CN109293776A - A kind of Fipronil binding proteins specific and its application - Google Patents
A kind of Fipronil binding proteins specific and its application Download PDFInfo
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- CN109293776A CN109293776A CN201811341754.8A CN201811341754A CN109293776A CN 109293776 A CN109293776 A CN 109293776A CN 201811341754 A CN201811341754 A CN 201811341754A CN 109293776 A CN109293776 A CN 109293776A
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- Prior art keywords
- fipronil
- solution
- albumen
- added
- hapten
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- 239000005899 Fipronil Substances 0.000 title claims abstract description 118
- 229940013764 fipronil Drugs 0.000 title claims abstract description 118
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- 108091008324 binding proteins Proteins 0.000 title abstract description 9
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- 238000002965 ELISA Methods 0.000 claims description 21
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 16
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 12
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- 235000021240 caseins Nutrition 0.000 claims description 2
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- 231100000716 Acceptable daily intake Toxicity 0.000 description 3
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 235000005979 Citrus limon Nutrition 0.000 description 2
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- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 1
- XJFXZQKJQGYFMM-GUBZILKMSA-N Val-Cys-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)O)N XJFXZQKJQGYFMM-GUBZILKMSA-N 0.000 description 1
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- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
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- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
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- 244000000013 helminth Species 0.000 description 1
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- 210000003205 muscle Anatomy 0.000 description 1
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- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000008048 phenylpyrazoles Chemical class 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- HZRSNVGNWUDEFX-UHFFFAOYSA-N pyraclostrobin Chemical compound COC(=O)N(OC)C1=CC=CC=C1COC1=NN(C=2C=CC(Cl)=CC=2)C=C1 HZRSNVGNWUDEFX-UHFFFAOYSA-N 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The embodiment of the invention discloses a kind of Fipronil binding proteins specific and its application, the amino acid sequence of the albumen is as shown in SEQ ID NO.1 or the sequence is through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.The above-mentioned albumen of the present invention is easily prepared, and can specifically bind with Fipronil, specificity with higher and sensitivity；Furthermore it is possible to the detection for Fipronil in sample.
Description
Technical field
The present invention relates to Fipronil detection technique fields, and in particular to a kind of Fipronil binding proteins specific and its answers
With.
Background technique
Fipronil (Fipronil, also known as Fipronil, No. CAS is 120068-37-3) is a kind of phenyl pyrazoles desinsection
Agent, insecticidal spectrum is wide, to pest based on stomach poison function, has concurrently and tags and certain systemic action, the mechanism of action be by with
The GABA receptor of insect combines, and blocks the chloride channel of γ-aminobutyric acid control, hinders chloride metabolism interference
Central nervous system causes insect nerves and muscles surexcitation to death.Fipronil can also be used to kill cat, the jump with dog
The helminths such as flea and lice.
The acceptable daily intake (ADI) for the Fipronil that European Food Safety Authority was recommended in 2006 is 0.0002mg/kg,
The ADI that China is recommended in " national food safety standard --- Pesticide maximum residue limit " (GB 2763-2016)
For 0.0002mg/kg." 50 kinds of agricultures such as pyraclostrobin in national food safety standard food that China issues in November, 2016
Medicine maximum residue limit (exposure draft) " in regulation Fipronil be in the maximum residue limit in meat of poultris be 0.01mg/kg,
It is 0.02mg/kg in eggs.In September, 2017, the Ministry of Agriculture issue No. 2583 bulletin, forbid Fipronil and related preparations
For food animal.
The detection of Fipronil at present mainly based on instrument analytical method, is based on high performance liquid chromatography or liquid chromatogram matter
Joint technology is composed to the Fipronil quantitative detection in Different categories of samples, instrument and equipment valuableness, can not be applied to cumbersome complexity
On-site test；There is the defects of Antibody preparation period is long, screening difficulty is big, process is complicated, costly again in immunological detection method.
Summary of the invention
The first of the embodiment of the present invention is designed to provide a kind of Fipronil binding proteins specific, which is easy to make
It is standby, and can be specifically bound with Fipronil, specificity with higher and sensitivity.
The second of the embodiment of the present invention, which is designed to provide a kind of Fipronil binding proteins specific and detects in Fipronil, to be tried
Application in agent box and colloidal gold colloidal gold detection test paper strip, the application operating is simple, and can fast and accurately detect in sample
Fipronil；In addition, realizing the on-site test to sample.
To achieve the above object, the embodiment of the present invention provides a kind of albumen for specifically binding Fipronil, the albumen
Amino acid sequence is as shown in SEQ ID NO.1 or the sequence is through replacement, missing or one or several amino acids formed tools of addition
There is the amino acid sequence of same function.
In the present invention stringent limitation is not done to the preparation method of the albumen, for example, artificial synthesized side can be passed through
Method obtains, and specifically, screens to obtain corresponding gene order as shown in SEQ ID NO.2 by bioinformatics means, and
It is reached by said gene sequence table.
On the other hand the embodiment of the present invention provides a kind of Fipronil detection kit, which includes that box body, setting exist
Dismountable ELISA Plate and reagent, each hole of the ELISA Plate are coated with the albumen in box body；The reagent includes enzyme mark
Fipronil, Fipronil standard solution, developing solution, terminate liquid, cleaning solution and the Sample dilution of note.
Preferably, the ELISA Plate is prepared by including the following steps:
The albumen is diluted to 0.5-2 μ g/ml using carbonate buffer solution, and adds 50- into every hole of ELISA Plate
Diluted protein solution described in 100 μ L, is incubated overnight under the conditions of 3-5 DEG C；Then, the diluted protein solution is dried, and with phosphoric acid
Salt Tween buffer board-washing 2-4 times, then confining liquid is added in ELISA Plate, 1.5-2.5h is incubated under the conditions of 35-39 DEG C, then
Confining liquid is dried up to the ELISA Plate；
Wherein, the pH value of the carbonate buffer solution is 9.4-10；Preferably 9.6；The confining liquid is to contain 0.05%
Bovine serum albumin(BSA), the 0.01M pH7.2PBS solution of 5% casein or the 0.01M pH7.2PBS solution containing 1% gelatin.
Preferably, the Fipronil of the enzyme label is the fipronil hapten of horseradish peroxidase-labeled.It is highly preferred that
The fipronil hapten of the horseradish peroxidase-labeled is prepared by including the following steps:
(1) fipronil hapten is dissolved in n,N-Dimethylformamide solution, and dilute using PBS buffer solution progress
It releases；Preferably, fipronil hapten and n,N-Dimethylformamide solution quality volume ratio are (25-28): 1mg/ml；Using
Fipronil hapten concentration after PBS buffer solution dilution is 3.1-3.5mg/ml；
(2) horseradish peroxidase, sufficiently dissolution are added into fipronil hapten solution；Preferably, horseradish peroxidating
The mass ratio of object enzyme additive amount and fipronil hapten is 25: (12-14)；
(3) glutaraldehyde solution is added into dissolved mixed liquor and is stirred, and then, is added dropwise under agitation
The saturated ammonium sulfate of volume is reacted；Preferably, the mass concentration of glutaraldehyde solution is 4%-5%, glutaraldehyde solution addition
Amount is 150 with fipronil hapten liquor capacity ratio: (6-10)；Whipping temp is room temperature, mixing time 3.5-4.5h；Reaction
Temperature is 3-5 DEG C, reaction time 0.8-1.2h；
(4) sediment is collected by centrifugation in the solution after reaction and cleaned, then, sediment is dissolved in PBS solution, and adopts
It is dialysed with bag filter, the fipronil hapten that supernatant marks up to enzyme is collected by centrifugation；Preferably cleaning solution is semi-saturation
Ammonium sulfate；The ph of PBS solution is 7.4, concentration 0.15mol/L；The molecular weight of bag filter is 10KDa；Centrifugal rotational speed is
3000-15000r/min, centrifugation time 25-35min.
Preferably, the Fipronil standard solution include concentration be respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml,
The Fipronil solution of 0.9ng/ml, 2.7ng/ml and 8.1ng/ml.
Preferably, the developing solution is one-component TMB developing solution.
Preferably, it is 0.2-2M sulfuric acid solution that the terminate liquid, which is concentration,.
Preferably, the cleaning solution is the PBS solution of the 0.01M pH7.2 containing 0.5% Tween-20.
Preferably, the Sample dilution is the PBS solution of 0.1M pH7.2.
The another aspect of the embodiment of the present invention provides a kind of Fipronil colloidal gold colloidal gold detection test paper strip, which includes
PVC backboard, nitrocellulose filter, gold-labelled pad, sample pad and water absorption pad are coated with the described of colloid gold label in the gold-labelled pad
Albumen；There is detection line and nature controlling line on the nitrocellulose filter, be coated with Fipronil antigen, the matter in the detection line
The anti-His tag antibody of mouse is coated on control line.
Preferably, nitrocellulose filter is pasted on PVC backboard in the test strip,；The inspection of nitrocellulose filter
Survey line (T line) end is pasted with gold-labelled pad, and the upper end of gold-labelled pad is 4-10mm at a distance from T line, and is overlapped with nitrocellulose filter
1-3mm；Sample pad is pasted onto gold-labelled pad, and is overlapped 1-3mm with gold-labelled pad；Nature controlling line (C line) end of nitrocellulose filter is viscous
Water absorption pad is posted, water absorption pad is 4-10mm at a distance from C line, and is overlapped 1-3mm with nitrocellulose filter.
In the present invention stringent limitation is not done to the source of colloidal gold, for example, can be commercially available by market, lemon can also be led to
Lemon acid sodium reduction is prepared；Specifically, 0.01% aqueous solution of chloraurate is heated to boiling, under continuous agitation
1% trisodium citrate aqueous solution is added, continues to be heated with stirring to solution in bright claret, room temperature is cooling, then uses deionization
It is 30-40nm colloid gold particle that water, which is restored to original volume up to partial size,；Wherein, sodium citrate aqueous solution and aqueous solution of chloraurate
Volume ratio is 1.5%-2.5%.
Preferably, the albumen of the colloid gold label is prepared by including the following steps:
(1) K is added into colloidal gold solution2CO3Solution；Preferably, the mass concentration of colloidal gold solution is 1%-2%；Carbon
Sour potassium solution concentration is 0.08-0.12mol/L；Colloidal gold solution and solution of potassium carbonate volume ratio are 500: (1-10)；
(2) protein solution is added in colloidal gold solution to be incubated for；Then, ox is added into the solution after incubation
Seralbumin mixes, sediment is collected by centrifugation；Preferably, the protein solution concentration is 0.03-0.05mg/ml, additive amount
It is 500 with colloidal gold solution volume ratio: (5-9)；Incubation temperature is room temperature, incubation time 18-22min；It is pure to add ox blood
The concentration of bovine serum albumin(BSA) is 0.2%g/ml in solution after albumen；Centrifugal rotational speed is 9000-12000r/min, when centrifugation
Between be 35-45min；
(3) albumen that sediment marks to get colloid gold particle is redissolved using phosphate buffer；Preferably, phosphorus
Phthalate buffer be containing 1% sucrose, 1% trehalose, 2%BSA, 0.02% Sodium azide 0.02M phosphate buffer.
Preferably, the preparation method of the Fipronil antigen includes the following steps:
(1) Fipronil, succinic anhydride, anhydrous pyridine are mixed, stirring；Preferably, the quality of Fipronil and succinic anhydride
Than for (34-36): 16；The mass volume ratio of Fipronil and anhydrous pyridine is (1.7-1.8): 40g/ml；
(2) mixture is heated to reflux, then adds ice water, adjusted pH, save overnight；Preferably, when being heated to reflux
Between be 3.5-4.5h, for removing pyridine；The volume ratio of ice water additive amount and anhydrous pyridine is 3: (3-5)；Adjust pH to 2.5-
3.5；Storage temperature is 3-5 DEG C；
(3) rear solution decompression overnight filtered, ice water washing, filtered again, repeated above-mentioned ice water and wash, filter operation 2- again
4 times, obtain fipronil hapten；
(4) fipronil hapten is dissolved in anhydrous N, it is then, molten to fipronil hapten in N '-dimethyl formamide
N is added in liquid, N'- Dicyclohexylcarbodiimide simultaneously stirs, and after stirring, then N- hydroxyl is added into fipronil hapten solution
Base succinimide and under room temperature, stirring condition, is protected from light；Preferably, fipronil hapten and anhydrous N, N '-two
The mass volume ratio of methylformamide is (80-81): 3mg/ml；Fipronil hapten, N, N'- Dicyclohexylcarbodiimide and N-
The mass ratio of HOSu NHS is (80-81): 31: 20；Mixing time is 9-11min；It is protected from light 7-9h；
(5) supernatant is collected by centrifugation in solution after reaction, and supernatant is added dropwise to buffer configuration
In carrier protein solution, it is stirred overnight；Preferably, carrier protein is ovalbumin or bovine serum albumin(BSA)；Buffer is
The phosphate buffer of 0.1mol/l pH 8.0；Carrier protein solution concentration is 9-11mg/ml；Carrier protein solution and step
(4) anhydrous N in, the volume ratio of N '-dimethyl formamide are 5: (2-4)；
(6) product after staying overnight is subjected to dialysis purification using bag filter to get Fipronil antigen is arrived；Preferably, it dialyses
The molecular cut off of bag is 10KDa.
The another aspect of the embodiment of the present invention provides a kind of kit or the test strip fluorine in test sample
The remaining application of worm nitrile.
The embodiment of the present invention has the advantages that
(1) it is easily prepared to provide a kind of Fipronil binding proteins specific by the present invention, and can be with Fipronil specificity knot
It closes, specificity with higher and sensitivity.
(2) Fipronil binding proteins specific of the present invention is in Fipronil detection kit and colloidal gold colloidal gold detection test paper strip
Application, the application operating is simple, and testing cost is low, and can fast and accurately detect the Fipronil in sample；And it realizes
To the on-site test of sample.
Detailed description of the invention
Fig. 1 is the canonical plotting for the Fipronil detection kit that the embodiment of the present invention 4 provides；
Fig. 2 is the structure chart for the Fipronil colloidal gold colloidal gold detection test paper strip that the embodiment of the present invention 7 provides.
In figure: 1- sample pad；2- gold-labelled pad；3- nitrocellulose filter；4- water absorption pad；5- detection line；6- nature controlling line；7-
PVC backboard.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products obtained can be bought by city.
Embodiment 1
The present embodiment is the preparation method that a kind of amino acid sequence is albumen shown in SEQ ID NO.1, the preparation method packet
Include following steps:
Screen to obtain corresponding gene order as shown in SEQ ID NO.2 by bioinformatics means；
SEQ ID NO.2 gene order is subjected to PCR amplification, specific reaction condition are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of changes
Property 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, then 30 circulations re-extend 5min, 4 DEG C of termination are reacted；
Pcr amplification product and pET28a are subjected to double digestion respectively, then the product after digestion is mixed, and add T4DNA
Overnight, then, connection product is converted into cloning host DH5 α competent cell at 4 DEG C for ligase, and by positive restructuring
Plasmid is named as pET28a-GABA-s α 1, then again converts the connection product after clone into expressive host BL21 competence
It is expressed；
1 glycerol stock of pET28a-GABA-s α for expressing GABA receptor protein is taken out from -20 DEG C, according to 1 after thawing:
1000 ratio is added in LB culture medium, and antibiotic is added in the ratio of 1:1000 again, then, in 37 DEG C, 220rpm item
Under part, culture 18h obtains seed liquor；
Seed liquor is transferred according to the ratio of 1:100, and antibiotic is added according to 1:1000 ratio, when OD value is 0.7,
IPTG is added in the culture medium to have spread cultivation according to the ratio of 1:1000, is induced overnight under the conditions of 18 DEG C, 180rpm, with
Afterwards, the bacterium of overnight induction is centrifuged 10min with 5000rpm revolving speed, collects sediment, then sediment is hanged using Buffer A
It rises, and moves into vessel, protease inhibitors is added in thallus and mix, then ultrasonication 30min, by broken thallus
It moves into centrifuge tube after trim, under the conditions of 4 DEG C, 30min is centrifuged with 15000rpm；Wherein, Buffer A is 50mM Hepes
500mM NaCl pH7.5；
Supernatant after collecting centrifugation, and removal of impurities is filtered by 0.22 μm of filter, filtered supernatant is through wriggling
Pump is loaded on the Ni column being balanced, with elution buffer Buffer B gradient elution, the gradient elution is 0% →
10% → 100%, the elution buffer Buffer B are 50mM Hepes 500mM NaCl 500mM imidazoles pH7.5, are collected
Sample is albumen shown in SEQ ID NO.1 up to amino acid sequence at detached peaks.
Embodiment 2
The present embodiment is the preparation of ELISA Plate, which includes the following steps:
It is that albumen shown in SEQ ID NO.1 uses pH9.6 carbonate buffer solution to be diluted to 1 μ g/ml by amino acid sequence,
And the 100 above-mentioned diluted protein solutions of μ L are added into every hole of ELISA Plate, it is incubated overnight under the conditions of 4 DEG C；Then, albumen is diluted
Liquid dries, and with phosphate Tween buffer board-washing 3 times, then the 0.01M pH7.2PBS solution containing 1% gelatin is added to
In each hole of ELISA Plate, 2h is incubated under the conditions of 37 DEG C, then confining liquid is dried up to ELISA Plate.
Embodiment 3
The present embodiment is the preparation method of the fipronil hapten of horseradish peroxidase-labeled, which includes such as
Lower step:
(1) fipronil hapten 26.86mg is dissolved in 1ml n,N-Dimethylformamide solution, and slow using PBS
Fliud flushing is diluted to 8ml；
(2) horseradish peroxidase 50mg, stirring to abundant dissolution are added into fipronil hapten solution；
(3) into dissolved mixed liquor, the 150 μ L of glutaraldehyde solution of addition 4.5% stirs 4h at room temperature, then,
Continue to stir, and isometric saturated ammonium sulfate is added dropwise, reacts 1h under the conditions of 4 DEG C；
(4) solution after reaction is collected into sediment with 3000r/min centrifugation 30min, and is rushed using semi-saturation ammonium sulfate
It washes 1-3 times, then, sediment is dissolved in the PBS of 0.15mol/LpH7.4, and use molecular weight for the progress of 10KDa bag filter
Dialysis collects the fipronil hapten that supernatant marks up to enzyme with 10000r/min centrifugation 30min.
Embodiment 4
The present embodiment is a kind of Fipronil detection kit, which includes box body, is arranged in box body removably
The ELISA Plate and reagent that 96 holes are prepared by embodiment 2；Reagent includes the fluorine worm for the enzyme label being prepared by embodiment 3
Nitrile, Fipronil standard solution, developing solution, terminate liquid, cleaning solution and Sample dilution；
Wherein, Fipronil standard solution include concentration be respectively 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml,
The Fipronil solution of 2.7ng/ml and 8.1ng/ml；Developing solution is one-component TMB developing solution；Terminate liquid is that concentration is that 1M sulfuric acid is molten
Liquid；Cleaning solution is the PBS solution of the 0.01M pH7.2 containing 0.5% Tween-20；Sample dilution is the PBS of 0.1M pH7.2
Solution.
Embodiment 5
The present embodiment is that the amino acid sequence of colloid gold particle label is the preparation method of albumen shown in SEQ ID No:1,
The preparation method includes the following steps:
(1) it takes the colloidal gold solution of 10ml 1.5% into centrifuge tube, and adds the 0.1mol/L K of 100 μ l2CO3Solution；
(2) protein solution prepared by the embodiment 1 that 140 μ l concentration are 0.04mg/ml is added in colloidal gold solution, room
Temperature is incubated for 20min；Then, bovine serum albumin(BSA) is added into the solution after incubation makes its final concentration of 0.2%g/ml, mixing,
Under the conditions of 4 DEG C, sediment is collected with 10000r/min centrifugation 40min；
(3) contain the 0.02mol/L phosphate of 1% sucrose, 1% trehalose, 2%BSA, 0.02% Sodium azide using 2ml
It is albumen shown in SEQ ID No:1 that buffer, which redissolves sediment to get the amino acid sequence that colloid gold particle marks,.
Embodiment 6
The present embodiment is the preparation method of Fipronil antigen, which includes the following steps:
(1) Fipronil 3.4972g, succinic anhydride 1.6g, anhydrous pyridine 81ml mixed, stirred evenly；
(2) mixture is carried out being heated to reflux 4h, then adds 60ml ice water, adjusting pH value to 3.0, mistake under the conditions of 4 DEG C
Night saves；
(3) rear solution decompression overnight filtered, ice water washing, filtered again, repeated aforesaid operations 2-4 times, obtain Fipronil
Haptens；
(4) fipronil hapten 80.58mg is dissolved in the anhydrous N of 3ml, in N '-dimethyl formamide, then, to fluorine worm
N is added in nitrile haptens solution, N'- Dicyclohexylcarbodiimide 31.0mg simultaneously stirs 10min, after stirring, then to fluorine worm
N-hydroxysuccinimide 20.0mg and under room temperature, stirring condition is added in nitrile haptens solution, carries out being protected from light 8h；
(5) supernatant is collected by centrifugation in solution after reaction, and supernatant is added dropwise to 0.1mol/l pH
The 5ml concentration of 8.0 phosphate buffer configuration is under the conditions of 4 DEG C, to stir in the solution of 10mg/ml bovine serum albumin(BSA)
Overnight；
(6) it uses molecular weight to carry out dialysis purification for 10KDa bag filter the product after staying overnight to resist to get to Fipronil
It is former.
Embodiment 7
The present embodiment is a kind of Fipronil colloidal gold colloidal gold detection test paper strip, which includes PVC backboard 7, nitric acid fibre
Plain film 3, gold-labelled pad 2, sample pad 1 and water absorption pad 4 are tieed up, the colloid gold particle label of the preparation of embodiment 5 is coated in gold-labelled pad 2
Amino acid sequence is albumen shown in SEQ ID No:1；There is detection line 5 and nature controlling line 6, in detection line on nitrocellulose filter 3
It is coated with the Fipronil antigen of the preparation of embodiment 6, is coated with the anti-His tag antibody of mouse on nature controlling line；
Wherein, nitrocellulose filter 3 is pasted on PVC backboard 7；The detection line 5 (T line) of nitrocellulose filter 3, which is held, pastes
There is gold-labelled pad 2, the upper end of gold-labelled pad 2 is 6mm at a distance from T line, and is overlapped 2mm with nitrocellulose filter 3；Sample pad 1 is pasted
2mm is overlapped in gold-labelled pad 2, and with gold-labelled pad 2；Nature controlling line 6 (C line) end of nitrocellulose filter 3 is pasted with water absorption pad 4, inhales
It is 6mm at a distance from water cushion 4 and C line, and is overlapped 2mm with nitrocellulose filter 3.
Experimental example 1
Fipronil in cow's milk sample is detected using the kit of the embodiment of the present invention 4；
Sample process: it will directly use Sample dilution according to 1: 50 dilution as to be detected after the concussion uniformly of cow's milk sample
Solution；
The preparation of Fipronil solution to be detected: being divided into 4 parts for liquid to be detected, adds fluorine worm respectively in 3 parts of liquid to be detected
Nitrile obtains the Fipronil solution to be detected that Fipronil concentration is respectively 0,0.5ng/ml, 2ng/ml, 5ng/ml；
Experimental method:
(1) room temperature 35min is placed after taking out the ELISA Plate got ready；
(2) standard items, Fipronil solution to be detected are added in ELISA Plate hole with the every hole 50 μ L, are incubated at room temperature 30min；
(3) board-washing 3 times after taking-up ELISA Plate, the Fipronil of 50 μ L enzymes label is added, is incubated at room temperature 15min；
(4) board-washing 3 times after taking-up ELISA Plate, 100 μ L of TMB developing solution is added, is incubated at room temperature 15min；
(5) 50 μ L of terminate liquid is added, OD value of each hole at wavelength 450nm, measurement result such as 1 institute of table are measured with microplate reader
Show；
(6) the OD value in the Fipronil standard solution hole containing 0ng/ml is positioned into B0；Remaining Fipronil standard solution hole OD value
Position B；With B/B0For ordinate, the logarithm logC of respective standard product concentration C is abscissa, draws Fipronil standard curve as schemed
Shown in 1；And the regression equation for obtaining standard curve is y=0.0845x3+0.0707x2- 0.4071x+0.3616；And it is related
Coefficients R2=0.9973.
(7) regression equation that the OD value in containing fipronil solution hole to be detected is substituted into standard curve can qualitative, quantitative judgement
Fipronil concentration in sample, testing result is referring to table 2.
Table 1
Table 2
| Fipronil concentration (ng/ml) | Testing result (ng/ml) | The rate of recovery (%) |
| 0 | — | — |
| 0.5 | 0.461 | 92.2 |
| 2 | 1.754 | 87.7 |
| 5 | 4.819 | 96.38 |
From table 1, table 2: detection kit detection sensitivity of the present invention reaches 0.5ng/ml, and the rate of recovery reaches
87.7%-96.38%.
Experimental example 2
The Fipronil residual in milk sample is detected using Fipronil colloidal gold colloidal gold detection test paper strip in embodiment 7,
Concrete operations are as follows:
Sample process: it will directly use Sample dilution according to 1: 50 dilution as to be detected after the concussion uniformly of cow's milk sample
Solution；
The preparation of Fipronil solution to be detected: being divided into 4 parts for liquid to be detected, adds fluorine worm respectively in 3 parts of liquid to be detected
Nitrile obtains the Fipronil solution to be detected that Fipronil concentration is respectively 0,0.5ng/ml, 2ng/ml, 5ng/ml；
Experimental method:
(1) colloidal gold strip got ready and micropore reagent are ready to, room temperature 35min is placed after taking-up；
(2) it takes 200 μ L to be added in micropore reagent the solution to be detected added with Fipronil, is inhaled up and down with pipettor and make a call to three
It is secondary, it mixes well, is stored at room temperature 5min；
(3) ready Fipronil colloidal gold colloidal gold detection test paper strip is taken out, the micropore reagent that the insertion of sample pad end is mixed is molten
In liquid, solution to be checked will creep along the sample pad end of test strips to water absorption pad end under siphonage, be stored at room temperature 5min；
(4) sample pad end is cut with scissors or will be blotted remaining solution to be checked thereon with blotting paper, and according to colloidal gold
The colour developing of C line and T line judges testing result in test strips.
When the C line color on colloidal gold strip is as T line, or than showing result for the positive when T line depth；When C line face
When color ratio T line is shallow, show result for feminine gender；C line does not develop the color, then result is invalid, needs to retest using new test strips.
The testing result of sample to be examined four addition concentration is as shown in table 3；
Table 3
| Fipronil concentration (ng/ml) | Testing result (ng/ml) |
| 0 | ————— |
| 0.5 | ————— |
| 2 | ————+ |
| 5 | +++++ |
As shown in Table 3: when addition concentration is 5ng/ml, Fipronil colloidal gold strip testing result is the positive, i.e., originally
Invention Fipronil colloidal gold strip can be identified as 5ng/ml to the detection limit of Fipronil.
Embodiment 3
Fipronil detection kit and colloidal gold colloidal gold detection test paper strip specific detection of the present invention
Imidacloprid, the 100 μ g/ for being 100 μ g/ml using Fipronil colloidal gold colloidal gold detection test paper strip detectable concentration in embodiment 7
The dieldrite of ml, the chlopyrifos of 100 μ g/ml, the endrin of 100 μ g/ml, 100 μ g/ml cypermethrin standard solution,
With embodiment 9, testing result is as shown in table 4 for concrete operations；
Table 4
| Drug and concentration (μ g/ml) | Testing result |
| 100 μ g/ml imidacloprids | ————— |
| 100 μ g/ml dieldrite | ————— |
| 100 μ g/ml chlopyrifos | ————— |
| 100 μ g/ml endrins | ————— |
| 100 μ g/ml cypermethrins | ————— |
As shown in Table 4, test strips of the invention are to above-mentioned each unrelated drug without obvious cross reaction；I.e. the present invention uses
The specificity of albumen and Fipronil is good.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Sequence table
<110>hundred Biotechnology Co., Ltd are received in Beijing
<120>a kind of Fipronil binding proteins specific and its application
<130> 2018
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 195
<212> PRT
<213>Artificial (artificial sequence)
<400> 1
Met Gly Ser Ser His His His His His His Ser Ser Gly Val Arg Gly
1 5 10 15
Ser His Met Ala Ser Met Thr Gly Gly Met Gly Arg Gly Ser Val Tyr
20 25 30
Val Cys Val Val Val Ser Trp Val Ser Trp His Arg Ala Thr Ser Asp
35 40 45
Arg Val Gly Gly Thr Thr Val Thr Ser Thr Ser Asp Ser Arg Thr Asp
50 55 60
Lys Val Arg Tyr Ala Thr Ala Asp Trp Met Ser Gly Tyr Cys Ala Thr
65 70 75 80
Ala Gly Val His Tyr Thr Lys Val Gly Ser Gly Trp Asn Asn Thr Asp
85 90 95
Tyr Asp Ala Cys Ser Val Ser Cys Ser Thr Val His Thr Asp Val Ser
100 105 110
Arg Arg Arg Ser Ser Cys Asp Val Tyr Gly Val Asn Ser Val Ser Tyr
115 120 125
Arg Gly Ser Met Thr Thr Val Ser Val Thr Ser Lys Ser Thr Met Arg
130 135 140
Thr Thr Trp Lys Trp Arg Tyr Cys Ala Gly Asp Ser Arg Lys Arg Arg
145 150 155 160
Arg Ala Ala Gly Asn Cys Arg Lys Arg Gly Lys Ala Arg His Asn Ser
165 170 175
Val Ser Tyr Asp Lys Val Ala Arg Val Ala Ser Gly Asn Val Cys Tyr
180 185 190
Trp Lys Leu
195
<210> 2
<211> 726
<212> DNA
<213>Artificial (artificial sequence)
<400> 2
gaattcgttt acgttccgtg cgttctgatc gttgttctgt cttgggtttc tttctggatc 60
caccgtgaag ctacctctga ccgtgttggt ctgggtatca ccaccgttct gaccctgtct 120
accatctctc tggactctcg taccgacctg ccgaaagttc gttacgctac cgctctggac 180
tggttcctgc tgatgtcttt cggttactgc atcgctaccc tgctggaatt cgctggtgtt 240
cactacttca ccaaagttgg ttctggtgaa atcccgctgg aagaagaaga atgggaaaac 300
gaaaacgaaa ccgactacca ggacgctttc tgctctatcg tttcttgctc tccgcagacc 360
gttcacccgg aaaccatcat cgacgtttct cgtcgtcgtt cttctctgat ctgcgacgtt 420
tacggtgtta acgaatctgt ttcttacgaa cgtggttcta tgaccgtttc tgttacctct 480
aaaccgtcta ccatggaacg tcagacccag accgaaatct ggatcccgaa atggcgtcag 540
ttcctgtact gcctggctgg tgacgaatct ttccgtaaac gtcgtcagcg tgaagctgct 600
ggtaactgcc gtaaacgtgg tgaaaaaatc gctcgtcaca tcaactctgt ttcttacatc 660
gacaaagttg ctcgtatcgt tttcccggct tctttcggtc tgctgaacgt ttgctactgg 720
aagctt 726
Claims (10)
1. a kind of albumen for specifically binding Fipronil, which is characterized in that the amino acid sequence of the albumen such as SEQ ID NO.1
The shown or sequence is through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.
2. albumen according to claim 1, which is characterized in that the albumen passes through the gene as shown in SEQ ID NO.2
Sequence table is reached.
3. a kind of Fipronil detection kit, including box body, setting dismountable ELISA Plate and reagent, feature in box body exist
In each hole of the ELISA Plate is coated with albumen of any of claims 1 or 2；The reagent include enzyme label Fipronil,
Fipronil standard solution, developing solution, terminate liquid, cleaning solution and Sample dilution.
4. kit according to claim 3, which is characterized in that the ELISA Plate is prepared by including the following steps:
The albumen is diluted to 0.5-2 μ g/ml using carbonate buffer solution, and adds 50-100 μ L into every hole of ELISA Plate
The diluted protein solution is incubated overnight under the conditions of 3-5 DEG C；Then, the diluted protein solution is dried, and with phosphate tween
Buffer board-washing 2-4 times, then confining liquid is added in ELISA Plate, 1.5-2.5h is incubated under the conditions of 35-39 DEG C, then will closing
Liquid dries up to the ELISA Plate；
The confining liquid be containing 0.05% bovine serum albumin(BSA), 5% casein 0.01M pH7.2PBS solution or contain 1%
The 0.01M pH7.2PBS solution of gelatin.
5. kit according to claim 3, which is characterized in that the Fipronil of the enzyme label is horseradish peroxidase
The fipronil hapten of label.
6. kit according to claim 5, which is characterized in that the Fipronil of the horseradish peroxidase-labeled half is anti-
Reason includes the following steps to be prepared:
(1) fipronil hapten is dissolved in n,N-Dimethylformamide solution, and is diluted using PBS buffer solution；
(2) horseradish peroxidase, sufficiently dissolution are added into fipronil hapten solution；
(3) glutaraldehyde solution is added into dissolved mixed liquor and is stirred, and then, is added dropwise under agitation isometric
Saturated ammonium sulfate reacted；
(4) sediment is collected by centrifugation in the solution after reaction and cleaned, then, sediment is dissolved in PBS solution and using saturating
Analysis bag is dialysed, and the fipronil hapten that supernatant marks up to enzyme is collected by centrifugation.
7. a kind of Fipronil colloidal gold colloidal gold detection test paper strip, including PVC backboard, nitrocellulose filter, gold-labelled pad, sample pad and water suction
Pad, which is characterized in that the albumen of any of claims 1 or 2 of colloid gold label is coated in the gold-labelled pad；The nitric acid is fine
Tieing up has detection line and nature controlling line on plain film, be coated with Fipronil antigen in the detection line, be coated with mouse on the nature controlling line
Anti- His tag antibody.
8. test strip according to claim 7, which is characterized in that the albumen of the colloid gold label is by including
Following steps are prepared:
(1) K is added into colloidal gold solution2CO3Solution；
(2) protein solution is added in colloidal gold solution to be incubated for；Then, cow's serum is added into the solution after incubation
Albumin mixes, sediment is collected by centrifugation；
(3) albumen that sediment marks to get colloid gold particle is redissolved using phosphate buffer.
9. test strip according to claim 7, which is characterized in that the Fipronil antigen is made by including the following steps
It is standby to obtain:
(1) Fipronil, succinic anhydride, anhydrous pyridine are mixed, stirring；
(2) mixture is heated to reflux, then adds ice water, adjusted pH, save overnight；
(3) rear solution decompression it will filter, ice water washing, filter overnight, and obtain fipronil hapten；
(4) fipronil hapten is dissolved in anhydrous N, in N'- dimethylformamide, then, into fipronil hapten solution
N is added, N'- Dicyclohexylcarbodiimide simultaneously stirs, and after stirring, then N- hydroxyl amber is added into fipronil hapten solution
Amber acid imide is simultaneously protected from light under room temperature, stirring condition；
(5) supernatant is collected by centrifugation in solution after reaction, and supernatant is added dropwise to the carrier of buffer configuration
In protein solution, it is stirred overnight；
(6) product after staying overnight is purified to get Fipronil antigen is arrived.
10. any kit of claim 3-6 or any test strip fluorine in test sample of claim 7-9
The remaining application of worm nitrile.
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| CN117497081B (en) * | 2023-12-29 | 2024-04-05 | 北京市农林科学院 | Logic gate construction based on porphyrin COF fipronil and application thereof |
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