CN103044526B - Antigenic mimic epitope of vardenafil and application thereof - Google Patents
Antigenic mimic epitope of vardenafil and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and relates to an antigenic mimic epitope of vardenafil. The amino acid sequence of the antigenic mimic epitope is CKFPSHPYC. The antigenic mimic epitope of the vardenafil, provided by the invention, can replace expensive vardenafil non-standard products, can be applied to the vardenafil immunological detection as a competitive antigen or solid-phase envelope antigen, has similar immune response characteristics to natural vardenafil non-molecular, can reduce the harm to human health due to vardenafil and save the cost, and has an excellent effect and very high application value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to Vardenafil antigenic epitope and application thereof.
Background technology
Vardenafil (vardenafil, vard) is a kind of phosphorus phosphodiesterase 5 inhibitor of being produced by Bayer A.G (PDE-5 inhibitors), is used for the treatment of male penis erection dysfunction.Natural establishing-Yang medicine is considered to safely, has no side effect, looked at by human consumer's parent all over the world, and in natural medea illegal interpolation PDE-5 inhibitor medicaments, can produce series of side effects to public health, as headache, flush, maldigestion, blurred vision and sore muscle.PDE-5 inhibitor and nitrate drug interaction can cause serious ypotension.Therefore, strengthen in natural Yang invigorating medicine the detection of the illegal PDE-5 of interpolation inhibitor significant to the mankind's health.
At present, the detection method of the Vardenafil of the illegal interpolation of examination comprises LC-MS, GC-MS and LC/ESI-MS/MS, although aforesaid method is sensitive, accurate, reliable, required equipment costliness and working cost are high, in being difficult to work in unit of primary level, carries out on a large scale.Immunological detection method has sensitive, special, quick and cheap feature, has been widely used in the rapid detection of Food and environment sample.But the method must be used vard standard substance, not only increase testing cost, and testing staff and environment have easily been worked the mischief, restrict to a certain extent application and the popularization of immunological method.
In view of this, people start to adopt antiidiotypic antibody and antigenic epitope technology to realize substituting of harmful small-molecule substance standard substance, and make some progress.The principal feature of phage display peptide library technology is can effectively filter out and the phage-displayed polypeptides of target target body specific combination, this technology exploring the binding site that interacts between acceptor and part, seek the bioactive ligand molecular of high-affinity, the aspect such as development of exploring agnoprotein matter space structure epi-position, new generation vaccine is widely used.Display technique of bacteriophage provides technical support for obtaining the substitute of vard.
The present invention is by using phage display peptide library technology, from peptide storehouse, filter out can with the polypeptide (antigenic epitope) of target molecule (anti-Vardenafil monoclonal antibody) specific binding, this antigenic epitope has the immune response characteristic with natural Vardenafil molecular mimicry, by the Vardenafil antigenic epitope obtaining, to replace the Vardenafil standard substance of expensive and strong toxicity, and be applied to the immunology detection of Vardenafil as competition antigen or solid-phase coating antigen.
Summary of the invention
The present invention, taking anti-Vardenafil monoclonal antibody as target molecule, on enzyme plate, drops into target molecule solid-phase coating phage random and shows ring seven peptide storehouse, carries out affine elutriation, has obtained the antigenic epitope of a kind of Vardenafil, and its aminoacid sequence is as follows:
Vardenafil antigenic epitope: the Vardenafil antigenic epitope (polypeptide) screening from phage random ring seven peptide storehouse, its aminoacid sequence is: CKFPSHPYC.
In above-mentioned antigenic epitope (polypeptide) structure, capitalization English letter represents respectively the one of 21 kinds of known natural L-type amino-acid residues or its D-type isomer, be that C represents cysteine residues, K represents lysine residue, F represents phenylalanine residue, and P represents proline residue, and S represents serine residue, H represents histidine residues, and Y represents tyrosine residues.Two cysteine residues of the N-terminal of antigenic epitope and C-terminal form intramolecular disulfide bond, are ring texture.
The nucleotide sequence that the invention still further relates to the above-mentioned antigenic epitope aminoacid sequence of coding, is preferably AAG TTT CCG TCG CAT CCT TAT.
The Vardenafil antigenic epitope (polypeptide) that the present invention mentions can be prepared in a large number by the recombinant expressed mode of phage amplification, chemosynthesis or genetically engineered.Phage amplification refers to the phage that displaying is had to Vardenafil antigenic epitope (polypeptide), the mode increasing by biology, and amount reproduction is produced and is shown the bacteriophage particles that has Vardenafil antigenic epitope (polypeptide).Chemosynthesis refers to the aminoacid sequence according to the mimic epitopes of announcing, and carries out polypeptide synthesize by the mode of chemically synthesized polypeptide.The recombinant expressed mode of genetically engineered refers to the gene of coding simulation epi-position, by being cloned into expression vector, carries out a large amount of preparations of Vardenafil antigenic epitope with the form of polypeptide-fusion rotein.
The invention still further relates to the application of described Vardenafil antigenic epitope in immunology detection is analyzed.The type of immunology detection comprises the immune analysis type of detection based on Ag-Ab specific reaction such as Enzyme-linked Immunosorbent Assay detection, colloidal gold immunochromatographimethod, immunodotting hybridization.
Vardenafil antigenic epitope of the present invention (CKFPSHPYC) is in the time of application, can be synthetic mimic epitopes for immunology detection analysis, there is the bacteriophage particles of Vardenafil antigenic epitope (polypeptide) to be directly used in analyzing and testing the displaying obtaining of increasing by phage, certainly, also Vardenafil antigenic epitope can be scaled off and replaces Vardenafil standard substance to carry out immunology detection analysis from phage.
Also relate to the application in immunology detection is analyzed with solid phase antigen or competition antigen of Vardenafil antigenic epitope.
Also relate to Vardenafil antigenic epitope and detect the application in analyzing as solid phase antigen at colloidal gold immunochromatographimethod.
Aforementioned Vardenafil antigenic epitope can be replaced expensive Vardenafil standard substance, and be applied to the immunology detection of Vardenafil as competition antigen or solid-phase coating antigen, this antigenic epitope has the immune response characteristic with natural Vardenafil molecular mimicry, and effect is very good.
The invention has the beneficial effects as follows: Vardenafil antigenic epitope of the present invention can be replaced expensive Vardenafil standard substance, and be applied to the immunology detection of Vardenafil as competition antigen or solid-phase coating antigen, this antigenic epitope has the immune response characteristic with natural Vardenafil molecular mimicry, and effect is very good.Reduce the harm of Vardenafil to HUMAN HEALTH, saved cost, there is very high using value.
Brief description of the drawings
The indirect competitive ELISA typical curve that Fig. 1 sets up with Vardenafil antigenic epitope.Linear detection range is (2.4-32) ng/mL, IC
50be 8.8 ng/mL.
Embodiment
Affine elutriation and the qualification thereof of embodiment 1. Vardenafil antigenic epitopes
1) the affine elutriation of Vardenafil antigenic epitope: concrete grammar is: dilute anti-Vardenafil monoclonal antibody with 10 mM PBS (pH 7.4), and with the coated 96 hole enzyme plates of final concentration 100 μ g/mL, 4 DEG C of overnight incubation.Within second day, wash after 10 times with TBST (50 mM NaCl, pH 7.5 comprises 0.1% Tween-20 (v/v)), add 4 DEG C of 300 μ l confining liquids (3% BSA-PBS) to hatch 2 hours.After 2 hours, abandon confining liquid, with TBST washing 5 times, every hole adds 100 μ l phage peptide libraries, and (phage display ring seven peptide storehouse or dodecapeptide storehouse, purchased from NEB company, with 10 times of dilution phage stostes of TBS, approximately 1.0 × 10
11pfu), 22-26 DEG C of oscillatory reaction 1 hour.Discard unconjugated phage, with TBST washing 10 times, in conjunction with 0.2 M Glycine-HCl (pH 2.2) wash-out for upper phage, and use immediately 15 μ l 1 M Tris-HCl (pH 9.1) neutralizations.Get 10 μ l wash-out bacteriophages and survey titres, remaining grows to logarithm early stage for infecting 20 mL
e. colieR2738 bacterial strain increases.The 3rd day with PEG/NaCl deposition and purification phage, and measures the titre of phage after amplification.
In the elutriation process of taking turns at second, third, coated anti-Vardenafil monoclonal anti bulk concentration is respectively 75 μ g/mL and 50 μ g/mL, and TBST concentration used is 0.25% and 0.5%, and all the other steps are the same.
2) qualification of positive phage clones: measure random 20 phage spots of picking in the flat board of phage titre from third round elutriation, carry out the amplification of phage, adopt indirect enzyme-linked immunosorbent absorption detection method (Indirect Enzyme Linked immunoasorbent assay, I-ELISA) carry out the qualification of positive phage clones, concrete grammar is: first, dilute anti-Vardenafil monoclonal antibody with 10 mM PBS (pH 7.4), 10 μ g/mL are coated with 96 hole enzyme plates, 4 DEG C of overnight incubation.Within second day, wash after 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), use the PBS that contains 3% skim-milk to seal, hatch 1 hour for 37 DEG C; Drop into 100 μ l phage spot amplification liquid (1.0 × 10
11pfu),, using original phage peptide library as negative control, hatch 1 hour for 37 DEG C; The anti-100 μ l of the anti-M13 phage two of HRP mark that add 1:5000 doubly to dilute, hatch 1 hour for 37 DEG C; Add 100 μ l tmb substrate liquid, lucifuge colour developing 5min, microplate reader (Thermo Scientific Multiskan FC) reads the absorption value at 450 nm places.Choose OD
450be greater than the positive clone of phage clone of 2 times of negative controls.
3) qualification of Vardenafil antigenic epitope: adopt the method for indirect competitive ELISA to carry out the qualification of Vardenafil antigenic epitope, concrete grammar is: dilute anti-Vardenafil monoclonal antibody with 10 mM PBS (pH 7.4), 10 μ g/mL coated elisa plates, 4 DEG C of overnight incubation; Within second day, wash after 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), use the PBS that contains 3% skim-milk to seal, hatch 1 hour for 37 DEG C; Drop into 50 μ l and be accredited as positive phage clone (1.0 × 10 through indirect ELISA
11pfu) and 50 μ l Vardenafil standard substance (concentration range is 0-20 ng/ml), hatch 1 hour for 37 DEG C; Add the anti-100 μ l of anti-M13 phage two of 1:5000 dilution HRP mark, hatch 1 hour for 37 DEG C; Add 100 μ l tmb substrate liquid, lucifuge colour developing 5min, reads OD
450, can be in conjunction with anti-Vardenafil monoclonal antibody, and the phage that can be blocked by Vardenafil standard substance, be accredited as the antigenic epitope of Vardenafil.
Determining of the order-checking of embodiment 2. Vardenafil antigenic epitope encoding genes and aminoacid sequence thereof
By having the phage of Vardenafil antigenic epitope to increase through indirect competitive ELISA qualification displaying, extract the DNA sequencing template of phage.Concise and to the point process is as follows: carry out phage amplification, after the first step is centrifugal, 800 μ l are proceeded to a new centrifuge tube containing phage supernatant.Add 200 μ l PEG/NaCl precipitation phages.After centrifugal, precipitation is resuspended in to 100 μ l iodide damping fluid (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 μ l dehydrated alcohols precipitations DNA, precipitate (DNA sequencing template) by 70% washing with alcohol again after centrifugal.Precipitation is finally resuspended in 20 μ l aqua sterilisas, gets 2 μ l and carries out agarose gel electrophoresis analysis; Get 5 μ L phage templates and carry out DNA sequencing, its-96 gIII sequencing primer is: 5 '-
hOcCC TCA TAG TTA GCG TAA CG-3 '.Can obtain the aminoacid sequence CKFPSHPYC of Vardenafil antigenic epitope according to DNA sequencing result and password sublist.
Embodiment 3. Vardenafil antigenic epitopes are the application in ELISA as competition antigen
(1) sample extraction
Take 5g testing sample, add methyl alcohol-PBS solution of 25 ml 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1 ml filtrate and add 4 ml PBS(phosphate buffered saline buffers, pH=7.2)
After mixing, be sample extracting solution, stand-by.
(2) coated and sealing
Dilute anti-Vardenafil monoclonal antibody, 10 μ g/ml coated elisa plates, 4 DEG C of overnight incubation with 10 mM PBS (pH 7.4).Second day with after PBST (10 mM PBS, 0.05% Tween-20 (v/v)) washing 3 times, seals with the PBS that contains 3% skim-milk, hatches after 1 hour for 37 DEG C, washes plate 6 times stand-by with PBST.
(3) foundation of typical curve
Take out the lath of handling well through step (2), every hole is dropped into respectively 50 μ l and is shown the phage (1.0 × 10 that has Vardenafil antigenic epitope
11pfu) and 50 μ l Vardenafil standard substance of a series of different concns, hatch 1 hour for 37 DEG C.Add the anti-M13 phage two of 1:5000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.Taking Vardenafil concentration logarithm as X-coordinate, combination rate (adds the OD in the hole of Vardenafil
450/ do not add the OD in the hole of Vardenafil
450× 100%) be ordinate zou, set up indirect competition typical curve (Fig. 1).
(4) detection of sample
Take out the lath of handling well through step (2), every hole is dropped into respectively 50 μ l and is shown the phage (1.0 × 10 that has Vardenafil antigenic epitope
11pfu) and testing sample extracting solution, hatch 1 hour for 37 DEG C.Add the anti-M13 phage two of 1:5000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450, calculations incorporated rate, and according to typical curve, the content of Vardenafil in the sample of retrodicting out.
The application in ELISA as solid phase antigen of embodiment 4. Vardenafil antigenic epitopes
(1) sample extraction
Take 5g sample, add methyl alcohol-PBS solution of 25 ml 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1ml filtrate and add 4ml PBS(phosphate buffered saline buffer, pH=7.2)
After mixing, be sample extracting solution, stand-by.
(2) coated and sealing
There is the phage (2.0 × 10 of Vardenafil antigenic epitope with 10 mM PBS (pH 7.4) dilution displaying
11pfu), 100 μ l are coated in enzyme plate, 4 DEG C of overnight incubation.Second day with after PBST (10 mM PBS, 0.05% Tween-20 (v/v)) washing 3 times, seals with the PBS that contains 3% skim-milk, hatches after 1 hour for 37 DEG C, washes plate 6 times stand-by with PBST.
(3) foundation of typical curve
Take out the lath of handling well through step (2), 50 μ l Vardenafil standard substance of the anti-Vardenafil monoclonal antibody of 50 μ l (0.5 ng/ml) and a series of different concns are dropped into respectively in every hole, hatch 1 hour for 37 DEG C.Add the sheep anti-mouse igg two of 1:2000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.Taking Vardenafil concentration logarithm as X-coordinate, combination rate (adds the OD in the hole of Vardenafil
450/ do not add the OD in the hole of Vardenafil
450× 100%) be ordinate zou, set up indirect competition typical curve.
(4) detection of sample
Take out the lath of handling well through step (2), 50 μ l Vardenafil standard substance of the anti-Vardenafil monoclonal antibody of 50 μ l (0.5 ng/ml) and a series of different concns are dropped into respectively in every hole, hatch 1 hour for 37 DEG C.Add the sheep anti-mouse igg two of 1:2000 dilution HRP mark anti-, hatch 1 hour for 37 DEG C.Then with tmb substrate colour developing, read OD
450.Taking Vardenafil concentration logarithm as X-coordinate, combination rate (adds the OD in the hole of Vardenafil
450/ do not add the OD in the hole of Vardenafil
450× 100%) be ordinate zou, set up indirect competition typical curve
The application in highly-pathogenic avian influenza as solid phase antigen of embodiment 5. Vardenafil antigenic epitopes
(1) sample extraction
Take 5g sample, add methyl alcohol-PBS solution of 25 ml 60 %, 200 rpm vibrations 5 minutes; After extracting solution is filtered with No. 1 filter paper of whatman, get 1 ml filtrate and add 4 milliliters of PBS(phosphate buffered saline buffers, pH=7.2)
After mixing, be sample extracting solution, stand-by.
(2) dot matrix of phage and control line
There is the phage (2.0 × 10 of Vardenafil antigenic epitope with 10 mM PBS (pH 7.4) dilution displaying
11pfu), phage is lined to (aperture 0.2-0.45 micron) on nitrocellulose filter with dot matrix instrument or micropipet, as detection line; By anti-the sheep anti-mouse igg of the HRP mark of 0.5 mg/ml two, line on same nitrocellulose filter and (be positioned at the top of detection line, apart from being greater than 5 millimeters) with dot matrix instrument or micropipette, as control line.
(3) colloid gold label Vardenafil antibody
Vardenafil antibody is dropwise added in colloidal gold solution (pH=8.2), stir while dripping, after 30 minutes, getting 1% PEG adds in above-mentioned solution, continue to stir the 10% BSA solution that adds 1/10th volumes after 15 minutes, stir after 15 minutes, leave standstill 30 minutes, after centrifugal, remove supernatant, obtain the Vardenafil antibody-solutions of colloid gold label.
(4) assembling of colloidal-gold detecting-card
The Vardenafil antibody point of colloid gold label is sprayed on to glue gold pad upper (1.0 ug/ml), by sample pad, glue gold pad, dot matrix nitrocellulose filter and the blotter of detection line and control line assemble, be cut into test strip, pack in test card stand-by.
(5) detection of sample
Sample extracting solution is added in sample pad, leave standstill 10 minutes, if contain Vardenafil in sample and exceed the detection threshold of colloidal gold test, do not develop the color in detection line region, and the colour developing of control line region; If do not contain Vardenafil and the detection threshold lower than colloidal gold test in sample, detection line region colour developing, also develop the color in control line region.If do not develop the color in control line region, show that test strip lost efficacy.
A large amount of preparations of embodiment 5 Vardenafil antigenic epitopes
(1) mode increasing with phage
Having the phage of Vardenafil antigenic epitope to be added to 20 ml inoculations displaying has in the culture of ER 2738,37 degree 220 rpm shaking culture 4.5 h.Culture is proceeded in another centrifuge tube, and 4 DEG C of 10000 centrifugal 10 min of rpm, proceeds to the top of supernatant 80 % in one fresh tube, adds the PEG/NaCl of 1/6 volume, leaves standstill 120 min at 4 DEG C.4 DEG C of 10000 centrifugal PEG/NaCL of rpm leaves standstill solution 15 min.Abandon supernatant, of short durationly suck residual supernatant liquor after centrifugal.Add 1mL TBS to carry out resuspended, be phage amplification liquid.
(2) be prepared in the mode of Vardenafil antigenic epitope-fusion rotein
A. the external source encoding gene of pcr amplification Vardenafil antigenic epitope
PCR reaction system: (50 μ L)
10 × Pyrobest Buffer (Mg2+ plus) 5 μL
dNTP Mixture (each for 2.5 mM) 4 μL
M13KE insert extension primer (10 mM） 1 μL
-96 gIII sequencing primer (10 mM) 1 μL
Phage DNA template 1 μ L
Pyrobest DNA Polymerase 0.5 μL
Sterilizing ddH2O 37.5 μ L
PCR reaction conditions:
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.
Adopt PCR product to reclaim test kit purifying PCR product, trace dna quantitative instrument is quantitative.The coding gene sequence of Vardenafil antigenic epitope is AAG TTT CCG TCG CAT CCT TAT.
B. the double digestion of external source encoding gene and expression vector
Adopt respectively the external source code gene of ACC65I and Eag I enzyme and expression vector (MBP fusion rotein can be expressed by pMAl-pIII, NEB company) to carry out double digestion.
C. enzyme is cut connection and the conversion of after product
By plasmid pMal-PIII and object fragment with 1: 10(mol ratio) mix, connect 12 h in 16 DEG C of water-baths, get 10 μ L and connect products and add in 100 μ L competent cell TB1, fully mix.After ice bath 30 min, 42 DEG C of water-bath heat shock 90 s, after ice bath 5 min, add immediately 600 μ L LB liquid mediums, 37 DEG C, 200 rpm cultivate 1 h, centrifugal 2 min of 10000 rpm, suck supernatant and leave and take approximately 200 μ L, coat in LB-A solid (Ampr) substratum, 37 DEG C of incubated overnight, obtain positive colony.
The expression of Vardenafil antigenic epitope-MBP fusion rotein
By positive colony of above-mentioned acquisition, choose a single colony inoculation in 5 mL LB-A from flat board, in 0.2% sucrose, 37 DEG C, 220 r/min, shaking culture is spent the night, overnight culture is inoculated in to the LB-A of 50 mL by 1 % inoculum size (v/v), in 0.2 % sucrose medium, inoculate respectively 3 bottles, 37 DEG C, 220 r/min shaking culture, in the time that culture bacterial concentration OD600 reaches 0.6, to add in three bottles of cultures IPTG to final concentration be 0.2 mmol/L, 220 r/min shaking culture, by inductor (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, under room temperature, shake 5-10 min, 8000 g, 4 DEG C, centrifugal 20 min, abandon supernatant, the 5 mM MgSO4 that precipitation is resuspended in 400 ml precoolings, shake 10 min on ice, 8000 g, 4 DEG C, centrifugal 20 min, retain supernatant, in supernatant liquor, add 8 mL 1 M Tris-HCl, pH 7.4, obtains Vardenafil antigenic epitope-MBP fusion rotein.
SEQUENCE LISTING
<110> University Of Nanchang
The antigenic epitope of <120> Vardenafil and application thereof
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> artificial sequence
<400> 1
Cys Lys Phe Pro Ser His Pro Tyr Cys
1 5
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
aagtttccgt cgcatcctta t 21
Claims (6)
1. the antigenic epitope of Vardenafil, is characterized in that aminoacid sequence is CKFPSHPYC.
2. the nucleotide sequence of antigenic epitope aminoacid sequence described in coding claim 1.
3. the preparation method of antigenic epitope as claimed in claim 1, is characterized in that preparing in a large number by the recombinant expressed mode of phage amplification, chemosynthesis or genetically engineered; Described phage amplification refers to the phage that displaying is had to Vardenafil antigenic epitope, the mode increasing by biology, and amount reproduction is produced and is shown the bacteriophage particles that has Vardenafil antigenic epitope; Described chemosynthesis refers to according to mimic epitopes mimic epitopes aminoacid sequence, carries out polypeptide synthesize by the mode of chemically synthesized polypeptide; The recombinant expressed mode of described genetically engineered refers to the gene of coding simulation epi-position, by being cloned into expression vector, carries out a large amount of preparations of Vardenafil antigenic epitope with the form of polypeptide-fusion rotein.
4. the application of antigenic epitope in immunology detection is analyzed described in claim 1.
5. application as claimed in claim 4, is characterized in that the application in immunology detection is analyzed with solid phase antigen or competition antigen of Vardenafil antigenic epitope.
6. as claimed in claim 4 apply, it is characterized in that Vardenafil antigenic epitope detects the application in analyzing as solid phase antigen at colloidal gold immunochromatographimethod.
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| CN101555512A (en) * | 2009-04-16 | 2009-10-14 | 南昌大学 | Preparation method for monoclonal antibody specifically binding vardenafil and analog thereof |
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