CN101845447A - Beta-agarase encoding gene and gene acquisition method - Google Patents
Beta-agarase encoding gene and gene acquisition method Download PDFInfo
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- CN101845447A CN101845447A CN 201010170690 CN201010170690A CN101845447A CN 101845447 A CN101845447 A CN 101845447A CN 201010170690 CN201010170690 CN 201010170690 CN 201010170690 A CN201010170690 A CN 201010170690A CN 101845447 A CN101845447 A CN 101845447A
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Abstract
The invention discloses an agarase and an acquisition method of an encoding gene thereof. The acquisition method utilizes various PCR methods to acquire the agarase gene aga41 from marine bacteria vibrio natriegens (the preservation number is CGMCC No.2428) for the first time and breaks through the conventional method which screens the agarase gene by constructing a library. The agarase gene aga41 belongs to a GH50 family in a beta-agarase. The whole length of the agarase gene aga41 is 2,973bp. 990 amino acids are encoded on the agarase gene aga41. The highest sequence similarity of the agarase gene aga41 with the existing agarase is 49 percent. The agarase gene aga41 is a novel beta-agarase. An activated recombinant agarase which is obtained by cloning the agarase gene of the invention on an expression vector pET28b, constructing a recombinant escherichia coli Rosetta bacterial strain and carrying out heterologous expression can be applied to the industrial production.
Description
Technical field
The present invention relates to the genetically engineered field, specifically, relate to a kind of β-agarase encoding gene and gene acquisition methods thereof.
Background technology
Agarase (agarase, EC 3.2.1.81) is a kind of glycoside hydrolase of the agar-agar of can degrading, and mainly is present in the microorganism, and is the most outstanding with marine bacteria.From seawater, be separated to a strain agar-agar degradation bacteria in 1902 by Gran at first, be cloned into an agarase gene first by people such as Buttner MJ after more than 80 year, opened human understanding for agarase.Up to the present the agarase of Fa Xianing has been formed a bigger family, according to its mode of action difference, can be divided into α-and β-agarase.α-1,3 glycosidic link of α-agarase degraded agar-agar, the fine jade oligosaccharides product of generation different polymerization degree; β-1,4 glycosidic link of β-agarase degraded agar-agar, the new fine jade oligosaccharides product of generation different polymerization degree.According to amino acid sequence similarity, agarase can be divided into different Glycoside Hydrolases (GHs) families again: the GH96 family of α-agarase; The GH86 of β-agarase, GH50 and GH16 family.Only reported two α-agarases so far, major part all is β-agarase, wherein occupies advantage with GH16 family again.
The zymogenic bacteria of agarase mostly is marine bacteria.Known to inspection information, literature search, some bacterial strain in the genus such as Alterococcus, Alteromonas, Bacillus, Cytophaga, Microscilla, Microbulbifer, Pseudoalteromonas, Pseudomonas, Streptomyces, Zobellia can produce agarase.
Chinese patent 200410023656.1 has been reported a kind of novel bacterial Alteromonas sp.nov.SY 37-12 (CCTCCM204009) from the isolating production agarase in marine site, China South Sea.Chinese patent 200810121997.0 has reported that novel bacterial that a strain separates the sedimental production agarase from the East Sea needs sodium vibrios (Vibrio natriegens or be abbreviated as V.natriegens), and culture presevation number is CGMCC No.2428.Above-mentioned patent emphasis is at bacterium producing multi enzyme preparation, and single bacterial strain often has the isozyme genes of multiple agarase.
Most researchers all is to adopt the method for library construction to obtain the agarase gene at present.But library construction need spend plenty of time and energy, just can obtain positive colony by screening thousands of clones, also will carry out links such as subclone afterwards.If there is a kind of method directly from genome, to go out the goal gene partial sequence, then can removes the loaded down with trivial details work in above-mentioned structure library from, thereby realize efficiently obtaining of goal gene based on homologous clone by pcr amplification.Yet since agarase family sequences many, that announce limited, differ greatly to each other, very difficultly obtain core sequence according to common cloning process.
Summary of the invention
An object of the present invention is to provide a kind of method of from the bacterial strain of producing agarase, cloning the agarase gene, open up new a, approach efficiently, and the agarase gene that obtains is used for the structure of recombinant bacterial strain and realizes heterogenous expression, finally realize the suitability for industrialized production agarase.
Another object of the present invention is to utilize this method to be cloned into a brand-new β-agarase gene.
Method of the present invention comprises the steps:
(1) known agarase is carried out the comparison of amino acid multisequencing, find the core conserved sequence;
(2) design degenerate primer;
(3) be template with the strain gene group, utilize the core DNA sequence of degenerate primer through PCR method amplification agarase gene;
(4) utilize the flanking sequence of site stepping PCR method amplification agarase core sequence, and then splicing obtains the agarase complete genome sequence.
All agarases that this method will be delivered at present carry out the comparison of amino acid multisequencing according to different families such as GH16, GH50, GH86 and GH96 etc., find the core conserved sequence, perhaps also can directly in software Block Maker (http://bioinformatics.weizmann.ac.il/blocks/blockmkr/www/make_b locks.html), compare and seek conservative region, utilize software CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html) to seek possible degenerate primer then.Fundamental principle and requirement according to primer: degeneracy is as far as possible little, the Tm value is high as far as possible, the distance of upstream and downstream primer TM value big as far as possible, the upstream and downstream primer is close as far as possible etc., selects suitable degenerate primer.
Utilize this method, we utilize degenerate primer (the upstream primer GH50F:5 '-AACTGGGGCTTCACCACCYTNGGNAAYTGG-3 ' of GH50 family; Downstream primer GH50R:5 '-ACACGAAGCCCACGTTCTARTTYTCNCC-3 '), with in the Chinese patent 200810121997.0 report need sodium vibrios (preserving number: be that template is carried out pcr amplification CGMCC No.2428), the result obtains the agarase gene core sequence (the 1939th of SeqID.NO:1 to 2878 bit base sequences) of one section 940bp.Through the Blastx comparison, (the GenBank accession number: BAG71428) similarity is 61% to the most similar known agarase gene order.Then, (site stepping PCR is a kind of gene or chromosome walking technology to utilize site stepping (SiteFinding) round pcr, utilize one section synthetic anchor series that configures to be attached to the genome specific site at low temperatures, special primer on special primer on the known sequence and synthetic anchor series carries out the PCR reaction, amplify aim sequence) amplify the flanking sequence of this core sequence, obtain the full length sequence of a kind of brand-new β-agarase gene aga41 through splicing, it is characterized in that total length 2973bp (nucleotide sequence as Seq ID.NO:1 as described in).990 amino acid of this genes encoding (aminoacid sequence as Seq ID.NO:2 as described in) belong to GH50 family, are low to moderate 49% with announcing agarase sequence (GenBank accession number BAG71428, aminoacid sequence is as described in the Seq ID.NO:3) homology.
Utilize gene clone technology, the agarase gene of being cloned into can be connected on the suitable carriers, and conversion or transfection are to prokaryotic organism or eukaryote host expresses preparation reorganization agarase.Suitable prokaryotic organism host comprises various bacteriums such as E.coli etc., and suitable eukaryote host comprises yeast (as methanol yeast) and mammalian cell (as Chinese hamster ovary cell) etc., preferably adopts prokaryotic expression system E.coli.
But protokaryon or carrier for expression of eukaryon that the various commercializations that suitable carriers is well known to those skilled in the art are bought.A preferred example is that β-agarase gene aga41 that the present invention is cloned into is connected on the coli expression carrier pET28b (Novagen), and is transformed among the intestinal bacteria Rosetta, goes out highly active recombinant beta-agarase through abduction delivering.
Method of the present invention can be by the design degenerate primer, through PCR from need sodium vibrios (preserving number: CGMCC No.2428) directly amplify β-agarase gene aga41 core sequence the genome, utilize the flanking sequence of site stepping pcr amplification core sequence then, and then splicing obtains complete genome sequence.The present invention has successfully made up recombinant expression plasmid and recombinant strains Rosetta with the β-agarase gene aga41 that obtains, the reorganization agarase of energy output activity expression.
Method of the present invention can be fast produced bacterial strain from newfound agarase and is cloned the gene of agarase and identify, overcomes to make up shortcomings such as complex steps, the success ratio of obtaining the agarase gene are low through the library in the past.The agarase gene that obtains can be cloned into and be realized heterogenous expression in the appropriate host, finally realizes the suitability for industrialized production agarase, for follow-up industrial application provides agarase parent material with low cost.
Description of drawings
Fig. 1 is that different PH conditions are to the active influence of β-agarase
Fig. 2 is that condition of different temperatures is to the active influence of β-agarase
Embodiment
Embodiment 1: the obtaining of β-agarase gene aga41 core sequence
The agarase of announcing is at present carried out the comparison of amino acid multisequencing according to the different GH16 of family, GH50, GH86 and GH96 in software ClusterW, find conserved sequence, simultaneously can in software Block Maker, compare and seek conservative region, utilize software CODEHOP to search the degenerate primer of conservative region, select suitable degenerate primer to carry out pcr amplification.According to said method by aminoacid sequence comparison, degenerate primer (the upstream primer GH50F:5 '-AACTGGGGCTTCACCACCYTNGGNAAYTGG-3 ' that designs from the conserved sequence NWGFTSFGNW and the ENYNVGFVSV of GH50 family; Downstream primer GH50R:5 '-ACACGAAGCCCACGTTCTARTTYTCNCC-3 '), need sodium vibrios (preserving number: be that template is carried out pcr amplification CGMCC No.2428) with report in the Chinese patent 200810121997.0, obtain one section β-agarase gene aga41 core sequence, be 940bp (the 1939th of Seq ID.NO:1 to 2878 bit base sequences).
Embodiment 2: the obtaining of β-agarase gene aga41 flanking sequence
According to the β that obtains among the embodiment 1-agarase gene aga41 core sequence, utilize the site stepping round pcr flanking sequence that increases.Specific practice is as follows:
3 specificity nested primerss based on core sequence design amplification 5 ' end upstream sequence:
GSP1：5’-ATACAAACCACTACGCACTGGCGATT-3’；
GSP2：5’-GCAATACCGTCAACAATACAAACCACTA-3’；
GSP3：5’-GATTACGGGAGACCACCAACGAGTTAGT-3’；
Synthetic anchor series site:
5’-CACGACACGCTACTCAACACACCACCTCGCACAGCGTCCTCAAGCGGCCGCNNNNNNGCCT-3’；
Synthetic specificity nested primers:
SFP1：5’-CACGACACGCTACTCAACAC-3’；
SFP2：5’-ACTCAACACACCACCTCGCACAGC-3’)；
Carry out 3 and take turns pcr amplification:
1, site stepping PCR.The PCR system is as follows: comprise LA Taq enzyme and the buffer thereof of 0.5U in the 20 μ L reaction systems, 10pmol synthesizes the anchor series site, and dNTP mixture and template need sodium vibrios (preserving number: genomic dna CGMCC No.2428).Alternating temperature PCR condition is as follows: 92 ℃ of pre-sex change 2min, and 95 ℃ of sex change 1min, 25 ℃ of annealing 1min, 68 ℃ of ramp to, 3min, last 68 ℃ are extended 10min.
2, first round primer amplification.In above-mentioned site stepping PCR system, add 5 μ L primer mixtures (50pmol SFP1 and 10pmol GSP1), increase according to following PCR program: 95 ℃ of sex change 1min, according to carrying out 30 two step PCR circulation (95 ℃ of sex change 10s, 6min is extended in 68 ℃ of annealing), last 72 ℃ are extended 10min.
3, the second take turns primer amplification.With first round PGR product dilution is template for 100 times, with primer GSP2 and SFP2 is carried out second and takes turns nest-type PRC, with primer GSP3 and SFP2 is carried out parallel nido amplification simultaneously.The PCR program is identical with first round primer amplification.Owing to differ 175bp between primer GSP2 and the GSP3, can confirm that further specific PCR increases by above-mentioned second standoff distance of taking turns between the electrophoretic band of primer extension product.
Confirm to obtain specific amplification products through above-mentioned many wheel PCR, back TA clone is reclaimed in rubber tapping, carries out sequencing, thereby amplifies 5 ' end upstream sequence.Equally, the specific gene primer of design amplification 3 ' end downstream sequence:
GSP4：5’-CTGATTGGGGGATAACACCTGAAG-3’；
GSP5：5’-GATGTGATGAGTTACAATCTTTACGCCA-3’；
GSP6：5’-AGTAGTGACAATCAACAAGGTCGTGC-3’)；
Amplify the fragment of 3 ' end downstream sequence according to above-mentioned same method.Pass through sequence assembly then, obtain the full length sequence of gene.The β that the present invention obtains-agarase gene aga41 total length is 2973bp, 990 amino acid of encoding altogether, its sequence is seen Seq ID.NO:1 and SeqID.NO:2 respectively, itself and agarase (the GenBank accession number of having announced: BAG71428, aminoacid sequence is as described in the Seq ID.NO:3) homology is lower than 50%.
Embodiment 3: the β-recombinant expression plasmid of agarase gene aga41 and the structure of recombinant bacterial strain
β-agarase gene aga41 that the present invention obtains is cloned on the expression vector, makes up recombinant strains.At first, design upstream primer aga41F (the 5 '-CACTAC of the full gene of amplification based on full length sequence
CATATGTACTGTTCGTTTTA-3 ', NdeI) and downstream primer aga41R (5 '-ATTCTCGAGTCATTTGTTAATAGATC-3 ', XhoI), pcr amplification is confirmed the full length gene sequence.Adopt the method construction expression plasmid of enzyme cutting clone, promptly use NdeI and XhoI double digestion PCR product, the fragment after enzyme is cut is reclaimed in rubber tapping, is connected with same plasmid pET28b through NdeI and XhoI double digestion, according to CaCl
2Conversion method is transformed in the bacillus coli DH 5 alpha, the kalamycin resistance screening positive clone.Adopt plasmid extraction test kit (Axygen) to extract the plasmid of attached sex clone, so NdeI and the evaluation of XhoI double digestion, obtain fragment near 3kb, be β-agarase gene aga41 through sequencing.Show successfully to have made up expression plasmid, this recombinant expression plasmid is transformed in the intestinal bacteria Rosetta expression strain, made up the express recombinant bacterial strain.
Embodiment 4: utilize recombinant strains express recombinant β-agarase gene aga41
The 3ml recombinant strains that builds is transferred to 100ml contains in the LB liquid nutrient medium of 20 μ g/ml kantlex and 34 μ g/ml paraxin, 37 ℃ of shaking culture are to 0D
600Reach 0.6, adding final concentration this moment is that the IPTG of 0.1mM carries out abduction delivering, changes 25 ℃ over to 150r/min shaking culture 9h.Centrifugal collection thalline is resuspended among the Mcilvane ' sbuffer (pH 7.5), carries out ultrasonic disruption on ice and handles.Low-temperature centrifugation is collected supernatant, carries out NTA-Ni then
2+Affinity column chromatography, because expressed recombinant protein contains 6 * His tag of N end, can affine being adsorbed onto in layer suction post, behind the imidazoles solution gradient wash-out through different concns (30,70,100 and 250mmol/L), collect elutriant.Detect through SDS-PAGE, obtain electrophoretically pure recombinant beta-agarase albumin A ga41.Measure protein concn with the Bradford method, obtain the expression amount of about 6mg/100ml fermented liquid.
Embodiment 5: the activity of recombinant beta-agarase Aga41 detects and the enzyme specificity analysis
Utilize DNS (3, the 5-dinitrosalicylic acid) method to measure the recombinant beta-agarase Aga41 activity of purifying.Concrete operations: comprise that in the reaction system of 1ml 990 μ L contain Mcilvane ' the s buffer (pH7.5) of 0.25% agarose and the purifying enzyme liquid of 10 μ L, in 40 ℃ of water-baths, react 30min, taking out 500 μ L solution adds in the 500 μ L DNS solution, cool off rapidly after boiling 5min, carry out light absorption value A
520Mensuration.Same quadrat method is used for zeroing in contrast with the enzyme liquid of inactivation.The agarase that records is active to be 1.51U/ml.In addition, the enzyme liquid of purifying is directly dripped on the solid agar plate, can observe tangible depression, with seeing transparent circle behind the iodine staining, show that this recombinant expressed β-agarase Aga41 has the agar-agar degrading activity, for follow-up research and application provide good material.
After measured, the optimal pH of β-agarase Aga41 is 7.5 (as Fig. 1), and optimal reactive temperature is 40 ℃ (as Fig. 2), and 0.5mM is to the Ca of 5mM
2+Zn with 0.5mM
2+, Mn
2+, Mg
2+, Co
2+Can promote enzymic activity.
Sequence table
＜110〉The Second Institute of Oceanograghy,SOA
＜120〉a kind of β-agarase encoding gene and gene acquisition methods
<160>3
<170>PatentIn?version?3.3
<210>Seq?ID?No.1
<211>2973
<212>DNA
<213>Vibrio?natriegens
<400>Seq?ID?No.1
atgtactgtt?cgttttattg?gttatccaat?aactacgtga?aggaaaggag?aatctcagtg 60
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attgccgcta?caaaaacacc?agacacatcc?aataacgaaa?caactcaaca?agatatgagt 180
agtagcgttc?aacctgttgt?catgacagat?gatgggtttc?tatcaaaaac?cgccaacaca 240
cacacctcat?ataatgtggc?tggcgaaaac?ctggaaatcg?tgttcgatgc?gatttctgag 300
tcagaagcaa?actctaaatg?gccaaacatg?aagtttcgcc?cggagtctgg?ttcttatgat 360
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gagatgaata?ttgaaggtta?ccgaggtggt?agtgagcttg?atttacgaaa?tattgctgag 600
ttccaattct?acgcggttgg?cccgataggt?gagcaaaaag?tagtagtaca?tgacattgat 660
tttattgaac?gcactggaga?ttttgttcta?tcagaagcgc?gtcaaggtca?agttattgag 720
gcgcaaattc?ccactatttt?aacagtaact?gactttgaaa?gtggtgttga?aggtattgtt 780
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caggcttggg?attggtcaaa?gtatggcgat?gttgccctag?catttgatgc?gaaaaacatt 960
ggtgattcag?gcatgcagct?atttgtgcgt?gtcgatgatg?cactagatga?aaagctgggt 1020
ggtacggcta?cgggggcagt?aaacagccgt?acgggttatg?tacagattcc?agcaaactca 1080
gaagacaaat?attacttcac?atttaaagac?ttggctgaag?gcttggattc?aggtatgcgt 1140
ggcgagccgc?caaagaaatc?cttctcagca?agccaagtgg?tgtttggttg?gggcgaatct 1200
gagcttgatc?tatcgaacat?tgtcagcgtg?cagctttaca?tgatgaaccc?tcaagaagag 1260
gcgacattgg?ttatcgataa?cctatcgttg?attcctaacc?taagtacgga?cactacgcga 1320
tacgccaatc?tgttggatga?gtttggtcaa?tatcaggaag?aaacgtggcc?agaaaaaatc 1380
accgatttgg?atcagctaaa?ggctcaagcc?aaagtcgaca?aaaaactgtt?gaagaatgcc 1440
tcgttgatga?gtgatcgttc?aaagttcggc?ggctgggcta?aaggtccgaa?actagaggca 1500
acaggttatt?tccgcacaga?gaaagtcaat?gataagtggg?cgctggtgga?ccctgaagga 1560
tacctctact?tcgctacagg?ggtagacaat?attcgtatgg?atgacagcta?cacagtgaca 1620
gggatggact?ttgctgatgc?ggtagaggca?gacactaagg?ggatgcgtcc?aagtcaggtt 1680
gccgctgagc?gttacattga?tgacaagcgt?gagcgtgtag?aagcctcaac?actccgccgt 1740
ggtatgtttg?attggttacc?tgatttcaat?gatccactgg?ctgacaacta?cagctatacg 1800
caaatggtgc?ataaaggacc?tctcaagcat?ggtgaagtgt?tcagcttcta?ttcggcgaac 1860
ttgcagcgta?agtatgatac?cgactctgca?caagaagcga?ttgagatttg?gaaggacgtg 1920
acacttgcac?gaatgcagga?ctggggtttc?acttccttag?ggaactggtc?cgatcctatg 1980
taccgaaaaa?acggtaaagt?gccgtataca?gcacatggtt?ggattacggg?agaccaccaa 2040
cgagttagta?caggtaacga?ctactggggg?gctatgcatg?acccatttga?cacaaatttt 2100
cgggtctctg?tggccataat?ggcgaaggag?ttgggtgaag?aggttgacaa?tgacccttgg 2160
tgtatcggtt?atttcgttga?caatgagttg?agctggggca?ataccgtcaa?caatacaaac 2220
cactacgcac?tggcgatttc?tggtttacgt?gccagcacaa?aagaaagctc?ggccaaagcg 2280
gcgtttgatg?cattacttaa?agcaaaatat?ggctcgatca?agaagttcaa?cgctgcatgg 2340
ggcgttgatg?ttgcctcatg?ggatgctttt?gctaaaggct?tcaattatca?aggtgaatac 2400
actgaaacag?tgaaagccga?tctttcagta?ttgttagatg?catttgctga?taagtatttt 2460
gcggttgtta?gtgaagagat?ggagaaagtg?ttacctaacc?atctatacat?gggagtgcga 2520
ttctctgatt?gggggataac?acctgaagct?gcgacagcag?ccgcacgtta?tgttgatgtg 2580
atgagttaca?atctttacgc?cactgatcta?aatgcaaaag?gggactggag?ccgtttaccg 2640
gagttggata?aacctagcat?tattggtgag?ttccactttg?gttctactga?ttcaggttta 2700
ttctaccctg?gtattatcag?tagtgacaat?caacaaggtc?gtgcgcaatc?ttacgctaag 2760
tatatggaaa?gcgtgattga?taacccgtac?tttgttggtg?cacattggtt?ccagtatatg 2820
gattcacccg?taacagggcg?tgcatgggat?ggtgaaaact?ataacgtggg?ttttgtgaca 2880
gtaacggata?caccatatga?gcccttggtg?aagtctgcca?aagagatcaa?ccgtaattta 2940
tacaatcgac?gattcggatc?tattaacaaa?tga 2973
<210>Seq?ID?No.2
<211>990
<212>PRT
<213>Vibrio?natriegens
<400>Seq?ID?No.2
mycsfywlsn?nyvkerrisv?rfnkntiala?iiastlaags?iaatktpdts?nnettqqdms 60
ssvqpvvmtd?dgflsktant?htsynvagen?leivfdaise?seanskwpnm?kfrpesgsyd 120
wntkgglqlt?lenpgdrevr?iemkvadnig?imgatthqld?lpiylpagkt?ttvdflfnga 180
emniegyrgg?seldlrniae?fqfyavgpig?eqkvvvhdid?fiertgdfvl?searqgqvie 240
aqiptiltvt?dfesgvegiv?erhsgsnvdi?vksdtgsaik?vhyttdddyp?tikfsagkdg 300
qawdwskygd?valafdakni?gdsgmqlfvr?vddaldeklg?gtatgavnsr?tgyvqipans 360
edkyyftfkd?laegldsgmr?geppkksfsa?sqvvfgwges?eldlsnivsv?qlymmnpqee 420
atlvidnlsl?ipnlstdttr?yanlldefgq?yqeetwpeki?tdldqlkaqa?kvdkkllkna 480
slmsdrskfg?gwakgpklea?tgyfrtekvn?dkwalvdpeg?ylyfatgvdn?irmddsytvt 540
gmdfadavea?dtkgmrpsqv?aaeryiddkr?erveastlrr?gmfdwlpdfn?dpladnysyt 600
qmvhkgplkh?gevfsfysan?lqrkydtdsa?qeaieiwkdv?tlarmqdwgf?tslgnwsdpm 660
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cigyfvdnel?swgntvnntn?hyalaisglr?astkessaka?afdallkaky?gsikkfnaaw 780
gvdvaswdaf?akgfnyqgey?tetvkadlsv?lldafadkyf?avvseemekv?lpnhlymgvr 840
fsdwgitpea?ataaaryvdv?msynlyatdl?nakgdwsrlp?eldkpsiige?fhfgstdsgl 900
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vtdtpyeplv?ksakeinrnl?ynrrfgsink 990
<210>Seq?ID?No.3
<211>955
<212>PRT
<213>Vibrio?sp.PO-303
<400>Seq?ID?No.3
mvevmkftkn?kiaallsltl?lgvygcgstp?sssdaegave?dvggtipdfe?saaffkkvkk 60
dhakaevvsd?qgvtsgssal?kvsfdsvsea?nkfkywpnvk?vhpdsgfwnw?nakgslsldi 120
tnptdspani?ilkladnvgv?mgsgdnqlny?avnvpagetv?pvemlfngtk?rkldgywgge 180
kinlrnivef?qifvggpmda?qtviidnfnl?vdatgdfiea?sgqevkvsgp?iptvasitsf 240
degqptfvaf?drsaaatvte?lktdmgglla?vklaatnayp?nitfkapqpw?dwseygdfsl 300
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idadatryeg?lidqygqftg?sewpkkited?eqletmgkla?kmslkstsqm?tgrsiyggwa 480
dgpklkgtgf?frtekvdgkw?slvdpqgnlf?fatgvdnirm?ddtvtitghd?fadkdkrsgk 540
evasevrrsm?ftwlpedddv?laenydyanw?vhsgalkkge?vfsfyganlq?rkyggtfsea 600
ekvwkditid?rmvdwgfttl?gnwadpmfyd?nkkvayvang?wifgdharis?tgndywgpih 660
dpfdpefvns?vkamtkklmt?evdkndpwmm?gvfvdneisw?gntkndanhy?glvvnalsyn 720
vkkspakaaf?tehlkekywa?iedlntswgv?kvaswaefek?sfdhrsrlsk?nmkkdyaeml 780
emlsakyfst?vraelkkvlp?nhlylgarfa?dwgvtpeiak?gaapyvdvms?ynlyaedlns 840
kgdwsklael?dkpsiigefh?fgstdsglfh?ggivsaasqq?drakkytnym?nsiadnpyfv 900
gahwfqyids?pttgrawdge?nynvgfvsit?dtpyvplvea?akkfnqdvym?lrykk 955
Claims (10)
1. the gene of the β-agarase of encoding, its nucleotide sequence is consistent with sequence shown in the Seq ID.NO:1.
2. β-the agarase of the described genes encoding of claim 1, its aminoacid sequence is consistent with sequence shown in the SeqID.NO:2.
3. carry the carrier of gene order according to claim 1.
4. carrier according to claim 3, it is escherichia coli vector pET28b.
5. host system, its by the described carrier of claim 3 through transforming or transfection prokaryotic organism or eukaryote host obtain.
6. host system according to claim 5, it is bacterium, yeast or mammalian cell.
7. host system according to claim 6, it is intestinal bacteria E.coli.
8. the method for clone's agarase gene from the bacterial strain of producing agarase comprises the steps:
(1) known agarase is carried out the comparison of amino acid multisequencing, find the core conserved sequence;
(2) design degenerate primer;
(3) be template with the strain gene group, utilize the core DNA sequence of degenerate primer through PCR method amplification agarase gene;
(4) utilize the flanking sequence of site stepping PCR method amplification agarase core sequence, and then splicing obtains the agarase complete genome sequence.
9. method according to claim 8, it is characterized in that, described step (1) is for carrying out the comparison of amino acid multisequencing according to different agarase GH16, GH50, GH86, GH96 family etc., find the core conserved sequence, perhaps also can directly in software Block Maker, compare and seek conservative region.
10. method according to claim 8, it is characterized in that, described step (2) is for utilizing software CODEHOP design degenerate primer, the fundamental principle of foundation is that degeneracy is as far as possible little, Tm value is high as far as possible, the distance of upstream and downstream primer as far as possible greatly, the Tm value of upstream and downstream primer is close as far as possible etc.
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