阅读说明：本技术 鸡滑液支原体抗原蛋白lp85及相应抗体的elisa检测方法及试剂盒 (Chicken synovial mycoplasma antigen protein LP85, ELISA detection method of corresponding antibody and kit ) 是由 于圣青 祁晶晶 李浩然 丁铲 王宇 王少辉 田明星 李涛 于 2019-09-20 设计创作，主要内容包括：本发明提供了一种鸡滑液支原体抗原蛋白LP85、建立的检测所述鸡滑液支原体相应抗体的ELISA方法、根据该方法制备的ELISA试剂盒和相应的使用方法,由于基于鸡滑液支原体抗原蛋白LP85,特别是其鸡滑液支原体重组表达蛋白LP85,使得相应的抗体的ELISA检测方法和根据该方法制备的ELISA试剂盒,相比现有的检测形式,具有特异性、敏感性更好的特点,且检测的重复性好。(The invention provides a chicken mycoplasma synoviae antigen protein LP85, an established ELISA method for detecting a corresponding antibody of the chicken mycoplasma synoviae, an ELISA kit prepared according to the method and a corresponding using method, and because the ELISA method based on the chicken mycoplasma synoviae antigen protein LP85, particularly the chicken mycoplasma synoviae recombinant expression protein LP85, the ELISA detection method of the corresponding antibody and the ELISA kit prepared according to the method have the characteristics of better specificity and sensitivity and good detection repeatability compared with the existing detection form.)

1. A mycoplasma synoviae antigen protein LP85, which can be used as a target for ELISA detection of corresponding antibodies of mycoplasma synoviae, is characterized in that:

the total length of the coding gene is 2319 bp;

is a lipoprotein with a molecular weight of about 85 kD;

the target protein sequence is:

MSCGAPQEEKKPTNPKDGNKDGGIIDLPPTIGGPGEGGDSPRTYGSIDPVQSKLVGSWADYTKLSIAKRYEVDNKAYLNGLKSQYDIDPKQEFVPSDIKNGFGTVQAYDDKATKLGLPDFVTSYLKGFTTYSGSGLEISPNVSGPALGFWNSEQSGFDGRSRFLPNDLYKNTALQTYSISYVNEVKGDYVNEVKGDIEGTGKNTLKSNKGTAWILDYVKPTDSSYPTKWYIATNIHVIGDLTFTKSQTDSFGSDFTSIYDEEREKPLIKEARKVKAQIDELQNEYNEVYRKLQTKEHENDQALKARADQLNFELAPPLQKKLKDLNAQISGLTKNVTLAILDSDVPLQTSLNTVSADSRMKFVTLPASAVNIVYTANDFLKTSPKDYLDTTSTHNKDYLNNQEMADFAVLEIDFSKVTGEFKYTKNSVGDKPAKEMTVNSAQELARVMTNAYASTEKQDKQIKFAKNSLFYQYKDLTNEKVTVTNDANKQKLQVSRIVANFVSLGYPIAKDDLVNLRAEFPAKYAGREGILLAENDNSLWTNKPQKGRNFYQEFGNRLNRSMILRNFVQYPGIYDLFITNPVINKGVGFNIKQIKDKTSSYQQGNYLNYGLAYSLESWRPAPGASGSSLRDLNNEVLGINFAIRAGAGSGTSLIQAFRSEGANYGGVYGSYNLPQYDLIYGGGKDQRTSYREALAKILKNGETTNLFTSGVNVIPEEYKFRTNALINFTNSSNPEEAGLITNTGGGVQALTDQNKKYGSTITNPVVLPEGTS。

2. the mycoplasma synoviae antigen protein LP85 of claim 1, wherein:

the homology of the mycoplasma synoviae antigen protein LP85 between isolates of mycoplasma synoviae was 96-100%.

3. The chicken mycoplasma synoviae antigen protein LP85 of claim 1 or 2, wherein:

the mycoplasma synoviae antigen protein LP85 is a mycoplasma synoviae recombinant expression protein LP 85.

4. The mycoplasma synoviae antigen protein LP85 of claim 3, wherein:

the chicken mycoplasma synoviae recombinant expression protein LP85 is prokaryotic expressed by a recombinant expression vector pCold LP85, and the recombinant expression vector pCold I-LP85 is constructed as follows:

selecting the screened mycoplasma synoviae antigen protein LP85, optimizing a termination codon TGA into TGG according to the gene sequence of the protein, adding enzyme cutting sites Sac I and Xho I at two ends of the protein respectively, and synthesizing a corresponding DNA sequence of the mycoplasma synoviae antigen protein LP 85;

connecting the synthesized DNA sequence with an expression vector pCold I after double enzyme digestion by Sac I and Xho I to construct a recombinant expression plasmid pCold I-LP85, transforming E.coli DH5 alpha competent cells, culturing for 12-15h on an LB agar plate containing 100 mu g/ml ampicillin, selecting a single colony, performing PCR identification by MS LP85 gene primers P1 and P2, performing amplification culture on the strains identified as positive, extracting plasmids, performing double enzyme digestion identification on the plasmids, and obtaining the recombinant expression vector pCold LP85 if the identification is correct,

wherein the content of the first and second substances,

primer P1 was: 5'-GAGCTCATGAGCTGTG-3' the flow of the air in the air conditioner,

primer P2 was: 5'-CTCGAGTTAGCTTGTACC-3' are provided.

5. The mycoplasma synoviae antigen protein LP85 of claim 4, wherein:

the chicken mycoplasma synoviae recombinant expression protein LP85 is obtained by performing prokaryotic expression and purification on the recombinant expression vector pCold LP85 as follows:

transforming E.coli BL21 with correctly identified recombinant vector pCold I-MS LP85 to obtain recombinant expression strain E.coli BL 21; and carrying out ultrasonic cracking on the recombinant expression strain after IPTG induction expression, and purifying a cracked supernatant by using a His Trap protein purification column to obtain the mycoplasma synoviae recombinant expression protein LP 85.

6. An ELISA detection method for detecting the mycoplasma synoviae antibody is characterized in that:

the mycoplasma gallisepticum antigen protein LP85 of any one of claims 1-5 as a target for detection of said mycoplasma synoviae antibody to detect said mycoplasma synoviae antibody.

7. The ELISA detection method of claim 6, wherein:

wherein, the chicken synovial fluid mycoplasma antigen protein LP85 is used as an enzyme label plate of the antigen, and the antigen coating concentration is 5 mug/ml and 100 mug/hole.

8. The ELISA detection method of claim 7, wherein:

wherein, the antigen coating conditions are as follows: incubate at 37 ℃ for 2 h.

9. The ELISA detection method of claim 6, wherein:

wherein the serum dilution factor is 1: 200.

10. The ELISA detection method of claim 6, wherein:

wherein the dilution ratio of the enzyme-labeled antibody is 1: 20000.

11. The ELISA detection method of claim 10, wherein:

wherein the enzyme-labeled antibody is goat anti-chicken IgG-HRP.

12. The ELISA detection method of claim 6, wherein:

wherein the reaction time of the substrate is 15 min.

13. The ELISA detection method of claim 6, wherein:

wherein, the serum sample diluent is 5% skim milk-PBST added with 30% BL21 whole mycoprotein.

14. The ELISA detection method of claim 6, wherein:

wherein, the negative-positive critical value for judging the negative and positive is 2.388, the S/N value of the serum to be detected is equal to or higher than 2.388 and is judged to be positive, and the S/N value is lower than 2.388 and is judged to be negative.

15. The ELISA detection method of claim 6, wherein:

wherein, 5% skim milk-PBS blocking solution is adopted for blocking.

16. An ELISA kit for detecting the mycoplasma synoviae antibody is characterized in that:

the ELISA detection method for detecting mycoplasma synoviae antibodies of claims 6-15.

17. The ELISA kit according to claim 15, comprising:

the kit comprises an ELISA plate, an enzyme-labeled antibody, a chicken mycoplasma synoviae standard positive serum and an SPF chicken serum, wherein the ELISA plate is used for coating the chicken mycoplasma synoviae antigen protein LP85 as an antigen.

18. The ELISA kit of claim 17, further comprising:

coating buffer diluent, washing buffer, blocking solution, antibody diluent, serum sample diluent, substrate solution and termination liquid.

19. A use method of an ELISA kit for detecting mycoplasma synoviae antibody is characterized in that:

the ELISA kit is according to any one of claims 16-17.

20. Use according to claim 19, characterized in that:

the specific use method is as follows:

antigen coating of an ELISA plate: diluting the antigen to 5 mu g/ml by using carbonate coating buffer solution, adding 100 mu l/hole into an enzyme label plate, incubating for 2h at 37 ℃, then washing for 3 times by using PBST, and patting dry;

sealing an enzyme label plate: adding 200 mul of 5% skim milk-PBS into each well of the coated enzyme label plate, sealing for 2h at 37 ℃, continuously washing for 3 times by PBST, and patting to dry;

serum diluted with serum sample diluent was added: adding 100 μ l/well of serum to be detected diluted by 1:200 times via serum sample diluent, adding MS standard positive and negative serum diluted by 1:200 times via serum sample diluent as positive and negative control, respectively, acting at 37 deg.C for 1.5h, washing with PBST for 4 times, and drying;

adding enzyme-labeled antibody diluted by antibody diluent: continuously adding goat anti-chicken IgG-HRP secondary antibody diluted by 1:20000 times of the antibody diluent into each hole, acting for 1h at 37 ℃, washing for 4 times, and patting dry;

adding a substrate for color development: continuously adding 100 μ l of TMB substrate color development solution into each well, and developing at 37 deg.C in dark for 15 min;

adding a stop solution to complete detection: adding 50 μ l of the solution into each wellStopping the solution, measuring OD on an enzyme-linked immunosorbent assay (ELIASA) within 10 minutes₄₅₀The S/N value of the serum to be detected is equal to or higher than 2.388, and the serum to be detected is judged to be positive, and the serum to be detected is judged to be negative when the S/N value is lower than 2.388.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a mycoplasma synoviae antigen protein LP85, an ELISA detection method of a corresponding antibody and a kit.

Background

Mycoplasma synoviae (MS, or Mycoplasma synoviae) is one of the important poultry pathogens, and can infect chickens and turkeys, causing synovitis and tenosynovitis of joints, and can also cause airsacculitis, and in severe cases, systemic infection is caused (1, 2), and in recent years, it has been reported that MS infects laying hen groups, and then the egg yield is reduced, and the egg quality and the hatching rate are reduced (3, 4, 5).

MS is prevalent worldwide and epidemiological surveillance in many countries indicates a high prevalence of MS in commercial chicken flocks (6, 7). The latest serological survey shows that in 2010 to 2015, chicken serum samples in a plurality of provinces of China including Guangxi, Guangdong, Jiangxi, Sichuan, Shandong, Beijing, Shaanxi and the like all detect higher MS antibody positive rate, and in 44395 chicken serum samples in China, the total positive rate is 41.19 percent, while the positive rate of chicken farms is as high as 80.99(8), which indicates that the chicken flocks in China have the serious MS situation. MS is often mixed with escherichia coli, newcastle disease virus, infectious bronchitis virus and the like to cause serious harm to chicken flocks, so that rapid and accurate diagnosis is necessary for controlling the spread of the disease.

Mycoplasma does not contain cell walls, has abundant lipoproteins on membranes, and has strong immunogenicity.

Due to the low and time consuming isolation of MS pathogens, serological testing is a commonly used method for clinical rapid diagnosis. At present, MS antibody detection is mainly carried out by using an MS plate agglutination antigen produced by Chinese veterinary medicine supervision and an MS serum antibody ELISA detection kit produced by American Edesx (IDEXX). The serum flat plate agglutination (SA) method is quick and convenient to operate, is commonly used for mycoplasma infection general investigation of chicken flocks, but is easy to generate cross reaction and high in false positive; the detection kit for the MS antibody of IDEXX company has stable results and high reliability, but is expensive. Therefore, the screening of an MS antigen with good immunoreactivity and high specificity for developing an MS diagnostic kit has very important significance.

Disclosure of Invention

The invention provides a mycoplasma synoviae antigen protein LP85, an established ELISA detection method of a corresponding mycoplasma synoviae antibody, an ELISA kit prepared according to the method and a corresponding using method, so as to provide an ELISA detection method and a kit which are suitable for MS serum antibody detection and have good specificity and high sensitivity.

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

one object of the present invention is to provide a mycoplasma synoviae antigen protein LP85, which can be used as a target for the ELISA detection of mycoplasma synoviae antibodies, characterized in that:

the total length of the coding gene is 2319 bp;

is a lipoprotein with a molecular weight of about 85 kD;

the target protein sequence is:

MSCGAPQEEKKPTNPKDGNKDGGIIDLPPTIGGPGEGGDSPRTYGSIDPVQSKLVGSWADYTKLSIAKRYEVDNKAYLNGLKSQYDIDPKQEFVPSDIKNGFGTVQAYDDKATKLGLPDFVTSYLKGFTTYSGSGLEISPNVSGPALGFWNSEQSGFDGRSRFLPNDLYKNTALQTYSISYVNEVKGDYVNEVKGDIEGTGKNTLKSNKGTAWILDYVKPTDSSYPTKWYIATNIHVIGDLTFTKSQTDSFGSDFTSIYDEEREKPLIKEARKVKAQIDELQNEYNEVYRKLQTKEHENDQALKARADQLNFELAPPLQKKLKDLNAQISGLTKNVTLAILDSDVPLQTSLNTVSADSRMKFVTLPASAVNIVYTANDFLKTSPKDYLDTTSTHNKDYLNNQEMADFAVLEIDFSKVTGEFKYTKNSVGDKPAKEMTVNSAQELARVMTNAYASTEKQDKQIKFAKNSLFYQYKDLTNEKVTVTNDANKQKLQVSRIVANFVSLGYPIAKDDLVNLRAEFPAKYAGREGILLAENDNSLWTNKPQKGRNFYQEFGNRLNRSMILRNFVQYPGIYDLFITNPVINKGVGFNIKQIKDKTSSYQQGNYLNYGLAYSLESWRPAPGASGSSLRDLNNEVLGINFAIRAGAGSGTSLIQAFRSEGANYGGVYGSYNLPQYDLIYGGGKDQRTSYREALAKILKNGETTNLFTSGVNVIPEEYKFRTNALINFTNSSNPEEAGLITNTGGGVQALTDQNKKYGSTITNPVVLPEGTS。

the mycoplasma synoviae antigen protein LP85 provided by the invention also has the following characteristics: the homology of the mycoplasma synoviae antigen protein LP85 between isolates of mycoplasma synoviae was 96-100%.

The mycoplasma synoviae antigen protein LP85 provided by the invention also has the following characteristics: the mycoplasma synoviae antigen protein LP85 is a mycoplasma synoviae recombinant expression protein LP 85.

The mycoplasma synoviae antigen protein LP85 provided by the invention also has the following characteristics: the chicken mycoplasma synoviae recombinant expression protein LP85 is prokaryotic expressed by a recombinant expression vector pCold LP85, and the recombinant expression vector pCold I-LP85 is constructed as follows: selecting the screened mycoplasma synoviae antigen protein LP85, optimizing a termination codon TGA into TGG according to the gene sequence of the protein, adding enzyme cutting sites Sac I and Xho I at two ends of the protein respectively, and synthesizing a corresponding DNA sequence of the mycoplasma synoviae antigen protein LP 85; connecting the synthesized DNA sequence with an expression vector pCold I after double digestion by Sac I and Xho I to construct a recombinant expression plasmid pCold I-LP85, transforming E.coli DH5 alpha competent cells, culturing for 12-15h on an LB agar plate containing 100 mu g/ml ampicillin, selecting a single colony, carrying out PCR identification by MS LP85 gene primers P1 and P2, carrying out amplification culture on a strain (2331bp) identified as positive, extracting the plasmid, carrying out double digestion identification on the plasmid, and obtaining the recombinant expression vector pCold LP85 if the identification is correct, wherein,

primer P1 was: 5'-GAGCTCATGAGCTGTG-3' the flow of the air in the air conditioner,

primer P2 was: 5'-CTCGAGTTAGCTTGTACC-3' are provided.

The mycoplasma synoviae antigen protein LP85 provided by the invention also has the following characteristics: the chicken mycoplasma synoviae recombinant expression protein LP85 is obtained by performing prokaryotic expression and purification on a recombinant expression vector pCold LP85 as follows: transforming E.coli BL21 with correctly identified recombinant vector pCold I-MS LP85 to obtain recombinant expression strain E.coli BL 21; and carrying out ultrasonic cracking on the recombinant expression strain after IPTG induction expression, and purifying a cracked supernatant by using a His Trap protein purification column to obtain the mycoplasma synoviae recombinant expression protein LP 85.

Another objective of the invention is to provide an ELISA detection method for detecting mycoplasma synoviae antibodies, which is characterized in that: the mycoplasma gallisepticum antigen protein LP85 is used as a target for detection of corresponding mycoplasma synoviae antibodies to detect mycoplasma synoviae antibodies.

The ELISA detection method provided by the invention also has the following characteristics: wherein, the antigen protein LP85 of the mycoplasma synoviae is used as an enzyme label plate of the antigen, and the antigen coating concentration is 5 mug/ml and 100 mug/hole.

The ELISA detection method provided by the invention also has the following characteristics: wherein, the antigen coating conditions are as follows: incubate at 37 ℃ for 2 h.

The ELISA detection method provided by the invention also has the following characteristics: wherein the serum dilution factor is 1: 200.

The ELISA detection method provided by the invention also has the following characteristics: wherein the dilution ratio of the enzyme-labeled antibody is 1: 20000.

The ELISA detection method provided by the invention also has the following characteristics: wherein the enzyme-labeled antibody is goat anti-chicken IgG-HRP.

The ELISA detection method provided by the invention also has the following characteristics: wherein the reaction time of the substrate is 15 min.

The ELISA detection method provided by the invention also has the following characteristics: wherein, the serum sample diluent is 5% skim milk-PBST added with 30% BL21 whole mycoprotein.

The ELISA method provided by the invention also has the following characteristics: wherein, the negative-positive critical value for judging the negative and positive is 2.388, the S/N value of the serum to be detected is equal to or higher than 2.388 and is judged to be positive, and the S/N value is lower than 2.388 and is judged to be negative.

The ELISA detection method provided by the invention also has the following characteristics: wherein, 5% skim milk-PBS blocking solution is adopted for blocking.

Still another object of the present invention is to provide an ELISA kit for detecting mycoplasma synoviae antibodies, characterized in that: the antibody is prepared according to the ELISA detection method for detecting the mycoplasma synoviae antibody.

The ELISA kit provided by the invention is also characterized by comprising: the kit comprises an ELISA plate which is used for coating mycoplasma synoviae antigen protein LP85 as an antigen, an enzyme-labeled antibody, mycoplasma synoviae standard positive serum and SPF chicken serum.

The ELISA kit provided by the present invention is also characterized by further comprising: coating buffer diluent, washing buffer, blocking solution, antibody diluent, serum sample diluent, substrate solution and termination liquid.

The invention also provides a using method of the ELISA kit for detecting the mycoplasma synoviae antibody, which is characterized by comprising the following steps: the ELISA kit is the ELISA kit.

The use method provided by the invention also has the following characteristics: the specific use method is as follows: antigen coating of an ELISA plate: diluting the antigen to 5 mu g/ml by using carbonate coating buffer solution, adding 100 mu l/hole into an enzyme label plate, incubating for 2h at 37 ℃, then washing for 3 times by using PBST, and patting dry; sealing an enzyme label plate: adding 200 mul of 5% skim milk-PBS into each well of the coated enzyme label plate, sealing for 2h at 37 ℃, continuously washing for 3 times by PBST, and patting to dry; serum diluted with serum sample diluent was added: adding 100 μ l/well of serum to be detected diluted by 1:200 times via serum sample diluent, adding MS standard positive and negative serum diluted by 1:200 times via serum sample diluent as positive and negative control, respectively, acting at 37 deg.C for 1.5h, washing with PBST for 4 times, and drying; adding enzyme-labeled antibody diluted by antibody diluent: continuously adding goat anti-chicken IgG-HRP secondary antibody diluted by 1:20000 times of the antibody diluent into each hole, acting for 1h at 37 ℃, washing for 4 times, and patting dry; adding a substrate for color development: continuously adding 100 mul of TMB substrate color development solution into each hole, and developing for 15min at 37 ℃ in a dark place; adding a stop solution to complete detection: add 50. mu.l stop buffer to each well and measure OD on microplate reader within 10 minutes₄₅₀The S/N value of the serum to be detected is equal to or higher than 2.388, and the serum to be detected is judged to be positive, and the serum to be detected is judged to be negative when the S/N value is lower than 2.388.

Action and Effect of the invention

According to the mycoplasma synoviae antigen protein LP85, the established corresponding ELISA method for detecting the mycoplasma synoviae antibody, the ELISA kit prepared according to the method and the corresponding using method, based on the mycoplasma synoviae antigen protein LP85, particularly the recombinant expression protein LP85 of the mycoplasma synoviae, the ELISA detection method of the corresponding antibody and the ELISA kit prepared according to the method have the characteristics of better specificity and sensitivity and good detection repeatability compared with the existing detection form.

Drawings

FIG. 1 is a schematic diagram showing the results of screening for MS immunogenic proteins using Western blot as described in example 1;

FIG. 2 shows the results of PCR and double digestion identification of the recombinant expression vector pCold I-MS LP85 of example 1;

FIG. 3 shows the results of expression and purification of Mycoplasma synoviae recombinant expression protein LP85(rMS LP85) according to example 1;

FIG. 4 shows Western blot reaction of recombinant expression protein LP85(rMS LP85 protein) of Mycoplasma gallisepticum in example 1 with positive sera of different MS clinical isolates;

FIG. 5 shows the Western blot reaction of recombinant expression protein of Mycoplasma synoviae (rMS LP85 protein) with positive chicken sera of different pathogens according to example 1;

FIG. 6 shows the results of Western blot analysis of clinical serum samples that did not match the IDEXX test results in example 3.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.

The experimental methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

The materials or reagents involved in the following examples are as follows:

(1) bacterial strain and serum

Mycoplasma gallisepticum (MS) WVU1853 strain (ATCC25204), Mycoplasma Gallisepticum (MG) R_lowStrain (CVCC 1651) and mycoplasma Chlamydomonas (MI)695 strain (CVCC364) were purchased from the chinese institute of veterinary medicine;

MS clinical isolates JS1, SD1, SH1 and HB1, and Escherichia Coli (EC) O1(APEC O1), O2(DE719) and O78(APEC94) serotype strains are isolated, identified and preserved by the laboratory, and the strains are preserved in respective culture media containing 25% of glycerol and frozen at-80 ℃.

Mycoplasma synoviae standard positive serum (MS standard positive serum), MG positive serum, salmonella gallinarum (SPG) positive serum, Newcastle Disease Virus (NDV) positive serum, Infectious Bronchitis Virus (IBV) positive serum, Infectious Bursal Disease Virus (IBDV) positive serum and SPF chicken serum (MS negative serum) are purchased from China veterinary medicine monitoring institute;

MS clinical isolates JS1, SD1, SH1 and HB1 positive serum, MG R_lowThe strain positive serum, the MI695 strain positive serum, the ECO1, the O2 and the O78 trivalent positive serum are prepared by the laboratory, and the test of the plate agglutination test is positive;

150 clinical chicken serum samples were collected from a certain poultry farm in Shanghai, and all sera were judged to be MS positive or negative by the Mycoplasma synoviae antibody detection kit (IDEXX).

(2) Culture medium, main reagent and reagent preparation

Mycoplasma liquid culture medium: weighing 33g of mycoplasma basal medium (Qingdao Haibo), adding proper double distilled water for dissolving, fixing the volume to 900ml, sterilizing at 121 ℃ for 15min, cooling, adding 100ml of inactivated pig serum (Gibco) and 10ml of 1% NAD (Roche, filtration sterilization), mixing uniformly, and storing at 4 ℃. LB medium was purchased from OXOID, UK.

The BCA protein concentration determination kit is purchased from Biyuntian biotechnology company; protein purification column His TraptM FF crude was purchased from GE Healthcare Life Sciences; skim milk was purchased from BBI Life Science; goat anti-chicken IgG-HRP was purchased from Abcam; the TMB substrate developing solution is purchased from Beijing Tiangen biology, Inc. The 96-hole enzyme label plate is a product of Corning corporation of America; the mycoplasma synoviae antibody detection kit is a product of IDEXX company in the United states. Other conventional reagents are imported or domestic analytical purifiers.

Solution preparation:

carbonate coating buffer (CBS, PH 9.6): na (Na)₂CO₃ 1.6g，NaHCO₃2.92g, distilled water is added to reach the constant volume of 1000ml, and the pH value is adjusted to 9.6 by 0.1M HCl or NaOH;

phosphate buffer (PBS, ph 7.2): KH (Perkin Elmer)₂PO4 0.24g，KCl 0.2g，Na₂HPO41.44g and NaCl 8g, adding deionized water to reach a constant volume of 1000ml, and adjusting the pH value to 7.2 by using 0.1M HCl or NaOH;

washing buffer solution: PBST, 0.5ml Tween-20 dissolved in 1000ml PBS;

sealing liquid: 5% skim milk-PBS, 5g skim milk dissolved in 100ml PBS;

antibody dilution: 5% skim milk-PBST, 5g skim milk dissolved in 100ml PBST;

serum sample diluent: adding 30% BL21 whole mycoprotein into 5% skim milk-PBST;

substrate solution: soluble single component TMB substrate solution;

stopping liquid: 2M H₂SO₄。

Example 1

Identification, cloning and expression of mycoplasma synoviae antigen protein LP85(MS LP 85).

1.1 screening of immunogenic proteins for MS

1.1.1MS Membrane protein extraction

The MS WVU1853 culture in logarithmic growth phase is transferred to mycoplasma liquid culture medium according to the proportion of 5%, and is statically cultured for 30h at 37 ℃, and then the thalli are collected by centrifugation of 12000 g. After washing with PBS for 2 times, resuspending with PBS of 1% bacterial liquid volume, and carrying out ultrasonication for 20min (power 40%, working time 5s, and pause 5s) to obtain the MS holomycoprotein. The membrane protein was extracted from the whole MS mycoprotein using Mycoplasma membrane protein extraction kit (Shanghai Bibo) according to the instructions, and the protein concentration was determined using BCA kit, which was stored at-80 ℃ after being dispensed.

1.1.2Western blot analysis of MS immunogenic proteins and Mass Spectrometry

And respectively carrying out SDS-PAGE electrophoresis on the MS whole bacterial protein and the membrane protein, preparing two pieces of electrophoresis gel, dyeing one piece of gel by Coomassie brilliant blue R250, transferring the other piece of gel to a nitrocellulose membrane (NC membrane), and carrying out Western blot detection. Blocking the transferred NC membrane by using 5% skim milk-PBS for 2h, adding MS standard positive serum (1:500), incubating for 1.5h at 37 ℃, washing for 3 times by using PBST, adding goat anti-chicken IgG-HRP (1:50000, Abcam in England), incubating for 1h at 37 ℃, and finally adding ECL substrate color development solution for color development reaction.

FIG. 1 is a schematic diagram showing the results of screening for MS immunogenic proteins using Western blot as described in example 1.

The result of Western blot screening for MS immunogenic proteins is shown in FIG. 1. Wherein:

a: coomassie brilliant blue staining pattern after SDS-PAGE electrophoresis;

b: western blot reaction result with MS infected serum;

m: a protein molecule standard; 1, MS WVU1853 membrane protein; 2, MS WVU1853 holomyces protein.

In contrast to the result of the Western blot positive bands, bands were excised from the gel at the corresponding positions (see the positions marked by black boxes in FIG. 1), and subjected to LCMSMS (NanoLC-QE) mass spectrometry by Shanghai Kagaku New Life technologies, Inc.

1.1.4 bioinformatics analysis

Bioinformatics analysis was performed on the obtained mass spectrum results, and mycoplasma synoviae lipoprotein (MS LP85 protein) with a molecular weight of approximately 85kD was selected as a candidate immunogenic protein for bioinformatics analysis.

Wherein, PSORTb (http:// www.psort.org/PSORTb /) is used for online prediction of subcellular localization of protein;

predicting the transmembrane region of the protein by using TMHMM server 2(http:// www.cbs.dtu.dk/services/TMHMM /);

the protein signal peptide was predicted using the online software SignalP 4.1Seve (http:// www.cbs.dtu.dk/services/SignalP /);

the Non-redundant protein sequences (nr) database was searched using online Blastp (https:// blast. ncbi. nlm. nih. gov/blast. cgi) to analyze the conservation and specificity of protein sequences.

Biological information analysis results:

TMHMM and SignalP predict that the protein has no transmembrane region and no signal peptide;

the PSORTb is used for performing sublocalization prediction, and the result shows that the PSORTb is outer membrane protein

The MS LP85 amino acid sequence is compared with an nr database through Blastp, and the result shows that MS LP85 is highly conserved in MS species, and the homology among different MS isolates is up to 96-100%; the homology with MG LP85 protein is 35% -36%, and the homology with other mycoplasma is only 28% -36%, which shows that the MS LP85 protein has good conservation among MS species, and can be used as a diagnostic target of mycoplasma synoviae.

In addition, the analysis shows that the total length of the coding gene of the MS LP85 protein is 2319 bp; is a lipoprotein with a molecular weight of about 85 kD; the target protein sequence is as follows: MSCGAPQEEKKPTNPKDGNKDGGIIDLPPTIGGPGEGGDSPRTYGSIDPVQSKLVGSWADYTKLSIAKRYEVDNKAYLNGLKSQYDIDPKQEFVPSDIKNGFGTVQAYDDKATKLGLPDFVTSYLKGFTTYSGSGLEISPNVSGPALGFWNSEQSGFDGRSRFLPNDLYKNTALQTYSISYVNEVKGDYVNEVKGDIEGTGKNTLKSNKGTAWILDYVKPTDSSYPTKWYIATNIHVIGDLTFTKSQTDSFGSDFTSIYDEEREKPLIKEARKVKAQIDELQNEYNEVYRKLQTKEHENDQALKARADQLNFELAPPLQKKLKDLNAQISGLTKNVTLAILDSDVPLQTSLNTVSADSRMKFVTLPASAVNIVYTANDFLKTSPKDYLDTTSTHNKDYLNNQEMADFAVLEIDFSKVTGEFKYTKNSVGDKPAKEMTVNSAQELARVMTNAYASTEKQDKQIKFAKNSLFYQYKDLTNEKVTVTNDANKQKLQVSRIVANFVSLGYPIAKDDLVNLRAEFPAKYAGREGILLAENDNSLWTNKPQKGRNFYQEFGNRLNRSMILRNFVQYPGIYDLFITNPVINKGVGFNIKQIKDKTSSYQQGNYLNYGLAYSLESWRPAPGASGSSLRDLNNEVLGINFAIRAGAGSGTSLIQAFRSEGANYGGVYGSYNLPQYDLIYGGGKDQRTSYREALAKILKNGETTNLFTSGVNVIPEEYKFRTNALINFTNSSNPEEAGLITNTGGGVQALTDQNKKYGSTITNPVVLPEGTS are provided.

1.2 expression purification of Mycoplasma synoviae antigen protein LP85(MS LP85)

1.2.1. Construction and identification of recombinant expression vector

Selecting candidate screened MS LP85, optimizing termination codon TGA into TGG according to gene sequences published in GenBank, adding enzyme cutting sites Sac I (GAGCTC) and Xho I (CTCGAG) at two ends respectively, and synthesizing DNA sequences by Shanghai Rui bare Biotech limited company.

The synthetic MS LP85 gene sequence is subjected to double digestion by Sac I and Xho I and then is connected with an expression vector pCold I (TaKaRa) to construct a recombinant expression plasmid pCold I-LP85, E.coli DH5 alpha competent cells are transformed and then are cultured on an LB agar plate containing 100 mu g/ml ampicillin for 12-15h, a single colony is picked up, and PCR identification is carried out by MS LP85 gene primers P1 and P2.

Wherein the content of the first and second substances,

primer P1 was: 5'-GAGCTCATGAGCTGTG-3', respectively;

primer P2 was: 5'-CTCGAGTTAGCTTGTACC-3', respectively;

the PCR system (20. mu.l) was: easy Taq PCR mix (2X) 10. mu.l, upstream and downstream primers 0.2. mu. mol/l each, colony template, dd H₂Make up to 20. mu.l. PCR amplification conditions: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 2 min; 10min at 72 ℃. And (3) carrying out amplification culture on the strains (2331bp) identified as positive, extracting plasmids, carrying out double enzyme digestion identification on the plasmids, and identifying the plasmids correctly to obtain the recombinant expression vector pCold I-LP 85.

FIG. 2 shows the results of PCR and double digestion of the recombinant expression vector pCold I-MS LP85 of example 1.

In fig. 2, (a) M: DNA molecular mass standard DL 5000; 1: MS LP85 synthetic gene (positive control); 2: coli DH5 alpha (pCold I-MS LP85) bacterial liquid sample; 3: negative control;

(B) m: DNA molecular standard DL 5000; 4: recombinant plasmid pCold I-MS LP85 double enzyme digestion product

As shown in A in FIG. 2, a single colony of the recombinant strain was identified as positive by PCR, and further subjected to double digestion with Sac I and Xho I to identify the recombinant expression plasmid pCold I-MS LP85, and the results are shown in B in FIG. 2, and B in FIG. 2 shows that a fragment corresponding to the expected size was obtained.

1.2.2 expression and purification of Mycoplasma synoviae recombinant expression protein LP85

E.coli BL21(DE3) was transformed with correctly identified recombinant vector pCold I-MS LP85 to obtain recombinant expression strain E.coli BL 21: the correctly identified recombinant expression vector pCold I-LP85 and the unloaded pCold I were transferred to E.coli BL21, respectively, containing 100 μ g/ml ampicillin in LB medium, 37 ℃, 200rpm/min to bacterial liquid OD₆₀₀0.4 to 0.6;

adding 0.1mM Isopropyl Thiogalactoside (IPTG) into a recombinant expression strain E.coli BL21, and continuously culturing at 16 ℃ and 110rpm/min for 24h to induce to obtain thalli;

and centrifugally collecting the induced thallus, washing the thallus with PBS for 2 times, then suspending the thallus in PBS, cracking the thallus by using an ultrasonicator, centrifugally collecting supernatant and precipitate, respectively detecting the expression of mycoplasma synoviae recombinant expression protein LP85 by using SDS-PAGE, and purifying the His-MS LP85 detected as soluble expression by using a His-tagged protein purification column to obtain the mycoplasma synoviae recombinant expression protein LP85 (represented by rMS LP85 protein).

FIG. 3 shows the expression and purification results of the recombinant expression protein LP85(rMS LP85) of Mycoplasma gallisepticum in example 1.

In fig. 3, M: a protein molecule standard; 1: IPTG-induced empty vector bacteria; 2: cracking the recombinant expression strain induced by IPTG to obtain supernatant; 3: purified recombinant protein rMS LP85

After purification, the BCA kit is used for determining the concentration of the rMS LP85 protein, SDS-PAGE electrophoretic analysis is carried out, the analysis result is shown in figure 3, the figure 3 result shows that the size of a recombinant protein band is about 90kD and is basically consistent with the expected molecular weight of the target protein, and the purified rMS LP85 protein band is pure and has no impurity band.

1.2.3 Western blot analysis of the specificity of the recombinant Mycoplasma synoviae recombinant expression protein LP85(rMS LP85 protein)

Purified rrms LP85 protein was tested for its reactivity to MS positive serum, non-MS positive serum and SPF chicken serum using Western blot: performing SDS-PAGE electrophoresis on rMS LP85 protein (1 mu g/hole), transferring the protein onto an NC membrane, and blocking the protein for 2 hours by 5% skim milk-PBS; then respectively mixing with MS standard positive serum, MS clinical isolates JS1, SD1, SH1 and HB1 positive serum, MG R_lowRespectively incubating strain positive serum, MI695 strain positive serum, EC O1, O2 and O78 trivalent positive serum, SPG positive serum, NDV positive serum, IBV positive serum, IBDV positive serum or SPF chicken negative serum (1:500) at room temperature for 2 hr, washing, adding goat-chicken resistant serum, goat-chickenIgG-HRP (1:50000, Abcam) is incubated for 1.5h at room temperature, and finally ECL substrate developing solution is added for developing reaction.

FIG. 4 shows the Western blot reaction of the recombinant expression protein LP85 of Mycoplasma gallisepticum (rMS LP85 protein) in example 1 with positive sera of different MS clinical isolates.

In fig. 4, M: a protein molecule standard; 1: MS WVU1853 standard positive chicken serum control; 2: MS JS1 positive chicken serum; 3: MS SD1 positive chicken serum; 4: MS SH1 positive chicken serum; 5: MS HB1 positive chicken serum; 6: SPF chicken negative serum control;

the purified rMS LP85 protein is respectively subjected to Western blot reaction with MS WVU1853 standard positive serum and MS clinical isolates JS1, SD1, SH1 and HB1 positive serum, and SPF chicken serum is used as a negative control. The results are shown in figure 4, the protein rMS LP85 (90kD position) and positive serum of different MS isolates can generate obvious reaction bands at corresponding positions, and have no reaction band with SPF chicken serum, and the MS LP85 has better immunoreactivity and better intracorporeal conservation.

FIG. 5 shows the Western blot reaction of the recombinant expression protein of Mycoplasma synoviae (rMS LP85 protein) of example 1 with positive chicken sera of different pathogens.

In fig. 5, M: a protein molecule standard; 1: MS WVU1853 positive serum; 2: MG-positive chicken serum; 3: MI695 strain positive chicken serum; 4: EC (O1, O2, and O78) trivalent positive sera; 5: SPG positive serum; 6: an IBV positive serum; 7: IBDV-positive sera; 8: NDV positive serum

Using purified rMS LP85 protein and MG R_lowWestern blot reaction is respectively carried out on strain chicken positive serum, MI695 strain positive serum, EC O1, O2 and O78 trivalent positive serum, SPG positive serum, NDV positive serum, IBV positive serum and IBDV positive serum, SPF chicken serum is used as negative control, MS WVU1853 standard positive serum is used as positive control, the result is shown in figure 5, the purified protein rMS LP85 has stronger reaction bands with the MS WVU1853 standard positive serum, and the purified protein rMS LP85 does not have reaction bands with other non-MS positive serum.

The immunoreactivity and specificity test results of example 1 indicate that mycoplasma synoviae antigen protein LP85 can be used as a target for ELISA detection of corresponding mycoplasma synoviae antibodies.

Example 2

This example illustrates the establishment of an ELISA assay for Mycoplasma gallisepticum antibodies using the Mycoplasma synoviae antigen protein LP85 screened and identified in example 1, and the corresponding ELISA kits and methods of use.

According to example 1, an ELISA detection method for Mycoplasma synoviae antibodies can be established as follows:

2.1 determination of optimum reaction conditions

2.2.1 determination of optimal antigen coating concentration and optimal serum dilution factor

Diluting rMS LP85 protein antigen with the carbonate coating buffer solution to 10. mu.g/ml, 7.5. mu.g/ml, 5.0. mu.g/ml, 2.5. mu.g/ml and 1.25. mu.g/ml, and coating the enzyme label plate according to the amount of 100. mu.l/hole respectively; respectively diluting MS standard positive serum and SPF chicken negative serum by 1:200 and 1:500 times with the antibody diluent to respectively serve as positive serum control and negative serum control; the enzyme-labeled antibody is goat anti-chicken IgG-HRP, and is diluted by the dilution recommended by Abcam company of a manufacturer; OD determination according to conventional ELISA procedure₄₅₀The value is obtained. Selection of Positive serum OD₄₅₀Value (P) greater than 1.0, negative serum OD₄₅₀The value (N) is less than 0.12, and the coating concentration and the serum dilution factor of the P/N maximum ratio are used as the optimum antigen coating concentration and the optimum serum dilution factor.

The results of the square matrix titration (Table 1) showed that MS LP85 recombinant protein was coated at a concentration of 5. mu.g/ml and positive serum OD was obtained at a 1:200 dilution of serum₄₅₀Value greater than 1.0, negative serum OD₄₅₀The value is less than 0.12 and the P/N value is maximum. Therefore, 5. mu.g/ml is determined as the optimal antigen coating concentration, and 1:200 is determined as the optimal serum dilution factor.

2.2.2 optimization of dilution factor of enzyme-labeled antibody

Coating with the selected antigenCoating enzyme label plate with concentration of 5 μ g/ml, adding MS standard positive serum, SPF chicken negative serum (diluted with the selected optimal dilution multiple of 1: 200), and goat anti-chicken IgG-HRP diluted with 1:10000, 1:20000 and 1:40000 times, and determining OD₄₅₀The value is obtained. Calculating P/N value, and selecting positive serum OD₄₅₀Value greater than 1.0, negative serum OD₄₅₀The value is less than 0.12, and the combination of the maximum ratio of P/N is the dilution of the enzyme-labeled antibody.

The result shows that when the dilution ratio of the enzyme-labeled secondary antibody is 1:20000, the OD of the positive serum₄₅₀Value greater than 1.0, negative serum OD₄₅₀The value is less than 0.12 and the P/N value is maximum. Therefore, the dilution of the enzyme-labeled secondary antibody is determined to be 1: 20000.

2.2.3 optimization of confining liquids

After 5% skim milk or BSA was added to the above PBST buffer as a blocking solution, ELISA was performed, P/N values were compared, and the group with the maximum P/N value was selected and determined as the optimal blocking solution. Specifically, 5% skim milk-PBS and 5% BSA-PBS are respectively used for blocking the ELISA plate, the ELISA plate acts for 2 hours at 37 ℃, and the detection result shows that (table 3) the P/N value of the 5% skim milk-PBS is larger than that of the 5% BSA-PBS blocking group, and the blocking effect is better.

2.2.4 antigen coating Condition optimization

The ELISA plate was coated with the selected antigen coating concentrations and incubated at 4 ℃ overnight and 37 ℃ for 30min, 1h and 2h, respectively. Performing conventional ELISA according to the determined dilution of serum and goat anti-chicken IgG-HRP, and determining OD₄₅₀Selecting the group with the maximum P/N ratio as the optimal antigen packageIs conditioned.

The antigen was coated under different conditions, and the results are shown in Table 4, in which the P/N value was maximal at 37 ℃ for 2 hours, i.e., 16.685, and the positive serum OD₄₅₀The value was 1.485, negative serum OD₄₅₀The value was 0.089, thus determining that the coating conditions were optimal for 2h incubation at 37 ℃.

2.2.5 optimal reaction time for the substrate

Under the optimized conditions, 5 mu g/ml rMS LP85 and 100 mu g/hole are added into an enzyme label plate for coating for 30min, and after washing, 5 percent skim milk is used for sealing for 2 h; adding MS positive serum and SPF chicken serum diluted at a ratio of 1:200, acting for 1.5h, washing, adding goat anti-chicken IgG-HRP diluted at a ratio of 1:20000 times, washing, adding TMB substrate, reacting at 37 deg.C for 5min, 10min, 15min and 20min, adding stop solution, comparing P/N value, and determining optimal reaction time.

The detection results (Table 5) showed that the P/N value was the largest when the color development time was 15min, and therefore, the color development time was determined to be 15 min.

2.2.6 optimization of serum sample dilutions

Adding 5%, 10%, 20%, 30%, 40%, 50% E.coli BL21(DE3) whole bacterial protein into 5% skim milk-PBST (i.e. the above-mentioned antibody diluent) as serum sample diluent, performing conventional ELISA operation according to the determined antigen coating amount and serum and goat anti-chicken IgG-HRP dilution, and determining OD₄₅₀Values, the group with the largest P/N ratio was selected as the serum sample dilution.

The test results (table 6) showed that the P/N value was the largest when 30% of e.coli BL21(DE3) whole bacterial protein was added to 5% skim milk-PBST as serum sample diluent, so that it was determined that 30% of e.coli BL21(DE3) whole bacterial protein was added to the serum sample diluent.

Preparation of BL21 holomyces protein: pcold I no-load (BL21) is transferred to LB culture medium containing ampicillin resistance according to 1 percent, and cultured for 8 hours at 37 ℃ and 220 rmp; collecting pcold I no-load (BL21) bacterial liquid, washing with PBS for 2 times, and adjusting the concentration of the bacterial liquid to OD₆₅₀1.0; after ultrasonication, 5% skim milk-PBST was added in proportion.

2.2.7 determination of Positive cut-off

42 parts of mycoplasma synoviae antibody negative serum are detected by using the kit, and the negative serum is determined according to OD₄₅₀Value calculation S/N values (table 7), and the average value X of the S/N values was 2.163; the standard deviation SD is 0.0761, and the negative-positive critical value point C is determined to be X +3SD 2.16+3X0.0761 2.388, that is, the S/N value of the serum to be detected is equal to or higher than 2.388, and the serum to be detected is judged to be positive, and the serum to be detected is judged to be negative below 2.388.

Calculating the formula: S/N-sample OD₄₅₀Value/negative control OD₄₅₀Value average

2.2.8 optimized ELISA method

The method comprises the following specific steps:

antigen coating of an ELISA plate: diluting the antigen to 5. mu.g/ml with carbonate coating buffer, adding 100. mu.l/well to a 96-well microplate (Corning, USA), incubating at 37 ℃ for 2h, then washing 3 times with PBST, and patting dry;

sealing an enzyme label plate: adding 200 mul of 5% skim milk-PBS into each well of the coated enzyme label plate, sealing for 2h at 37 ℃, continuously washing for 3 times by PBST, and patting to dry;

serum diluted with serum sample diluent was added: adding 100 μ l/well of serum to be detected diluted by 1:200 times via serum sample diluent, adding MS standard positive and negative serum diluted by 1:200 times via serum sample diluent as positive and negative control, respectively, acting at 37 deg.C for 1.5h, washing with PBST for 4 times, and drying;

adding enzyme-labeled antibody diluted by antibody diluent: continuously adding goat anti-chicken IgG-HRP secondary antibody diluted by 1:20000 times of the antibody diluent into each hole, acting for 1h at 37 ℃, washing for 4 times, and patting dry;

adding a substrate for color development: continuously adding 100 mul of TMB substrate color development solution into each hole, and developing for 15min at 37 ℃ in a dark place;

adding a stop solution to complete detection: add 50. mu.l stop buffer to each well and measure OD on microplate reader within 10 minutes₄₅₀The value is obtained.

According to the ELISA method for detecting mycoplasma synoviae antibody established in this embodiment, a corresponding ELISA detection kit can be prepared, which naturally includes the mycoplasma synoviae antigen protein LP85 that can be used as a diagnosis target in example 1, preferably the mycoplasma synoviae recombinant expression protein LP85 that is recombinantly expressed and optimized in example 1, and of course, the kit can further include an ELISA plate, an ELISA antibody, mycoplasma synoviae standard positive serum and mycoplasma synoviae standard negative serum (SPF chicken serum), wherein the ELISA plate can be coated with the mycoplasma synoviae antigen protein LP85 that can be used as a diagnosis target at a certain coating concentration, or can be coated with the coating buffer diluent and the mycoplasma synoviae antigen LP85 that are provided by the ELISA kit during use, and the coating concentration is provided to dilute the ELISA antibody and serum, and complete detection, and can also include coating buffer diluent, washing buffer, confining liquid, antibody diluent, serum sample diluent, substrate solution, termination liquid and other solvents.

In short, the ELISA kit corresponding to the ELISA detection method may be the ELISA protein LP85 only provided for the mycoplasma synoviae antigen as a diagnosis target, or may be an ELISA plate coated with the antigen, which is not listed here, and the kit may be specifically configured according to actual needs.

Example 3

This example is a specific test, a sensitivity test and a reproducibility test of the ELISA detection method and the corresponding kit provided in example 2.

3.1 specificity test

The indirect ELISA detection method established by the optimized conditions is respectively used for MS clinical isolates JS1, SD1, SH1 and HB1 positive serum and mycoplasma gallisepticum MG R_lowDetecting strain positive serum, positive serum of Chlamydomonas Mi695 strain, trivalent positive serum of Escherichia coli EC O1, O2 and O78, SPG positive serum of Salmonella pullorum, NDV positive serum of newcastle disease virus, IBV positive serum of infectious bronchitis virus, and IBDV positive serum of infectious bursal disease virus, setting MS standard positive serum and SPF chicken serum (MS negative serum) control, determining OD₄₅₀Value, commercial Mycoplasma synoviae antibody detection kit (IDEXX) was used as a method control, and the procedure was performed with reference to the instructions.

The detection result of the ELISA detection method shows that as shown in Table 8, the result of the positive serum of mycoplasma synoviae is positive, while the positive serum of mycoplasma gallisepticum, mycoplasma chlamydia, Escherichia coli O1, O2, O78 trivalent serum, the positive serum of salmonella gallinarum bivalent positive chicken, the positive serum of mycoplasma gallinarum, the positive serum of newcastle disease and the negative serum of SPF chicken are negative results, and the results are consistent with the detection result of the IDEXX kit, so that the ELISA method established in the laboratory and the detection of the IDEXX kit have better specificity.

3.2 sensitivity test

The serum to be detected is diluted from the initial dilution by multiple times and is OD₄₅₀The serum maximum dilution factor with the value S/N larger than the critical value is judged as the antibody titer of the serum. Taking MS standard positive serum, diluting with serum sample diluent at 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240 and 1:20480 times respectively, and then using the hair conditionerSerum titers were determined by well established ELISA methods. Meanwhile, antibody titers were measured with a commercial Mycoplasma synoviae antibody detection kit (IDEXX) as a control, and the sensitivities of the two measurement methods were compared.

Randomly taking 5 samples from the sera which are detected to be positive after immunization, detecting by using 3 batches of kits prepared by the ELISA method established in the example 2, comparing the antibody titer with the IDEXX kit, wherein the detection results of the 3 batches and the detection results of the IDEXX kit are respectively shown in tables 9, 10, 11 and 12, and the results of comparing the serum titer detected by two kits in the results of tables 9-12 are shown in Table 13.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.121; the S/N is more than or equal to 2.388, and the mycoplasma synoviae antibody is judged to be positive; S/N<And 2.388, judging the mycoplasma synoviae antibody to be negative.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.129; the S/N is more than or equal to 2.388, and the mycoplasma synoviae antibody is judged to be positive; S/N<And 2.388, judging the mycoplasma synoviae antibody to be negative.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.132; S/N is not less than 2.388, and the judgment is madePositive synovial bursa mycoplasma antibody; S/N<And 2.388, judging the mycoplasma synoviae antibody to be negative.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/P ═ sample OD₆₅₀Value-negative control mean)/(positive control mean-negative control mean); the result shows that S/P is more than or equal to 0.5 and is positive according to the judgment of an IDEXX kit.

The results show that the reagent kit prepared in the laboratory and the reagent kit of IDEXX company are used for simultaneously detecting the mycoplasma synoviae antibody, 30 parts of serum are respectively detected before immunization, 21 days after immunization and 48 days after immunization, and the test reagent kit of the serum before immunization and the IDEXX detection are negative; in the serum 21 days after immunization, 30 positive parts are detected by a trial kit, and 28 positive parts are detected by an IDEXX kit; and (3) detecting positive 30 parts by using a trial kit and positive 29 parts by using an IDEXX kit in serum 42 days after immunization. The coincidence rate of the two kits is 90%. 3 batches of kit and IDEXX prepared in the laboratory detect 5 parts of known positive serum at the same time, the sensitivity of the trial-made kit to 5 parts of positive serum is superior to that of the IDEXX kit, and the sensitivity of the laboratory trial-made kit is better.

3.3 in-batch repeatability test

The same kit was prepared according to the ELISA assay of example 2, and 24 sera were assayed simultaneously, and each sample was run through 5 replicates in each batch and the coefficient of variation was calculated. The results are shown in table 14 and show that: the intra-batch variation coefficient is between 0.88% and 5.19%, which indicates that the ELISA kit has better intra-batch repeatability.

Note: (+) denotes detectionThe result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.088; the S/N is more than or equal to 2.388, and the mycoplasma synoviae antibody is judged to be positive; S/N<And 2.388, judging the mycoplasma synoviae antibody to be negative.

3.4 run-to-run repeatability test

24 sera were tested using 5 batches of kits prepared according to the ELISA test method of example 2, and the batch-to-batch repeat experiments were performed and the coefficient of variation was calculated. The results are shown in Table 15 and show that: the inter-batch variation coefficient is between 2.06% and 5.9%, which shows that the ELISA kit prepared according to the ELISA detection method has better inter-batch repeatability.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.108; the S/N is more than or equal to 2.388, and the mycoplasma synoviae antibody is judged to be positive; S/N<And 2.388, judging the mycoplasma synoviae antibody to be negative.

3.5 detection of clinical samples

150 clinical serum samples were simultaneously tested using the kit prepared according to the ELISA test method of example 2 and the IDEXX kit.

Note: (+) indicates that the detection result is positive; (-) indicates that the detection result is negative; S/N-sample OD₄₅₀Value/negative control OD₄₅₀A value; negative control mean OD₄₅₀0.159; the S/N is more than or equal to 2.388, and the mycoplasma synoviae antibody is judged to be positive; S/N<2.388, judging the mycoplasma synoviae antibody is negative; S/P ═ sample OD₆₅₀Value-negative control mean)/(positive control mean-negative control mean); the result shows that S/P is more than or equal to 0.5 and is positive according to the judgment of an IDEXX kit.

150 clinical serum samples are detected to be positive 104 parts by using the kit related to the embodiment, 65 parts by using the IDEXX kit, 111 parts of serum with consistent detection results and 39 parts of serum with inconsistent detection results of the two kits, and the coincidence rate reaches 74%.

3.6Western blot verification of clinical serum samples not in accordance with IDEXX detection results

Western blot was used to verify 8 clinically negative sera detected in 3.5 that did not match the IDEXX assay.

FIG. 6 shows the results of Western blot analysis of clinical serum samples that did not match the IDEXX test results in example 3.

The results are shown in fig. 6, 8 serum samples all showed positive reactions with prokaryotic expression of rLP85, which was consistent with the detection results of the ELISA detection kit for mycoplasma synoviae antibodies prepared according to the ELISA method of example 2, indicating that the ELISA detection kit is more sensitive and accurate than the IDEXX detection kit.

Examples effects and effects

The respiratory disease of poultry is the most main problem facing the poultry industry in China at present, and mycoplasma gallisepticum and mycoplasma synoviae often cause the persistent infection of chicken flocks, are in a clinical or subclinical state for a long time, cause immunosuppression, cause other important infectious diseases such as the complications and secondary diseases of newcastle disease, avian influenza, infectious bronchitis and the like, and cause serious economic loss. In recent years, the epidemic situation of mycoplasma gallisepticum and mycoplasma synoviae is compared, and the mycoplasma synoviae has increasing harm to the poultry industry, and even the harm may exceed that of mycoplasma gallisepticum (6, 9). Therefore, the method has important significance for the development of poultry industry in China by strengthening the research on the prevention and control of mycoplasma synoviae while preventing and controlling other diseases.

The ELISA detection method based on the mycoplasma synoviae antigen protein LP85, in particular to the ELISA detection method of the corresponding antibody of the mycoplasma synoviae recombinant expression protein LP85 and the kit prepared by the method have the characteristics of better specificity and sensitivity and good detection repeatability. Compared with the MS flat plate agglutination antigen produced by Chinese veterinary drug supervision and the positive serum of the mycoplasma gallisepticum and the mycoplasma chlamydia, the invention can detect the MS antibody more specifically; in addition, the sensitivity of the present invention is 32 times that of the us IDEXX kit; the repeatability test shows that the variation coefficient in batches of the method is less than 6 percent, the variation coefficient in batches is less than 10 percent, and the method has better repeatability.

The method and the ELISA kit for detecting the mycoplasma synoviae antibody prepared by the method are used for detecting clinical serum, the coincidence rate with the American IDEXX kit reaches 74%, and Western blot verification results show that the ELISA kit for detecting the mycoplasma synoviae antibody is more sensitive and accurate and has better clinical application potential.

Sequence listing

<110> Shanghai veterinary research institute of Chinese academy of agricultural sciences

<120> ELISA detection method and kit for mycoplasma synoviae antigen protein LP85 and corresponding antibody

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<170> SIPOSequenceListing 1.0

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Met Ser Cys Gly Ala Pro Gln Glu Glu Lys Lys Pro Thr Asn Pro Lys

1 5 10 15

Asp Gly Asn Lys Asp Gly Gly Ile Ile Asp Leu Pro Pro Thr Ile Gly

20 25 30

Gly Pro Gly Glu Gly Gly Asp Ser Pro Arg Thr Tyr Gly Ser Ile Asp

35 40 45

Pro Val Gln Ser Lys Leu Val Gly Ser Trp Ala Asp Tyr Thr Lys Leu

50 55 60

Ser Ile Ala Lys Arg Tyr Glu Val Asp Asn Lys Ala Tyr Leu Asn Gly

65 70 75 80

Leu Lys Ser Gln Tyr Asp Ile Asp Pro Lys Gln Glu Phe Val Pro Ser

85 90 95

Asp Ile Lys Asn Gly Phe Gly Thr Val Gln Ala Tyr Asp Asp Lys Ala

100 105 110

Thr Lys Leu Gly Leu Pro Asp Phe Val Thr Ser Tyr Leu Lys Gly Phe

115 120 125

Thr Thr Tyr Ser Gly Ser Gly Leu Glu Ile Ser Pro Asn Val Ser Gly

130 135 140

Pro Ala Leu Gly Phe Trp Asn Ser Glu Gln Ser Gly Phe Asp Gly Arg

145 150 155 160

Ser Arg Phe Leu Pro Asn Asp Leu Tyr Lys Asn Thr Ala Leu Gln Thr

165 170 175

Tyr Ser Ile Ser Tyr Val Asn Glu Val Lys Gly Asp Tyr Val Asn Glu

180 185 190

Val Lys Gly Asp Ile Glu Gly Thr Gly Lys Asn Thr Leu Lys Ser Asn

195 200 205

Lys Gly Thr Ala Trp Ile Leu Asp Tyr Val Lys Pro Thr Asp Ser Ser

210 215 220

Tyr Pro Thr Lys Trp Tyr Ile Ala Thr Asn Ile His Val Ile Gly Asp

225 230 235 240

Leu Thr Phe Thr Lys Ser Gln Thr Asp Ser Phe Gly Ser Asp Phe Thr

245 250 255

Ser Ile Tyr Asp Glu Glu Arg Glu Lys Pro Leu Ile Lys Glu Ala Arg

260 265 270

Lys Val Lys Ala Gln Ile Asp Glu Leu Gln Asn Glu Tyr Asn Glu Val

275 280 285

Tyr Arg Lys Leu Gln Thr Lys Glu His Glu Asn Asp Gln Ala Leu Lys

290 295 300

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

305 310 315 320

Lys Leu Lys Asp Leu Asn Ala Gln Ile Ser Gly Leu Thr Lys Asn Val

325 330 335

Thr Leu Ala Ile Leu Asp Ser Asp Val Pro Leu Gln Thr Ser Leu Asn

340 345 350

Thr Val Ser Ala Asp Ser Arg Met Lys Phe Val Thr Leu Pro Ala Ser

355 360 365

Ala Val Asn Ile Val Tyr Thr Ala Asn Asp Phe Leu Lys Thr Ser Pro

370 375 380

Lys Asp Tyr Leu Asp Thr Thr Ser Thr His Asn Lys Asp Tyr Leu Asn

385 390 395 400

Asn Gln Glu Met Ala Asp Phe Ala Val Leu Glu Ile Asp Phe Ser Lys

405 410 415

Val Thr Gly Glu Phe Lys Tyr Thr Lys Asn Ser Val Gly Asp Lys Pro

420 425 430

Ala Lys Glu Met Thr Val Asn Ser Ala Gln Glu Leu Ala Arg Val Met

435 440 445

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

450 455 460

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

465 470 475 480

Val Thr Val Thr Asn Asp Ala Asn Lys Gln Lys Leu Gln Val Ser Arg

485 490 495

Ile Val Ala Asn Phe Val Ser Leu Gly Tyr Pro Ile Ala Lys Asp Asp

500 505 510

Leu Val Asn Leu Arg Ala Glu Phe Pro Ala Lys Tyr Ala Gly Arg Glu

515 520 525

Gly Ile Leu Leu Ala Glu Asn Asp Asn Ser Leu Trp Thr Asn Lys Pro

530 535 540

Gln Lys Gly Arg Asn Phe Tyr Gln Glu Phe Gly Asn Arg Leu Asn Arg

545 550 555 560

Ser Met Ile Leu Arg Asn Phe Val Gln Tyr Pro Gly Ile Tyr Asp Leu

565 570 575

Phe Ile Thr Asn Pro Val Ile Asn Lys Gly Val Gly Phe Asn Ile Lys

580 585 590

Gln Ile Lys Asp Lys Thr Ser Ser Tyr Gln Gln Gly Asn Tyr Leu Asn

595 600 605

Tyr Gly Leu Ala Tyr Ser Leu Glu Ser Trp Arg Pro Ala Pro Gly Ala

610 615 620

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

625 630 635 640

Phe Ala Ile Arg Ala Gly Ala Gly Ser Gly Thr Ser Leu Ile Gln Ala

645 650 655

Phe Arg Ser Glu Gly Ala Asn Tyr Gly Gly Val Tyr Gly Ser Tyr Asn

660 665 670

Leu Pro Gln Tyr Asp Leu Ile Tyr Gly Gly Gly Lys Asp Gln Arg Thr

675 680 685

Ser Tyr Arg Glu Ala Leu Ala Lys Ile Leu Lys Asn Gly Glu Thr Thr

690 695 700

Asn Leu Phe Thr Ser Gly Val Asn Val Ile Pro Glu Glu Tyr Lys Phe

705 710 715 720

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

725 730 735

Ala Gly Leu Ile Thr Asn Thr Gly Gly Gly Val Gln Ala Leu Thr Asp

740 745 750

Gln Asn Lys Lys Tyr Gly Ser Thr Ile Thr Asn Pro Val Val Leu Pro

755 760 765

Glu Gly Thr Ser

770

<210> 2

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagctcatga gctgtg 16

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctcgagttag cttgtacc 18