CN101979571A - Ridgeia piscesae chitin binding protein and preparation method thereof - Google Patents
Ridgeia piscesae chitin binding protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses ridgeia piscesae chitin binding protein and a preparation method thereof and relates to chitin binding protein. A ridgeia piscesae chitin binding protein gene is a cbpA, cbpB, cbpC or cbpD gene. A method for obtaining a nucleotide sequence of the ridgeia piscesae chitin binding protein gene comprises the following steps of: extracting RNA of ridgeia piscesae; reversely transcribing the RNA into cDNA; and performing recombinant expression in insect cells high5 by using the cDNA as a template and using a composition sequence as a primer through polymerase chain reaction (PCR) amplification so as to prove that polypeptide encoded by the cbpA, cbpB, cbpC or cbpD gene has chitin binding activity. The preparation method comprises the following steps of: extracting the RNA of the ridgeia piscesae; performing cDNA reverse transcription on the ridgeia piscesae; performing PCR amplification and sequence analysis on the ridgeia piscesae chitin binding protein gene; and performing expression on the ridgeia piscesae chitin binding protein.
Description
Technical field
The present invention relates to a kind of chitin-binding protein, especially relate to a kind of tubulose worm chitin-binding protein and preparation method thereof.
Background technology
Chitin be by the N-acetyl-glucosamine monomer with β-1, the polymer that the 4-glycosidic link couples together is mainly produced by insect, fungi, Crustacean and other marine organisms.At present, found to have 3 kinds of chitin crystal types, i.e. α-chitin, β-chitin and γ-chitin at occurring in nature.In biosphere, the annual chitin that produces several hundred million tons is that occurring in nature content is only second to cellulosic macromolecular substance, also be the macromolecular substance of output maximum in the ocean, so chitin is the wide energy substance of DEVELOPMENT PROSPECT.Present stage can only handle by strong acid and strong base chitinous utilization, has both wasted resource, and contaminate environment is applied to biological enzyme in the production process again, will greatly solve this difficult problem.In the process of chitinase degrade chitin, chitin-binding protein plays a part can not ignore, it not only can be in conjunction with chitin, it is diffusing that its structure is fluffed, can also strengthen the chitinase activity, improve degradation efficiency (Chamoy, L., Nicolai greatly, M., Ravaux, J., Quennedey, B., Gail, F.and Delachambre J. (2001) .A Novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specifically with β-chitin.J Biol Chem 276 (11), 8051-8058).
Summary of the invention
First purpose of the present invention provides tubulose worm Ridgeia piscesae chitin-binding protein gene.
Second purpose of the present invention provides tubulose worm Ridgeia piscesae chitin-binding protein.
The 3rd purpose of the present invention provides the preparation method of described tubulose worm Ridgeia piscesae chitin-binding protein.
Tubulose worm Ridgeia piscesae chitin-binding protein gene of the present invention is tubulose worm Ridgeia piscesae chitin-binding protein cbpA gene, tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene, tubulose worm Ridgeia piscesae chitin-binding protein cbpC gene or tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene.
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpA gene is DNA, sequence signature: length is 564bp, type is a nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpA gene is as follows, is designated as SEQ ID No.1:
1 ATGTCGAAGC?TTCTCGCAGC?CCTTGTGGCA?CTTAGCATCG?CCTACTCCGT?GCAAGGGTGC
61?TCGGATTCGT?GCGTCGGCAG?ACAGAACGGC?CGCTGCTATC?CTTCGGCGTG?TGGTCCGTGC
121?AACTATTACT?TCAAGTGTCA?ACGTACGATC?GTTGTCGAGC?AATACTGTAG?AGCGTCATCG
181?TACTGCTACC?TCAACGGACG?ATGCCGACCA?TGTGGTGACT?CTTGCGAGAG?AAAAAGGGAT
241?GGCGTCTACC?CATCAGTGAA?CCGGGCCCGA?CCCTACTACT?ACCAGTGCGA?AGCCGGACAA
301?CTGTACTACC?GCGAGTGCCA?GCCCTTCCAG?ATCTTCAACG?CGTGGACCAG?GAAATGCGAA
361?TGCTTCGAGG?GCGCCTGTCT?GCACGGAAAC?GGATGGAGAT?CTTCCTTCTG?CCGAGGCAAG
421?GGATGGCGTG?TGTTCTGCAG?CGGTGGTTTC?GCCAGGTATT?ACAGGCAATG?CCCTCGACCC
481?CGACCTCACG?TGTGCTGTCA?CACCTACACG?TGCGTGTCCA?CCTGCACCTC?GTACTATGGA
541?CGTGGTCACT?GCTGCAGAGG?ATAG。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene is DNA; Sequence signature: length is 582bp, and type is a nucleic acid, and chain is double-stranded, and topological framework is linear; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene is as follows, is designated as SEQ ID No.2:
1 ATGTCGAAGC?TTATCATTGC?CCTGGCGGTT?CTTTCCGCCT?GCATCGCCGT?CCAAGCCCGC
61 GGATGGGAGG?GATGTTCTCA?GGACAAATGC?GACGGCAGAC?GTACCGGCAG?GAGATACCAG
121?TCTCTCTGCG?CCCCTGTCAG?CGTCTACTAC?AGATGTCGCC?ACAGTCGCCC?CGTCTACGAC
181?AACTGCCGTG?AGCGCAACTA?TTGCGTCGTC?AACGGCCGAT?GCTGCCGATG?CGGTGACACC
241?TGTGAGGGCA?AATGCGACGG?CGTCTACCCG?TCCGTGAACC?GACCCCGTCC?CGCCTACTAC
301?CAGTGTGACG?GAGGAGAGCT?GTACTACCGC?ACGTGCCAGC?CCTTCCAGAT?CTTCAACCCC
361?TGGACCAAGC?AATGCGAGTG?CTACGAGGGT?TCGTGTCTCC?ATGGCAACGG?GTACAGGGCG
421?TCCTTCTGCC?GCGGCAAGGA?ATGGCGCGTG?TACTGCCGGG?CTGGATTCCC?CCAGTTCTAC
481?GAGCGCTGTC?CCCGACCCAG?GCCCTACGTG?TGCTGCAAGA?CTTACACCTG?CATCTCCACG
541?TGCAGCGTGT?TCCAGGGCAG?AAACGGATGT?TGCCACGCCT?AG。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpC gene is DNA; Sequence signature: length is 591bp, and type is a nucleic acid, and chain is double-stranded, and topological framework is linear; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpC gene is as follows, is designated as SEQ ID No.3:
1 ATGGCGAAGT?TTTTCATCGC?ATTGTTGGTG?CTCGTCGCTG?TATATGCAAC?TGAAGCACTC
61 CCTGGTAACG?GTTACGAAGG?TTGTTTCAAC?GGCTACAACT?ACGATTGCTG?CAGGCGTCGT
121?AGACCCAACT?TCTGCTACCC?GTCGCTGGTG?AAGCCACTGC?CTTACTACTA?CCGATGTGTC
181?GGCAGGAGAG?TTGTGTACGG?CAGATGTGAG?ACGCACCTTT?GCGTCACAGT?AAAGGGAAAT
241?TGCGGATCGT?GCAGTGATCG?TTGTCGCTAC?ACTCGTCATT?CACACTATTA?CTACTCGAGC
301?TACTTCTACG?GTCGCTACTA?CTACCGATGC?AACCGCGGAG?CACTGAACTA?CCGCTCGTGT
361?GGACTCAACC?AGTCTTACTA?TCCGGGACGC?GGCCGTTGCG?GCTGCAGAGA?GAATTACTGT
421?GAGAGGCACA?ACGGCTGGCA?GTCGACCGTT?TGCAACGGAT?GCGGAAGCTA?CGTCTTCTGC
481?AGATATGGAC?GTCCCATTCG?CTACAGGAGA?TGCCCCTGGA?GCAGACGCCA?CTACGACTGT
541?CATCGCCATC?GTTGTGTCAG?CCATTGCAAC?GGTTGCTGTG?GCCATCGATA?A。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene is DNA; Sequence signature: length is 453bp, and type is a nucleic acid, and chain is double-stranded, and topological framework is linear; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene is as follows, is designated as SEQ ID No.4:
1 ATGCAGAAGT?TGGTCTTCAT?AGTGGTAGCA?ATCGTCATGG?CCTTGGCGAG?TTGTACCTCC
61 GCTAGGAAGA?AAAAACCGTG?CGTGGAGTCG?TGCGTGGGCC?GACCGGACGG?CGAGTACCAA
121?GCATCCTGCA?CCTACGACTG?TCGCTTCTTC?GTCAAGTGTG?TCTTCGGCTT?CTCCTTCCTG
181?TTGAAATGCC?GCGAGGGACA?TTTCTATGAC?GTCGACGTGA?GAGACTGTGT?CAAAGGATGT
241?CCGACTGGAC?CACGCGGAAT?CCTCGACTGC?TACGACCGCC?CTGACGGCCA?CTGGCAGCAT
301?TGCTGCTACT?GCGACTTGTA?CGCCACTCGT?GCGAGCAGGG?GTATGTTCTT?CCACCGCCGC
361?TGTGAGCCGG?GCGGTCCGGT?CTGGCACGAT?GACAAACACA?AGTGTCTCTG?GACGTCGGGA
421?ACATGTCCCG?GGGTACACCG?ATACTACGCT?TAA。
The nucleotide sequence of tubulose worm Ridgeia piscesae chitin-binding protein gene of the present invention can adopt following method to obtain: the RNA that extracts tubulose worm Ridgeia piscesae, and the RNA reverse transcription become cDNA, it further is template with cDNA, respectively with composition sequence SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQID No.13, SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16 are as primer, obtained SEQID No.1 through pcr amplification, SEQ ID No.2, the sequence of SEQ ID No.3 and SEQ ID No.4.Carry out recombinant expressedly then in insect cell high5, the polypeptide (SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8) of biological method proof cbpA, cbpB, cbpC and cbpD genes encoding has chitin in conjunction with activity.
Described SEQ ID No.9:
GGATCCGGTGCTCGGATTCGTGCGTC;
Described SEQ ID No.10:
CTCGAGCTATCCTCTGCAGCAGTGAC;
Described SEQ ID No.11:
GGATCCCCCGCGGATGGGAGGGATGT;
Described SEQ ID No.12:
CTCGAGCTAGGCGTGGCAACATCCGT;
Described SEQ ID No.13:
GGATCCCACTCCCTGGTAACGGTTAC;
Described SEQ ID No.14:
TCTAGATTATCGATGGCCACAGCAAC;
Described SEQ ID No.15:
GGATCCCTAGGAAGAAAAAACCGTGC;
Described SEQ ID No.16:
CTCGAGTTAAGCGTAGTATCGGTGTA;
Described SEQ ID No.5:
1 MSKLLAALVA?LSIAYSVQGC?SDSCVGRQNG?RCYPSACGPC?NYYFKCQRTI?VVEQYCRASS
61 YCYLNGRCRP?CGDSCERKRD?GVYPSVNRAR?PYYYQCEAGQ?LYYRECQPFQ?IFNAWTRKCE
121?CFEGACLHGN?GWRSSFCRGK?GWRVFCSGGF?ARYYRQCPRP?RPHVCCHTYT?CVSTCTSYYG
181?RGHCCRG；
Described SEQ ID No.6:
1 MSKLIIALAV?LSACIAVQAR?GWEGCSQDKC?DGRRTGRRYQ?SLCAPVSVYY?RCRHSRPVYD
61 NCRERNYCVV?NGRCCRCGDT?CEGKCDGVYP?SVNRPRPAYY?QCDGGELYYR?TCQPFQIFNP
121?WTKQCECYEG?SCLHGNGYRA?SFCRGKEWRV?YCRAGFPQFY?ERCPRPRPYV?CCKTYTCIST
181?CSVFQGRNGC?CHA；
Described SEQ ID No.7:
1 MAKFFIALLV?LVAVYATEAL?PGNGYEGCFN?GYNYDCCRRR?RPNFCYPSLV?KPLPYYYRCV
61 GRRVVYGRCE?THLCVTVKGN?CGSCSDRCRY?TRHSHYYYSS?YFYGRYYYRC?NRGALNYRSC
121?GLNQSYYPGR?GRCGCRENYC?ERHNGWQSTV?CNGCGSYVFC?RYGRPIRYRR?CPWSRRHYDC
181?HRHRCVSHCN?GCCGHR；
Described SEQ ID No.8:
1 MQKLVFIVVA?IVMALASCTS?ARKKKPCVES?CVGRPDGEYQ?ASCTYDCRFF?VKCVFGFSFL
61 LKCREGHFYD?VDVRDCVKGC?PTGPRGILDC?YDRPDGHWQH?CCYCDLYATR?ASRGMFFHRR
121?CEPGGPVWHD?DKHKCLWTSG?TCPGVHRYYA。
Tubulose worm Ridgeia piscesae chitin-binding protein of the present invention is tubulose worm Ridgeia piscesae chitin-binding protein CbpA, tubulose worm Ridgeia piscesae chitin-binding protein CbpB, tubulose worm Ridgeia piscesae chitin-binding protein CbpC or tubulose worm Ridgeia piscesae chitin-binding protein CbpD.
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpA is a protein; Sequence signature: length is 187aa, and type is an amino acid; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpA is as follows, is designated as SEQ ID No.5:
1 MSKLLAALVA?LSIAYSVQGC?SDSCVGRQNG?RCYPSACGPC?NYYFKCQRTI?VVEQYCRASS
61 YCYLNGRCRP?CGDSCERKRD?GVYPSVNRAR?PYYYQCEAGQ?LYYRECQPFQ?IFNAWTRKCE
121?CFEGACLHGN?GWRSSFCRGK?GWRVFCSGGF?ARYYRQCPRP?RPHVCCHTYT?CVSTCTSYYG
181?RGHCCRG。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpB is a protein; Sequence signature: length is 193aa, and type is an amino acid; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpB is as follows, is designated as SEQ ID No.6:
1 MSKLIIALAV?LSACIAVQAR?GWEGCSQDKC?DGRRTGRRYQ?SLCAPVSVYY?RCRHSRPVYD
61 NCRERNYCVV?NGRCCRCGDT?CEGKCDGVYP?SVNRPRPAYY?QCDGGELYYR?TCQPFQIFNP
121?WTKQCECYEG?SCLHGNGYRA?SFCRGKEWRV?YCRAGFPQFY?ERCPRPRPYV?CCKTYTCIST
181?CSVFQGRNGC?CHA。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpC is a protein; Sequence signature: length is 196aa, and type is an amino acid; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpC is as follows, is designated as SEQ ID No.7:
1 MAKFFIALLV?LVAVYATEAL?PGNGYEGCFN?GYNYDCCRRR?RPNFCYPSLV?KPLPYYYRCV
61 GRRVVYGRCE?THLCVTVKGN?CGSCSDRCRY?TRHSHYYYSS?YFYGRYYYRC?NRGALNYRSC
121?GLNQSYYPGR?GRCGCRENYC?ERHNGWQSTV?CNGCGSYVFC?RYGRPIRYRR?CPWSRRHYDC
181?HRHRCVSHCN?GCCGHR。
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpD is a protein; Sequence signature: length is 150aa, and type is an amino acid; The sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpD is as follows, is designated as SEQ ID No.8:
1 MQKLVFIVVA?IVMALASCTS?ARKKKPCVES?CVGRPDGEYQ?ASCTYDCRFF?VKCVFGFSFL
61 LKCREGHFYD?VDVRDCVKGC?PTGPRGILDC?YDRPDGHWQH?CCYCDLYATR?ASRGMFFHRR
121?CEPGGPVWHD?DKHKCLWTSG?TCPGVHRYYA。
These albumen have the polypeptide of aminoacid sequence shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and the SEQ ID No.8; Or replacement, disappearance or the interpolation through one or more amino-acid residues of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 aminoacid sequence formed, and has polypeptide with the aminoacid sequence identical function shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and the SEQ ID No.8.
The preparation method of tubulose worm Ridgeia piscesae chitin-binding protein of the present invention may further comprise the steps:
1) extraction of tubulose worm Ridgeia piscesae RNA;
2) tubulose worm Ridgeia piscesae cDNA reverse transcription;
3) pcr amplification and the sequential analysis of tubulose worm Ridgeia piscesae chitin-binding protein gene;
4) expression of tubulose worm Ridgeia piscesae chitin-binding protein.
The present invention is by extracting the RNA of tubulose worm Ridgeia piscesae, pass through reverse transcription, therefrom clone and identified 4 chitin-binding protein gene cbpA, cbpB, cbpC and cbpD, and in insect cell high5, carry out recombinant expressed, further recombinant protein has been carried out property testing, for this proteic practical application provides the foundation.
The present invention expresses 4 kinds of chitin-binding proteins by homologous recombination, can overcome deficiencies such as the tubulose worm is difficult for gathering, separation and purification process complexity is loaded down with trivial details, for the widespread use of this albumen in research fields such as biology, food, medicine, agriculturals lays the foundation.
Description of drawings
Fig. 1 is that the recombinant expressed western blot of tubulose worm Ridgeia piscesae chitin-binding protein detects collection of illustrative plates.In Fig. 1,1: the expression of recombinant vectors pIZ-FLAG-CbpA in high5; 2: the expression of recombinant vectors pIZ-FLAG-CbpB in high5; 3: the expression of recombinant vectors pIZ-FLAG-CbpC in high5; 4: the expression of recombinant vectors pIZ-FLAG-CbpD in high5.
Fig. 2 is that the affine property analysis western of tubulose worm Ridgeia piscesae chitin-binding protein blot detects collection of illustrative plates.In Fig. 2,1: the expression of recombinant vectors pIZ-FLAG-CbpA in high5; 2: experiment that recombinant C bpA and α-chitin are affine detects; 3: experiment that recombinant C bpA and β-chitin are affine detects; 4: the expression of recombinant vectors pIZ-FLAG-CbpB in high5; 5: experiment that recombinant C bpB and α-chitin are affine detects; 6: experiment that recombinant C bpB and β-chitin are affine detects; 7: the expression of recombinant vectors pIZ-FLAG-CbpC in high5; 8: experiment that recombinant C bpC and α-chitin are affine detects; 9: experiment that recombinant C bpC and β-chitin are affine detects; 10: the expression of recombinant vectors pIZ-FLAG-CbpD in high5; 11: experiment that recombinant C bpD and α-chitin are affine detects; 12: experiment that recombinant C bpD and β-chitin are affine detects.
Embodiment
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 such as molecular cloning laboratory manual (Sa nurse Brooker, Russell's (work), Huang Peitang (translating), " molecular cloning experiment guide ", Science Press, 2002, the third edition .) described in experiment condition, or the condition of being advised according to reagent or instrument production firm.
For achieving the above object, the present invention adopts following technical measures, and its concrete steps are:
1. the extraction of tubulose worm Ridgeia piscesae RNA
Get 100mg tubulose worm Ridgeia piscesae tissue sample, add 1mL TRI Reagent (MRC company), with the thorough dispersion tissue of electronic refiner, room temperature leaves standstill the 5min lysing cell; Add the 0.2mL chloroform, thermal agitation 15s, room temperature leaves standstill 10min; 4 ℃, the centrifugal 15min of 12000g; Supernatant liquid is transferred in the new centrifuge tube, adds the 0.5mL Virahol, mixing, room temperature is placed 10min; 4 ℃, the centrifugal 10min of 12000g; Remove liquid, add 1mL 75% ethanol rinsing; 4 ℃, the centrifugal 5min of 7500g; Remove liquid, RNA precipitates a little and is dissolved in an amount of DEPC treated water after the drying; Measure total RNA at OD with ultraviolet spectrophotometer
230, OD
260, OD
280Absorbancy, judge concentration and the purity of total RNA.
2. tubulose worm Ridgeia piscesae cDNA reverse transcription
Prepare the cDNA inverse transcription reaction liquid at the 0.5mL centrifuge tube, comprise the total RNA of 2 μ g, 1 μ L Oligo (dT)
20(50 μ M), 1 μ L dNTP (10mM), the H that handled with DEPC
2O mends cumulative volume to 13 μ L; Reaction system is handled 5min for 65 ℃, puts into ice bath 1min immediately; Centrifugal slightly, add following component: 4 μ L5 * First-Strand Buffer, 1 μ LDTT (0.1M), 1 μ L RNase Inhibitor (40U/ μ L) (TaKaRa company) and 1 μ L SuperScript
TMIII reverse transcriptase (200U/ μ L) (Invitrogen company); Reaction system is flicked mixing, and is centrifugal slightly; 50 ℃ of incubation 60min; Handle the 15min termination reaction for 70 ℃.
3. the pcr amplification and the sequential analysis of tubulose worm Ridgeia piscesae chitin-binding protein gene
With tubulose worm Ridgeia piscesae cDNA is template, with primer AF and AR, BF and BR, CF and CR, DF and DR increase respectively chitin-binding protein gene cbpA, cbpB, cbpC and cbpD gene (not containing signal peptide).
AF：
GGATCCGGTGCTCGGATTCGTGCGTC(SEQID?No?9)；
AR：
CTCGAGCTATCCTCTGCAGCAGTGAC(SEQ?ID?No.10)。
BF：
GGATCCCCCGCGGATGGGAGGGATGT(SEQ?ID?No.11)；
BR：
CTCGAGCTAGGCGTGGCAACATCCGT(SEQ?ID?No.12)。
CF：
GGATCCCACTCCCTGGTAACGGTTAC(SEQ?ID?No.13)；
CR：
TCTAGATTATCGATGGCCACAGCAAC(SEQ?ID?No.14)。
DF：
GGATCCCTAGGAAGAAAAAACCGTGC(SEQ?ID?No.15)；
DR：
CTCGAGTTAAGCGTAGTATCGGTGTA(SEQ?ID?No.16)。
Line part GGATCC represents BamH I restriction enzyme site, and CTCGAG represents Xho I restriction enzyme site, and TCTAGA represents the XbaI enzyme cutting site.
In the reaction system of 50 μ L, contain 2 μ L templates, 1 μ L primer AF (or BF, CF, DF) (10 μ M), 1 μ L primer AR (or BR, CR, DR) (10 μ M), 4 μ LdNTP (each 2.5mM), 10 μ L, 5 * PrimerSTAR
TMBuffer, 1 μ L PrimerSTAR
TMHS DNA Polymerase (2.5U/ μ L) (TaKARa company) uses H
2O mends cumulative volume to 50 μ L.The PCR reaction conditions is: 98 ℃ of 10s, 68 ℃ of 1min, 68 ℃ of 7min (30cycles); 4 ℃ of preservations.The purified back of PCR product is connected with the T carrier, is transformed among the competent escherichia coli cell Top10, and picking has the positive colony order-checking of inserting dna fragmentation.
The aminoacid sequence of CbpA, CbpB, CbpC and the CbpD that derives according to the nucleotide sequence that obtains, contain 187,193,196 and 150 amino-acid residues respectively, its aminoacid sequence sees SEQ ID No.5, SEQ ID No.6, SEQID No.7, SEQ ID No.8 for details.
4. tubulose worm Ridgeia piscesae chitin-binding protein expression of gene
The plasmid that will contain cbpA, cbpB, cbpC and cbpD gene with primer separately carry out enzyme with the restriction enzyme of restriction enzyme site and cut, glue reclaims cbpA, cbpB, cbpC and cbpD gene fragment, simultaneously plasmid pIZ-FLAG is also carried out enzyme with identical enzyme and cuts.Both connections are spent the night (12～16h).Connect product and be transformed among the competent escherichia coli cell DH5 α, extract plasmid, sequence verification.
Recovery insect cell high5 is by 10
6Cells/well is seeded to 6 porocyte culture plates, and adds 1ml SFX substratum (Hyclone company), 27 ℃ of overnight incubation in advance.Recombinant expression plasmid pIZ-FLAG-CbpA/B/C/D is prepared A liquid with 200 μ l SFX substratum mixings respectively; Get 200 μ l SFX substratum and 4 μ l cell fectin transfection reagent (Invitrogen company) mixings prepare B liquid; Again two kinds of liquid mixing of A, B are prepared C liquid, put preheating 30min in 27 ℃ of incubators.Substratum among the insect cell high5 of recovery overnight incubation is removed, and washed once with the SFX substratum; Add 400 μ l SFX substratum in C liquid, mixing joins in the Tissue Culture Plate then; Cultivate 5h for 27 ℃, change the SFX substratum; After cultivating 60h, with Lysis buffer (20mM Tris-HCl (pH 7.4), 100mM KCl, 2mM EDTA, 0.1%NP40,10%Glycerol, 0.5mM PMSF, 3mM DTT) the cracking culturing cell prepares protein sample, carries out western blot and detect behind the SDS-PAGE electrophoresis.The result shows that tubulose worm Ridgeia piscesae chitin-binding protein has obtained recombinant expressed (Fig. 1) in insect cell high5.
5. the affine property analysis of tubulose worm Ridgeia piscesae chitin-binding protein
Prepare 4 kinds of recombinant expressed samples of chitin-binding protein of tubulose worm Ridgeia piscesae respectively according to step 4; With protein sample and the equivalent α-chitin, β-chitin mixing of preparation, put vertical mixing tank respectively, in 4 ℃ in conjunction with 4h; Lysis Buffer with 100 times of volumes carries out rinsing to binding mixture, removes non-specific binding; Add the last sample buffer of 100 μ l, 2 * SDS (20mM Tris-HCl pH8.2,2mM EDTA, 2%SDS, 10% beta-mercaptoethanol, 16% glycerine, 0.05% tetrabromophenol sulfonphthalein), carry out the SDS-PAGE electrophoresis behind the mixing; Further carrying out western blot detects.The result shows that four kinds of chitin-binding proteins are α-chitin-binding protein (Fig. 2).
Claims (6)
1. tubulose worm Ridgeia piscesae chitin-binding protein gene is characterized in that being tubulose worm Ridgeia piscesae chitin-binding protein cbpA gene, tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene, tubulose worm Ridgeia piscesae chitin-binding protein cbpC gene or tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene.
2. tubulose worm Ridgeia piscesae chitin-binding protein gene as claimed in claim 1 is characterized in that,
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbp gene is DNA, sequence signature: length is 564bp, type is a nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpA gene is as follows, is designated as SEQ ID No.1:
1 ATGTCGAAGC?TTCTCGCAGC?CCTTGTGGCA?CTTAGCATCG?CCTACTCCGT?GCAAGGGTGC
61 TCGGATTCGT?GCGTCGGCAG?ACAGAACGGC?CGCTGCTATC?CTTCGGCGTG?TGGTCCGTGC
121?AACTATTACT?TCAAGTGTCA?ACGTACGATC?GTTGTCGAGC?AATACTGTAG?AGCGTCATCG
181?TACTGCTACC?TCAACGGACG?ATGCCGACCA?TGTGGTGACT?CTTGCGAGAG?AAAAAGGGAT
241?GGCGTCTACC?CATCAGTGAA?CCGGGCCCGA?CCCTACTACT?ACCAGTGCGA?AGCCGGACAA
301?CTGTACTACC?GCGAGTGCCA?GCCCTTCCAG?ATCTTCAACG?CGTGGACCAG?GAAATGCGAA
361?TGCTTCGAGG?GCGCCTGTCT?GCACGGAAAC?GGATGGAGAT?CTTCCTTCTG?CCGAGGCAAG
421?GGATGGCGTG?TGTTCTGCAG?CGGTGGTTTC?GCCAGGTATT?ACAGGCAATG?CCCTCGACCC
481?CGACCTCACG?TGTGCTGTCA?CACCTACACG?TGCGTGTCCA?CCTGCACCTC?GTACTATGGA
541?CGTGGTCACT?GCTGCAGAGG?ATAG；
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene is DNA, sequence signature: length is 582bp, type is a nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpB gene is as follows, is designated as SEQ ID No.2:
1 ATGTCGAAGC?TTATCATTGC?CCTGGCGGTT?CTTTCCGCCT?GCATCGCCGT?CCAAGCCCGC
61 GGATGGGAGG?GATGTTCTCA?GGACAAATGC?GACGGCAGAC?GTACCGGCAG?GAGATACCAG
121?TCTCTCTGCG?CCCCTGTCAG?CGTCTACTAC?AGATGTCGCC?ACAGTCGCCC?CGTCTACGAC
181?AACTGCCGTG?AGCGCAACTA?TTGCGTCGTC?AACGGCCGAT?GCTGCCGATG?CGGTGACACC
241?TGTGAGGGCA?AATGCGACGG?CGTCTACCCG?TCCGTGAACC?GACCCCGTCC?CGCCTACTAC
301?CAGTGTGACG?GAGGAGAGCT?GTACTACCGC?ACGTGCCAGC?CCTTCCAGAT?CTTCAACCCC
361?TGGACCAAGC?AATGCGAGTG?CTACGAGGGT?TCGTGTCTCC?ATGGCAACGG?GTACAGGGCG
421?TCCTTCTGCC?GCGGCAAGGA?ATGGCGCGTG?TACTGCCGGG?CTGGATTCCC?CCAGTTCTAC
481?GAGCGCTGTC?CCCGACCCAG?GCCCTACGTG?TGCTGCAAGA?CTTACACCTG?CATCTCCACG
541?TGCAGCGTGT?TCCAGGGCAG?AAACGGATGT?TGCCACGCCT?AG；
The molecule type of described tubulose worm Ridgeiapiscesae chitin-binding protein cbpC gene is DNA, sequence signature: length is 591bp, type is a nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpC gene is as follows, is designated as SEQ ID No.3:
1 ATGGCGAAGT?TTTTCATCGC?ATTGTTGGTG?CTCGTCGCTG?TATATGCAAC?TGAAGCACTC
61 CCTGGTAACG?GTTACGAAGG?TTGTTTCAAC?GGCTACAACT?ACGATTGCTG?CAGGCGTCGT
121?AGACCCAACT?TCTGCTACCC?GTCGCTGGTG?AAGCCACTGC?CTTACTACTA?CCGATGTGTC
181?GGCAGGAGAG?TTGTGTACGG?CAGATGTGAG?ACGCACCTTT?GCGTCACAGT?AAAGGGAAAT
241?TGCGGATCGT?GCAGTGATCG?TTGTCGCTAC?ACTCGTCATT?CACACTATTA?CTACTCGAGC
301?TACTTCTACG?GTCGCTACTA?CTACCGATGC?AACCGCGGAG?CACTGAACTA?CCGCTCGTGT
361?GGACTCAACC?AGTCTTACTA?TCCGGGACGC?GGCCGTTGCG?GCTGCAGAGA?GAATTACTGT
421?GAGAGGCACA?ACGGCTGGCA?GTCGACCGTT?TGCAACGGAT?GCGGAAGCTA?CGTCTTCTGC
481?AGATATGGAC?GTCCCATTCG?CTACAGGAGA?TGCCCCTGGA?GCAGACGCCA?CTACGACTGT
541?CATCGCCATC?GTTGTGTCAG?CCATTGCAAC?GGTTGCTGTG?GCCATCGATA?A；
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene is DNA, sequence signature: length is 453bp, type is a nucleic acid, chain is double-stranded, topological framework is linear, the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein cbpD gene is as follows, is designated as SEQ ID No.4:
1 ATGCAGAAGT?TGGTCTTCAT?AGTGGTAGCA?ATCGTCATGG?CCTTGGCGAG?TTGTACCTCC
61 GCTAGGAAGA?AAAAACCGTG?CGTGGAGTCG?TGCGTGGGCC?GACCGGACGG?CGAGTACCAA
121?GCATCCTGCA?CCTACGACTG?TCGCTTCTTC?GTCAAGTGTG?TCTTCGGCTT?CTCCTTCCTG
181?TTGAAATGCC?GCGAGGGACA?TTTCTATGAC?GTCGACGTGA?GAGACTGTGT?CAAAGGATGT
241?CCGACTGGAC?CACGCGGAAT?CCTCGACTGC?TACGACCGCC?CTGACGGCCA?CTGGCAGCAT
301?TGCTGCTACT?GCGACTTGTA?CGCCACTCGT?GCGAGCAGGG?GTATGTTCTT?CCACCGCCGC
361?TGTGAGCCGG?GCGGTCCGGT?CTGGCACGAT?GACAAACACA?AGTGTCTCTG?GACGTCGGGA
421?ACATGTCCCG?GGGTACACCG?ATACTACGCT?TAA。
3. tubulose worm Ridgeia piscesae chitin-binding protein gene as claimed in claim 1 is characterized in that the nucleotide sequence of described tubulose worm Ridgeia piscesae chitin-binding protein gene adopts following method to obtain:
Extract the RNA of tubulose worm Ridgeia piscesae, and the RNA reverse transcription become cDNA, further be template, respectively with composition sequence SEQ ID No.9 with cDNA, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ IDNo.13, SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16 are as primer, obtained SEQ IDNo.1 through pcr amplification, SEQ ID No.2, the sequence of SEQ ID No.3 and SEQ ID No.4; Carry out recombinant expressedly then in insect cell high5, polypeptide SEQ ID No.5, SEQ IDNo.6, SEQ ID No.7 and the SEQ ID No.8 of biological method proof cbpA, cbpB, cbpC and cbpD genes encoding have chitin in conjunction with activity;
Described SEQ ID No.9:
GGATCCGGTGCTCGGATTCGTGCGTC;
Described SEQ ID No.10:
CTCGAGCTATCCTCTGCAGCAGTGAC;
Described SEQ ID No.11:
GGATCCCCCGCGGATGGGAGGGATGT;
Described SEQ ID No.12:
CTCGAGCTAGGCGTGGCAACATCCGT;
Described SEQ ID No.13:
GGATCCCACTCCCTGGTAACGGTTAC;
Described SEQ ID No.14:
TCTAGATTATCGATGGCCACAGCAAC;
Described SEQ ID No.15:
GGATCCCTAGGAAGAAAAAACCGTGC;
Described SEQ ID No.16:
CTCGAGTTAAGCGTAGTATCGGTGTA;
Described SEQ ID No.5:
1 MSKLLAALVA?LSIAYSVQGC?SDSCVGRQNG?RCYPSACGPC?NYYFKCQRTI?VVEQYCRASS
61 YCYLNGRCRP?CGDSCERKRD?GVYPSVNRAR?PYYYQCEAGQ?LYYRECQPFQ?IFNAWTRKCE
121?CFEGACLHGN?GWRSSFCRGK?GWRVFCSGGF?ARYYRQCPRP?RPHVCCHTYT?CVSTCTSYYG
181?RGHCCRG；
Described SEQ ID No.6:
1 MSKLIIALAV?LSACIAVQAR?GWEGCSQDKC?DGRRTGRRYQ?SLCAPVSVYY?RCRHSRPVYD
61 NCRERNYCVV?NGRCCRCGDT?CEGKCDGVYP?SVNRPRPAYY?QCDGGELYYR?TCQPFQIFNP
121?WTKQCECYEG?SCLHGNGYRA?SFCRGKEWRV?YCRAGFPQFY?ERCPRPRPYV?CCKTYTCIST
181?CSVFQGRNGC?CHA；
Described SEQ ID No.7:
1 MAKFFIALLV?LVAVYATEAL?PGNGYEGCFN?GYNYDCCRRR?RPNFCYPSLV?KPLPYYYRCV
61 GRRVVYGRCE?THLCVTVKGN?CGSCSDRCRY?TRHSHYYYSS?YFYGRYYYRC?NRGALNYRSC
121?GLNQSYYPGR?GRCGCRENYC?ERHNGWQSTV?CNGCGSYVFC?RYGRPIRYRR?CPWSRRHYDC
181?HRHRCVSHCN?GCCGHR；
Described SEQ ID No.8:
1 MQKLVFIVVA?IVMALASCTS?ARKKKPCVES?CVGRPDGEYQ?ASCTYDCRFF?VKCVFGFSFL
61 LKCREGHFYD?VDVRDCVKGC?PTGPRGILDC?YDRPDGHWQH?CCYCDLYATR?ASRGMFFHRR
121?CEPGGPVWHD?DKHKCLWTSG?TCPGVHRYYA。
4. tubulose worm Ridgeia piscesae chitin-binding protein is characterized in that being tubulose worm Ridgeia piscesae chitin-binding protein CbpA, tubulose worm Ridgeia piscesae chitin-binding protein CbpB, tubulose worm Ridgeiapiscesae chitin-binding protein CbpC or tubulose worm Ridgeia piscesae chitin-binding protein CbpD.
5. tubulose worm Ridgeia piscesae chitin-binding protein as claimed in claim 4 is characterized in that,
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpA is a protein, sequence signature: length is 187aa, type is an amino acid, and the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpA is as follows, is designated as SEQ ID No.5:
1 MSKLLAALVA?LSIAYSVQGC?SDSCVGRQNG?RCYPSACGPC?NYYFKCQRTI?VVEQYCRASS
61 YCYLNGRCRP?CGDSCERKRD?GVYPSVNRAR?PYYYQCEAGQ?LYYRECQPFQ?IFNAWTRKCE
121?CFEGACLHGN?GWRSSFCRGK?GWRVFCSGGF?ARYYRQCPRP?RPHVCCHTYT?CVSTCTSYYG
181?RGHCCRG；
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpB is a protein, sequence signature: length is 193aa, type is an amino acid, and the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpB is as follows, is designated as SEQ ID No.6:
1 MSKLIIALAV?LSACIAVQAR?GWEGCSQDKC?DGRRTGRRYQ?SLCAPVSVYY?RCRHSRPVYD
61 NCRERNYCVV?NGRCCRCGDT?CEGKCDGVYP?SVNRPRPAYY?QCDGGELYYR?TCQPFQIFNP
121?WTKQCECYEG?SCLHGNGYRA?SFCRGKEWRV?YCRAGFPQFY?ERCPRPRPYV?CCKTYTCIST
181?CSVFQGRNGC?CHA；
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpC is a protein, sequence signature: length is 196aa, type is an amino acid, and the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpC is as follows, is designated as SEQ ID No.7:
1 MAKFFIALLV?LVAVYATEAL?PGNGYEGCFN?GYNYDCCRRR?RPNFCYPSLV?KPLPYYYRCV
61 GRRVVYGRCE?THLCVTVKGN?CGSCSDRCRY?TRHSHYYYSS?YFYGRYYYRC?NRGALNYRSC
121?GLNQSYYPGR?GRCGCRENYC?ERHNGWQSTV?CNGCGSYVFC?RYGRPIRYRR?CPWSRRHYDC
181?HRHRCVSHCN?GCCGHR；
The molecule type of described tubulose worm Ridgeia piscesae chitin-binding protein CbpD is a protein, sequence signature: length is 150aa, type is an amino acid, and the sequence of described tubulose worm Ridgeia piscesae chitin-binding protein CbpD is as follows, is designated as SEQ ID No.8:
1 MQKLVFIVVA?IVMALASCTS?ARKKKPCVES?CVGRPDGEYQ?ASCTYDCRFF?VKCVFGFSFL
61 LKCREGHFYD?VDVRDCVKGC?PTGPRGILDC?YDRPDGHWQH?CCYCDLYATR?ASRGMFFHRR
121?CEPGGPVWHD?DKHKCLWTSG?TCPGVHRYYA。
6. the preparation method of tubulose worm Ridgeia piscesae chitin-binding protein as claimed in claim 4 is characterized in that may further comprise the steps:
1) extraction of tubulose worm Ridgeia piscesae RNA;
2) tubulose worm Ridgeia piscesae cDNA reverse transcription;
3) pcr amplification and the sequential analysis of tubulose worm Ridgeia piscesae chitin-binding protein gene;
4) expression of tubulose worm Ridgeia piscesae chitin-binding protein.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042104A (en) * | 2019-04-28 | 2019-07-23 | 自然资源部第三海洋研究所 | Deep-sea Ridgeia AprA gene and its expression |
| CN112430691A (en) * | 2020-11-27 | 2021-03-02 | 江南大学 | Method for efficiently degrading insect egg shells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1379088A (en) * | 2002-04-27 | 2002-11-13 | 陈燕娜 | Radish chitin bindin and its preparing process |
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| CN1379088A (en) * | 2002-04-27 | 2002-11-13 | 陈燕娜 | Radish chitin bindin and its preparing process |
Non-Patent Citations (2)
| Title |
|---|
| 《中国博士学位论文全文数据库 基础科学辑》 20091115 阮灵伟 深海热液区管状蠕虫的分子特征及对虾microRNA的初探 A006-16 , 第11期 2 * |
| 《齐齐哈尔医学院学报》 20080228 张可勇等 几丁质亲和层析纯化蛋白的方法改良 , 第04期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042104A (en) * | 2019-04-28 | 2019-07-23 | 自然资源部第三海洋研究所 | Deep-sea Ridgeia AprA gene and its expression |
| CN112430691A (en) * | 2020-11-27 | 2021-03-02 | 江南大学 | Method for efficiently degrading insect egg shells |
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