CN113683674B - Antigen epitope polypeptide of anti-snakehead Nramp antibody and application of antigen epitope polypeptide in antibody preparation - Google Patents

Abstract

The invention provides an epitope polypeptide of an anti-snakehead Nramp antibody and application thereof in antibody preparation, belonging to the technical fields of immunochemistry and molecular biology. The invention provides a polypeptide sequence of snakehead Nramp with dominant antigen epitope, the polypeptide consists of 11 amino acid residues, the epitope of the polypeptide is 318-328 amino acid sequences of snakehead Nramp protein, and the polypeptide has high immunogenicity and specificity. Therefore, the method can be used for preparing the anti-snakehead Nramp antibody, thereby laying a foundation for fish Nramp protein related research, and having good practical application value.

Description

Antigen epitope polypeptide of anti-snakehead Nramp antibody and application of antigen epitope polypeptide in antibody preparation

Technical Field

The invention belongs to the technical fields of immunochemistry and molecular biology, and particularly relates to an epitope polypeptide of an anti-snakehead Nramp antibody and application thereof in antibody preparation.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

In recent years, with the development of aquaculture industry, the aquaculture scale is continuously enlarged, but the aquaculture environment is worsened, diseases exploded in the aquaculture process of the aquatic fishes are more and more, great economic loss is brought to the aquaculture industry, and the sustainable development of the aquaculture industry is severely restricted. Therefore, the cultivation of new fish strains with certain disease resistance is an effective way for solving the disease problem, and is also a trend for realizing healthy development of the aquaculture industry.

The natural resistance-related megaphaga protein (Nramp) gene is taken as a disease resistance candidate gene, is closely related to the innate immunity of fish, and has important value in the disease resistance research of fish. The natural resistance-associated megaphaga protein (Nramp) family is a class of natural immune-related proteins that inhibit intracellular pathogen infection. Two naturally resistant megaphaga proteins, nramp1 and Nramp2, have been found to have different functions in mammals such as humans and mice. However, in fish, except rainbow trout (Nramp alpha and Nramp beta), only one type of Nramp protein is found, and the amino acid sequence comparison analysis research shows that the homology of the Nramp gene of fish and the mammalian Nramp2 is higher than that of the Nramp1, but the function of the Nramp gene is similar to that of the Nramp 1. However, the inventors found that antibodies and proteins for laboratory studies against fish Nramp are still freshly reported.

Disclosure of Invention

Based on the defects of the prior art, the invention provides an epitope polypeptide of an anti-snakehead Nramp antibody and application thereof in antibody preparation. According to the invention, research shows that the dominant epitope polypeptide of the anti-snakehead Nramp antibody is successfully obtained, and the snakehead Nramp polyclonal antibody is successfully prepared by taking the dominant epitope polypeptide as an antigen, so that a foundation is laid for further researching the effect and the function of fish Nramp, and the method has a good practical application value.

In a first aspect of the invention, there is provided an epitope polypeptide of an anti-snakehead Nramp antibody, the amino acid sequence of said polypeptide being selected from the group consisting of: comprising or consisting of the sequence of SEQ ID NO. 1.

The epitope of the polypeptide is 318 th to 328 th amino acid sequences of the snakehead Nramp protein.

In a second aspect, the invention provides application of the epitope polypeptide in preparation of an anti-snakehead Nramp antibody.

Wherein, the anti-snakehead Nramp antibody can be a monoclonal antibody or a polyclonal antibody.

The antibody can be used for detecting the snakehead Nramp protein.

In a third aspect of the present invention, there is provided an anti-snakehead Nramp antibody which specifically binds to a snakehead Nramp protein and specifically recognizes the above epitope polypeptide.

In a fourth aspect of the invention, there is provided a nucleic acid molecule encoding an anti-snakehead Nramp antibody as described above.

In a fifth aspect of the present invention, there is provided a method for preparing the above-mentioned anti-snakehead Nramp antibody, the method comprising: and immunizing a non-human animal against an antigen formed by the epitope polypeptides.

In a sixth aspect of the invention, there is provided a kit for detecting an snakehead Nramp protein, the kit comprising the above-described anti-snakehead Nramp antibody.

In a seventh aspect of the invention, there is provided a method of detecting a snakehead Nramp protein, the method comprising: and detecting the sample to be detected by adopting the anti-snakehead Nramp antibody and/or the kit.

The beneficial technical effects of one or more of the technical schemes are as follows:

the technical scheme provides a polypeptide sequence of the snakehead Nramp with dominant antigen epitope, the polypeptide consists of 11 amino acid residues, the epitope of the polypeptide is 318-328 amino acid sequences of snakehead Nramp protein, and the polypeptide has high immunogenicity and specificity. Therefore, the method can be used for preparing the anti-snakehead Nramp antibody, thereby laying a foundation for fish Nramp related research, and having good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application.

FIG. 1 is a graph showing the analysis of membrane-spanning protein of snakehead Nramp in the embodiment of the invention;

FIG. 2 is a diagram showing the analysis of the signal peptide of the snakehead Nramp protein in the embodiment of the invention;

FIG. 3 is a diagram showing the hydrophobicity analysis of the snakehead Nramp protein in the embodiment of the invention;

FIG. 4 is a chart showing unordered sequence analysis of snakehead Nramp proteins in the embodiment of the invention;

FIG. 5 is a chart showing an antigenic analysis of the snakehead Nramp protein in the embodiment of the invention; wherein, the light area with the score of more than 0.5 is the predicted epitope;

FIG. 6 is a diagram showing the homology analysis of the snakehead Nramp protein in the embodiment of the invention;

FIG. 7 is a domain analysis diagram of the snakehead Nramp protein in the embodiment of the invention;

FIG. 8 is a sequence antigenicity analysis chart of an epitope polypeptide in an embodiment of the present invention;

FIG. 9 is a SDS-PAGE verification of antigens in an embodiment of the invention; wherein M: m. marker (5. Mu.L, 0.1 mg/mL); 1: PA1-21050016 polypeptide coupled to BSA;

FIG. 10 is a graph showing the results of antibody purification in the examples of the present invention, wherein M: marker (5. Mu.L, 0.1 mg/mL); 1: rabbit G1638 purified antibody; 2: rabbit G1639 purified antibody.

Fig. 11 is a diagram of the results of rabbit polyclonal antibody assay in an embodiment of the present invention, wherein M: a Marker;1: snakehead spleen; 2: snakehead kidney.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In one exemplary embodiment of the present invention, there is provided an epitope polypeptide of an anti-snakehead Nramp antibody, the amino acid sequence of which is selected from the group consisting of: comprising or consisting of the sequence of SEQ ID NO. 1.

In yet another embodiment of the present invention, the epitope of the polypeptide is amino acid sequence 318-328 of the snakehead Nramp protein.

In still another embodiment of the present invention, there is provided the use of the above epitope polypeptide in the preparation of an anti-snakehead Nramp antibody.

In yet another embodiment of the invention, the application is: the antigen epitope polypeptide is used as an antigen to prepare the anti-snakehead Nramp antibody.

Wherein, the anti-snakehead Nramp antibody can be a monoclonal antibody or a polyclonal antibody.

The antibody can be used for detecting the snakehead Nramp protein.

In yet another embodiment of the present invention, there is provided an anti-snakehead Nramp antibody which specifically binds to a snakehead Nramp protein and specifically recognizes the above epitope polypeptide.

The anti-snakehead Nramp antibody can be a monoclonal antibody or a polyclonal antibody.

In yet another embodiment of the present invention, there is provided a nucleic acid molecule encoding the above-described anti-snakehead Nramp antibody.

In still another embodiment of the present invention, there is provided a method for preparing the above-mentioned anti-snakehead Nramp antibody, the method comprising: and immunizing a non-human animal against an antigen formed by the epitope polypeptides.

The anti-snakehead Nramp antibody can be a monoclonal antibody or a polyclonal antibody.

When preparing the snakehead Nramp polyclonal antibody, the non-human animal is a rabbit. Therefore, the rabbit polyclonal antibody of the antigen epitope polypeptide of the anti-snakehead Nramp antibody is finally prepared.

In still another embodiment of the present invention, there is provided a kit for detecting an snakehead Nramp protein, the kit comprising the above-mentioned anti-snakehead Nramp antibody.

In yet another embodiment of the present invention, there is provided a method for detecting an snakehead Nramp protein, the method comprising: and detecting the sample to be detected by adopting the anti-snakehead Nramp antibody and/or the kit.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Examples

1. Sequence analysis of Nramp

The amino acid sequence of the Nramp protein is selected from KAF3689712.1 (Natural resistance-associated macrophage protein 2[ Channa argus ]), and proteins with homology up to 100% are found by alignment in a Uniprot database, and the amino acid sequence is derived from the species Channa argus (SEQ ID NO. 2), wherein the Uniprot ID is A0A6G1PHJ9. Analysis of the transmembrane region, signal peptide, hydrophobicity analysis, disordered sequence, antigenicity and structural domain shows that: the protein codes 562 amino acids, 10 times of transmembrane and no signal peptide sequence; as an immunogen, the full-length protein antigen index score is moderate, so that better immunity can be caused; from the perspective of homology analysis and antibody preparation requirements, the protein has stronger specificity in the species Channa argus, has low homology with rabbits and has high immunogenicity. Peptide fragments are selected according to the antigen epitope to carry out polypeptide synthesis and antibody preparation.

2. Peptide fragments are selected according to the epitope analysis result and the transmembrane analysis result, the peptide fragments are synthesized in a chemical synthesis mode, and antibodies are prepared after immunization.

Polypeptide sequence: DKTNIEVSEKC (SEQ ID NO. 1) is the 318 th-328 th amino acid sequence of the snakehead Nramp protein.

3. Experimental protocol

Polypeptide synthesis, hapten coupling, quality control, animal immunity, titer detection, antiserum preparation, antibody purification and antibody verification

(1) Antigen preparation

Polypeptide synthesis and complete antigen preparation, polypeptide synthesis and coupling carrier protein, naked peptide providing MS and HPLC quality control report, purity more than 90%, yield 100%.

(2) Antigen quality control

SDS-PAGE detection, complete antigen (-BSA or-OVA or-KLH) 6-7mg, purity greater than 90%, and two rabbits were generally immunized.

(3) Immunization of animals

After emulsification of the antigen 2 experimental rabbits were immunized. One was given a second 3 weeks later, three 2 weeks later at intervals, and three 2 weeks later at intervals. A fourth step of avoiding the need for a special device, the four exemptions were performed five exemptions 2 weeks later.

(4) Potency detection

Blood is taken after three-immunity to measure titer, and at most 6 times of immunization are carried out to improve antiserum titer, and serum WB detection positive tissue lysate is taken during immunization.

(5) Antibody purification

Antibodies were affinity purified using ProteinA/G.

(6) WB validation

The antibody after antiserum purification can be used for detecting recombinant protein or positive tissue lysate by WB respectively.

4. Detailed description of the invention

1. Antigen SDS-PAGE validation

1.1 antigen SDS-PAGE validation:

1.2 conclusion: SDS-PAGE detection result: meets the immunization requirement, and can arrange subsequent immunization.

2. Immunization

2.1 immunization protocol

Number of immunizations	Immune cycle	Immunization time	Immunization dose	Immunoadjuvant	Immunization of animals
						First immunization	For 1 day	2021/06/03	0.5mg	Complete Freund's adjuvant	Good quality
Second immunization	14 days	2021/06/17	0.5mg	Incomplete Freund's adjuvant	Good quality
						Third immunization	For 28 days	2021/07/01	0.5mg	Incomplete Freund's adjuvant	Good quality
Blood taking with three-free function	For 35 days	2021/07/08	/	/	Normal blood sampling
						Fourth immunization	42 days	2021/07/15	0.5mg	Incomplete Freund's adjuvant	Good quality
Four-way blood taking	49 days	2021/07/22	/	/	Normal blood sampling
						Blood collection of immunized animals	For 56 days	2021/07/29	/	/	Normal blood sampling

2.2 antiserum ELISA detection

(1) Antigen coating: the antigen was diluted to 2ug/ml with coating solution, 100. Mu.L/well was added to a 96-well enzyme-labeled reaction plate and coated overnight at 4 ℃.

(2) Washing: the next day the liquid in the wells was discarded and the wash liquid was washed 3 times.

(3) Closing: 200. Mu.L/well of blocking solution was added and incubated at 37℃for 2h.

(4) Washing: washing with the washing liquid for 3 times.

(5) Adding a sample to be tested (primary antibody): antisera (blood was collected at 4 ℃ C. And 4000rmp was centrifuged for 10min to collect supernatant), and the serum was incubated with the diluent at 1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000.

(6) Washing: washing with the washing liquid for 3 times.

(7) Adding enzyme-labeled anti-antibody: HRP-labeled IgG secondary antibody was added and incubated at 37℃for 40min at 100. Mu.l/well. ( Goat anti-mouse-HRP 1:5000-1:10000, goat anti-rabbit-HRP 1:5000-1:10000 )

(8) Washing: washing with washing liquid for 5 times, and drying.

(9) Color development: adding 100 mu L/hole of freshly prepared substrate solution, and shading and standing for 5-20 min at 37 ℃.

(10) Termination reaction, colorimetric: add 50. Mu.L/Kong Zhongzhi solution. The color turns yellow; the absorbance of each well at 450nm was measured with a microplate reader.

2.3 antiserum titers

Two rabbits had serum titers exceeding 1:50K.

3. Antibody purification

3.1 antibody purification

(1) Pretreatment of a chromatographic column: the deionized water with 10 times of column bed volume is washed 3-5 times, the 0.02M PB+0.3M NaCl with 1ml/min and 10 times of column volume is washed 3-5 times, and the flow rate is 1ml/min.

(2) Sample loading: 4-10ml of antiserum/ascites, diluted with 0.02M PB, filtered through a 0.22um filter and applied to a column, the flow rate was adjusted to 5-7 s/drop.

(3) Washing: 0.02M PB wash until no protein was run off (G250 does not change blue), flow rate 2 s/drop.

(4) Antibody elution: 0.1M ph=3.0 glycine was eluted at 3-5 s/drop through the column, the eluate was collected and the eluted product was detected with G250 until it did not turn blue.

(5) pH value adjustment: saturated sodium carbonate adjusts the pH of the eluted product to neutral.

(6) Ultrafiltration concentration: the 10kDa ultrafiltration tube is subjected to ultrafiltration concentration to about 1-3 ml.

(7) And (3) dialysis: 5L,0.01M PH=7.4 PBS dialyses overnight, after changing liquid 1 time the next day, dialyses about 4-6 hours, runs SDS gel, and packs into the marked EP tube after measuring the antibody concentration, can be temporarily stored at 4 ℃ for standby or directly stored at-20 ℃.

(8) Washing and storing the chromatographic column: washing the chromatographic column with deionized water, washing the chromatographic column with 20% ethanol with 5 times of column bed volume, and sealing at 4deg.C

3.2 antibody purification results

The purity of the immunized rabbit G1638/G1639 antibody is more than 90 percent.

4.Western Blot

4.1 preparation of Polyacrylamide gel

4.1.1.1 preparation of a 12% strength (10 ml) solution of the separating gel 3.3ml of deionized water, 4ml of 30% acrylamide, 2.5ml of Tris at pH8.8, 100. Mu.L of 10% ammonium persulfate, 100. Mu. L, TEMED 6. Mu.L of 10% SDS (after TEMED addition, the separating gel starts to polymerize immediately and should be mixed immediately and rapidly).

4.1.2 rapidly add the gum solution to the gap between the two glass plates, leaving room for the concentrated gum to be poured, carefully add 1ml isopropyl alcohol (wall-mounted addition) to the gum solution.

4.1.3 after the completion of the polymerization of the separating gel (about 30min at 25 ℃), the liquid on the gel was drained as much as possible, and the residual liquid was sucked off by the edges of the filter paper.

4.1.4 preparation of concentrated gum solution (4 ml) at 5% strength: 2.7ml of deionized water, 670 mu L, PH 6.8.8 of 30% acrylamide, 500 mu L of Tris, 40 mu L of 10% ammonium persulfate and 40 mu L, TEMED mu L of 10% SDS are added in sequence (after TEMED is added, the separation gel starts to polymerize immediately and should be quickly mixed).

4.1.5 quick pouring of concentrated gel directly onto the polymerized separating gel, immediately insert a clean comb (care was taken to avoid mixing in air bubbles) into the concentrated gel solution.

4.1.6 after the gel concentrate has been completely polymerized (about 30min at 25 ℃), the comb is carefully removed and protein running buffer is added to the running tank.

4.2 preparation of samples

Snakehead is obtained from a culture base of a freshwater fishery institute of Shandong province, 50g of spleen and front kidney respectively and 15ul of extracted sample lysate are evenly mixed with 1-6ul 6X Loading buffer, and then the mixture is subjected to boiling water bath for 5-10min and centrifugation at 2500rpm for 2min, and tube wall solution is centrifuged.

4.3 electrophoresis

4.3.1 the sample mixture is slowly added to the sample well using a pipette, and the prestaining Marker is typically added at 10. Mu.L.

4.3.2 turning on the power supply, adopting 80V electrophoresis for 30min, changing the voltage to 120V electrophoresis until bromophenol blue loading buffer solution migrates to the bottom of the gel in the gel.

4.4 Wet rotating film

4.4.1 taking out the glue and immersing in a transfer film buffer.

4.4.2 soaking PVDF film (6% -12% of the adhesive film is 0.45um,15% of the adhesive is 0.2 um) in methanol for 1min, then sucking the solution with filter paper, transferring the solution into a transfer film buffer, and immersing the filter paper into the transfer film buffer (the PVDF film and the filter paper are sheared into the same size as the adhesive, and 5.5cm is 8.5 cm).

4.4.3 making transfer film sandwiches: (negative black surface) transfer paper 3 sheets+gel+PVDF film+transfer paper 3 sheets (positive white surface), and each layer should be completely removed.

4.4.5 placing the transfer sandwiches in a transfer tank with black facing black and white facing red, constant current 200mA 60min (12% glue 200mA 60min,15% glue 100mA 45min,6% glue 360mA 120 min).

4.5 closure

4.5.1 membranes were removed and TBS washed 1 time for 5min each (shake on horizontal shaker).

4.5.2 membranes were removed and immersed in a blocking solution at 25℃for 1h or at 4℃overnight (blocking solution was 500ml of 1 XTBS+5 g casein).

4.6 binding antibodies

4.6.1 the membranes were removed and washed 1 time with TBST for 5min each (shaking on a horizontal shaker).

4.6.2 the membranes were removed and immersed in primary anti-dilution diluted with dilution at 25℃for 2h or at 4℃overnight. (typically, antiserum was diluted 1:2000 antibody to 1-10 ug/ml). (500 ml of 1×TBS+2.5g casein+250 ul Tween as diluent).

4.6.3 the membranes were removed and washed 5 times with TBST for 5min each (shaking on a horizontal shaker).

4.6.4 the membrane was removed and immersed in a secondary antibody diluted with a diluent at 25℃for 1h. Goat anti-rabbit-HRP 1:5000).

4.7ECL Exposure

4.7.1 diluting and mixing A, B luminous solutions in equal proportion (500 ul/film respectively), placing the film on a preservative film, uniformly dripping AB mixed solution on the film, covering the preservative film, and carrying out light-shielding reaction for 1min;

4.7.2 placing the film on filter paper to suck the liquid (no liquid flows out, the film can not be dried), transferring the film into a clean preservative film to be wrapped (keeping the front surface flat), and fixing the film in a cassette;

4.7.3 placing the film cassette in a darkroom, taking out the film and rapidly placing the film on the film in the film cassette, closing the film cassette, and exposing according to the visible fluorescence intensity (generally, the exposure time is 1min, and tabletting overnight can be selected if the strip is weaker);

4.7.4 opening the cassette, taking out the film and completely invading the developing solution for 1min immediately;

4.7.5 taking out the film, rinsing with deionized water, and then immersing into the fixing solution for 1min;

4.7.6 taking out the film, rinsing with deionized water, airing, calibrating a Marker, and analyzing the experimental result.

4.8Western Blot results

The prepared rabbit antibody has weaker suspected signals with spleen, has stronger prorenal positive reaction and accords with the expected signal of 62 kDa.

Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.

SEQUENCE LISTING

<110> Shandong province fresh water fishery institute (Shandong province fresh water fishery monitoring center)

<120> an epitope polypeptide of an anti-snakehead Nramp antibody and application thereof in antibody preparation

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> artificial sequence

<400> 1

Asp Lys Thr Asn Ile Glu Val Ser Glu Lys Cys

1 5 10

<210> 2

<211> 562

<212> PRT

<213> artificial sequence

<400> 2

Met Lys Thr Glu Gln Asp Gly Glu Val Phe Glu Glu Asp Ser Pro Glu

1 5 10 15

Glu Asn Gly Val His Thr Asn Gln Tyr Ser Ser Ile Ser Pro Pro Ala

20 25 30

Ser Pro Glu Ala Gln Glu Asn Ala Phe Ser Thr Tyr Phe Glu Asp Lys

35 40 45

Val Pro Ile Pro Glu Ser Val Asn Gln Ser Phe Ser Phe Arg Lys Leu

50 55 60

Trp Ala Phe Thr Gly Pro Gly Phe Leu Met Ser Ile Ala Tyr Leu Asp

65 70 75 80

Pro Gly Asn Ile Glu Ser Asp Leu Gln Ser Gly Ala Lys Ala Gly Phe

85 90 95

Lys Leu Leu Trp Val Leu Leu Val Ser Thr Ile Ile Gly Leu Leu Leu

100 105 110

Gln Arg Leu Ala Ala Arg Leu Gly Val Val Thr Gly Met His Leu Ala

115 120 125

Glu Val Cys Asn Arg Gln Tyr Pro Thr Val Pro Arg Ile Ile Leu Trp

130 135 140

Leu Met Val Glu Leu Ala Ile Ile Gly Ser Asp Met Gln Glu Val Ile

145 150 155 160

Gly Cys Ala Ile Ala Leu Asn Leu Leu Ser Val Gly Arg Ile Pro Leu

165 170 175

Trp Ala Gly Val Leu Ile Thr Ile Thr Asp Thr Phe Val Phe Leu Phe

180 185 190

Leu Asp Lys Tyr Gly Leu Arg Lys Leu Glu Ala Phe Phe Gly Phe Leu

195 200 205

Ile Thr Val Met Gly Leu Ser Phe Gly Tyr Glu Tyr Val Leu Val Lys

210 215 220

Pro Asp Gln Gly Glu Leu Leu Lys Gly Met Phe Leu Pro Tyr Cys Ala

225 230 235 240

Gly Cys Gly Pro Val Gln Leu Glu Gln Ala Val Gly Val Val Gly Ala

245 250 255

Val Ile Met Pro His Asn Ile Tyr Leu His Ser Ala Leu Val Lys Ser

260 265 270

Arg Glu Val Asp Arg Lys Asn Lys Lys Glu Val Lys Glu Ala Asn Lys

275 280 285

Tyr Phe Phe Ile Glu Ser Ser Ile Ala Leu Phe Val Ser Phe Leu Ile

290 295 300

Asn Val Phe Val Val Ala Val Phe Ala Glu Ala Phe Tyr Asp Lys Thr

305 310 315 320

Asn Ile Glu Val Ser Glu Lys Cys Asn Ala Ser Gly Ile Pro His Thr

325 330 335

Asn Val Phe Pro Tyr Asn Asn Asp Thr Leu Glu Val Asp Ile Tyr Lys

340 345 350

Gly Gly Val Val Leu Gly Cys Val Phe Gly Pro Ala Ala Leu Tyr Ile

355 360 365

Trp Ala Ile Gly Ile Leu Ala Ala Gly Gln Ser Ser Thr Met Thr Gly

370 375 380

Thr Tyr Ser Gly Gln Phe Val Met Glu Gly Phe Leu Asn Leu Arg Trp

385 390 395 400

Ser Arg Phe Ala Arg Val Leu Leu Thr Arg Ser Ile Ala Ile Thr Pro

405 410 415

Thr Leu Leu Val Ala Ile Phe Gln Asp Val Arg His Leu Thr Gly Met

420 425 430

Asn Asp Phe Leu Asn Val Leu Gln Ser Met Gln Leu Pro Phe Ala Leu

435 440 445

Ile Pro Ile Leu Thr Phe Thr Ser Met Thr Ser Ile Met Asn Asp Phe

450 455 460

Ala Asn Ala Leu Trp Trp Lys Ile Ser Gly Gly Val Val Ile Leu Val

465 470 475 480

Val Cys Ala Ile Asn Val Tyr Phe Val Val Val Tyr Val Thr Ser Leu

485 490 495

Asn Ser Val Leu Leu Tyr Val Phe Ala Ala Leu Leu Ser Val Ala Tyr

500 505 510

Leu Cys Phe Val Gly Tyr Leu Ala Trp Tyr Cys Leu Ile Ala Leu Gly

515 520 525

Val Ser Cys Leu Asp Phe Gly Ser Arg Met Gln Thr Gly Leu Ser Arg

530 535 540

Pro Ile Asp Ile Tyr Leu Leu Asn Asp Met Asp Thr Asp Ala Leu Val

545 550 555 560

Glu Arg

Claims (1)

1. An application of an epitope polypeptide of an anti-snakehead Nramp antibody in preparing the anti-snakehead Nramp antibody, which is characterized in that the amino acid sequence of the epitope polypeptide is selected from the following: a sequence consisting of SEQ ID NO. 1;

the application is as follows: preparing the anti-snakehead Nramp antibody by using the epitope polypeptide as an antigen;

the anti-snakehead Nramp antibody is a polyclonal antibody;

the anti-snakehead Nramp antibody is used for detecting snakehead Nramp proteins;

the anti-snakehead Nramp antibody is obtained by immunizing rabbits with an antigen consisting of the antigen epitope polypeptide.