CN114324859A - Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a coating antigen for detecting mycoplasma synoviae antibody, a kit and a detection method thereof. Detecting the envelope antigen of the mycoplasma synoviae antibody as a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises the amino acid sequence as set forth in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein includes the nucleotide sequence set forth in SEQ ID NO: 2. The recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, and can be used for detecting the clinical mycoplasma synoviae serum antibody and efficiently detecting the mycoplasma synoviae antibody in a farm; the recombinant EFG protein is used as a coating antigen of an ELISA method for detecting the mycoplasma synoviae antibody, so that the detection method has good reactogenicity, specificity, sensitivity and repeatability, can be used for clinical detection of the mycoplasma synoviae serum antibody, and provides certain guidance for formulation of a mycoplasma synoviae vaccine immunization program, thereby achieving the purpose of preventing and controlling the infection of the mycoplasma synoviae.

Description

Coating antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a coating antigen for detecting mycoplasma synoviae antibody, a kit and a detection method thereof.

Background

Mycoplasma Synoviae (MS) is one of the main pathogenic Mycoplasma of poultry, the main infection group is chickens or turkeys, and poultry infected with MS mainly shows synovial cyst membrane and tendon sheath synovitis with joint exudation, and can also cause subclinical respiratory symptoms and air sacculitis. MS infects both chickens and turkeys at all ages, but chicks are the most infectious and become sexually germ-carrying once infected. Most of MS infections are chronic or recessive infections, and the acute cases are few, and the MS infections are clinically often expressed as mixed infections with NDV, IBDV, Escherichia coli and other pathogens. MS is spread horizontally, mainly by contact between healthy and sick chickens, and also by vertical spread, spread through eggs, and is extremely harmful.

At present, the prevention and control of MS are mainly realized through drug prevention and vaccine immunization. In the aspect of vaccine use, foreign parental breeding chickens are immunized with the live vaccine once at the age of 4-6 weeks, and the immunization program of 'one live and one dead' or 'one live and two dead' is mainly adopted at home. The immune effect of the vaccine needs to be effectively detected by an antibody to achieve the aim of preventing and controlling the infection of the mycoplasma synoviae, but the existing detection method has poor antigen specificity and sensitivity and cannot effectively evaluate the antibody of the vaccine.

Disclosure of Invention

In view of the above, there is a need to provide a coating antigen, a kit and a method for detecting mycoplasma synoviae antibodies.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a coating antigen for detecting antibodies to mycoplasma synoviae, wherein the coating antigen is a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises the amino acid sequence as set forth in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein includes the nucleotide sequence set forth in SEQ ID NO: 2.

Further, the amino acid sequence of the recombinant EFG protein comprises a sequence identical to SEQ ID NO: 1 sequences having at least 90% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO: 2 having at least 90% homology.

Further, the amino acid sequence of the recombinant EFG protein comprises a sequence identical to SEQ ID NO: 1 sequences having at least 95% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO: 2 with at least 95% homology.

Preferably, the amino acid sequence of the recombinant EFG protein is SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is SEQ ID NO: 2.

Further, the amplification primer sequence of the recombinant EFG protein is shown in SEQ ID NO: 3 and SEQ ID NO: 4:

SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；

SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’。

in a second aspect, the present invention provides a kit for detecting a mycoplasma synoviae antibody, wherein the kit comprises a coating antigen using the recombinant EFG protein as an elisa plate.

Furthermore, the kit also comprises an enzyme label plate, a coating solution, a confining liquid, an enzyme-labeled secondary antibody, a TMB color development solution, a stop solution, a washing solution and a serum diluent.

Further, the coating solution is carbonate buffer solution.

Further, the wash solution is PBST.

Further, the enzyme-labeled secondary antibody is a goat anti-rabbit enzyme-labeled secondary antibody.

Further, the confining liquid is 5% skimmed milk powder.

Further, the serum diluent is 5% skimmed milk powder.

In a third aspect, the present invention provides a detection method for detecting mycoplasma synoviae antibodies, comprising the following steps:

s1; diluting the coating antigen with a coating solution, putting the diluted coating antigen into an ELISA plate, and coating the antigen overnight;

s2: removing the coating solution in the step S1, cleaning the cleaning solution, drying the cleaning solution, adding a sealing solution, and sealing;

s3: removing the sealing liquid in the S2, cleaning the washing liquid, drying by beating, adding the serum sample to be tested diluted by the serum diluent, and incubating;

s4: removing the serum sample in S3, washing with a washing solution, drying by beating, adding a diluted enzyme-labeled secondary antibody, and incubating;

s5: removing the enzyme-labeled secondary antibody in the S4, washing with a washing solution, drying by beating, adding a TMB color development solution for light-shielding reaction, and adding a stop solution after the reaction is finished to terminate the reaction;

s6: testing liquid OD in plate hole of enzyme label on enzyme label instrument_450nmValue, OD_450nmWhen the value is not less than 0.3998, the detection result is judged to be positive; when OD of the sample to be tested_450nmWhen the value is < 0.3998, the result of the detection is judged to be negative.

Furthermore, the concentration of the coating antigen coating solution diluted in S1 is 0.8. mu.g/mL, 100. mu.L of the coating antigen is added into each well of the ELISA plate hole, and the mixture is coated overnight at 4 ℃.

Further, the washing solution in S2 is washed for 4 times, each time for 2 min; the blocking solution is added into each well in an amount of 100. mu.L, and blocked at 4 ℃ for 16 h.

Further, the washing solution in S3 is washed for 4 times, each time for 2 min; add 100. mu.L of serum dilutions per well at a rate of 1: and incubating the serum sample to be detected after 800-3200 times of dilution at 37 ℃ for 90 min.

Further, the washing solution in S4 is washed for 4 times, each time for 2 min; add 100. mu.L per well in PBS at 1: the enzyme-labeled secondary antibody after 2000-fold dilution is incubated for 120min at 37 ℃.

Further, washing with a washing solution in S4 for 5 times, each for 2 min; adding 100 μ L of TMB color developing solution into each well, reacting for 10min in dark, adding stop solution into each well at a rate of 100 μ L, and stopping reaction.

The invention has the beneficial effects that:

(1) the recombinant EFG protein is used as the coating antigen of the ELISA method for detecting the mycoplasma synoviae antibody, and can be used for detecting the clinical mycoplasma synoviae serum antibody and efficiently detecting the mycoplasma synoviae antibody in a farm.

(2) The recombinant EFG protein is used as the envelope antigen of the ELISA method for detecting the mycoplasma synoviae antibody, so that the detection method has good reactogenicity, specificity, sensitivity and repeatability, can be used for detecting the mycoplasma synoviae serum antibody clinically, provides a certain guidance for the formulation of the mycoplasma synoviae vaccine immunization program, and achieves the purpose of preventing and controlling the infection of the mycoplasma synoviae.

Drawings

FIG. 1 is a SDS-PAGE analysis of purified recombinant EFG protein: in the figure 1, M is a protein molecular mass standard, 1 is a cracked bacteria supernatant lysate which is induced and collected by EFG-pSmartI, 2 is after the EFG-pSmartI passes through a nickel column, 3 is after impurity washing by 50mMol imidazole, and 4-7 are after elution by 250mMol imidazole;

FIG. 2 is a graph showing the results of the antigen reactivity analysis of the recombinant EFG protein in example 4;

FIG. 3 is a graph showing the results of antigen specificity analysis of the recombinant EFG protein in example 5: wherein, the antibody positive serum Po. (+) is a positive standard substance, and the Neg. (-) is a negative standard substance;

FIG. 4 is a graph showing the results of the antigen sensitivity test for recombinant EFG protein coating in example 6;

FIG. 5 is OD of clinical serum samples tested for different immune backgrounds in example 8_450nmA data plot of values.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further clearly and completely described below with reference to the embodiments of the present invention. It should be noted that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A coating antigen for detecting mycoplasma synoviae antibodies, wherein the coating antigen is a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein is shown as SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is shown as SEQ ID NO: 2, the sequence is optimized by GC content, and is convenient for expression.

The preparation method of the coating antigen comprises the following steps:

1. EFG gene primer design:

the EFG target gene Sequence is obtained by referring to the full-length Sequence of the Mycoplasma synoviae EFG protein gene (NCBI gene Sequence number: Sequence ID: WP-011283211.1) and performing codon optimization. Designing a pair of primers to amplify the target gene sequence of the EFG, wherein the primer sequence is shown as SEQ ID NO: 3 and SEQ ID NO: 4:

SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；

SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’；

the size of the EFG protein target gene sequence is 2088 bp;

2. construction of recombinant plasmid:

connecting the EFG target gene sequence to a pSmart-I skeleton vector, transforming and purifying to obtain a recombinant plasmid pSmartI-EFG with correct sequencing, wherein the sequence of the recombinant plasmid pSmartI-EFG is shown as SEQ ID NO: 5 is shown in the specification;

3. expression and purification of recombinant EFG protein:

3.1 expression of recombinant EFG protein: the recombinant plasmid pSmartI-EFG with correct sequencing result is transformed into an expression bacterium Rosetta, aseptically coated on an LB/Kan plate culture medium, and cultured for 18h at 37 ℃. The monoclonal with positive PCR identification result is selected and inoculated into 10mL LB/Kan liquid culture medium according to the ratio of 1:100, and when the monoclonal is cultured at 37 ℃ and 200r/min of a shaking table until the OD600nm value is about 0.6, IPTG with the final concentration of 0.4mmol/L is added, and the monoclonal is induced and expressed for 6h at 37 ℃ and 150 r/min. Centrifuging 1mL of bacterial liquid at 8000r/min for 5min, discarding the supernatant, resuspending with 50 μ L of PBS, adding 50 μ L of 2 × protein loading buffer, boiling at 100 deg.C for 5min, centrifuging, collecting the supernatant, and performing SDS-PAGE analysis to select strains with positive protein expression;

selecting a strain with positive protein expression, carrying out shake culture overnight, inoculating the strain into a liquid culture medium containing 300mL LB/Kan 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. Centrifuging the ultrasonic thallus at 4 ℃ at 12000r/min for 10min, and collecting bacterial supernatant lysate;

3.2 purification of recombinant EFG protein:

adding nickel into a purification column, balancing the nickel column by using a Tris-HCl buffer solution until the nickel column is completely discolored, adding a cracked bacterial supernatant lysate after the nickel column is balanced, and slowly passing through the column for adsorption. Elution with 50mmol of imidazole followed by elution with 250mmol of imidazole after adsorption gave the purified recombinant EFG protein, the results are shown in FIG. 1, indicating successful purification of the recombinant EFG protein.

Example 2

A kit for detecting mycoplasma synoviae antibodies comprises a coating antigen, an ELISA plate, a coating solution, a confining solution, an ELISA secondary antibody, a TMB (tetramethylbenzidine) color development solution, a stop solution, a washing solution and a serum diluent, wherein the recombinant EFG protein purified in example 1 is used as the coating antigen of the ELISA plate; the coating solution is carbonate buffer solution; the washing solution is PBST; the enzyme-labeled secondary antibody is an enzyme-labeled goat anti-rabbit antibody; the confining liquid is 5% of skimmed milk powder; the serum diluent is 5% skimmed milk powder.

Example 3

The method for detecting the mycoplasma synoviae antibody by using the kit in the embodiment 2 comprises the following steps:

s1; diluting the coated antigen with carbonate buffer solution to a concentration of 0.8. mu.g/mL, adding 100. mu.L/well into an ELISA plate, and coating overnight at 4 ℃;

s2: removing the coating solution in S1, washing with PBST for 4 times (2 min each time), beating, adding 100 μ L of 5% skimmed milk powder sealing solution into each well, and sealing at 4 deg.C for 16 h.

S3: the blocking solution in S2 was removed, PBST was washed 4 times for 2min each, patted dry, 100 μ L per well with 5% skim milk powder at 1: incubating the serum sample to be tested after 800 times of dilution for 90min at 37 ℃;

s4: serum samples were removed in S3, washed 4 times with PBST for 2min each, patted dry, and mixed with PBS at 1: incubating the goat anti-rabbit enzyme-labeled secondary antibody diluted by 2000 times for 120min at 37 ℃;

s5: removing the enzyme-labeled secondary antibody in S4, washing with PBST for 5 times (2 min each time), drying, reacting with 100 μ L of TMB developing solution in dark for 10min, adding stop solution at 100 μ L/hole, and stopping reaction;

s6: testing liquid OD in plate hole of enzyme label on enzyme label instrument_450nmValue, OD_450nmWhen the value is not less than 0.3998, the detection result is judged to be positive; when OD of the sample to be tested_450nmWhen the value is < 0.3998, the result of the detection is judged to be negative.

Determination of negative and positive cut-off values: detecting 29 negative serums, repeating 3 holes for each sample in parallel, and calculating the OD of the negative sample_450nmThe mean (X) and Standard Deviation (SD) of the values were calculated as the threshold value X +3SD, and the resulting ELISA OD was calculated_450nmThe threshold is 0.3998. When OD of the sample to be tested_450nmWhen the value is greater than 0.3998, the result is judged to be positive; when OD of the sample to be tested_450nmWhen the value is less than 0.3998, the result is judged as negative.

Example 4

Detecting the positive standard substance and the negative standard substance respectively according to the detection method of the embodiment 3; the positive standard substance is immune serum or maternal antibody serum;

the immune antibody serum is obtained by injecting attenuated vaccine into muscle at 3 weeks old, injecting inactivated vaccine into muscle at 7 weeks old and injecting inactivated vaccine into muscle at 17 weeks old, and collecting serum two weeks after immunization;

the maternal antibody serum is serum of 1-day-old chicks which are hatched by laying eggs of immunized hens;

the negative standard substance is a serum sample which is not subjected to vaccine immunization injection and has the same culture condition.

The detection result is shown in fig. 2, the recombinant EFG protein antigen reacts well with the immunized serum antibody or maternal antibody serum, and does not react with the negative standard, indicating that the reactivity of the detection method is good.

Example 5

According to the detection method of example 3, different serum samples were respectively detected, the detected serum samples were Newcastle Disease Virus (NDV) antibody positive serum, Avian Influenza (AIV) antibody positive serum, Mycoplasma Gallisepticum (MG) antibody positive serum, avian escherichia coli (e.coli) antibody positive serum, Mycoplasma Anatis (MA) antibody positive serum, and Mycoplasma Bovis (MB) antibody positive serum, and the negative and positive standards in example 4 were used as controls. The results of the detection are shown in FIG. 3. As can be seen from FIG. 3, the recombinant EFG protein-coated antigen of the present invention did not react with other antibody sera, and had good antigen specificity.

Example 6

Taking 3 parts of the positive standard substance in the example 4, and carrying out detection according to the detection method of the example 3, wherein the dilution ratio in S3 is respectively from 1: when the dilution ratio of 800 is up to 1:12800, the detection result is shown in fig. 4, and the detection method of the invention can still detect the antibody positivity after the positive standard serum is diluted up to 3200 times, which indicates that the recombinant EFG protein coated antigen of the invention has high sensitivity.

Example 7

The same batch of protein-coated ELISA plates were used to detect 3 positive sera with different antibody titers, each sample was run in duplicate through 4 wells, and the OD per well was determined_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%. Under the same experimental environment, enzyme labels are coated in three different time periodsPlate, separately detect 3 positive sera of different antibody titers, replicate 4 wells per serum in parallel, detect OD per well_450nmThe results of calculating the lot-to-lot variation coefficient for each sample are shown in table 1. 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.

TABLE 1 results of the within-batch and between-batch reproducibility tests of sera from different positive samples

Example 8

Clinical serum samples of different immune backgrounds were tested according to the test method of example 3, and the immunization program was 3 weeks old intramuscular injection of attenuated vaccine, 7 weeks old intramuscular injection of inactivated vaccine, and 17 weeks old intramuscular injection of inactivated vaccine. The results of the measurements are shown in table 2 and fig. 5.

TABLE 2

Group	Immune background	Age of day	Positive rate
				2018	After 10 weeks of autoimmunity	27 weeks	93.75％
2019	After 10 weeks of autoimmunity	27 weeks	100％
				2020	After 10 weeks of autoimmunity	27 weeks	100％
2021	After 10 weeks of autoimmunity	27 weeks	100％
				2026	12 weeks after secondary immunization	For 19 weeks	100％
2101	12 weeks after secondary immunization	For 19 weeks	93.75％
				2103	4 weeks after primary immunization	11 weeks	100％
2105	1 week after one time of immunization	30 days	56.25％
				2106	Non-immune	16 days	25％
2107	Non-immune	16 days	0％

As can be seen from fig. 5 and table 2, the detection method of the present invention can specifically detect the level of mycoplasma synoviae antibody, wherein the positive rate of the spot test 10 weeks after the triple immunization is 100%, the positive rate of the spot test 12 weeks after the double immunization reaches 93.75%, the positive rate of one group of the first immunization 4 weeks after the first immunization reaches 100%, the positive rate of the first immunization 1 week is only 56.25%, and the positive rate of one group of the two groups of non-immunized chickens reaches 25%, which may indicate the infection with mycoplasma synoviae.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Win food group Ltd

South China university of agriculture

<120> envelope antigen for detecting mycoplasma synoviae antibody, kit and detection method thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 696

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Arg Asp Tyr Asp Leu Lys Asp Tyr Arg Asn Ile Gly Ile Met

1 5 10 15

Ala His Ile Asp Ala Gly Lys Thr Thr Thr Thr Glu Arg Ile Leu Tyr

20 25 30

His Thr Gly Lys Ile His Lys Ile Gly Glu Thr His Asp Gly Val Ser

35 40 45

Gln Met Asp Trp Met Glu Gln Glu Lys Glu Arg Gly Ile Thr Ile Thr

50 55 60

Ser Ala Ala Thr Thr Ala Tyr Trp Lys Asn Lys Arg Ile Asn Ile Ile

65 70 75 80

Asp Thr Pro Gly His Val Asp Phe Thr Val Glu Val Glu Arg Ser Leu

85 90 95

Arg Val Leu Asp Gly Ala Val Ala Val Leu Asp Ala Gln Ser Gly Val

100 105 110

Glu Pro Gln Thr Glu Thr Val Trp Arg Gln Ala Thr Asn Tyr Lys Val

115 120 125

Pro Arg Ile Val Tyr Val Asn Lys Met Asp Lys Ala Gly Ala Asp Phe

130 135 140

Glu Ala Ala Val Ala Ser Val Lys Ser Arg Leu Gly Gly Asn Ala Val

145 150 155 160

Ala Ile Gln Trp Pro Ile Gly Ser Glu Ser Asn Phe Asn Gly Ile Ile

165 170 175

Asp Leu Val Thr Met Thr Ala Thr Thr Tyr Asn Gly Glu Ser Ala Glu

180 185 190

Glu Glu Phe Pro Met Glu Ile Pro Thr Asp Leu Leu Asp Val Ala Lys

195 200 205

Ala Lys Arg Gln Glu Leu Leu Glu Ala Ala Ala Asn Phe Asp Glu Glu

210 215 220

Val Met Met Met Val Leu Glu Gly Ala Asp Val Asp Ile Asp Thr Phe

225 230 235 240

Lys Asn Thr Ile Arg Lys Ala Thr Leu Thr Ser Glu Phe Phe Pro Val

245 250 255

Val Cys Gly Thr Ser Phe Lys Asn Lys Gly Val Lys Lys Met Ile Asp

260 265 270

Ala Val Val Asp Tyr Leu Pro Ser Pro Leu Asp Ile Pro Pro Ile Lys

275 280 285

Ala Tyr Leu Asn Asp Gln Glu Thr Asp Val Val Ala Thr Asp Asp Gly

290 295 300

Glu Phe Ala Ala Leu Ala Phe Lys Val Met Thr Asp Pro Phe Val Gly

305 310 315 320

Ser Leu Thr Phe Phe Arg Val Tyr Arg Gly Val Leu Glu Lys Gly Ser

325 330 335

Tyr Val Tyr Asn Ser Thr Lys Glu Gln Lys Glu Arg Ile Gly Arg Ile

340 345 350

Leu Gln Met His Ala Asn Asn Arg Val Glu Ile Asp Glu Cys Arg Ala

355 360 365

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

370 375 380

Thr Leu Val Gly Glu Lys Ser Pro Lys Val Val Leu Glu Lys Met Val

385 390 395 400

Phe Pro Glu Pro Val Ile Ser Gln Ala Leu Glu Pro Glu Ser Lys Ala

405 410 415

Ala Asn Glu Lys Leu Ser Leu Gly Leu Gln Lys Leu Ser Ala Glu Asp

420 425 430

Pro Thr Phe Arg Thr Tyr Thr Asp Glu Glu Thr Gly Gln Thr Ile Ile

435 440 445

Ser Gly Met Gly Glu Leu His Leu Asp Ile Ile Val Asp Arg Leu Lys

450 455 460

Arg Glu Phe Gly Val Lys Val Lys Val Gly Ala Pro Gln Val Ser Tyr

465 470 475 480

Arg Glu Thr Ile Thr Lys Ser Ala Glu Val Glu Gly Lys His Ile Lys

485 490 495

Gln Ser Gly Gly Lys Gly Gln Tyr Gly His Val Trp Leu Lys Phe Glu

500 505 510

Pro Asn His Asp Gln Gly Phe Glu Phe Ile Asp Lys Ile Val Gly Gly

515 520 525

Lys Ile Pro Lys Glu Tyr Ile Lys Pro Ile Gln Lys Gly Leu Glu Glu

530 535 540

Lys Met Ala Val Gly Ile Leu Ala Gly Tyr Pro Met Ile Asp Val Lys

545 550 555 560

Ala Thr Leu Phe Asp Gly Ser Tyr His Asp Val Asp Ser Ser Glu Leu

565 570 575

Ala Tyr Lys Ile Ala Ala Ser Lys Ala Leu Thr Lys Ala Lys Asp Leu

580 585 590

Ile Gly Thr Val Leu Leu Glu Pro Ile Met Asp Val Ser Val Val Val

595 600 605

Pro Ser Asp His Met Gly Asp Val Ile Gly Asp Leu Ser Arg Arg Arg

610 615 620

Gly Leu Ile Ser Asp Gln Glu Gln Arg Asn Asp Gly Ala Val Ile Val

625 630 635 640

Arg Ala Lys Val Pro Leu Ser Glu Met Phe Gly Tyr Ser Thr Glu Leu

645 650 655

Arg Ser Met Thr Ser Gly Arg Gly Thr Tyr Gln Met Gln Phe Asp His

660 665 670

Tyr Glu Lys Cys Pro Lys Asn Ile Ser Asp Glu Ile Ile Lys Lys Arg

675 680 685

Asn Ile Gln Asn Lys Asp Glu Glu

690 695

<210> 2

<211> 2088

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcacgtg attatgatct gaaagattat cgtaacatcg gtattatggc gcatattgat 60

gcgggtaaaa ccaccaccac cgaacgcatt ctgtatcata ccggtaaaat tcataaaatc 120

ggtgaaacac atgatggtgt ttctcagatg gattggatgg aacaggaaaa agaacgtggc 180

attacaatca ccagcgcagc aaccacagca tattggaaaa ataaacgtat taacatcatc 240

gataccccgg gtcatgttga ttttaccgtt gaagttgaac gtagcctgcg tgttctggat 300

ggcgccgttg cagttctgga tgcacagagc ggtgtggaac cgcagaccga aaccgtgtgg 360

cgccaggcaa ccaattataa agttccgcgt attgtgtatg ttaataaaat ggataaagca 420

ggtgcggatt ttgaagcggc agtagcaagc gttaaaagcc gtctgggtgg caatgcggtt 480

gcaattcagt ggccgatcgg tagcgaaagc aattttaatg gcatcattga tctggttaca 540

atgaccgcga caacctataa tggtgaaagt gcagaagaag aatttccgat ggaaattccg 600

accgatctgc tggatgtggc aaaagcaaaa cgtcaggaac tgctggaagc agcggccaac 660

tttgatgaag aagttatgat gatggtgctg gaaggtgcag atgttgatat tgataccttt 720

aaaaacacca tccgtaaagc aacactgacc agcgaatttt ttccggtagt ttgtggcacg 780

agctttaaaa ataaaggtgt caaaaagatg atcgatgcag tggtggatta tctgccgagc 840

ccgctggata ttccgccgat taaagcatat ctgaatgatc aggaaaccga tgttgtcgca 900

acagatgatg gtgaatttgc agcactggca tttaaagtta tgaccgatcc ttttgttggt 960

agcctgacct tttttcgtgt ctatcgcggt gttctggaaa aaggcagcta tgtttataat 1020

agcacgaaag aacagaaaga acgtattggt cgtattctgc agatgcatgc aaataatcgt 1080

gttgaaattg atgaatgtcg tgcaggtgat attgcagcag cagtgggtct gaaatttacc 1140

accaccggtg atacactggt tggcgaaaaa tcacctaaag ttgttctgga aaaaatggtt 1200

tttccggaac cggttattag ccaggcactg gaaccggaaa gcaaagcagc aaatgaaaaa 1260

ctgagcctgg gtctgcagaa actgagcgca gaagatccga cctttcgtac ctataccgat 1320

gaagaaaccg gtcagaccat tattagcggt atgggtgaac tgcatctgga tattattgtt 1380

gatcgtctga aacgtgaatt tggtgttaaa gttaaagttg gtgcaccgca ggttagctat 1440

cgtgaaacca ttaccaaaag cgcagaagtt gaaggtaaac atattaaaca gagcggtggt 1500

aaaggtcagt atggtcatgt ttggctgaaa tttgaaccga atcatgatca gggttttgaa 1560

tttattgata aaattgttgg tggtaaaatt ccgaaagaat atattaaacc gattcagaaa 1620

ggtctggaag aaaaaatggc agttggtatt ctggcaggtt atccgatgat tgatgttaaa 1680

gcaaccctgt ttgatggtag ctatcatgat gttgatagca gcgaactggc atataaaatt 1740

gcagcaagca aagcactgac caaagcaaaa gatctgattg gtaccgttct gctggaaccg 1800

attatggatg ttagcgttgt tgttccgagc gatcacatgg gtgatgttat tggtgatctg 1860

agccgtcgtc gtggtctgat tagcgatcag gaacagcgta atgatggtgc agttattgtt 1920

cgtgcaaaag ttccgctgag cgaaatgttt ggttatagca ccgaactgcg tagcatgacc 1980

agcggtcgtg gtacctatca gatgcagttt gatcattatg aaaaatgtcc gaaaaatatt 2040

agcgatgaaa ttattaaaaa acgtaatatt cagaataaag atgaagaa 2088

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcacgtg attatgatct gaaag 25

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttcttcatct ttattctgaa tattac 26

<210> 5

<211> 7651

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggtcacc atcaccatca ccatatgtcg 5100

gactcagaag tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact 5160

cacatcaatt taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc 5220

actcctttaa gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc 5280

ttaagattct tgtacgacgg tattagaatt caagctgatc agacccctga agatttggac 5340

atggaggata acgatattat tgaggctcac agagaacaga ttggtggcca aggatccatg 5400

gcacgtgatt atgatctgaa agattatcgt aacatcggta ttatggcgca tattgatgcg 5460

ggtaaaacca ccaccaccga acgcattctg tatcataccg gtaaaattca taaaatcggt 5520

gaaacacatg atggtgtttc tcagatggat tggatggaac aggaaaaaga acgtggcatt 5580

acaatcacca gcgcagcaac cacagcatat tggaaaaata aacgtattaa catcatcgat 5640

accccgggtc atgttgattt taccgttgaa gttgaacgta gcctgcgtgt tctggatggc 5700

gccgttgcag ttctggatgc acagagcggt gtggaaccgc agaccgaaac cgtgtggcgc 5760

caggcaacca attataaagt tccgcgtatt gtgtatgtta ataaaatgga taaagcaggt 5820

gcggattttg aagcggcagt agcaagcgtt aaaagccgtc tgggtggcaa tgcggttgca 5880

attcagtggc cgatcggtag cgaaagcaat tttaatggca tcattgatct ggttacaatg 5940

accgcgacaa cctataatgg tgaaagtgca gaagaagaat ttccgatgga aattccgacc 6000

gatctgctgg atgtggcaaa agcaaaacgt caggaactgc tggaagcagc ggccaacttt 6060

gatgaagaag ttatgatgat ggtgctggaa ggtgcagatg ttgatattga tacctttaaa 6120

aacaccatcc gtaaagcaac actgaccagc gaattttttc cggtagtttg tggcacgagc 6180

tttaaaaata aaggtgtcaa aaagatgatc gatgcagtgg tggattatct gccgagcccg 6240

ctggatattc cgccgattaa agcatatctg aatgatcagg aaaccgatgt tgtcgcaaca 6300

gatgatggtg aatttgcagc actggcattt aaagttatga ccgatccttt tgttggtagc 6360

ctgacctttt ttcgtgtcta tcgcggtgtt ctggaaaaag gcagctatgt ttataatagc 6420

acgaaagaac agaaagaacg tattggtcgt attctgcaga tgcatgcaaa taatcgtgtt 6480

gaaattgatg aatgtcgtgc aggtgatatt gcagcagcag tgggtctgaa atttaccacc 6540

accggtgata cactggttgg cgaaaaatca cctaaagttg ttctggaaaa aatggttttt 6600

ccggaaccgg ttattagcca ggcactggaa ccggaaagca aagcagcaaa tgaaaaactg 6660

agcctgggtc tgcagaaact gagcgcagaa gatccgacct ttcgtaccta taccgatgaa 6720

gaaaccggtc agaccattat tagcggtatg ggtgaactgc atctggatat tattgttgat 6780

cgtctgaaac gtgaatttgg tgttaaagtt aaagttggtg caccgcaggt tagctatcgt 6840

gaaaccatta ccaaaagcgc agaagttgaa ggtaaacata ttaaacagag cggtggtaaa 6900

ggtcagtatg gtcatgtttg gctgaaattt gaaccgaatc atgatcaggg ttttgaattt 6960

attgataaaa ttgttggtgg taaaattccg aaagaatata ttaaaccgat tcagaaaggt 7020

ctggaagaaa aaatggcagt tggtattctg gcaggttatc cgatgattga tgttaaagca 7080

accctgtttg atggtagcta tcatgatgtt gatagcagcg aactggcata taaaattgca 7140

gcaagcaaag cactgaccaa agcaaaagat ctgattggta ccgttctgct ggaaccgatt 7200

atggatgtta gcgttgttgt tccgagcgat cacatgggtg atgttattgg tgatctgagc 7260

cgtcgtcgtg gtctgattag cgatcaggaa cagcgtaatg atggtgcagt tattgttcgt 7320

gcaaaagttc cgctgagcga aatgtttggt tatagcaccg aactgcgtag catgaccagc 7380

ggtcgtggta cctatcagat gcagtttgat cattatgaaa aatgtccgaa aaatattagc 7440

gatgaaatta ttaaaaaacg taatattcag aataaagatg aagaataact cgagcaccac 7500

caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 7560

gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 7620

tttttgctga aaggaggaac tatatccgga t 7651

Claims (10)

1. An envelope antigen for detecting antibodies to M.synoviae, wherein said envelope antigen is a recombinant EFG protein; the amino acid sequence of the recombinant EFG protein comprises the amino acid sequence as set forth in SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein includes the nucleotide sequence set forth in SEQ ID NO: 2.

2. The coating antigen for detecting mycoplasma synoviae antibodies of claim 1, wherein the amino acid sequence of said recombinant EFG protein comprises a sequence identical to the sequence of SEQ ID NO: 1 sequences having at least 90% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO: 2 having at least 90% homology.

3. The coating antigen for detecting mycoplasma synoviae antibodies of claim 1, wherein the amino acid sequence of said recombinant EFG protein comprises a sequence identical to the sequence of SEQ ID NO: 1 sequences having at least 95% homology; the recombinant EFG protein nucleotide sequence comprises a nucleotide sequence identical to SEQ ID NO: 2 with at least 95% homology.

4. The coating antigen for detecting mycoplasma synoviae antibodies of claim 1, wherein the amino acid sequence of said recombinant EFG protein is SEQ ID NO: 1; the nucleotide sequence of the recombinant EFG protein is SEQ ID NO: 2.

5. The coating antigen for detecting mycoplasma synoviae antibodies of claim 1, wherein the amplification primer sequence of the recombinant EFG protein is as shown in SEQ ID NO: 3 and SEQ ID NO: 4:

SEQ ID NO：3：5’-ATGGCACGTGATTATGATCTGAAAG-3’；

SEQ ID NO：4：5’-TTCTTCATCTTTATTCTGAATATTAC-3’。

6. a kit for detecting a Mycoplasma synoviae antibody, comprising the coating antigen for detecting a Mycoplasma synoviae antibody according to claim 1 to 5.

7. The kit for detecting the mycoplasma synoviae antibody according to claim 6, wherein the kit further comprises an ELISA plate, a coating solution, a confining solution, an ELISA secondary antibody, a TMB color development solution, a stop solution, a washing solution and a serum diluent.

8. The kit for detecting mycoplasma synoviae antibodies of claim 7, wherein the coating solution is carbonate buffer; the washing solution is PBST; the enzyme-labeled secondary antibody is a goat anti-rabbit enzyme-labeled secondary antibody; the confining liquid is 5% of skimmed milk powder; the serum diluent is 5% skimmed milk powder.

9. The detection method for detecting the mycoplasma synoviae antibody by using the kit according to claims 6-8 is characterized by comprising the following steps:

s1; diluting the coating antigen with a coating solution, putting the diluted coating antigen into an ELISA plate, and coating the antigen overnight;

s2: removing the coating solution in the step S1, cleaning the cleaning solution, drying the cleaning solution, adding a sealing solution, and sealing;

s3: removing the sealing liquid in the S2, cleaning the washing liquid, drying by beating, adding the serum sample to be tested diluted by the serum diluent, and incubating;

s4: removing the serum sample in S3, washing with a washing solution, drying by beating, adding a diluted enzyme-labeled secondary antibody, and incubating;

s5: removing the enzyme-labeled secondary antibody in the S4, washing with a washing solution, drying by beating, adding a TMB color development solution for light-shielding reaction, and adding a stop solution after the reaction is finished to terminate the reaction;

s6: testing liquid OD in plate hole of enzyme label on enzyme label instrument_450nmValue, OD_450nmWhen the value is not less than 0.3998, the detection result is judged to be positive; when OD of the sample to be tested_450nmWhen the value is < 0.3998, the result of the detection is judged to be negative.

10. The detection method according to claim 9, wherein the concentration of the coating antigen in S1 is 0.8 μ g/mL after dilution, 100 μ L of the coating antigen is added to each well of the microplate, and the coating antigen is coated overnight at 4 ℃.

Images