CN104119425B - The antigenic epitope of simulation norfloxacin and application thereof - Google Patents
The antigenic epitope of simulation norfloxacin and application thereof Download PDFInfo
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Abstract
The invention belongs to biological technical field, relate to the antigenic epitope of norfloxacin, its aminoacid sequence is DHAYWPKFWGKA.Norfloxacin antigenic epitope of the present invention can replace the expensive and norfloxacin standard substance of strong toxicity, and the immunology detection of norfloxacin it is applied to as competition antigen or solid-phase coating antigen, this antigenic epitope has and the immunoreation characteristic of natural norfloxacin molecular mimicry, and effect is the best.Decrease the norfloxacin harm to health, saved cost, there is the highest using value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to norfloxacin antigenic epitope and application thereof.
Background technology
Norfloxacin (Norfloxacin, NOR) is a kind of broad ectrum antibiotic, can absorb moisture in atmosphere, meet photochromic
Gradual change is deep, and the most molten in dimethylformamide, soluble,very slightly in water or ethanol, in acetic acid, hydrochloric acid or sodium hydroxide solution
Readily soluble, it is high to the antibacterial activity of aerobic gram negative bacilli, and most antibacterials are had good antibacterial action in vitro, such as enterobacteria
Most of antibacterial of section, including bacillus citrate genus, enterobacter cloacae, clostridium perfringen etc. Enterobacter, escherichia coli, gram
The primary Pseudomonas of thunder, proteus, Salmonella, Shigella, vibrio, yersinia etc..Norfloxacin is external to multidrug resistant
Bacterium also has antibacterial activity.Diplococcus gonorrhoeae, hemophilus influenza and the moraxelle catarrhalis of Penicillin-resistant is also had the most antibacterial
Effect.Norfloxacin is antibacterial, by acting on the A subunit of DNA of bacteria helicase, the suppression synthesis of DNA and duplication and
Cause bacterial death.Yet with the abuse of antibiotic, for being particularly important of detection of Determination of norfloxacin residue.
At present, in detection food, the method for norfloxacin mainly has high performance liquid chromatography, gas chromatogram, thin layer chromatography and exempts from
The methods such as epidemiology detection, the advantages such as immunological detection method is highly sensitive with it, easy to detect, with low cost are at norfloxacin
Detection is widely used.But, during setting up immunological detection method, it is necessary to use norfloxacin standard
Product are that competition antigen or solid-phase coating antigen prepared by raw material, and norfloxacin is the most expensive but also has extremely strong carcinogenic
Property, health and environment to testing staff cause threat greatly, thus constrain immunological detection method to a certain extent
Application and popularization.In view of this, people begin with anti-idiotype antibody and antigenic epitope technology realizes harmful little
The replacement of molecular substance standard substance, and make some progress.Being mainly characterized by of phage display peptide library technology can be effective
Filter out and the phage-displayed polypeptides of target target body specific bond, this technology is exploring the interphase interaction of receptor and part
Binding site, seek the bioactive ligand molecular of high-affinity, explore agnoprotein matter space structure epi-position, new generation vaccine
The aspects such as development are widely used.
The present invention is by using phage display peptide library technology, and filtering out from peptide storehouse can be with target molecule (anti-norfloxacin list
Clonal antibody) specific binding polypeptide (antigenic epitope), this antigenic epitope has and natural norfloxacin molecule
Similar immunoreation characteristic, by the norfloxacin antigenic epitope obtained, to replace the promise of expensive and strong toxicity
Flucloxacillin standard substance, and the immunology detection of norfloxacin it is applied to as competition antigen or solid-phase coating antigen.
Summary of the invention
The present invention is with anti-norfloxacin monoclonal antibody as target molecule, by target molecule solid-phase coating in ELISA Plate, respectively
Put into phage random and show ring seven, dodecapeptide storehouse, carry out affine elutriation respectively, it is thus achieved that the antigen mimicking of four kinds of norfloxacins
Epi-position (ring seven peptide and each a kind of dodecapeptide).Their aminoacid sequence is as follows:
Norfloxacin antigenic epitope 1: the norfloxacin antigen mimicking table of screening from phage random ring seven peptide storehouse
Position (polypeptide), its aminoacid sequence is: CKFGFEPFC.
Norfloxacin antigenic epitope 2: the norfloxacin antigen mimicking table of screening from phage random ring seven peptide storehouse
Position (polypeptide),
Its aminoacid sequence is: CNTARWPFC.
Norfloxacin antigenic epitope 3: the norfloxacin antigen mimicking table of screening from phage random dodecapeptide storehouse
Position (polypeptide),
Its aminoacid sequence is: DHAYWPKFWGKA.
Norfloxacin antigenic epitope 4: the norfloxacin antigen mimicking table of screening from phage random dodecapeptide storehouse
Position (polypeptide),
Its aminoacid sequence is: SRMGPENWDKWY.
The invention still further relates to encode the nucleotide sequence of above-mentioned antigenic epitope aminoacid sequence, preferably AAG TTT
GGG TTT GAG CCG TTT； GTT AAT ACG GCG AGG TGG CCG TTT；GAT CAT GCG TAT TGG CCG
AAG TTT TGG GGT AAG GCT；AGT CGG ATG GGT CCG GAG AAT TGG GAT AAG TGG TAT.
In above-mentioned antigenic epitope (polypeptide) structure, capitalization English letter represents 21 kinds of known natural L-form respectively
Amino acid residue or the one of its D-type isomer, i.e. C represents cysteine residues, and D represents asparagicacid residue, and P represents dried meat
Histidine residue, R represents arginine residues, and K represents lysine residue, and H represents histidine residues, and I represents isoleucine residues, V
Representing valine residue, Y represents tyrosine residue, and S represents serine residue, and F represents phenylalanine residue, and E represents glutamic acid
Residue, M represents methionine residues.The N-terminal of mimic epitope 1 and the two of C-terminal cysteine residues form intramolecular two
Sulfide linkage, for circulus.
The norfloxacin antigenic epitope (polypeptide) that the present invention mentions can pass through Phage amplification, chemosynthesis or gene
The recombinant expressed mode of engineering is prepared in a large number.Phage amplification refers to that displaying has norfloxacin antigenic epitope (many
Peptide) phage, by the way of biology expands, amount reproduction produce displaying have norfloxacin antigenic epitope (polypeptide)
Bacteriophage particles.Chemosynthesis refers to according to the mimic epitope 1 announced, mimic epitope 2, mimic epitope 3, the ammonia of mimic epitope 4
Base acid sequence, carries out Peptide systhesis by the way of chemically synthesized polypeptide.The recombinant expressed mode of genetic engineering refers to encode
Mimic epitope 1, mimic epitope 2, mimic epitope 3 or the gene of mimic epitope 4, by being cloned into expression vector, with polypeptide-fusion
The form of albumen carries out a large amount of preparations of norfloxacin antigenic epitope.
The invention still further relates to the application in immunology detection is analyzed of the described norfloxacin antigenic epitope.Immunology is examined
The type surveyed includes that MBP enzyme linked immuno-adsorbent assay, colloidal gold immunochromatographimethod, immunodotting hybridization etc. are based on Ag-Ab specificity
The immune analysis detection type of reaction.
Norfloxacin antigenic epitope of the present invention (CKFGFEPFC, CNTARWPFC, DHAYWPKFWGKA,
SRMGPENWDKWY) in use, Phage amplification can will be passed through the mimic epitope of synthesis for immunology detection analysis
The displaying obtained has the bacteriophage particles of norfloxacin antigenic epitope (polypeptide) to be directly used in analysis detection, certainly, it is possible to
Norfloxacin standard substance are replaced to divide to carry out immunology detection to be scaled off from phage by norfloxacin antigenic epitope
Analysis.
Further relate to norfloxacin antigenic epitope and with solid phase antigen or compete antigen answering in immunology detection is analyzed
With.
Further relate to norfloxacin antigenic epitope as solid phase antigen answering in colloidal gold immunochromatographimethod detection is analyzed
With.
Aforementioned norfloxacin antigenic epitope can replace the expensive and norfloxacin standard substance of strong toxicity, and makees
Be applied to the immunology detection of norfloxacin for competition antigen or solid-phase coating antigen, this antigenic epitope has and sky promise
The immunoreation characteristic of Flucloxacillin molecular mimicry, effect is the best.
The invention has the beneficial effects as follows: norfloxacin antigenic epitope of the present invention can replace expensive and strong toxicity
Norfloxacin standard substance, and be applied to the immunology detection of norfloxacin as competition antigen or solid-phase coating antigen, this resists
Former mimic epitope has and the immunoreation characteristic of natural norfloxacin molecular mimicry, and effect is the best.Decrease norfloxacin
Harm to health, has saved cost, has the highest using value.
Accompanying drawing explanation
Fig. 1. the indirect competitive ELISA standard curve set up with norfloxacin antigenic epitope.(numbering c7-13,
C7-15,12-24,12-29 represent four kinds of antigenic epitopes of norfloxacin respectively, and its aminoacid sequence is respectively
CKFGFEPFC、CNTARWPFC、DHAYWPKFWGKA、SRMGPENWDKWY)
Detailed description of the invention
The affine elutriation of embodiment 1. norfloxacin antigenic epitope and qualification thereof
1) the affine elutriation of norfloxacin antigenic epitope: method particularly includes: dilute with 10 mM PBS (pH 7.4)
Anti-norfloxacin monoclonal antibody, and it is coated 96 hole ELISA Plate, 4 DEG C of overnight incubation with final concentration 100 μ g/mL.Second day use
After TBST (50 mM NaCl, pH 7.5 comprise 0.1% Tween-20 (v/v)) washs 10 times, add 300 μ l confining liquids
(3% BSA-PBS) 4 DEG C hatches 2 hours.Abandoning confining liquid after 2 hours, wash 5 times with TBST, every hole adds 100 μ l phage display peptides
Storehouse (phage display ring seven peptide storehouse or dodecapeptide storehouse, purchased from NEB company, dilute phage stock solution with TBS 10 times, about 1.0 ×
1011Pfu), 22-26 DEG C of oscillating reactions 1 hour.Discard unconjugated phage, with TBST wash 10 times, in conjunction with on phagocytosis
Body 0.2 M Glycine-HCl (pH 2.2) eluting, and neutralize with 15 μ l 1 M Tris-HCl (pH 9.1) immediately.
Taking 10 μ l wash-out bacteriophages and survey titre, remaining grows to logarithm early stage for infecting 20 mLE. coliER2738 bacterial strain
Expand.Within 3rd day, by PEG/NaCl deposition and purification phage, and measure the titre of phage after amplification.
In second, third panning process taken turns, coated anti-norfloxacin MAb concentration is respectively 75 μ g/
ML and 50 μ g/mL, TBST concentration used is 0.25% and 0.5%, and remaining step is ibid.
2) qualification of positive phage clones: measure random picking the flat board of phage titre after third round elutriation
20 phage speckles, carry out the amplification of phage, use Immunofluorescent antibody detection method (Indirect Enzyme
Linked immunoasorbent assay, I-ELISA) carry out the qualification of positive phage clones, method particularly includes: first
First, diluting anti-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), 10 g/mL are coated 96 hole ELISA Plate, incubate for 4 DEG C
Educate overnight.After second day washs 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), with containing 3% defat
The PBS of milk powder closes, and hatches 1 hour for 37 DEG C；Put into 100 l phage speckle amplification liquid (1.0 × 1011Pfu), with original
Phage peptide library, as negative control, hatches 1 hour for 37 DEG C；Add the 1:5000 times of HRP labelling anti-M13 phage two diluted
Anti-100 l, hatch 1 hour for 37 DEG C；Add 100 l tmb substrate liquid, lucifuge colour developing 5min, microplate reader (Thermo
Scientific Multiskan FC) read the absorption value at 450 nm.Choose OD450Phage more than negative control 2 times
Clone is positive colony.
3) qualification of norfloxacin antigenic epitope: use the method for indirect competitive ELISA to carry out norfloxacin antigen
The qualification of mimic epitope, method particularly includes: with 10 mM PBS (pH 7.4) dilute anti-norfloxacin monoclonal antibody, 10
G/mL coated elisa plate, 4 DEG C of overnight incubation；Within second day, wash with PBST (10 mM PBS, 0.05% Tween-20 (v/v))
After washing 3 times, close with the PBS containing 3% defatted milk powder, hatch 1 hour for 37 DEG C；Put into 50 l to identify through indirect ELISA
For positive phage clone (1.0 × 1011Pfu) and 50 l norfloxacin standard substance (concentration range is 0-20 ng/ml),
Hatch 1 hour for 37 DEG C；The anti-M13 phage two adding 1:5000 dilution HRP labelling resists 100 l, hatches 1 hour for 37 DEG C；Add
100 l tmb substrate liquid, lucifuge colour developing 5min, reads OD450, can be in conjunction with anti-norfloxacin monoclonal antibody, and can be husky by promise fluorine
The phage that star standard substance are blocked, is accredited as the antigenic epitope of norfloxacin.
The order-checking of embodiment 2. norfloxacin antigenic epitope encoding gene and the determination of aminoacid sequence thereof
By identifying that displaying has the phage of norfloxacin antigenic epitope to expand through indirect competitive ELISA, carry
Take the DNA sequencing template of phage.Simplified process is as follows: carry out Phage amplification, after the first step is centrifugal, by 800 l containing biting
Thalline supernatant proceeds to a new centrifuge tube.Add 200 l PEG/NaCl precipitating phage.After Li Xin, precipitation is resuspended in 100 l
Iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), adds 250 l dehydrated alcohol
Precipitation DNA, again by 70% washing with alcohol precipitation (DNA sequencing template) after being centrifuged.Precipitation is finally resuspended in 20 l aquesterilisa, takes 2
L carries out agarose gel electrophoresis analysis；Taking 5 L phage templates and carry out DNA sequencing, its-96 gIII sequencing primer is:
5’-HOCCC TCA TAG TTA GCG TAA CG-3’.Norfloxacin antigen can be obtained according to DNA sequencing result and password sublist
The aminoacid sequence of mimic epitope: CKFGFEPFC, CNTARWPFC, DHAYWPKFWGKA and SRMGPENWDKWY.
Embodiment 3. norfloxacin antigenic epitope is as competition antigen application in ELISA
(1) sample extraction
Weigh 5g sample (corn and relevant food thereof), add the methanol-PBS solution of 25 milliliter of 60 %, 200 rpm
Vibrate 5 minutes；After No. 1 filter paper of extracting solution whatman is filtered, take 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate
Buffer, pH=7.2)
After mixing, it is sample extracting solution, stand-by.
(2) it is coated and closes
With 10 mM PBS (pH 7.4) dilute anti-norfloxacin monoclonal antibody, 10 g/mL coated elisa plates, 4 DEG C
Overnight incubation.After second day washs 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), take off with containing 3%
The PBS of fat milk powder closes, and after 37 DEG C hatch 1 hour, washes plate 6 times with PBST stand-by.
(3) foundation of standard curve
Taking out the lath handled well through step (2), every hole puts into 50 l respectively and shows there is norfloxacin antigenic epitope
Phage (1.0 × 1011Pfu) and 50 l norfloxacin standard substance of a series of variable concentrations, 1 hour is hatched for 37 DEG C.Add
The anti-M13 phage two entering 1:5000 dilution HRP labelling resists, and hatches 1 hour for 37 DEG C.Then develop the color with tmb substrate, read
OD450.With blood concentration norfloxacin logarithm as abscissa, combination rate (adds the OD in the hole of norfloxacin450/ do not add norfloxacin
The OD in hole450× 100%) it is vertical coordinate, sets up indirect competition standard curve (Fig. 1).
(4) detection of sample
Taking out the lath handled well through step (2), every hole puts into 50 l respectively and shows there is norfloxacin antigenic epitope
Phage (1.0 × 1011Pfu) and testing sample extracting solution, 1 hour is hatched for 37 DEG C.Add 1:5000 dilution HRP labelling
Anti-M13 phage two resists, and hatches 1 hour for 37 DEG C.Then develop the color with tmb substrate, read OD450, calculations incorporated rate, and according to mark
Directrix curve, the content of norfloxacin in sample of retrodicting out.
Embodiment 4. norfloxacin antigenic epitope is as solid phase antigen application in ELISA
(1) sample extraction
Weigh 5g sample (corn and relevant food thereof), add the methanol-PBS solution of 25 milliliter of 60 %, 200 rpm
Vibrate 5 minutes；After No. 1 filter paper of extracting solution whatman is filtered, take 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate
Buffer, pH=7.2)
After mixing, it is sample extracting solution, stand-by.
(2) it is coated and closes
Show there is the phage (2.0 × 10 of norfloxacin antigenic epitope with 10 mM PBS (pH 7.4) dilution11
Pfu), 100 microlitres are coated in ELISA Plate, 4 DEG C of overnight incubation.Second day with PBST (10 mM PBS, 0.05% Tween-20
(v/v), after) washing 3 times, close with the PBS containing 3% defatted milk powder, after 37 DEG C hatch 1 hour, wash plate 6 times with PBST
Stand-by.
(3) foundation of standard curve
Taking out the lath handled well through step (2), every hole puts into 50 l anti-norfloxacin monoclonal antibodies (0.5 respectively
Ng/ml) and 50 l norfloxacin standard substance of a series of variable concentrations, 1 hour is hatched for 37 DEG C.Add 1:2000 and dilute HRP
The sheep anti-mouse igg two of labelling resists, and hatches 1 hour for 37 DEG C.Then develop the color with tmb substrate, read OD450.With blood concentration norfloxacin pair
Number is abscissa, and combination rate (adds the OD in the hole of norfloxacin450/ do not add the OD in hole of norfloxacin450× 100%) it is vertical
Coordinate, sets up indirect competition standard curve.
(4) detection of sample
Taking out the lath handled well through step (2), every hole puts into 50 l anti-norfloxacin monoclonal antibodies (0.5 respectively
Ng/ml) and 50 l norfloxacin standard substance of a series of variable concentrations, 1 hour is hatched for 37 DEG C.Add 1:2000 and dilute HRP
The sheep anti-mouse igg two of labelling resists, and hatches 1 hour for 37 DEG C.Then develop the color with tmb substrate, read OD450.With blood concentration norfloxacin pair
Number is abscissa, and combination rate (adds the OD in the hole of norfloxacin450/ do not add the OD in hole of norfloxacin450× 100%) it is vertical
Coordinate, sets up indirect competition standard curve
Embodiment 5. norfloxacin antigenic epitope is as solid phase antigen application in highly-pathogenic avian influenza
(1) sample extraction
Weigh 5g sample (corn and relevant food thereof), add the methanol-PBS solution of 25 milliliter of 60 %, 200 rpm
Vibrate 5 minutes；After No. 1 filter paper of extracting solution whatman is filtered, take 1 milliliter of filtrate and add 4 milliliters of PBS(phosphate
Buffer, pH=7.2)
After mixing, it is sample extracting solution, stand-by.
(2) phage and the dot matrix of control line
Show there is the phage (2.0 × 10 of norfloxacin antigenic epitope with 10 mM PBS (pH 7.4) dilution11
Pfu), with dot matrix instrument or micropipettor, phage is lined (aperture 0.2-0.45 micron) on nitrocellulose filter, as
Detection line；The sheep anti-mouse igg two of the HRP labelling of 0.5 mg/ml is resisted, lines same nitric acid with dot matrix instrument or micropipette
(it is positioned at the top of detection line, distance is more than 5 millimeters), as control line on cellulose membrane.
(3) colloid gold label norfloxacin antibody
Norfloxacin antibody is added dropwise in colloidal gold solution (pH=8.2), drips while stir, after 30 minutes, take 1%
PEG adds in above-mentioned solution, adds 10% BSA solution of 1/10th volumes, stir 15 minutes after continuing stirring 15 minutes
After, stand 30 minutes, remove supernatant after being centrifuged, obtain the norfloxacin antibody-solutions of colloid gold label.
(4) assembling of colloidal-gold detecting-card
The norfloxacin antibody point of colloid gold label is sprayed on (1.0 mcg/ml) on glue gold pad, by sample pad, glue gold
Pad, dot matrix detection line and the nitrocellulose filter of control line and absorption paper assemble, and are cut into test strips, load in detection card
Stand-by.
(5) detection of sample
Sample extracting solution is added in sample pad, stand 10 minutes, if sample containing norfloxacin and exceeding gold colloidal
The detection threshold value of Test paper, then do not develop the color in detection line region, and the colour developing of control line region；If sample does not contains norfloxacin
And less than the detection threshold value of colloidal gold test, then detection line region colour developing, also develops the color in control line region.If control line region
Do not develop the color, show that test strips lost efficacy.
A large amount of preparations of embodiment 5 norfloxacin antigenic epitope
(1) in the way of Phage amplification
The phage addition that displaying has norfloxacin antigenic epitope is inoculated with the culture of ER 2738 to 20 ml
In, 37 degree of 220 rpm shaken cultivation 4.5 h.Being proceeded to by culture in another centrifuge tube, 4 DEG C of 10000 rpm is centrifuged 10
Min, proceeds in a fresh tube by top 80 % of supernatant, adds the PEG/NaCl of 1/6 volume, stand 120 min at 4 DEG C.4
DEG C 10000 rpm are centrifuged PEG/NaCL and stand solution 15 min.Abandon supernatant, of short duration centrifugal after suck residual supernatant.Add
1mL TBS carries out resuspended, is Phage amplification liquid.
(2) it is prepared in the way of norfloxacin antigenic epitope-fusion protein
The external source encoding gene of A.PCR amplification norfloxacin antigenic epitope
PCR reaction system: (50 L)
10 × Pyrobest Buffer (Mg2+ plus) 5 µL
dNTP Mixture (each for 2.5 mM) 4µL
M13KE insert extension primer (10 mM) 1 L
-96 gIII sequencing primer (10 mM) 1µL
Phage DNA template 1 L
Pyrobest DNA Polymerase 0.5 µL
Sterilizing ddH2O 37.5 L
PCR reaction condition:
95℃ 5 min
Then 95 DEG C of 30 sec, 55 DEG C of 30 sec, 72 DEG C of 40sec, 72 DEG C of 10 min totally 30 cycles.
Using PCR product to reclaim kits PCR product, trace dna quantitative instrument is quantitative.Norfloxacin antigen
The coding gene sequence of mimic epitope 1 is AAG TTT GGG TTT GAG CCG TTT；Norfloxacin antigenic epitope 2
Coding gene sequence is: GTT AAT ACG GCG AGG TGG CCG TTT;The coding base of norfloxacin antigenic epitope 3
Because sequence is GAT CAT GCG TAT TGG CCG AAG TTT TGG GGT AAG GCT；Norfloxacin antigenic epitope 4
Coding gene sequence be: AGT CGG ATG GGT CCG GAG AAT TGG GAT AAG TGG TAT.
B. external source encoding gene and the double digestion of expression vector
Be respectively adopted ACC65I and Eag I enzyme external source code gene and expression vector (pMAl-pIII, NEB company,
MBP fusion protein can be expressed) carry out double digestion.
C. the connection of enzyme action afterproduct and conversion
By plasmid pMal-PIII and purpose fragment with 1: 10(mol ratio) mixing, connect 12 h in 16 DEG C of water-baths,
Take 10 μ L connection products to add to, in 100 μ L competent cell TB1, fully mix.After ice bath 30 min, 42 DEG C of water-bath heat shocks
90 s, add 600 μ L LB liquid mediums immediately after ice bath 5 min, 37 DEG C, 200 rpm cultivate 1 h, 10000 rpm from
The heart 2 min, sucks supernatant and leaves and takes about 200 μ L, coats in LB-A solid (Ampr) culture medium, and 37 DEG C of incubated overnight obtain
Positive colony.
The expression of norfloxacin antigenic epitope-MBP fusion protein
By the positive colony of above-mentioned acquisition, from flat board, choose a single colony inoculation in 5 mL LB-A, in 0.2% sucrose,
37 DEG C, 220 r/min, overnight culture overnight, is inoculated in the LB-A of 50 mL by shaken cultivation by 1 % inoculum concentration (v/v), and 0.2
In % sucrose medium, inoculation 3 bottles respectively, 37 DEG C, 220 r/min shaken cultivation, when culture bacterial concentration OD600 reaches 0.6
Time, in three bottles of cultures, adding IPTG to final concentration of 0.2 mmol/L, 220 r/min shaken cultivation, by inducer (PEG
Solution) in 4 DEG C, 4000 g, centrifugal 20 min collect bacterial sediment, abandon supernatant.Re-suspended cell is in 400 mL 30 mM Tris-
HCl, 20% sucrose, pH 8.0 (80 mL/g wet cell weight), add EDTA to 1 mM, concussion 5-10 min under room temperature, 8000
G, 4 DEG C, centrifugal 20 min, abandon supernatant, precipitation is resuspended in 5 mM MgSO4 of 400 ml pre-coolings, shakes 10 min on ice, and 8000
G, 4 DEG C, centrifugal 20 min, retain supernatant, in supernatant, add 8 mL 1 M Tris-HCl, pH 7.4, it is thus achieved that promise fluorine is husky
Star antigenic epitope-MBP fusion protein.
SEQUENCE LISTING
<110>University Of Nanchang
<120>antigenic epitope and the application thereof of norfloxacin are simulated
<130> 1
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213>artificial sequence
<400> 1
Cys Lys Phe Gly Phe Glu Pro Phe Cys
1 5
<210> 2
<211> 9
<212> PRT
<213>artificial sequence
<400> 2
Cys Asn Thr Ala Arg Trp Pro Phe Cys
1 5
<210> 3
<211> 12
<212> PRT
<213>artificial sequence
<400> 3
Asp His Ala Tyr Trp Pro Lys Phe Trp Gly Lys Ala
1 5 10
<210> 4
<211> 12
<212> PRT
<213>artificial sequence
<400> 4
Ser Arg Met Gly Pro Glu Asn Trp Asp Lys Trp Tyr
1 5 10
<210> 5
<211> 21
<212> DNA
<213>artificial sequence
<400> 5
aagtttgggt ttgagccgtt t 21
<210> 6
<211> 24
<212> DNA
<213>artificial sequence
<400> 6
gttaatacgg cgaggtggcc gttt 24
<210> 7
<211> 36
<212> DNA
<213>artificial sequence
<400> 7
gatcatgcgt attggccgaa gttttggggt aaggct 36
<210> 8
<211> 36
<212> DNA
<213>artificial sequence
<400> 8
agtcggatgg gtccggagaa ttgggataag tggtat 36
Claims (6)
1. simulate the antigenic epitope of norfloxacin, it is characterised in that aminoacid sequence is SRMGPENWDKWY.
2. the nucleotide of antigenic epitope aminoacid sequence described in coding claim 1.
3. nucleotide as claimed in claim 2, sequence is AGT CGG ATG GGT CCG GAG AAT TGG GAT AAG
TGG TAT。
4. the application in immunology detection is analyzed of the antigenic epitope described in claim 1.
Apply the most as claimed in claim 4, it is characterised in that norfloxacin antigenic epitope is with solid phase antigen or competition antigen
Application in immunology detection is analyzed.
Apply the most as claimed in claim 4, it is characterised in that norfloxacin antigenic epitope as solid phase antigen at gold colloidal
Application in immunochromatography detection analysis.
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| CN102212136A (en) * | 2011-03-25 | 2011-10-12 | 商务部流通产业促进中心 | ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof |
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|---|---|---|---|---|
| CN1861632A (en) * | 2006-03-14 | 2006-11-15 | 山东大学 | Coupling compound of Norfloxacin, preparation process and application thereof |
| CN101609073A (en) * | 2009-08-04 | 2009-12-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of Norfloxacin antibiotic residual quantity in a kind of milk |
| CN102212136A (en) * | 2011-03-25 | 2011-10-12 | 商务部流通产业促进中心 | ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof |
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