CN102653768A - GH (Glycoside hydrolases) 18th family chitinase gene cloning method - Google Patents
GH (Glycoside hydrolases) 18th family chitinase gene cloning method Download PDFInfo
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Abstract
The invention discloses a GH (Glycoside hydrolases) 18th family chitinase gene cloning method. According to the method, a special culture medium with a single carbon source is used for screening to obtain bacteria for generating chitinase, a unique and effective semi-nested degenerate primer is utilized to perform the amplification on bacterial GH 18th family chitinase gene segments, full-length chitinase genes are obtained by virtue of a site-finding PCR (Polymerase Chain Reaction) method or an inverse PCR method, expression plasmids of the chitinase genes are constructed, and the inducible expression of the genes is performed in escherichia coli. The novel GH 18th family chitinase gene cloning method is effective and reliable and lays the foundation for finally constructing chitinase engineering strains and realizing the industrialized production of the chitinase.
Description
Technical field
The invention belongs to genetically engineered and microbial technology field, be specifically related to a kind of cloning process of GH18 family members chitinase gene.
Background technology
Mierocrystalline cellulose, Regitex FA are the abundantest polysaccharide of nature content, are containing huge biological available stock, and they are formed by connecting through β-1,4 glycosidic link by Glucopyranose, and difference is the group that connected on No. 2 carbon.Wherein Regitex FA is to be made up of D-acetylglucosamine residue, and at occurring in nature, in-vivo contents such as fungi, insect are very abundant.Scientific circles extremely pay close attention to Regitex FA, chitosan and hydrolyzate thereof because they have many important with the biological function of potentiality to be exploited is arranged, such as sterilization, anticancer etc., some function has been applied to agricultural, food and medicine industry.
Chitinase (EC 3.2.1.14) is the enzyme that nature exist a type can catalytic hydrolysis Regitex FA β-1,4 glycosidic link, and it exists bacterium to fungi, even in virus and even the plant.According to its amino acid sequence analysis, chitinase can be divided into No. 18 and No. 19 two family members, and different family members' enzyme is different to specificity, the mode of action of substrate.
With respect to physical chemistry method hydrolysis Regitex FA, enzymatic hydrolysis has incomparable advantage such as energy-saving and environmental protection, efficient, safety, low cost, can quality products be provided for industries such as the food of Regitex FA raw material, pharmacy.The key of this problem is the cloning process of chitinase gene.
Summary of the invention
An object of the present invention is to provide a kind of method of from the bacterial strain that produces chitinase, fast cloning chitinase gene.
Method of the present invention comprises the steps:
(1) known GH18 family members chitinase gene is carried out sequence alignment, find out the core conserved sequence;
(2) design the segmental degenerated primer of chitinase gene that to increase;
(3) be template with the genome that produces the chitinase bacterial strain, utilize degenerated primer to pass through half nested PCR method amplification bacterium chitinase portion gene fragment;
(4) utilize the portion gene sequence that obtains, design nested primers, move through sitefinding PCR method or inverse PCR method step and obtain the total length chitinase gene.
Chitinase can be divided into No. 18 and No. 19 two family members, can find out GH18 family members' core conserved sequence through amino acid sequence analysis.Compare known GH18 family members' chitinase gene, find out its conserved sequence SIGGWT, FDGIDID and NIMTYD, design half nested degenerated primer.Software Block Maker also capable of using (http://bioinformatics.weizmann.ac.il/blocks/blockmkr/www/make_b locks.html) compares and seeks conservative region, utilizes software CODEHOP (http://bioinformatics.weizmann.ac.il/blocks/codehop.html) to seek possible degenerated 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 degenerated primer.
According to mentioned above principle, the contriver has designed three degenerated primers:
GH18FP?1：5’-AGYATHGGNGGNTGGCA-3’；
GH18FP2：5’-TTYGAYGGNATHGAYATHGA-3’；
GH18RP：5’-TCRTANGTCATDATRTT-3’；
Utilize only to contain Regitex FA and can obtain the bacterial strain that produces chitinase as the special selective medium of sole carbon source, and its genomic dna of extracting.With bacterial genomes DNA is that template is carried out PCR.The portion gene fragment (250-280bp) that amplification is obtained checks order, then according to specific separately nested primers FP1, FP2 and RP1, the RP2 of various chitinase gene fragments sequence designs that obtains.Wherein, FP1, FP2 and RP1, RP2 are special chitinase gene fragment (250-280bp) sequences Design that obtain according to said step (3) amplification, and every kind of its primers F of chitinase gene P1, FP2, RP1 and RP2 can adjust accordingly according to special chitinase gene fragment sequence.
Genomic dna with each bacterium is a template, carries out that single enzyme is cut and recirculation, utilizes FP1 and RP1 primer to carry out inverse PCR then, and the product that obtains with the gained amplification again utilizes primers F P2 to carry out nested PCR with RP2 as template.Perhaps utilize SFP1 (5 '-CACGACACGCTACTCAACAC-3 ') and FP1 (or RP1) primer to carry out Sitefinding PCR; The PCR product that obtains with gained amplification again is as template, utilizes primer SFP2 (5 '-ACTCAACACACCACCTCGCACAGC-3 ') to carry out nested PCR with FP2 (or RP2).To the chitinase gene fragment upstream and downstream sequence that the obtains amplification of moving one's steps, and to the gene fragment that is obtained check order, gene splicing.
Result and existing chitinase gene according to order-checking, splicing are compared, and the analysis according to homology etc. can obtain novel GH18 family members chitinase gene.
Compare according to sequencing result and with existing chitinase gene; Find that the present invention has obtained a kind of novel chitinase gene chi9; Its dna sequence dna and amino acid and announce that chitinase sequence (BAK53879) homology is 60.5% respectively shown in Seq ID No.2 and Seq ID No.3.
Do not influencing under the active prerequisite of chitinase protein, can carry out various replacements, interpolation and/or lack one or several amino acid obtaining to have the active derived protein of chitinase the aminoacid sequence shown in the SEQ ID NO:3.According to the common practise of art technology, proteinic BA is closely-related with its functional domain.In general, the site mutation that only occurs in functional domain possibly exert an influence to proteinic 2 and 3 dimensional organization, thereby influences its BA.And for the amino acid sites that occurs in away from functional domain; Because the protein function conformation is not participated in this zone; Thereby the sudden change of amino acid whose individual point can not produce substantial effect to proteinic BA, thereby can keep the biological function of crude protein basically.
Thereby; Preferably; Can be according to common practise and reasoning from logic; Thereby obtain the basic protein mutant that keeps the chitinase BA through the outer amino acid sites of chitinase protein BA structural domain being carried out various replacements, interpolation and/or lacking one or several amino acid, obtain the basic protein mutant that keeps the chitinase BA thereby preferably the chitinase amino acid sites is carried out point mutation.
In addition, consider the degeneracy of codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or do not influencing under the condition that chitinase gene expresses at its non-coding region, can make amendment to the above-mentioned proteic gene order of encoding.Therefore, thus the present invention also protects replacement, the interpolation that the nucleotide sequence shown in the SEQ ID NO.2 is carried out and/or lacks one or several Nucleotide and obtains the dna molecular that coding can keep the chitinase protein BA basically.
Utilize gene clone technology, can the chitinase gene of being cloned into be received on the suitable carriers, and conversion or transfection prepare recombinant chitinase to prokaryotic organism or eukaryote host expresses.Suitable prokaryotic organism host comprises various bacteriums such as E.coli etc., and suitable eukaryote host comprises yeast (like methanol yeast) and mammalian cell etc., preferably adopts prokaryotic expression system E.coli.
A preferred example is that NdeI and XhoI recombination site that the chitinase gene that the present invention is cloned into adopts design to insert are connected on the coli expression carrier pET28a; And change in e. coli bl21 (DE3) bacterial strain, carry out abduction delivering with 0.1mmol/L IPTG.Obtain chitinase through nickel post affinity chromatography, have higher enzymic activity through analyzing.
The invention provides a kind of from biologies such as bacterium clone GH18 family members chitinase gene, and carried out the method for expressing.Method of the present invention is effectively reliable, has set up the effective novel Regitex FA gene clone method of a cover, for the final suitability for industrialized production chitinase of realizing lays the first stone.
Description of drawings
Fig. 1 is the schema of clone, expression chitinase gene.
After Fig. 2 was engineering bacteria pET28a-chi9/BL21 abduction delivering, the SDS-PAGE that contains Chi9 target protein sample analyzed
Embodiment
Embodiment 1 produces the acquisition of chitinase bacterium
Utilization is substratum (0.5%colloidal chitin, the 0.07%K of sole carbon source with the Regitex FA
2HPO
4, 0.03%KH
2PO
4, 0.05%MgSO
4, 0.03%peptone, 0.03%yeast extract and 1.5%agar, pH 7.2) cultivate and screen the bacterium that obtains expressing the generation chitinase.For example Serratia sp.J1, Staphyloccus sp.J2, Rheinheimera sp.J4, Stenotrophomonas sp.J5, Lysobacter sp.J6, Pseudomonas sp.J8, Comamonas sp.J9, Paenibacillus sp.A5-1, Bacillus sp.SH3 etc., the similarity of these bacterial strains and known bacterial strain is more than 99%.
The clone of embodiment 2 chitinase gene chi9
Extract the bacterium Comamonas sp.J9 genomic dna of embodiment 1, inverse PCR (I-PCR) method obtains chitinase gene.
At first the highly conserved sequence through glycoside hydrolase the 18th family members' chitinase designs half nested degenerate primer:
GH18FP?1：5’-AGYATHGGNGGNTGGCA-3’；
GH18FP2：5’-TTYGAYGGNATHGAYATHGA-3’；
GH18RP：5’-TCRTANGTCATDATRTT-3’)；
With the genomic dna that screens the product chitinase bacterium that obtains is that template is carried out half nested PCR, and program is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 1min, 72 ℃ of constant temperature 1min, 30 circulations, last 72 ℃ of extension 10min.260bp fragment to obtaining checks order (Seq ID No.1), and utilizes base sequence speculating acid sequence, carries out the Blast comparison, confirms the segmental novelty of chitinase of acquisition, further designs nested primers according to the gene fragment order that obtains then
9FP1：5’-TGGACTGGGTCAACATCAT-3’
9FP2：5’-AAYATHATGACNTAYGA-3
9RP1：5’-GCGGCGGTCAGATAGAAT-3’
9RP2：5’-CGACCACATTGTTGCCTAT-3’
Cutting digested genomic dna with the HindIII enzyme then, and carry out self connection, is template to form annular DNA; Utilize nested primer to carry out inverse PCR (I-PCR), program is 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s; 56 ℃ of annealing 1min; 72 ℃ of constant temperature 3min, 30 circulations, last 72 ℃ are extended 10min.Obtain the segmental upstream and downstream sequence of chitinase 3.5kb fragment, finally obtain new chitinase gene chi9 (Seq ID No.2) and protein C hi9 (Seq ID No.3) complete sequence.
The clone of embodiment 3 chitinase gene chiA1
Extract the bacterium Paenibacillus sp.A5-1 genomic dna of embodiment 1, utilize degenerated primer GH18FP1, GH18FP2 and GH18RP to carry out half nested PCR (method is with embodiment 2).Obtain 280bp fragment (Seq ID No.4), then according to the gene design primer that obtains.
A5FP1：5’-GGACAGAAGGACGGCAAGCA-3’
A5FP2：5’-TGCCA?ACTCCACTTTCGTAGC-3’
A5RP1：5’-CGTTTGCAGCAGCAGGATGT-3’
A5RP2：5’-CGATGGCGTTCGGATACTC-3’
Utilize SFP1 and FP1 (or RP1) primer to carry out Sitefinding PCR; The product that obtains with gained amplification again is as template; Utilize primer SFP2 to carry out nested PCR with FP2 (or RP2); To the chitinase gene sequencing fragment that obtains, further design primer A5RP3 (5 '-GTTGCCCGCAGCGTCTTTCG-3 ') and A5RP4 (5 '-CCGTATAAGAGGTCGTGGTG-3 '), to the amplification of moving one's steps of the chitinase gene fragment upstream sequence of acquisition; And to the gene fragment that is obtained check order, gene splicing, obtain chitinase gene chiA1 (Seq ID No.5) and protein C hiA-1 (Seq ID No.6) complete sequence.With announce that chitinase sequence (EGL13634) homology is 95.5%.The structure of embodiment 4 chitinase Chi9 expression vectors
The chitinase complete encoding sequence (SEQ ID NO.2) that obtains according to gene annotation among the embodiment; Design can amplify complete coding and read the primer J9SP of frame (5 '-CGACATATGAAAGAGATGAAATACC-3 ') and J9AP (5 '-ATTCTCGAGCTTCACTTCAGCGATTGC-3 '), construction of expression vector.Comamonas sp.J9 genomic dna is a template, amplification chitinase full-length gene.Under guaranteeing the correct prerequisite of reading frame, recombinate to expression vector pET28a, again recombinant vectors is converted in the bacillus coli DH 5 alpha competent cell, with kalamycin resistance labelling method screening male reorganization bacterium pET28a-chi9/DH5 α.Picking list bacterium colony contains in the LB substratum of 30 μ g/ml kantlex jolting in 5ml and cultivates 37 ℃ and spend the night; Be converted in e. coli bl21 (DE3) competent cell after extracting recombinant plasmid, express bacterium pET28a-chi9/BL21 with kalamycin resistance labelling method screening male recombination high efficiency.
Embodiment 5: the expression of recombinant chitinase and purifying
The engineering bacteria pET28a-chi9/BL21 of picking list bacterium colony contains in the LB substratum of 30 μ g/ml kantlex jolting in 5ml and cultivates 37 ℃ and spend the night; Draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 30 μ g/ml kantlex), cultivated about 3 hours, to OD
600After reaching 0.5, add IPTG, cultivated respectively 5 hours in 25 ℃ of continuation to final concentration 0.1mmol/L.It is centrifugal to get 1ml bacterium liquid, in the bacterial precipitation thing, adds lysate, the suspension bacterial precipitation; Boiled in the boiling water bath 5 minutes; The centrifugal 1min of 12000rpm, supernatant adds electrophoresis in the 10%SDS-PAGE glue, observes the albumen great expression (Fig. 2) of expection molecular weight 66.1kDa after the dyeing.
Behind the engineering bacteria of abduction delivering desirable proteins,, add 8ml PBS Buffer as stated above by every 100ml bacterium with bacterium centrifugation; After the ultrasonication, 15000rpm gets supernatant after centrifugal 30 minutes, adds 2ml Ni-NTA resin (Invitrogen); 30 ℃ of joltings combine 30 minutes, staticly settle, and abandon supernatant; After deposition was washed 2 times with 10ml Wash Buffer, last 2ml chromatography column was used Elution Buffer wash-out target protein then; With 1ml is that unit is in charge of collection, merges enzyme the highest several pipes alive.Be stored in-80 ℃ behind the glycerine of the supernatant interpolation 50% of wash-out, and carry out chitinase analysis alive.
Claims (10)
1. the method for clone's chitinase gene from the bacterial strain that produces chitinase comprises the steps:
(1) known GH18 family members chitinase gene is carried out sequence alignment, find out the core conserved sequence;
(2) design the segmental degenerated primer of chitinase gene that to increase;
(3) be template with the genome that produces the chitinase bacterial strain, utilize degenerated primer to pass through half nested PCR method amplification bacterium chitinase portion gene fragment;
(4) utilize the portion gene sequence that obtains, design nested primers, move through sitefinding PCR method or inverse PCR method step and obtain the total length chitinase gene.
2. method according to claim 1 is characterized in that: the core conserved sequence is SIGGWT, FDGIDID and NIMTYD in the said method steps (1).
3. method according to claim 1 is characterized in that: degenerated primer is in the said method steps (2):
GH18FP?1：5’-AGYATHGGNGGNTGGCA-3’；
GH18FP2：5’-TTYGAYGGNATHGAYATHGA-3’；
GH18RP：5’-TCRTANGTCATDATRTT-3’。
4. method according to claim 1; It is characterized in that: nested primers FP1, FP2 and RP1, the RP2 in the said method steps (4) is special chitinase gene fragment (250-280bp) sequences Design that obtains according to said step (3) amplification, and every kind of its primers F of chitinase gene P1, FP2, RP1 and RP2 can adjust accordingly according to special chitinase gene fragment sequence.
5. chitinase protein is the protein that has as follows (1) or (2) characteristic:
(1), its aminoacid sequence is consistent with sequence shown in the Seq ID NO.3;
(2), with the amino acid residue sequence of Seq ID NO.3 through replacement, disappearance and/or the interpolation of one or several amino-acid residue and to have a chitinase active by (1) deutero-protein.
6. the dna molecular of coding claim 5 said chitinase protein, its nucleotide sequence is shown in SEQ ID NO.2; Thereby or for the nucleotide sequence shown in the SEQ ID NO.2 being replaced, is added and/or lacks the dna molecular that one or several Nucleotide acquisition coding can keep the chitinase protein BA basically.
7. carry the carrier of the said dna molecular of claim 6.
8. host system, its by the described carrier of claim 7 through transforming or transfection prokaryotic organism or eukaryote host obtain.
9. host system according to claim 8, it is bacterium, yeast or mammalian cell.
10. host system according to claim 9, it is intestinal bacteria E.coli.
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Cited By (4)
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| CN110878299A (en) * | 2019-11-27 | 2020-03-13 | 湖北工业大学 | Method for cloning isopentenyl transferase gene family member group |
| CN111286498A (en) * | 2020-03-26 | 2020-06-16 | 汪利平 | High-efficiency chitinase for producing acetylglucosamine |
| WO2021138799A1 (en) * | 2020-01-07 | 2021-07-15 | 上海交通大学 | Isolated or purified trichoderma antagonistic representative gene and application, and gene sequence-based trichoderma antagonistic evaluation amplification primer and application |
| CN116536289A (en) * | 2023-06-21 | 2023-08-04 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110878299A (en) * | 2019-11-27 | 2020-03-13 | 湖北工业大学 | Method for cloning isopentenyl transferase gene family member group |
| WO2021138799A1 (en) * | 2020-01-07 | 2021-07-15 | 上海交通大学 | Isolated or purified trichoderma antagonistic representative gene and application, and gene sequence-based trichoderma antagonistic evaluation amplification primer and application |
| CN111286498A (en) * | 2020-03-26 | 2020-06-16 | 汪利平 | High-efficiency chitinase for producing acetylglucosamine |
| CN111286498B (en) * | 2020-03-26 | 2024-03-29 | 汪利平 | Efficient chitinase for producing acetamido glucose |
| CN116536289A (en) * | 2023-06-21 | 2023-08-04 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
| CN116536289B (en) * | 2023-06-21 | 2023-09-15 | 中国农业科学院生物技术研究所 | Chitinase with lysozyme activity, mutant and application thereof |
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