CN112180087A

CN112180087A - ELISA method for detecting riemerella anatipestifer antibody, kit and application thereof

Info

Publication number: CN112180087A
Application number: CN202011143764.8A
Authority: CN
Inventors: 周祺; 麦凯杰; 杨德鸿; 罗翠芬; 严专强; 周庆丰; 陈丽
Original assignee: Wens Foodstuff Group Co Ltd
Current assignee: Wens Foodstuff Group Co Ltd
Priority date: 2020-10-23
Filing date: 2020-10-23
Publication date: 2021-01-05
Anticipated expiration: 2040-10-23
Also published as: CN112180087B

Abstract

The invention discloses an ELISA method for detecting a riemerella anatipestifer antibody, a kit and application thereof. The ELISA method is characterized in that the envelope antigen is recombinant OmpA protein, and the amino acid sequence of the recombinant OmpA protein comprises the amino acid sequence shown in SEQ ID NO: 1; the nucleotide sequence includes the nucleotide sequence shown as SEQ ID NO: 2. The method has good reactogenicity, specificity, sensitivity and repeatability, can be used for clinical riemerella anatipestifer serum antibody detection, and can be used for efficiently detecting riemerella anatipestifer antibodies in farms, so that outbreaks of riemerella anatipestifer infection can be prevented and controlled.

Description

ELISA method for detecting riemerella anatipestifer antibody, kit and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an ELISA method for detecting a riemerella anatipestifer antibody, a kit and application thereof.

Background

Riemerella Anatipestifer (RA) infection is one of main bacterial infectious diseases of domestic ducks, infectious septicemia characterized by cellulosic pericarditis, air sacculitis, perihepatitis and caseous salpingitis is clinically characterized, and the fatality rate is as high as 90%. And RA serotypes are complex, 21 serotypes are reported at present, cross protection among the serotypes is lacked, and vaccine control is mostly carried out on a single serotype or a few serotypes, so that effective control is difficult to obtain in areas with unknown epidemic serotypes. The clinical symptoms of RA infection are not specific, RA is difficult to distinguish from the infection of bacterial pathogens (including escherichia coli, salmonella enterica and pasteurella multocida) only by symptoms, and meanwhile, antibiotics are widely used clinically, the cost is increased rapidly, and drug-resistant strains are induced, which bring great difficulty and high economic loss to the clinical diagnosis and control of RA.

RA infection frequently outbreaks in waterfowl culture areas in China, RA vaccination is more and more extensive, and aiming at the current situations that the technologies of livestock farms and basic veterinary quarantine departments are deficient and equipment is simple, a simple and efficient antibody detection method needs to be established to monitor the RA antibody level so as to prevent and control outbreaks of Riemerella anatipestifer infection.

Disclosure of Invention

The invention aims to provide an ELISA method for detecting a riemerella anatipestifer antibody, a kit and application thereof, so that the riemerella anatipestifer antibody level can be efficiently detected and monitored, and outbreak of riemerella anatipestifer infection can be prevented and controlled.

According to one aspect of the present invention, there is provided an ELISA method for detecting a riemerella anatipestifer antibody, the envelope antigen of the ELISA method being a recombinant OmpA protein, the amino acid sequence of which comprises the amino acid sequence set forth in SEQ ID NO: 1; the nucleotide sequence of the recombinant OmpA protein comprises the nucleotide sequence shown as SEQ ID NO: 2.

In certain embodiments, the amino acid sequence of the recombinant OmpA protein as a coating antigen comprises a sequence identical to SEQ ID NO: 1 sequences having at least 90% homology; the nucleotide sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 2 having at least 90% homology.

In certain embodiments, the amino acid sequence of the recombinant OmpA protein as a coating antigen comprises a sequence identical to SEQ ID NO: 1 sequences having at least 95% homology; the nucleotide sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 2 with at least 95% homology.

In certain embodiments, the amino acid sequence of the recombinant OmpA protein as a coating antigen is SEQ ID NO: 1; the nucleotide sequence of the recombinant OmpA protein is SEQ ID NO: 2.

In some embodiments, the amplification primer sequences of the gene sequence of the recombinant OmpA protein as a coating antigen are as follows:

OmpAcut-up(SEQ ID NO：3)：5’-ATGAACAGAGATAAGGATAAAGATGGAGT-3’；

OmpAcut-dw(SEQ ID NO：4)：5’-TTATTGAACTGCATACGCTTCCTGAGGCTT-3’。

in certain embodiments, the ELISA method comprises the steps of:

1) recombinant OmpA protein was used as coating antigen:

the amino acid sequence of the recombinant OmpA protein comprises the amino acid sequence shown as SEQ ID NO: 1, and the nucleotide sequence comprises a sequence shown as SEQ ID NO: 2;

or the amino acid sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 1, the nucleotide sequence comprising a sequence having at least 90% homology to SEQ ID NO: 2 sequences having at least 90% homology;

or the amino acid sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 1, the nucleotide sequence comprising a sequence having at least 95% homology to SEQ ID NO: 2 sequences having at least 95% homology;

or the amino acid sequence of the recombinant OmpA protein is SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2;

or the recombinant OmpA protein gene sequence amplification primer sequence is as follows:

OmpAcut-up(SEQ ID NO：3)：5’-ATGAACAGAGATAAGGATAAAGATGGAGT-3’；

OmpAcut-dw(SEQ ID NO：4)：5’-TTATTGAACTGCATACGCTTCCTGAGGCTT-3’。

2) putting the envelope antigen into an enzyme label plate for envelope and sealing to obtain sealing liquid;

3) throwing off the confining liquid in the step 2), incubating with a primary antibody, and then incubating with a secondary antibody;

4) throwing away the secondary antibody incubation liquid in the step 3), developing by adopting TMB developing solution, adding developing termination solution, and terminating the reaction;

5) measuring the OD of the liquid in the hole of the ELISA plate in the step 4) by using an ELISA reader_450nmValue according to OD_450nmAnd (5) judging whether the sample is positive or negative by using a critical value.

In certain embodiments, OD_450nmThe critical value is 0.4855.

In certain embodiments, the coating concentration of the coating antigen is 2.5. mu.g/mL.

According to another aspect of the invention, an ELISA kit for detecting riemerella anatipestifer antibody is provided, and the coating antigen of the kit is recombinant OmpA protein:

or the amino acid sequence of the recombinant OmpA protein is SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2.

According to the third aspect of the invention, the ELISA kit for detecting the riemerella anatipestifer antibody is applied to the detection of the riemerella anatipestifer antibody.

The invention has the beneficial effects that:

1. the recombinant OmpA protein is used as the envelope antigen of the ELISA method for detecting the riemerella anatipestifer antibody, so that the riemerella anatipestifer can be efficiently and simply detected and monitored, and outbreak of riemerella anatipestifer infection can be prevented and controlled.

2. The recombinant OmpA protein is used as a coating antigen of an ELISA method for detecting the riemerella anatipestifer antibody, so that the method has good reactogenicity, specificity, sensitivity and repeatability, can be used for detecting clinical riemerella anatipestifer serum antibody, can efficiently detect the riemerella anatipestifer antibody in a farm, and can prevent and control outbreak of riemerella anatipestifer infection.

3. The recombinant OmpA protein is used as a coating antigen of an ELISA method for detecting the riemerella anatipestifer antibody, and is suitable for detecting various serotypes of riemerella anatipestifer.

Drawings

FIG. 1 shows SDS-PAGE analysis of purified OmpA fusion protein: m is a protein molecular mass standard, 1 is a supernatant after ultrasonic treatment, 2 is an inclusion body after ultrasonic treatment, and 3 is a purified OmpA fusion protein;

FIG. 2 shows the results of the antigen reactivity analysis of the recombinant OmpA protein: wensA-wensH are respectively RA8 different serotypes, and Neg (-) is a negative standard control;

FIG. 3 shows the results of antigen specificity analysis of recombinant OmpA protein: wherein PM is poultry pasteurella multocida antibody positive serum, MA is duck mycoplasma antibody positive serum, E.coli is poultry escherichia coli antibody positive serum, Mix is parvovirus, reovirus, viral hepatitis virus, AIV, duck viral enteritis virus antibody positive serum, Po. (+) is positive control, Neg. (-) is negative control;

FIG. 4 shows the determination of ELISA cut-off values: wherein the abscissa is the serum sample number and the ordinate is OD_450nmA value;

FIG. 5 shows the results of ELISA sensitivity tests: the abscissa represents the positive serum dilution and the ordinate represents OD_450nmA value;

FIG. 6 shows an inactivated vaccineDetermination results of antibody growth-reduction rule: the abscissa is the antibody detection time point and the ordinate is OD_450nmValues, ABC is a triple inactivated vaccine containing three serotypes of wensA, B and C, and FK is a double inactivated vaccine containing two serotypes of wensF and K.

Detailed Description

The specific embodiment is as follows:

design of 1 OmpA Gene primer

The OmpA gene full-length Sequence (NCBI gene Sequence number: Sequence ID: CP041029.1) of Riemerella anatipestifer is referenced, DNA STAR series software is used for carrying out Sequence shearing comparison and homology analysis to obtain a target Sequence with high homology, SignalP-5.0 and Bepipred 1.0 are used for respectively carrying out signal peptide and antigen epitope analysis on the target Sequence, and finally the OmpA truncated target Sequence is obtained. Designing a pair of primers to amplify an OmpA truncated gene sequence, wherein the primer sequences are as follows:

OmpAcut-up(SEQ ID NO：3)：

5’-ATGAACAGAGATAAGGATAAAGATGGAGT-3’；

OmpAcut-dw(SEQ ID NO：4)：

5’-TTATTGAACTGCATACGCTTCCTGAGGCTT-3’。

the size of the OmpA truncated target sequence is 375bp (the amino acid sequence is shown as SEQ ID NO: 1; the nucleotide sequence is shown as SEQ ID NO: 2)).

2 inducible expression of recombinant plasmid pGEX-OmpA

Construction of pGEX-OmpA plasmid: connecting the OmpA truncated target sequence fragment to pGEX-4T-1 framework vector, transforming and purifying to obtain recombinant plasmid pGEX-OmpA with correct sequencing, wherein the sequence of the recombinant plasmid pGEX-OmpA is shown as SEQ ID NO: 5, respectively.

Prokaryotic expression of recombinant OmpA protein: the plasmid pGEX-OmpA with correct sequencing result is transformed into an expression bacterium Rosetta, aseptically coated on an LB/AMP plate culture medium, and cultured for 18h at 37 ℃. Selecting the monoclonal with positive PCR identification result, inoculating the monoclonal with the positive PCR identification result into 10mL LB/AMP liquid culture medium according to the ratio of 1:100, and culturing the monoclonal with the positive PCR identification result to OD at the temperature of 200r/min on a shaking table_600nmWhen the value is about 0.6, IPTG with the final concentration of 0.4mmol/L is added, and the expression is induced for 6h at the temperature of 25 ℃ and at the speed of 150 r/min. Centrifuging 1mL of bacterial solution at 8000r/min for 5min, and discarding the supernatantResuspending with 50. mu.L of LPBS, adding 50. mu.L of 2 XProteins loading buffer, boiling at 100 ℃ for 5min, centrifuging, collecting the supernatant, and performing SDS-PAGE analysis to select strains with positive protein expression.

Large-Scale expression of 3 recombinant OmpA protein

Selecting the strain with positive protein expression, carrying out shake culture overnight, inoculating the strain into a liquid culture medium containing 300mL LB/AMP according to the proportion of 1:100, and carrying out induced expression under the optimized condition. Centrifuging the bacterial liquid at 8000r/min for 5min, blowing and beating thallus with sterilized PBS, centrifuging again, and repeating for several times until the supernatant becomes clear. And blowing off the finally obtained thalli by using 30mL of sterilized PBS, and carrying out ultrasonic crushing in an ice bath, wherein the ultrasonic program is as follows: sonication was performed 3 times 3s with intervals of 8s, ten minutes each. The ultrasonic thallus is centrifuged for 10min at 12000r/min at 4 ℃, and bacterial supernatant lysate is collected.

Purification of 4 recombinant OmpA protein

The purification method of the recombinant OmpA protein is as follows:

after equilibrating GST-tag Purification Resin 2-3 times with a buffer equal in volume to the gel, 4mL of bacterial lysate supernatant was added to each 0.5mL of gel (1: 8), and the mixture was slowly shaken for 60min at 4 ℃ on a side-shaking table or a horizontal shaking table. The mixture of lysate and GST-tag Purification Resin was loaded into an appropriate empty column tube, the cap at the bottom of the Purification column was opened, the liquid in the column was drained by gravity, the column was washed 5 times, and 1-2 column volumes of lysis buffer were added each time. After GST tag protein is combined with a purification column and is fully washed by a washing solution, elution is repeated for three times by using an eluent containing glutathione, the eluent is the target protein, and the concentration of the protein is measured by a nucleic acid protein concentration measuring instrument.

As a result, as shown in FIG. 1, the purification of the recombinant OmpA protein was successful.

5 recombinant OmpA protein antigen reactivity assay

(1) The purified recombinant OmpA protein is used as a coating antigen;

(2) diluting the coating antigen in the step (1) to 2.5 mu g/mL by using carbonate coating solution, adding 100 mu L/hole into an enzyme label plate, and coating for 30min at 37 ℃;

(3) removing the coating solution in the holes of the ELISA plate in the step (2), PBST cleaning for 4 times, each time for 2min, patting to dry, adding 5% skimmed milk powder 200 muL/hole, and sealing at 37 ℃ for 30 min;

(4) removing the sealing liquid in the holes in the step (3), washing with PBST for 4 times, each time for 2min, patting to dry, adding primary antibodies (8 rabbit polyclonal antibodies resisting different serotypes of RA and healthy non-immune rabbit serum stored in a laboratory are respectively used as primary antibodies) diluted by 5% skimmed milk powder 1:100 according to 100 mu L/hole, and incubating for 90min at 37 ℃;

(5) and (4) throwing away primary antibody liquid in the holes in the step (4), washing with PBST for 4 times, each time for 2min, beating to dry, adding PBS 1 at 100 mu L/hole: incubating goat anti-rabbit enzyme-labeled secondary antibody diluted by 2000 for 120min at 37 ℃;

(6) throwing off the secondary antibody in the hole in the step (5), washing for 5 times by PBST (Poly-p-phenylene benzobisoxazole) (2 min each time), beating to dry, adding TMB (Tetramethylbenzidine) color development solution according to 100 mu L/hole, reacting for 10min in a dark place, adding color development termination solution according to 100 mu L/hole, and terminating the reaction;

(7) measuring the OD of the liquid in the hole in the step (6) on a microplate reader_450nmThe value is obtained.

As shown in the result of the attached figure 2, the recombinant OmpA protein antigen has good reaction with the anti-RA rabbit polyclonal antibody and does not react with the negative standard substance, which indicates that the reactivity is good.

The preparation and identification method of the anti-RA rabbit polyclonal antibody comprises the following steps:

1) preparation of riemerella anatipestifer inactivated antigen

a. The method comprises the steps of streaking and inoculating riemerella anatipestifer to a TSA plate containing 5% newborn bovine serum, culturing at 37 ℃ for 24 hours, selecting a single colony to be inoculated to a TSB culture medium containing 5% newborn bovine serum, performing shake culture at 37 ℃ and 160rpm/min for 16-24 hours to obtain a seed solution, inoculating the seed solution to the TSB culture medium containing 5% newborn bovine serum according to the volume ratio of 1:100, performing enlarged culture, and performing shake culture at 37 ℃ and 160rpm/min for 16 hours to obtain a bacterial solution after enlarged culture.

b. Adding a formaldehyde solution into the enlarged and cultured bacterial liquid according to the volume ratio of 0.5 percent, inactivating the solution for 24 hours at 37 ℃, streaking the inactivated bacterial liquid on a TSA plate containing 5 percent newborn calf serum, and culturing the solution for 24 hours at 37 ℃, wherein if no bacteria grow, the inactivation is determined to be complete.

c. Washing the inactivated bacterial liquid at 5000rpm/min for 10min with sterilized PBSNext, the OD of the bacterial liquid was adjusted_600nmThe value was 1.0.

2) Immunization

Immunization was performed according to the following conditions: on day 1, 0.1ml of the bacterial solution was injected intravenously into rabbits; on day 3, 0.2ml of the bacterial solution was injected intravenously into rabbits; on day 7, 0.5ml of the bacterial solution was injected intravenously into rabbits; on day 10, 1ml of the bacterial solution was injected intravenously into rabbits; on day 14, 1.5ml of the bacterial solution was injected intravenously into rabbits; on day 20, 2.0ml of the bacterial solution was injected intravenously into rabbits; 7 days after the last immunization, blood was collected via the marginal vein of the ear, serum was separated, and the antibody level was examined.

3) Antibody level detection and collection preservation

Detecting the serum antibody level by using the agar-agar gel method, collecting blood by heart when the antibody level reaches 1:64, separating serum, packaging, labeling and storing at-70 deg.C for use.

6 OmpA protein antigen specificity assay

And (2) analyzing the antigen specificity of the OmpA protein by referring to an ELISA step in the '5 recombinant OmpA protein antigen reactivity analysis', wherein the primary antibody in the '5 recombinant OmpA protein antigen reactivity analysis' step (4) is changed into antibody positive serum (such as common pathogenic microorganisms such as parvovirus, reovirus, viral hepatitis virus, avian influenza, duck mycoplasma, avian escherichia coli, avian pasteurella multocida and duck viral enteritis virus) of clinically different duck pathogenic microorganisms, and a negative standard substance primary antibody and a positive standard substance primary antibody are used as controls.

The results are shown in figure 3, the recombinant OmpA protein antigen does not react with antibodies of avian pasteurella multocida, duck mycoplasma, avian Escherichia coli, parvovirus, reovirus, viral hepatitis virus, AIV, duck viral enteritis virus and the like, and the antigen specificity is good.

7 preparation of ELISA negative and positive standard substance primary antibody

The experiment selects 10 healthy and non-immune ducklings with the age of 9 days, and the ducklings are divided into an experimental group (5) and a control group (5): experiment group intramuscular injection serosity-Duck infectious serositis bivalent inactivated vaccine (type 1 SG4 strain + type 2 ZZY7 strain) (approval document: veterinary drug New word 190022211, manufacturing enterprise: Zhaoqing Dahua pesticide Co., Ltd.), and serum is collected one week after immunization, namely positive standard substance; and meanwhile, setting a non-immune control group, feeding and collecting serum under the same condition, and obtaining a negative standard substance.

8 determination of ELISA cut-off values

46 parts of clinical non-immune duck serum, 87 parts of test RA negative serum and 133 parts of serum are detected by referring to an ELISA step in '5 recombinant OmpA protein antigen reactivity analysis', each sample is repeated for 3 holes in parallel, and negative sample OD is calculated_450nmAverage value of values

And variance (SD) as calculated

Calculation of the resulting ELISA OD_450nmThe threshold value is 0.4855 (shown in fig. 4). When OD of the sample to be tested_450nmWhen the value is greater than 0.4855, the result is judged to be positive; when OD of the sample to be tested_450nmWhen the value is less than 0.4855, the result is judged as negative.

9 ELISA sensitivity test

3 positive sera were taken from 1:100 was diluted to 1:12800 at the start, and the detection was performed by referring to the ELISA procedure in "5 recombinant OmpA protein antigen reactivity assay", and from the results (FIG. 5), the method still detected antibody positivity by diluting the clinically positive serum 800 times, indicating high sensitivity.

10 ELISA within and between batches repeatability test

Performing within-batch and within-batch repeatability tests by referring to ELISA step in '5 recombinant OmpA protein antigen reactivity analysis', coating the ELISA plate with the same batch of protein, detecting 3 positive serums with different antibody titers, repeating 4 wells for each sample in parallel, and measuring OD of each well_450nmThe standard deviation of each sample was calculated, and the intra-batch coefficient of variation (C.V) was calculated for each sample, C.V ═ SD/MN × 100%. Coating an enzyme label plate in three different time periods under the same experimental environment, respectively detecting 3 parts of positive serum with different antibody titers, repeating 4 holes in each part of serum in parallel, and detecting OD of each hole_450nmValue, calculate each sampleThe coefficient of variation between batches. As can be seen from the results in Table 1, the variation coefficient of the repeatability tests in batch and between batches is lower than 10%, which shows that the method has good repeatability and stable results.

11 inactivated vaccine antibody length-reduction rule determination

Three serotypes of wensA, B and C triple inactivated vaccines and two serotypes of wensF and K are respectively used for immunizing a 9-day-old healthy duck group, blood is collected every other week, the law of antibody growth and elimination after immunization of inactivated vaccines is detected by referring to an ELISA step in a 5-recombinant OmpA protein antigen reactivity analysis, and the result shows that after the immunization of RA inactivated vaccines of different serotypes of the double triple, antibodies are converted to be positive basically in 1 week, gradually reduced after 4 weeks, basically negative after 6 weeks, and the antibody peak period is 2 weeks to 3 weeks after the immunization, so that the law of antibody growth and elimination of the inactivated vaccines is met (the result is shown in figure 6).

12 detection of clinical serum

The detection is carried out by using the established RA-OmpA-ELISA method, and the specific steps are as follows:

1) designing a pair of primers by using a Riemerella anatipestifer whole gene sequence to amplify a truncated OmpA gene, constructing a recombinant expression vector, obtaining a soluble recombinant OmpA expression protein through induced expression, and purifying and quantifying the protein to be used as a coating antigen;

2) diluting the coating antigen in the step 1) to 2.5 mu g/mL by using carbonate coating solution, adding 100 mu L/hole into an enzyme label plate, and coating for 30min at 37 ℃;

3) removing the coating solution in the holes of the ELISA plate in the step 2), PBST cleaning for 4 times, each time for 2min, patting to dry, adding 5% skimmed milk powder 200 muL/hole, and sealing at 37 ℃ for 30 min;

4) removing the sealing liquid in the holes in the step 3), washing with PBST for 4 times, each time for 2min, patting to dry, adding clinical serum (as primary antibody) diluted by 5% skimmed milk powder 1:100 into each hole at a concentration of 100 μ L, and incubating at 37 deg.C for 90 min;

5) and (3) throwing off the clinical serum-primary antibody liquid in the holes in the step 4), washing with PBST for 4 times, each time for 2min, beating to dry, adding PBS 1 according to 100 mu L/hole: incubating goat anti-duck enzyme-labeled secondary antibody diluted by 2000 for 120min at 37 ℃;

6) throwing away the secondary antibody in the holes in the step 5), washing for 5 times by PBST (Poly-p-phenylene benzobisoxazole) (2 min each time), beating to dry, adding TMB (Tetramethylbenzidine) color development solution according to 100 mu L/hole, reacting for 10min in a dark place, adding color development termination solution according to 100 mu L/hole, and terminating the reaction;

7) measuring the OD of the liquid in the hole in the step 6) on a microplate reader_450nmThe value is determined according to the critical value, when OD is positive_450nmValues above 0.4855 were positive and below 0.4855 were negative.

The results of the tests shown in table 2 above show: the method can specifically detect the antibody level of riemerella anatipestifer, wherein the RA positive in 48 parts of serum after spot-check immunization is 42 parts, the positive rate reaches 87.5%, the RA positive in 46 parts of serum without immunization is 1 part, and the positive rate reaches 2.2%.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

The present invention is not limited to the above description of the embodiments, and those skilled in the art should, in light of the present disclosure, appreciate that many changes and modifications can be made without departing from the spirit and scope of the invention.

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ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1860

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1920

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 1980

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 2040

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 2100

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 2160

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 2220

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 2280

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 2340

aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 2400

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 2460

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 2520

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 2580

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 2640

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 2700

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2760

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2820

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2880

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 2940

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 3000

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 3060

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 3120

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 3180

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 3240

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 3300

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 3360

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 3420

ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 3480

ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 3540

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca 3600

ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat 3660

tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct 3720

cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg 3780

aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac 3840

tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc 3900

cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg 3960

tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg 4020

cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg 4080

tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca 4140

tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg 4200

cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc 4260

tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac 4320

gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg 4380

gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg 4440

ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata 4500

ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc 4560

tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca 4620

atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa 4680

ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 4740

tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 4800

caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 4860

tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg 4920

tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 4980

cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 5040

cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg 5100

gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca 5160

cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt 5220

tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg 5280

gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaatt 5326

Claims

1. An ELISA method for detecting riemerella anatipestifer antibody, wherein the coating antigen of the ELISA method is recombinant OmpA protein, and the amino acid sequence of the recombinant OmpA protein comprises the amino acid sequence shown as SEQ ID NO: 1; the nucleotide sequence of the recombinant OmpA protein comprises a nucleotide sequence shown as SEQ ID NO: 2.

2. The ELISA method for detecting a Riemerella anatipestifer antibody according to claim 1, wherein the amino acid sequence of said recombinant OmpA protein comprises a sequence identical to SEQ ID NO: 1 sequences having at least 90% homology; the nucleotide sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 2 having at least 90% homology.

3. The ELISA method for detecting a Riemerella anatipestifer antibody according to claim 1, wherein the amino acid sequence of said recombinant OmpA protein comprises a sequence identical to SEQ ID NO: 1 sequences having at least 95% homology; the nucleotide sequence of the recombinant OmpA protein comprises a nucleotide sequence similar to SEQ ID NO: 2 with at least 95% homology.

4. The ELISA method for detecting a Riemerella anatipestifer antibody according to claim 1, wherein the amino acid sequence of the recombinant OmpA protein is SEQ ID NO: 1; the nucleotide sequence of the recombinant OmpA protein is shown as SEQ ID NO: 2.

5. The ELISA method for detecting Riemerella anatipestifer antibody of claim 1, wherein the gene sequence PCR amplification primer of the recombinant OmpA protein is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

6. The ELISA method for detecting Riemerella anatipestifer antibodies of any of claims 1-5, wherein the ELISA method comprises the following steps:

1) recombinant OmpA protein is used as a coating antigen;

5) measuring the OD of the liquid in the hole of the ELISA plate in the step 4) by using an ELISA reader_450nmValue according to OD_450nmAnd (5) judging the negative and positive by using a critical value.

7. The ELISA method for detecting Riemerella anatipestifer antibody of claim 6, wherein the OD_450nmThe critical value is 0.4855.

8. The ELISA method for detecting Riemerella anatipestifer antibody of claim 6, wherein the coating concentration of the coating antigen is 2.5 μ g/mL.

9. An ELISA kit for detecting a Riemerella anatipestifer antibody, wherein the envelope antigen of the kit is the recombinant OmpA protein according to any one of claims 1 to 4.

10. The use of the ELISA kit of claim 8 to detect riemerella anatipestifer antibodies.