addition, the invention also provides an amino acid sequence and a functional equivalent of the polypeptide with the high-temperature resistant dihydroorotate synthase activity. Meanwhile, the invention also provides a method for preparing, separating and purifying the polypeptide with the high-temperature resistant dihydroorotate synthase activity.
Description
Technical field
The present invention relates to field of genetic engineering, particularly, relate to a kind of refractory dihydroorate synthase gene and encoded polypeptides thereof and prepare the method for this polypeptide.
Background technology
Dihydroorate synthase (Dihydropteroate synthase) is a kind of enzyme that plays an important role during folic acid biological synthesizes.Its effect is catalysis 2-amino-4 a hydroxyl 6-methylol 7, and the amino benzoate of 8-dihydropteridine diphosphate and 4-generates bisphosphate and dihydroorotate.
Dihydroorate synthase is a kind of enzyme that plays an important role in folic acid biological is synthetic.Though higher eukaryotic cell can not synthesize folic acid again, therefore can not produce DHPS (dihydroorate synthase).DHPS is as the amino benzoate hydroxylase of 4-, and it acts on and produces the path that mixture and media are in relation to the neurodegeneration confusion.Folic acid deficiency has been used in the neurodegenerative confusion of research road syndrome and acquired immune deficiency syndrome (AIDS) generation.
Now very urgently to develop new antimalarial drug, because make human lethal malaria produce resistance to common antimalarial drug such as sulphormethoxine.By research, have been found that its drug-fast generation and dihydroorate synthase, the encoding gene of dihydroorate synthase is relevant.And, embodied by sulfa drugs and 2 hydrogen folic acid reduction mould (DHFR) inhibitor.Use molecule, it is to transmit by DHPS (dihydroorate synthase) that heredity and biochemical method have disclosed the opposing sulfa drugs.
Sulphormethoxine suppresses dihydroorate synthase, and this is to occur in a kind of parasite in malaria (malaria parasite), and still, it does not occur in the people.The mechanism of hiding of lethal effect that parasite hides two kinds of antimalarial drugs has been illustrated in research, promptly interrupts the synthetic of DNA by anti-folic acid, so that medicine is produced resistance.
This enzyme can be applied to the research and development of novel antimalarial drug, the synthetic of folic acid, the research and the treatment of the neurodegenerative confusion that road syndrome and acquired immune deficiency syndrome (AIDS) produce.Therefore aspect pharmaceutical sector, have broad application prospects.
The Tengchong thermophilc anaerobe that the present invention relates to (Thermoanaerobacter tangcongensis), it is a kind of microorganism that lives in the hot spring of Yunnan Province of China province Tengchong County, it is a kind of thermophilic eubacterium (eubacteria), optimum growth temperature is 75 degrees centigrade, anaerobic growth, the gramstaining reaction is positive.It is at first to be found and carried out the analysis on the taxonomy by Microbe Inst., Chinese Academy of Sciences.Bacterial classification is kept at Chinese microorganism and preserves center MB4
T(Chinese collection of microorganisms AS 1.2430
T=JCM 11007
T).This thermophilc anaerobe is the distinctive species of China, and the refractory dihydroorate synthase that is had in its body also has own its specific structure.
Summary of the invention
One of purpose of the present invention provides a kind of isolating, and coding has the nucleotide sequence of the active polypeptide of refractory dihydroorate synthase.
Two of purpose of the present invention provides a kind of isolating, has the refractory dihydroorate synthase active polypeptide.
Another order of the present invention is the recombinant vectors that the DNA that contains the refractory dihydroorate synthase of encoding also is provided, the host cell that contains aforementioned recombinant vectors, and this proteic method of production.
One aspect of the present invention provides a kind of coding to have the nucleotide sequence of the active polypeptide of refractory dihydroorate synthase.This nucleotide sequence can encode the polypeptide with the aminoacid sequence among the sequence table SEQ ID NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with dihydroorate synthase.Nucleotide sequence has the polynucleotide sequence of sequence table SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
The present invention provides a kind of refractory dihydroorate synthase active polypeptide on the other hand.This polypeptide has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the sequence table SEQ ID No.2.
The invention provides the method for preparing refractory dihydroorate synthase, it may further comprise the steps:
1) isolates the gene of the coding refractory dihydroorate synthase of nucleotide sequence shown in sequence table SEQ ID NO.1;
2) make up the expression vector that contains just like the nucleotide sequence of the described gene of step 1);
3) with step 2) in expression vector change host cell over to, formation can be produced the reconstitution cell of refractory dihydroorate synthase;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain refractory dihydroorate synthase.
The present invention relates to the separation and the expression of the dihydroorate synthase gene of thermophilc anaerobe.Based on Tengchong thermophilc anaerobe genome sequencing and analysis, clone and separate refractory dihydroorate synthase gene.Use the transgenic microorganism or the animals and plants of this genes produce refractory dihydroorate synthase, and reclaim the enzyme that obtains this genes encoding.In addition, the present invention also provides and has had active amino acid sequence of polypeptide of refractory dihydroorate synthase and functional equivalent body.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the active polypeptide of refractory dihydroorate synthase.
Description of drawings
Fig. 1 is an order-checking library construction flow chart of steps;
Fig. 2 is order-checking and data analysis schema.
Embodiment
The invention provides isolating, the nucleic acid molecule of the active polypeptide of coding refractory dihydroorate synthase, this nucleic acid molecule has the nucleotide sequence shown in the sequence table SEQ .ID NO.1 by Tengchong thermophilc anaerobe genome sequencing and analysis are obtained.It has been encoded and has had that refractory dihydroorate synthase is active 270 amino acid whose polypeptide, and the supposition molecular weight of this polypeptide is 29815 dalton.
The invention still further relates to a kind of recombinant vectors, this carrier comprises isolating nucleic acid molecule of the present invention, and the host cell that includes recombinant vectors.Simultaneously, the present invention includes the method that makes up this recombinant vectors and host cell, and utilize the recombined engineering technology to produce the method for dihydroorate synthase.
The present invention provides a kind of isolating refractory dihydroorate synthase or polypeptide further, it is characterized in that having the aminoacid sequence shown in the sequence table SEQ .ID NO.2, or at least 70% is similar, more preferably, have at least 90%, 95%, 99% identical.
In the present invention, " isolating " DNA is meant that this DNA or fragment have been arranged in its both sides under native state sequence separates, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, " refractory dihydroorate synthase gene " refers to encode and has the nucleotide sequence of the active polypeptide of refractory dihydroorate synthase, as nucleotide sequence and the degenerate sequence thereof of sequence table SEQ .ID NO.1.This degenerate sequence be meant have one or more codons to be encoded in this sequence the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of known codon, so be low to moderate about 70% the degenerate sequence described aminoacid sequence of sequence table SEQ ID NO.2 of also encoding out with SEQ IDNO.1 nucleotide sequence homology.This term also comprises can be under the rigorous condition of moderate, more preferably under highly rigorous condition with the nucleotide sequence of the nucleotide sequence hybridization of sequence table SEQ ID NO.1.This term also comprises and sequence table SEQ ID NO.1 nucleotide sequence homology 70% at least, preferably at least 80%, more preferably at least 90%, and at least 95% nucleotide sequence best.
In the present invention, " isolating " proteic polypeptide is meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as uses column chromatography, and PAGE or HPLC method are measured the purity of polypeptide.Isolated polypeptide is substantially free of the component of following it under the native state.
In the present invention, " refractory dihydroorate synthase " refers to have the active sequence table SEQ ID NO.2 of refractory dihydroorate synthase polypeptide of sequence.This term also comprises the varient of sequence table SEQ ID NO.2 sequence, and these varients have and natural refractory dihydroorate synthase identical functions.These varients include, but is not limited to several amino acid whose disappearances, insert and/or replace, and add one or several amino acid at C latter end and/or N-terminal, also can be the difference that does not influence on the modified forms of sequence.For example, for known in the field, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C latter end and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of refractory dihydroorate synthase.
In the present invention, can select various carrier known in the art for use, as various plasmids, clay, phage and the retrovirus of selling on the market etc.When producing refractory dihydroorate synthase of the present invention, the refractory dihydroorate synthase gene sequence operationally can be connected in expression regulation sequence, thereby form the refractory dihydroorate synthase expression vector.This expression vector contains replication origin and expression regulation sequence, promotor, enhanser and necessary machining information site.Expression vector also must contain alternative marker gene, as a) providing to microbiotic or other toxicant (penbritin, the protein or the b of resistance kantlex, methotrexate etc.)) complementary auxotroph protein or c) protein of the essential nutritive ingredient that does not have in the complex medium is provided.Various different hosts' appropriate flags gene is well known in the art or production firm's specification sheets indicates.These expression vectors can be with recombinant DNA technology known in those skilled in the art preparation, as can be with reference to people's such as Sambrook way (1989), or people's such as Ausubel way (1992).
Recombinant expression vector can be introduced host cell with method well known in the art, and these methods comprise: electrotransformation, Calcium Chloride Method, particle bombardment etc.The process that the external source recombinant vectors is imported host cell is called " conversion ".By cultivating host cell, induce the expression of desirable proteins, and by protein separation technology known in the art, obtain required protein as column chromatography etc.Also can adopt these protein of synthetic such as solid phase technique.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic cell prokaryocyte such as intestinal bacteria commonly used, Bacillus subtilus etc.Eukaryotic cell such as yeast cell commonly used, or various animal and plant cells.Refractory dihydroorate synthase gene full length sequence of the present invention or its fragment can be used PCR (polymerase chain reaction) TRAP usually, recombination method, or the method for synthetic obtains.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, is template with the thermophilc anaerobe complete genome DNA of ordinary method preparation well known by persons skilled in the art, increases and obtains relevant sequence.In case obtained relevant sequence, just it can be cloned into relevant carrier, change host cell again over to, from the host cell after the propagation, separate obtaining large batch of relevant sequence then by ordinary method.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: make up the order-checking library
The structure in order-checking library adopts full genome shotgun approach (shotgun) to carry out.At first cultivate the Tengchong thermophilc anaerobe, cultural method is pressed Marmur (1961) method and is collected bacterium by (Yanfen Xue, 2000) improved MB substratum (Balch et al., 1979), extracts total DNA.For the randomness of the library construction that guarantees to check order, farthest avoid producing the problem of breakage hot spot, that adopts several different methods, different condition builds the storehouse principle.Adopt earlier physics cutting method (comprise supersonic method and shear with Hydroshear Machine), next is selected for use AluI to carry out the random partial enzyme according to this bacterium genome signature and cuts.Adopt varying strength to handle sample when physics is sheared, handle sample by enzyme amount gradient is set when enzyme is cut.Sample after the processing adopts electrophoresis fraction collection 1.5-4kb dna fragmentation after flat terminal the processing, be connected with the dephosphorylized pUC18 that cuts through the SmaI enzyme, connects product has made up random sequencing by electric Transformed E .coli DH5 α library.Simultaneously, (cut genomic dna in the order-checking library that has also made up long insertion fragment (about 10kb) for the ease of the later overlap joint of contig (contig) with Sau3AI random partial enzyme, electrophoresis is collected the fragment about 10kb, is connected, makes up the library with the dephosphorylized pUC18 that cuts through the BamHI enzyme).The order-checking of these two ends in library can obtain the relation between the contig (contig) in structure is finished the process of figure (finishing), and can solve the difficulty that cause filling-up hole in bigger hole (gap).Build storehouse flow process such as (see figure 1).
Embodiment 2: gene order-checking
When finishing the genomic order-checking of Tengchong thermophilc anaerobe, two kinds of full-automatic sequenator: ABI377 and MegaBACE 1000 have mainly been used.These two kinds of sequenators all are to utilize the principle of electrophoresis (see figure 2) that checks order, and can finish 96 samples at every turn.ABI377 is the product of ABI company, is a kind of of ABI series.It belongs to the plate gel electrophoresis sequenator.MegaBACE 1000 is products of Pharmacia Corp, belongs to the capillary gel electrophoresis sequenator.
Embodiment 3: data analysis 1) Basecalling and sequencing quality monitoring
So-called Basecalling is meant the process that obtains correct base sequence from the raw data file that sequenator obtains.Because that obtain on the sequenator is A, T, G, the light intensity variation track (trace) of the different wave length that four kinds of base pairs of C are answered need take certain algorithm therefrom correctly to identify the base of different track correspondences with computer.What we used is Phred software (Ewing B, Hillier L, 1998), and reason is that its result is more reliable, and other programs that its result exports in the same software package of being more convenient for are further analyzed.
Phred carries out the algorithm principle of Basecalling, is the shape according to each peak in the track, and spacing, and factor such as signal to noise ratio are judged the base type, simultaneously this base are provided reliability information, i.e. the sequencing quality of base.
In large scale sequencing, the monitoring of sequencing quality is crucial, and it directly influences the decision-making to order-checking, comprises the structure in library, the size of fraction of coverage.Can in time feed back the error that may occur in the order-checking experiment simultaneously.2) sequence assembly
So-called sequence assembly, the sample sequence that full genome shotgun approach (claiming shotgun again) random sequencing is obtained is assembled into successive contig (contig) exactly, mainly utilizes the overlap between them for referencial use.Consider the influence that has carrier in the order-checking, need earlier sample sequence to be unloaded body and handle.Here used software cross_match and the back used software Phrap of splicing are the software (Gordon D, Abajian C, 1998) of U.S. Washington university, and its ultimate principle is Swith-Waterman algorithm (WatermanMS, 1990).This is a kind of dynamic algorithm, after having considered the comparison between the sequence in twos, can obtain the publicly-owned sequence (consensus sequence) of one group of sequence.Sample sequence behind the removal carrier splices with Phrap again.In when splicing, the sequencing quality of base also has been considered, and the confidence level of resulting publicly-owned each base of sequence is calculated by the sequencing quality of the sample of forming this publicly-owned sequence.3): gene annotation
After obtaining genomic most of sequence (frame diagram of finishing the work) substantially, just need carry out note, comprise and carry out open reading frame (Open Reading Frame, prediction ORF) genome, the prediction of gene function, and the segmental analysis of special RNA etc.
The first step adopts the GLIMMER2.0 (Delcher of default parameter, A.L., Harmon, D.1999) and ORPHEUS (Frishman, D.1998) software prediction gene coded sequence, open frame and the non-coding region (intergenic region) of all predictions all use BLAST software (Altschul, S.F.et al. 1997) and the irredundant albumen database (non-redundant proteindatabase) of NCBI (American National biotechnology information center) relatively to find the gene that may miss then.When judging the starting point of a gene, will be with reference to various relevant informations, as sequence homology, ribosome bind site, possible signal peptide sequence and promoter sequence etc.If when in an open reading frame, a plurality of promotor occurring, generally adopt the starting point of first promotor as gene.(Ermolaeva M.D.2000) predicts the transcription terminator that does not rely on Rho (ρ) factor at non-coding region to adopt TransTerm software.If this terminator be positioned at a gene the catchment too at a distance, then may hint a minigene lose or the mistake that checks order has shortened this gene artificially, can be used as the reference of further analysis.When determining to move frame sudden change and point mutation, main basis is judged with the proteinic similarity in the database.If protein is corresponding to the situation of two encoding sequences adjacent one another are, then be considered to a non-activity gene (pseudogene seudogenes), produce the abort phenomenon because this illustrates between these two encoding sequences owing to suddenling change, and then gene is lost activity.All analytical resultss use Artemis sequence viewer software (Rutherford, K.et al.2000) to carry out manual analysis again.Some are obviously shown eclipsed with other code sequence and open frame, and length does not have homology and wherein do not have tangible promotor or open reading frame that termination is regional will be removed less than 150 base pairs and in the data with existing storehouse.
Proteinic function fragment (motif) and functional area (domain) employing and Pfam, PRINTS, PROSITE, ProDom and SMART database respectively compare, the result uses InterPro database (Apweiler, R.et al.2001) to carry out Macro or mass analysis again.According to the COGs database (Tatusov, R.L.et al.2001) of NCBI and with reference to other result of querying database determine protein in the COGs classification functional classification and possible pathways metabolism.Confirm membranin, abc transport albumen and stride the film functional domain with TMHMM software (Krogh, A.et al.2001).The employing Gram-negative bacteria is a parameter, with SIGNALP2.0 software (Nielsen, H.et al.1999) analytical signal peptide zone.4) filling-up hole
After finishing genomic work frame chart, will carry out the filling-up hole work of difficulty more, promptly finish the order-checking of whole genome 100%, obtain an annular genome.Groundwork is exactly that the contig that obtains is previously coupled together.This is a very concrete and numerous and diverse job.Main method comprises:
A. utilize forward and reverse order-checking sample message in the order-checking in the order-checking process, we have carried out two-way order-checking to some sample intentionally, check order simultaneously promptly that certain inserts segmental two ends, institute's calling sequence are spliced with other sequences again.Because the relation of this a pair of sequence on genome is certain, distance between them is roughly known, according to this information, one can confirm whether certain section contig is reliable, the 2nd, when this a pair of sequence lays respectively on the different contig, can determine direction relations and the position relation of these two contig, for further contrived experiment provides reference.
B. long insertion fragment and equipment type carrier clay (Cosmid) end sequencing are based on same principle, and we can make up the insertion fragment library of different lengths, and only to its two ends order-checking, its particular location is analyzed in splicing then.These libraries comprise that length is the long Cosmid library of inserting about sheet phase library and 20-40Kb of 9-12Kb.Specific analytical method is same as above.
C. PCR and terminal direction and position relation of extending the contig that (Walking) experimental basis above-mentioned steps A and B provided, further Biochemistry Experiment just can have been carried out.As design a pair of primer and carry out pcr amplification, or carry out end extension (Walking) with a certain contig end sequence synthetic primer and come filling-up hole etc.Embodiment 4: the preparation of dihydroorate synthase and purification
The dihydroorate synthase gene complete encoding sequence (SEQ IDNO.1) that obtains according to gene annotation among the embodiment, design and to amplify the primer that complete coding is read frame, and on positive anti-primer, introduce restriction endonuclease sites respectively, so that construction of expression vector.Plasmid DNA with the order-checking library that obtains among the embodiment 1 is a template, behind pcr amplification, guarantee to read recombinate under the correct prerequisite of frame to the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Again recombinant vectors is transformed into that (method for transformation is CaCL in the bacillus coli DH 5 alpha
2Method or electrotransformation).Screening and Identification to the engineering bacteria DH5 α-pGEX-2T-FolP that contains expression vector.
The engineering bacteria DH5 α-pGEX-2T-FolP of picking list bacterium colony contains in the LB substratum of 100 μ g/ml penbritins jolting in 3ml and cultivates 37 ℃ and spend the night, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD
600After reaching 0.5, add IPTG, continue at 37 ℃ and cultivated respectively 0,1,2,3 hours to final concentration 1mmol/L.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing desirable proteins.
As stated above behind the engineering bacteria of abduction delivering desirable proteins; With bacterium centrifugation; Add 50% saturated Triptide Sepharose 4B of 20ml PBS by every 400ml bacterium; 37 ℃ of of of of of of joltings were in conjunction with 30 minutes; 10000rpm precipitated the Triptide Sepharose 4B that combines desirable proteins in centrifugal 10 minutes; Abandoned supernatant.Add 100 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation; Room temperature was put 10 minutes; In-80 ℃ of of of of of and supernatant is the albumen of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored; and carries out SDS-PAGE SEQ ID NO.1 electrophoresis, detects purification effect.Protein band at 29815 dalton place is dihydroorate synthase.＜110〉＜120〉＜140〉＜141〉＜160〉2＜170〉PatentIn Version 3.1＜210〉1＜211〉813＜212〉DNA＜213〉 ( Thermoanaerobacter tangcongensis )＜220〉＜221〉gene＜222〉 ( 1 ) … ( 813 )＜223〉n＝agct＜400〉1atgattttaa agtgcaggga taagacttta gagattggca aaaggaccta cataatgggt 60attttgaaca tgactcctga ttccttttca gacggaggca aatacaatac tttggaaaaa 120gggattgaaa gagctttgca gatgattgaa gagggagcag atataataga tgtaggagga 180gagtctacaa ggcccggata cactcctgtc gatgaagagg aagaaattag aagggttata 240cctgtaatcg aaaggctttc taaaatttca gatgtgataa tttctgtgga tactatgaag 300gcgaatgtag ctttaagggc gctggaggca ggagcccata tagtaaatga tgtgtggggg 360cttcagagag accccaagat ggcagaagta gttgccaaat ataaagcagg ggttgtcatg 420atgcacaaca gcaatgtggc tgagtacggc gatgtagtaa aatctatcat agaatttttg 480aaaaagagca tagaaattgc tgagaaagct ggaattgaaa gaaaccagat gatagttgac 540cctggcatag gctttggaaa gactttagag cataatttgg aagtcatgaa tcgcctggag 600gaattaaaag ttctgggact tcctgtcctt ttgggtactt ccagaaaatc tatgataggc 660aaagtcttaa acgtagatgt agaggataga ttggagggga cagcggcgac tgtagctgtg 720ggaatagtaa aaggagtgga tataataaga gtccacgatg taaaagagat gtcaagagta 780gctaaaatga ctgatgcgat ggtgagaaga tga 813＜210〉2＜211〉270＜212〉PRT＜213〉 ( Thermoanaerobacter tangcongensis )＜400〉2Met Ile Leu Lys Cys Arg Asp Lys Thr Leu Glu Ile Gly Lys Arg Thr1 5 10 15Tyr Ile Met Gly Ile Leu Asn Met Thr Pro Asp Ser Phe Ser Asp Gly
20 25 30Gly?Lys?Tyr?Asn?Thr?Leu?Glu?Lys?Gly?Ile?Glu?Arg?Ala?Leu?Gln?Met
35 40 45Ile?Glu?Glu?Gly?Ala?Asp?Ile?Ile?Asp?Val?Gly?Gly?Glu?Ser?Thr?Arg
50 55 60Pro?Gly?Tyr?Thr?Pro?Val?Asp?Glu?Glu?Glu?Glu?Ile?Arg?Arg?Val?Ile65 70 75 80Pro?Val?Ile?Glu?Arg?Leu?Ser?Lys?Ile?Ser?Asp?Val Ile?Ile?Ser?Val
85 90 95Asp?Thr?Met?Lys?Ala?Asn?Val?Ala?Leu?Arg?Ala?Leu?Glu?Ala?Gly?Ala
100 105 110His?Ile?Val?Asn?Asp?Val?Trp?Gly?Leu?Gln?Arg?Asp?Pro?Lys?Met?Ala
115 120 125Glu?Val?Val?Ala?Lys?Tyr?Lys?Ala?Gly?Val?Val?Met?Met?His?Asn?Ser
130 135 140Asn?Val?Ala?Glu?Tyr?Gly?Asp?Val?Val?Lys?Ser?Ile?Ile?Glu?Phe?Leu145 150 155 160Lys?Lys?Ser?Ile?Glu?Ile?Ala?Glu?Lys?Ala?Gly?Ile?Glu?Arg?Asn?Gln
165 170 175Met?Ile?Val?Asp?Pro?Gly?Ile?Gly?Phe?Gly?Lys?Thr?Leu?Glu?His?Asn
180 185 190Leu?Glu?Val?Met?Asn?Arg?Leu?Glu?Glu?Leu?Lys?Val?Leu?Gly?Leu?Pro
195 200 205Val?Leu?Leu?Gly?Thr?Ser?Arg?Lys?Ser?Met?Ile?Gly?Lys?Val?Leu?Asn
210 215 220Val?Asp?Val?Glu?Asp?Arg?Leu?Glu?Gly?Thr?Ala?Ala?Thr?Val?Ala?Val225 230 235 240Gly?Ile?Val?Lys?Gly?Val?Asp?Ile?Ile?Arg?Val?His?Asp?Val?Lys?Glu
245 250 255Met?Ser?Arg?Val?Ala?Lys?Met?Thr?Asp?Ala?Met?Val?Arg?Arg
260 265 270
Claims (8)
1. an isolated DNA molecule is characterized in that: be the nucleotide sequence that coding has the polypeptide of refractory dihydroorate synthase protein-active.
2. dna molecular as claimed in claim 1, it is characterized in that: the polypeptide of the aminoacid sequence among the said nucleotide sequence coded SEQ.ID of the having NO.2 or the modified forms of this polypeptide, on this modified forms function quite or relevant with refractory dihydroorate synthase.
3. dna molecular as claimed in claim 1 is characterized in that: said nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
4. polypeptide separated, it is characterized in that: it has the refractory dihydroorate synthase activity.
5. polypeptide as claimed in claim 4 is characterized in that: it has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ ID No.2.
6. carrier, it is characterized in that: it contains the DNA in the claim 1.
7. host cell is characterized in that: it is prokaryotic cell prokaryocyte or the eukaryotic cell that transforms with the described carrier of claim 6.
8. a method for preparing refractory dihydroorate synthase is characterized in that this method comprises the steps:
1) isolate the gene of the coding refractory dihydroorate synthase of nucleotide sequence shown in SEQ ID NO.1:
2) make up the expression vector that contains just like the nucleotide sequence of the described gene of step 1);
3) with step 2) in expression vector change host cell over to, formation can be produced the reconstitution cell of refractory dihydroorate synthase;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain refractory dihydroorate synthase.
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| CN109810991B (en) * | 2019-03-02 | 2021-11-12 | 昆明理工大学 | Application of dihydropteroate synthase gene folP |
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