CN110305882A - A kind of zymoprotein of the gene and its coding of tetracycline antibiotics of degrading - Google Patents
A kind of zymoprotein of the gene and its coding of tetracycline antibiotics of degrading Download PDFInfo
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- CN110305882A CN110305882A CN201910614364.1A CN201910614364A CN110305882A CN 110305882 A CN110305882 A CN 110305882A CN 201910614364 A CN201910614364 A CN 201910614364A CN 110305882 A CN110305882 A CN 110305882A
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- gene
- tetracycline
- tetracycline antibiotics
- zymoprotein
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13008—Flavin-containing monooxygenase (1.14.13.8), i.e. dimethylaniline-monooxygenase
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
Abstract
The present invention relates to the zymoproteins of the gene and its coding of a kind of tetracycline antibiotics of degrading, belong to technical field of biological genetic engineering, the gene has the nucleotide sequence as shown in SEQ ID NO:1, the zymoprotein has the amino acid sequence as shown in SEQ ID NO:4, the zymoprotein of the gene and its coding is expected to the tetracycline antibiotics being applied in degradation environment, degradation problem to solve tetracycline antibiotics from gene level and albumen level provides new approach, it is degraded by way of gene or albumen to tetracycline antibiotics, advantageously reduce the risk that drug resistant gene is propagated in environment, it avoids caused by degradation bacteria strains directly launchtdeGene travels to pathogenic bacteria bring human health security threat from engineering bacteria.
Description
Technical field
The present invention relates to the zymoproteins of the gene and its coding of a kind of tetracycline antibiotics of degrading, belong to biological gene work
Journey technical field.
Background technique
Tetracycline antibiotics are output and the maximum a kind of antibiotic of usage amount in China's feeding industry, are only existed
1999, China's tetracycline antibiotics usage amount was as high as 9413 tons, and to the yield of China's terramycin in 2003, to account for the world total
The 65% of yield, Doxycycline yield come the first in the world.Total usage amount of China's tetracycline antibiotics in 2013 has been up to
12000 tons.Since absorptivity is lower, rear a large amount of tetracycline antibiotics are taken in the form of parent or active metabolite with urine
Enter environment with excrement, so that Animal manure and farm's surrounding enviroment have accumulated a large amount of tetracycline antibiotics.Tetracycline
Class antibiotic largely uses the generation and propagation that tetracycline resistant bacterium and related drug resistant gene are promoted to discharge.Tetracyclines
Antibiotic is transmitted into the human body step by step by food chain, the antibiotic intake of long-term low dose, normal in human body in addition to that can interfere
Intestinal flora, it is also possible to lead to the generation of clinical " superbacteria ", some drugs also contain the work such as teratogenesis, carcinogenic, mutagenesis
With seriously endangering the physiological health of people.
And the method for solving the antibiotic accumulated in environment at present is mainly biological degradation method and two kinds of non-biodegradation method.
Non-biodegradation method includes During High-Temperature Composting degradation, photoelectric catalysis degrading, oxidative degradation etc., but has specificity poor, at high cost, easy
The disadvantages of leading to secondary pollution.Biological degradation method is the Major degradation pathways of tetracycline antibiotics, and both economical at present
Effective method.Conventional biological degradation method is usually to screen degradation bacteria strains, but degradation bacteria strains easily cause the diffusion of drug resistant gene,
There are certain risks for this method.Therefore, it excavates with the degradation active biological substance of tetracycline antibiotics with important
Meaning.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the invention proposes it is a kind of degrade tetracycline antibiotics gene and
Its zymoprotein encoded, it is intended to find the new direction of degradation tetracycline antibiotics, from gene level and albumen level to solve
The residue problem of tetracycline antibiotics provides the solution of new safety in environment, to avoid tetracycline antibiotics resistance to
The biological risk of medicine genetic transmission.
To achieve the goals above, the present invention provides a kind of gene of tetracycline antibiotics of degrading, the gene tools
Just like nucleotide sequence shown in SEQ ID NO:1.
In the technical scheme, the overall length of amplification said gene or the primer pair of its any segment is also disclosed, it is described to draw
The sequence of object pair is as shown in SEQ ID NO:2 and SEQ ID NO:4.
Meanwhile the present invention also provides the zymoprotein of said gene coding, the zymoprotein has such as SEQ ID NO:4
Shown in amino acid sequence.
It has been investigated that being a kind of by the gene that the nucleotide formstet(X)-like gene, but withtet(X) gene has
Relatively big difference, only 89% nucleotide similarity (tet(X) a kind of flavine monooxygenase is encoded, tetracycline medication can be made
Hydroxylating occurs, unstable intermediate product is generated, so that quick spontaneous degradation occur), therefore willtet(X)-like gene is ordered
It is entitledtdeThe albumen of (Tetracycline degrading enzyme), coding are named as TDE.
In the technical scheme, the recombinant vector containing said gene, expression cassette, transgenic cell line or recombination are further related to
Bacterium.
In the technical scheme, said gene can be applied to the tetracycline antibiotics in degradation environment.
Preferably, said gene is applied to the tetracycline in degradation environment.
In the technical scheme, above-mentioned zymoprotein can be applied to the tetracycline antibiotics in degradation environment.
Preferably, above-mentioned zymoprotein is applied to the tetracycline in degradation environment.
Compared with prior art, the beneficial effects of the present invention are:
The zymoprotein of the gene and its coding of a kind of tetracycline antibiotics of degrading provided by the invention, wherein the gene has
The nucleotide sequence as shown in SEQ ID NO:1, the zymoprotein have the amino acid sequence as shown in SEQ ID NO:4, should
The zymoprotein of gene and its coding is expected to the tetracycline antibiotics being applied in degradation environment, is from gene level and albumen
The degradation problem that level solves tetracycline antibiotics provides new approach, to Tetracyclines by way of gene or albumen
Antibiotic is degraded, and the risk that drug resistant gene in environment is propagated is advantageously reduced, and is avoided degradation bacteria strains and is directly launched and causes
'stdeGene travels to pathogenic bacteria bring human health security threat from engineering bacteria.
Detailed description of the invention
Fig. 1 is bacterial strain HNS2-5-2 to the color changeable effect containing tetracycline broth；
Fig. 2 is LC-MS quantitative detection result (the Theoretical max in abscissa of bacterial strain HNS2-5-2 degradation tetracycline
Indicate tetracycline concentration initial in culture medium；M9 media-only indicates negative control group；HNS2-5-2 expression is added to bacterium
The test group of strain HNS2-5-2；" * * * " indicates P < 0.0001；Concentration in ordinate indicates Fourth Ring in culture medium
The concentration of element)；
(swimming lane 1 is that BL21-pET32a-tde is lured through IPTG to the Western blot qualification result that Fig. 3 is bacterial strain pET32a-tde
The expression product led；Swimming lane 2 is BL21-pET32a empty carrier；M is Protein Marker)；
Fig. 4 is the LC-MS testing result of tetracycline in vegetable tissue (abscissa is time d, and ordinate is concentration ng/g).
Specific embodiment
Specific embodiments of the present invention will be further explained below.It should be noted that for these implementations
The explanation of mode is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, invention described below
Technical characteristic involved in each embodiment can be combined with each other as long as they do not conflict with each other.
The acquisition of the zymoprotein of the gene and its coding of the degradation tetracycline antibiotics of embodiment 1
Using Guangzhou from the excrement for changing certain pig farm as laboratory sample, LB solid medium is coated on after normal saline dilution
In (Guangdong Huan Kai Biotechnology Co., Ltd), 37 DEG C of 12 h of culture, the single bacterium for selecting all different shapes on culture medium is dropped into
The single colonie purified is carried out strain number and preservation by row purifying.By all strains examined being separated to according to clinical trial standard
(Guangdong ring is triumphant using MH agar for the guideline of the change committee (Clinical and Laboratory Intitute, CLSI)
Biotechnology Co., Ltd) dilution method does tetracycline medication (tetracycline, terramycin, aureomycin, Doxycycline, minocycline)
Minimal inhibitory concentration test (MICs), with screen have tetracycline medication drug-resistant phenotype bacterial strain.Wherein, Tetracyclines medicine
Object is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.
By the above-mentioned strain inoculated with tetracycline medication drug-resistant phenotype in 4 mL LB test tube meat soup (the triumphant lifes of Guangdong ring
Object Science and Technology Ltd.) in, 37 DEG C, 180 rpm shaken cultivation, 12 h, next day take 200 μ L overnight cultures to be added to 100
In the LB meat soup of mL, and the tetracycline of final concentration of 100 mg/L is added in meat soup.Then culture is placed in 37 DEG C of dark
Shaken cultivation is carried out with the revolving speed of 220 rpm in environment, does blank control with the culture for being not added with tetracycline.It was found that bacterial strain
The overnight culture of HNS2-5-2 can make the culture medium containing tetracycline become sepia (see figure 1) from yellow.
Bacterial strain HNS2-5-2 is then inoculated in M9 culture medium (1 × M9 trace salt, the 2 mM MgSO of 4 mL4With 100 μM
CaCl2), the glucose of 9 g/L concentration, the thiamine of 100 mg/L concentration, the bright ammonia of 100 mg/L concentration are added in culture medium
The tetracycline of acid and 8 μ Lg/mL concentration (reagent is purchased from Shanghai Yuan Ye Biotechnology Co., Ltd).By bacterial strain in 37 DEG C of dark
With 24 h of speed oscillation culture of 200 rpm in environment.Then, centrifuging and taking supernatant, in the filter filtering in 0.22 μm of aperture
Filtered supernatant is diluted 10 times and carries out liquid chromatography-mass spectrography (LC-MS) quantitative detection by clear liquid.LC-MS quantitative result
Display: the Fourth Ring cellulose content after 24 h of experimental group of addition bacterial strain HNS2-5-2 is 2.39 × 10-3Mg/mL, negative control group 24
Fourth Ring cellulose content after h is 8.43 × 10-3mg/mL.Compared with negative control group, the content of tetracycline has dropped in experimental group
71.6%(is shown in Fig. 2), show that bacterial strain HNS2-5-2 has efficient degrading activity to tetracycline.
Using the bacterial genomes extracts kit of TIANGEN Biotech (Beijing) Co., Ltd., and grasp to specifications
Make, extract the genomic DNA of bacterial strain HNS2-5-2, PCR amplification, the system of PCR amplification then are carried out to its 16S rDNA gene
(25 uL) are as follows: 18.375 μ L ddH2O, 2.5 μ L 10 × Buffer, 2 μ L dNTPs, 0.5 μ L upstream primer (SEQ ID NO:
5), 0.5 μ L downstream primer (SEQ ID NO:6), 0.125 μ L rTaq enzyme and 1 μ L DNA profiling.Wherein, 16S rDNA base is expanded
The upstream primer and downstream primer of cause are synthesized by Guangzhou Qing Ke Bioisystech Co., Ltd, other PCR reagents are purchased from precious biological work
Journey (Dalian) Co., Ltd.The program of PCR amplification are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of 45 s of denaturation, 53 DEG C of annealing 45
S, 72 DEG C of 90 s of extension are recycled 30 times；72 DEG C re-extend 10 min, finally by 1% Ago-Gel of resulting PCR product
Carry out electrophoresis.
According to agarose gel electrophoresis as a result, by PCR positive sample be sent to Guangzhou Qing Ke Bioisystech Co., Ltd into
Row sequencing, obtains the nucleotide sequence of the 16S rDNA gene of bacterial strain HNS2-5-2, using ncbi database to the 16S of the bacterium
RDNA gene carries out nucleotide sequence comparison analysis,
Compare analysis the result shows that, bacterial strain HNS2-5-2 is accredited asWautersiella falseniiBacterium, bacterial strain HNS2-5-2
16S rDNA gene nucleotide sequence as shown in SEQ ID NO:7.Preservation finally is carried out to bacterial strain HNS2-5-2.
The preservation information of bacterial strain HNS2-5-2 is as follows:
The preservation time: on May 30th, 2019；
Depositary institution's title: Guangdong Province's Culture Collection；
Deposit number: GDMCC No:60678；
Depositary institution address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100；
Classification naming:Wautersiella falsenii HNS2-5-2。
Meanwhile using above-mentioned PCR product as sample, respectively using MiSeq platform (China silver health) to bacterial strain HNS2-5-2 into
Row genome sequencing, and by SPAdes(version 3 .12.0) initial sequence information is assembled, and pass through RAST
(http://rast.nmpdr.org/) carries out Sequence annotation and analysis.
Genome sequencing result compares display online, and bacterial strain HNS2-5-2 is carriedtet(X)-like gene, andtet
(X) a kind of flavine monooxygenase is encoded, can make tetracycline medication that hydroxylating occur, generate unstable intermediate product, from
And quick spontaneous degradation occurs.Present invention discover thattet(X)-like gene withtet(X) gene has relatively big difference, only 89%
Nucleotide similarity, therefore we willtet(X)-like unnamed gene istde(Tetracycline degrading
Enzyme), the albumen of coding is named as TDE,tdeThe nucleotide sequence of gene is as shown in SEQ ID NO:1, the ammonia of TDE albumen
Base acid sequence is as shown in SEQ ID NO:4.
The degradation of embodiment 2 tetracycline antibiotics gene (tde) clone
In order to incite somebody to actiontdeGene carries out clonal expression, selects pET32a as protein expression vector, uses SnapGene software first
Retrieval is comparedtdeGene (nucleotide sequence as shown in SEQ ID NO:1) carries out design of primers, to expand purpose
The full length sequence of gene, while being added at primer both endsBamHI andEcoTwo restriction enzyme sites of RI and protection base, it is ensured that gene
Forward direction expression.The upstream primer of designed primer pair as shown in SEQ ID NO:2, downstream primer as shown in SEQ ID NO:3,
Guangzhou Qing Ke Bioisystech Co., Ltd is transferred to synthesize designed primer.
Using the genome of bacterial strain HNS2-5-2 as template, using above-mentioned primer pair as primer, using Ex-Taq archaeal dna polymerase
(purchased from precious bioengineering (Dalian) Co., Ltd) carries out PCR amplification, the program of PCR amplification are as follows: 94 DEG C of 5 min of initial denaturation；94
DEG C denaturation 45 s, 55 DEG C of annealing 45 s, 72 DEG C of 90 s of extension, circulation 30 times；72 DEG C re-extend 10 min.The body of PCR amplification
It is (25 μ L) are as follows: 18.375 μ L ddH2O, 2.5 μ 10 × Ex-Taq of L Buffer, 2 μ L dNTPs, 0.5 μ L upstream primer, 0.5
μ L downstream primer, 0.125 μ L Ex-taq enzyme and 1 μ L DNA profiling then recycle resulting PCR product takara segment
Kit (precious bioengineering (Dalian) Co., Ltd) carries out purification and recovery, and restriction enzyme is used after recyclingBamHI andEcoNEB company, the U.S. RI() target fragment of recovery purifying and protein expression vector pET32a were carried out at 37 DEG C respectively
Night double digestion.After the completion of digestion, reuses takara segment QIAquick Gel Extraction Kit and product is recycled respectively, and use T4Connection
Carrier after the recovery and target fragment are placed at 4 DEG C by enzyme (NEB company, the U.S.) according to the molar ratio of 1:3 to carry out staying overnight connection.
Take 10 μ L connection product pET32a-tdeBacillus coli DH 5 alpha competent cell (precious biology is transformed into using chemical transformation
Engineering (Dalian) Co., Ltd) in, then use the small extraction reagent kit of bacterial plasmid of TIANGEN Biotech (Beijing) Co., Ltd.
It is stripped to successful bacterial strain plasmid is converted.Obtained plasmid will be extracted and send to sequencing company progress sequence verification, sequencing is just
True plasmid rotates into BL21 competent cell (precious bioengineering (Dalian) Co., Ltd) using chemical transformation, will finally turn
Change successful bacterial strain BL21-pET32a-tdeIt is stored in the LB meat soup containing 30% glycerol.
Embodiment 3 degrade tetracycline antibiotics zymoprotein gene (tde) expression
By protein expression engineered strain BL21-pET32a-tdeIt is inoculated in (the ammonia containing 50 mg/L in 4 ml LB liquid mediums
Benzyl XiLin), it is placed in 8 h of shaken cultivation at 37 DEG C；It is added in 100 mL LB meat soups in the ratio of 1:100 (containing 50 mg/L's
Ampicillin), 37 DEG C of shaken cultivations to OD600Value is 0.4-0.6；Addition inducer isopropylthio thiogalactoside
Isopropyl β-D-Thiogalactoside, IPTG(treasured bioengineering (Dalian) Co., Ltd) ] extremely final concentration of 0.5
It is pure using the HIS label protein purification kit progress albumen that health is ShiJi Co., Ltd after mmol/L, 16 DEG C of 16 h of shaken cultivation
Change.Purified albumen is subjected to quantitative analysis using the BCA protein quantification kit that health is ShiJi Co., Ltd's production, and uses health
One-step method Fast W B kit for ShiJi Co., Ltd's production carries out Western blot identification to albumen after purification.BCA method is surveyed
Determine BL21-pET32a-tdeThe content of expressed destination protein be 1.413 mg/mL, Western blot the results show that
BL21-pET32a-tdeThere is purpose band at 60 kDa, it is in the same size with expected albumen, illustrate that fusion protein obtains correctly
Expression, BL21-pET32a empty carrier are negative control, see Fig. 3.
The functional verification of the zymoprotein (TDE) of the degradation tetracycline antibiotics of embodiment 4
It is verifying originally by BL21-pET32a-tdeThe protease of expression activity in vitro and to tetracycline medication in environment
Degradation effect, experimental group, negative control group and blank control group, every group of setting 6 are separately designed by vegetable aquaculture culture solution
It is a parallel.Added respectively in experimental group and negative control group vegetable aquaculture culture solution final concentration of 10 mg/L tetracycline and
The Shanghai the NADPH(Yuan Ye Biotechnology Co., Ltd of final concentration of 0.2 mM), blank control group is not added then.Meanwhile it will be pure
The zymoprotein changed is added into the vegetable aquaculture culture solution of experimental group, makes its final concentration of 1 μ g/ml, after adding tetracycline, in
Two periods of 0 d and 3 d are using the Fourth Ring cellulose content in LC-MS method detection vegetable tissue.The results show that experimental group when 0 d,
Tetracycline is not detected in negative control group and blank control group；Fourth Ring cellulose content is 153 in negative control group vegetable tissue when 3 d
Ng/g, and the content of tetracycline in the experimental group vegetable tissue of TDE zymoprotein has been added to be the 57 [ Fourth Rings compared with the control group ng/g
Cellulose content reduces 62.7% (P < 0.0001) ], tetracycline is not detected in blank control group；Show that TDE zymoprotein has external drop
Solve the activity of tetracycline.As a result see that (tetracycline is not detected in blank control group to Fig. 4, therefore only provides experimental group in figure and feminine gender is right
According to the testing result of group).
Above the embodiments of the present invention are described in detail, but the present invention is not limited to described embodiments.It is right
For those skilled in the art, in the case where not departing from the principle of the invention and spirit, these embodiments are carried out more
Kind change, modification, replacement and modification, still fall in protection scope of the present invention.
Sequence table
<110>Guangdong provincial experimental middle school
<120>a kind of zymoprotein of the gene and its coding of tetracycline antibiotics of degrading
<141> 2019-06-21
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<170> SIPOSequenceListing 1.0
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gacgtttcag tttacgaaag agacaacgac cgagaggcaa gaatttttgg tggaaccctt 180
gacctacaca aaggttcagg tcaggaagca atgaaaaaag cgggattgtt acaaacttat 240
tatgacttag ccttaccaat gggtgtaaat attgctgatg aaaaaggcaa tattttatcc 300
acaaaaaatg taaagcccga aaatcgattt gacaatcctg aaataaacag aaatgactta 360
agggctatct tgttgaatag tttagaaaac gacacggtta tttgggatag aaaacttgtt 420
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gcagatttgg ttattcttgc caatggtgga atgtcgaaaa taaggagctt tgttaccgac 540
acgcaagttg aagaaaccgg tactttcaac atccaagctg atattcttca accggaaata 600
aactgtcccg gattttttca gctatgcaac ggcaaccgat taatggcggg acatcagggc 660
attttattgt ttgccaatcc caataataat ggtgcattgt atttaggaat tagttttaaa 720
acgcccgatg aatggaaaaa taaaattccc ttagattttc aggacagaaa cagcgttgcc 780
gattttttat tgaaaagatt ttccaaatgg agtgaagttt acaaacaatt aatacgttcg 840
gtatcaacat ttcaatgctt gcccacaagg aaatttcctt tgaacaatga ttggaaaagt 900
aaccgtccat tacccataac aatgattggc gatgctgctc atttgatgcc tccttttgca 960
ggacaaggcg taaacagtgg gttgatggat gccttgatat tgtcggataa tctgaccaat 1020
gggaaattta acagcattga agaggctatt gaaaattatg aacagcaaat gtttgcttat 1080
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1 5 10 15
Lys Asn Val Ala Ile Ile Gly Gly Gly Pro Val Gly Leu Thr Met Ala
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Lys Leu Leu Gln Gln Asn Gly Ile Asp Val Ser Val Tyr Glu Arg Asp
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Asn Asp Arg Glu Ala Arg Ile Phe Gly Gly Thr Leu Asp Leu His Lys
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Tyr Asp Leu Ala Leu Pro Met Gly Val Asn Ile Ala Asp Glu Lys Gly
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Asn Ile Leu Ser Thr Lys Asn Val Lys Pro Glu Asn Arg Phe Asp Asn
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Pro Glu Ile Asn Arg Asn Asp Leu Arg Ala Ile Leu Leu Asn Ser Leu
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Glu Asn Asp Thr Val Ile Trp Asp Arg Lys Leu Val Met Leu Glu Pro
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Gly Lys Lys Lys Trp Thr Leu Thr Phe Glu Asn Lys Pro Ser Glu Thr
145 150 155 160
Ala Asp Leu Val Ile Leu Ala Asn Gly Gly Met Ser Lys Ile Arg Ser
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Phe Val Thr Asp Thr Gln Val Glu Glu Thr Gly Thr Phe Asn Ile Gln
180 185 190
Ala Asp Ile Leu Gln Pro Glu Ile Asn Cys Pro Gly Phe Phe Gln Leu
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Cys Asn Gly Asn Arg Leu Met Ala Gly His Gln Gly Ile Leu Leu Phe
210 215 220
Ala Asn Pro Asn Asn Asn Gly Ala Leu Tyr Leu Gly Ile Ser Phe Lys
225 230 235 240
Thr Pro Asp Glu Trp Lys Asn Lys Ile Pro Leu Asp Phe Gln Asp Arg
245 250 255
Asn Ser Val Ala Asp Phe Leu Leu Lys Arg Phe Ser Lys Trp Ser Glu
260 265 270
Val Tyr Lys Gln Leu Ile Arg Ser Val Ser Thr Phe Gln Cys Leu Pro
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Thr Arg Lys Phe Pro Leu Asn Asn Asp Trp Lys Ser Asn Arg Pro Leu
290 295 300
Pro Ile Thr Met Ile Gly Asp Ala Ala His Leu Met Pro Pro Phe Ala
305 310 315 320
Gly Gln Gly Val Asn Ser Gly Leu Met Asp Ala Leu Ile Leu Ser Asp
325 330 335
Asn Leu Thr Asn Gly Lys Phe Asn Ser Ile Glu Glu Ala Ile Glu Asn
340 345 350
Tyr Glu Gln Gln Met Phe Ala Tyr Gly Arg Glu Ala Gln Ala Glu Ser
355 360 365
Ile Ile Asn Glu Thr Glu Met Phe Ser Leu Asp Phe Ser Phe Gln Lys
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Leu Met Asn Leu
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aggaggatta atactttata acagatttaa tggcatcatt agattttgaa agatttatcg 180
cagtaggata ggcatgcgta agattagtta gttggtgagg taacggctca ccaagacgat 240
gatctttagg gggcctgaga gggtgaaccc ccacactggt actgagacac ggaccagact 300
cctacgggag gcagcagtga ggaatattgg acaatgggtg gaagcctgat ccagccatcc 360
cgcgtgtagg atgacggcct tatgggttgt aaactacttt tatctgggga taaacctact 420
tacgtgtaag tagctgaagg taccagaaga ataagcaccg gctaactccg tgccagcagc 480
cgcggtaata cggagggtgc aagcgttatc cggatttatt gggtttaaag ggtccgtagg 540
cggattaatc agtcagtggt gaaatctcat agcttaacta tgaaactgcc attgatactg 600
ttagtcttga gtgatgttga agttgctgga atgtgtagtg tagcggtgaa atgcttagat 660
attacgcaga acaccaattg cgaaggcagg tgactaaaca ttaactgacg ctgatggacg 720
aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatggata 780
cttgctgttg gattttcgga ttcagtggct aagcgaaagt tataagtatc ccacctgggg 840
agtacgttcg caagaatgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgatgat acgcgaggaa ccttaccaag gcttaaatgc aatttgacag 960
aactagaaat agttttttct tcggacagaa tgcaaggtgc tgcatggctg tcgtcagctc 1020
gtgccgtgag gtgttaggtt aagtcctgca acgagcgcaa cccctatcat tagttgccag 1080
cgtttaaaga cggggactct aatgagactg ccggtgcaaa ccgcgaggaa ggtggggacg 1140
acgtcaagtc atcacggccc ttacgtcttg ggctacacac gtgctacaat ggtaagtaca 1200
gagggcagct acttggcaac aagatgcgaa tctcaaaaac ttatctcagt tcggattgga 1260
gtctgcaact cgactctatg aagctggaat cgctagtaat cgcatatcag ccatgatgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaag ccatggaagc tgggggtacc 1380
tgaagtcggt gaccgtaata ggagctgcct aggctagacc cgtcg 1425
Claims (8)
1. a kind of gene for tetracycline antibiotics of degrading, it is characterised in that: the gene has as shown in SEQ ID NO:1
Nucleotide sequence.
2. the primer of the overall length of gene described in pair for amplification claim 1 or its any segment, it is characterised in that: the primer
Sequence is as shown in SEQ ID NO:2 and SEQ ID NO:3.
3. a kind of zymoprotein of the coding of the gene as described in claim 1, it is characterised in that: the zymoprotein has such as SEQ ID
Amino acid sequence shown in NO:4.
4. a kind of recombinant vector containing gene described in claim 1, expression cassette, transgenic cell line or recombinant bacterium.
5. application of the gene as described in claim 1 in the tetracycline antibiotics in degradation environment.
6. application of the gene according to claim 5 in the tetracycline antibiotics in degradation environment, it is characterised in that:
The tetracycline antibiotics are tetracycline.
7. application of the zymoprotein as claimed in claim 3 in the tetracycline antibiotics in degradation environment.
8. application of the zymoprotein according to claim 7 in the tetracycline antibiotics in degradation environment, feature exist
In: the tetracycline antibiotics are tetracycline.
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| CN201910543175 | 2019-06-21 | ||
| CN201910543175X | 2019-06-21 |
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| CN110305882A true CN110305882A (en) | 2019-10-08 |
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| CN201910614364.1A Withdrawn CN110305882A (en) | 2019-06-21 | 2019-07-09 | A kind of zymoprotein of the gene and its coding of tetracycline antibiotics of degrading |
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Cited By (5)
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| CN111172180A (en) * | 2020-01-06 | 2020-05-19 | 华南农业大学 | Optimization of tetracycline antibiotic degradation gene tet (X) and application thereof in eukaryotic expression system |
| CN113005211A (en) * | 2019-12-20 | 2021-06-22 | 中国农业大学 | LAMP primer and method for detecting tigecycline high-level drug resistance gene tet (X) and variant thereof |
| CN113832117A (en) * | 2021-09-23 | 2021-12-24 | 盐城师范学院 | Enzyme for degrading oxytetracycline, and coding gene and application thereof |
| CN115536158A (en) * | 2022-09-21 | 2022-12-30 | 华南农业大学 | Complex enzyme and application of complex enzyme in tetracycline degradation |
| CN115536158B (en) * | 2022-09-21 | 2024-05-10 | 华南农业大学 | Complex enzyme and application thereof in degradation of tetracycline |
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| CN115536158A (en) * | 2022-09-21 | 2022-12-30 | 华南农业大学 | Complex enzyme and application of complex enzyme in tetracycline degradation |
| CN115536158B (en) * | 2022-09-21 | 2024-05-10 | 华南农业大学 | Complex enzyme and application thereof in degradation of tetracycline |
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