CN112979767A

CN112979767A - Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof

Info

Publication number: CN112979767A
Application number: CN202110171271.3A
Authority: CN
Inventors: 李书光; 刘吉山; 沈志强; 肖跃强; 曲光刚; 杨立芳; 赵家磊
Original assignee: Shandong Binzhou Animal Science & Veterinary Medicine Academy
Current assignee: Shandong Binzhou Animal Science & Veterinary Medicine Academy
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-18
Anticipated expiration: 2041-02-08
Also published as: CN112979767B

Abstract

The application discloses an antigen composition for detecting mycoplasma bovis antibodies, a kit and application thereof. The antigen composition comprises a first antigen, and the amino acid sequence of the first antigen comprises a sequence shown in SEQ ID No. 1. The antigen composition also comprises a second antigen, and the amino acid sequence of the second antigen comprises a sequence shown in SEQ ID No. 2. The antigen composition of the invention is used as the coating antigen of the ELISA method, and the detection rate of the domestic mycoplasma bovis epidemic serotype is obviously higher than that of the existing imported kit.

Description

Antigen composition for detecting mycoplasma bovis antibody, kit and application thereof

Technical Field

The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and discloses an antigen composition for detecting mycoplasma bovis antibodies, a kit and application thereof.

Background

2015 + 2019, epidemiological investigation shows that the bovine mycoplasma disease has higher and higher incidence in China, can cause the disease of dairy cows and beef cattle, and causes serious economic loss to part of cattle farms. Mycoplasma bovis can cause various diseases such as arthritis, pneumonia and abortion of cattle, and is currently and mostly seen in pneumonia form in China.

The common antigenic proteins of mycoplasma bovis and mycoplasma agalactiae, mycoplasma alcaligenes, mycoplasma filamentous subspecies caprine, mycoplasma filamentous subspecies fil, etc., especially the high homology exists between mycoplasma bovis and Variable surface lipoproteins (Vsps) which are a group of membrane proteins with adhesion function and play a key role in adhesion of mycoplasma bovis to host cells, and the functions may depend on C-terminal antigenic determinant and adhesion structure of Vsps. The common antigen protein causes certain cross agglutination phenomenon of mycoplasma bovis and antiserum of the mycoplasma, and causes misjudgment of clinical serum antibody diagnosis. The existing imported or domestic detection kit has the condition of insufficient sensitivity when detecting the serum antibody of the domestic epidemic strain.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an antigen composition for detecting mycoplasma bovis antibodies, a kit and application thereof.

In one aspect, the present application provides an antigenic composition for the specific detection of antibodies to Mycoplasma bovis comprising a first antigen,

the amino acid sequence of the first antigen comprises a sequence shown in SEQ ID No.1 or a sequence which has more than 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of homology with the sequence shown in SEQ ID No.1 and has the same or similar function with the sequence shown in SEQ ID No. 1.

In the antigen composition, a second antigen is further included, and the amino acid sequence of the second antigen comprises a sequence shown in SEQ ID No.2, or a sequence which has more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of homology with the sequence shown in SEQ ID No.2 and has the same or similar function with the sequence shown in SEQ ID No. 2;

preferably, the mass ratio of the first antigen to the second antigen is 1: (1-20), preferably, 1: (1-10), more preferably, 1: (2-7), more preferably, 1: (3-5).

In another aspect, the present application provides a polynucleotide capable of encoding an antigenic composition of a mycoplasma bovis antibody as described above.

In the above-mentioned polynucleotide, the polynucleotide sequence encoding the first antigen includes the sequence shown in SEQ ID No.3, or a sequence having a homology of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with the sequence shown in SEQ ID No.3, or a complementary sequence of the above-mentioned sequence,

the polynucleotide sequence encoding the second antigen comprises a sequence shown in SEQ ID No.4, or a sequence with homology of more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the sequence shown in SEQ ID No.4, or a complementary sequence of the above sequences.

In another aspect, the present application also provides an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell comprising any of the polynucleotides described above.

In another aspect, the application also protects the application of any one of the antigen composition, the antigen, the polynucleotide, or the expression cassette, the recombinant vector, the recombinant bacterium or the recombinant cell in the preparation of a mycoplasma bovis detection product,

preferably, the method for detecting mycoplasma bovis is ELISA method, and can also be other methods such as immune colloidal gold method,

preferably, the target of mycoplasma bovis detection is mycoplasma bovis antibody, and the mycoplasma bovis antibody comprises polyclonal antibody or monoclonal antibody, more preferably, the polyclonal antibody is from animal body fluid, more preferably, the polyclonal antibody is from animal blood, and more preferably, serum.

In another aspect, the present application further provides an ELISA kit for specifically detecting mycoplasma bovis antibodies, comprising a coating antigen, wherein the coating antigen is any one of the above antigen compositions or the first antigen.

In the above kit, when the coating antigen is the first antigen, the coating concentration of the first antigen is 0.5 to 3.5. mu.g/ml, preferably, 1.5 to 2.5. mu.g/ml, more preferably, 2.0. mu.g/ml;

when the antigenic composition comprises the first antigen and the second antigen,

the first antigen in the coating antigen is coated at a concentration of 0.5-1.5. mu.g/ml, more preferably, 0.8-1.2. mu.g/ml, more preferably, 1.0. mu.g/ml,

the concentration of the second antigen in the coating antigen is 2.0-6.0. mu.g/ml, more preferably, 3.0-5.0. mu.g/ml, more preferably, 4.0. mu.g/ml.

The kit also comprises a solid phase carrier (generally polystyrene or polyvinyl chloride materials, such as PVC plates in test strips or the inner walls of reaction holes of ELISA plates) for fixing the coated antigen, and/or an enzyme-labeled secondary antibody (the secondary antibody is staphylococcal protein A or immune serum IgG, the immune serum IgG is immunoglobulin, the immunoglobulin can be rabbit anti-bovine IgG, sheep anti-bovine IgG and/or mouse anti-bovine IgG, the labeled enzyme used by the enzyme-labeled secondary antibody can be horseradish peroxidase), and/or a coating buffer solution, and/or a sample diluent, and/or a blocking solution, and/or a positive control, and/or a negative control, and/or a color solution (TMB), and/or a stop solution.

In another aspect, the application also protects the use of any one of the above antigen compositions, the first antigen, the polynucleotide, the expression cassette, recombinant vector, recombinant bacterium or recombinant cell, or the kit for the specific detection of Mycoplasma bovis,

preferably, the specific detection method of mycoplasma bovis is ELISA method, and can also be other methods such as immune colloidal gold method,

preferably, the target for specifically detecting mycoplasma bovis is mycoplasma bovis antibody, and the mycoplasma bovis antibody comprises polyclonal antibody or monoclonal antibody, and more preferably, the polyclonal antibody comprises animal serum.

In another aspect, the present application also provides a method for specifically detecting mycoplasma bovis antibodies, comprising the step of contacting any one of the above antigen compositions or the first antigen with antibodies in a test sample, preferably, the test sample is derived from a body fluid, more preferably, blood, more preferably, serum of an animal;

preferably, the detection method comprises ELISA, and other methods such as an immunogold method can be used.

The invention has the beneficial effects that:

on the basis of researching the whole genome sequence of mycoplasma bovis at home and abroad, 2 variable surface lipoproteins are screened, and are mixed according to a certain proportion by a method combining computer prediction analysis and immunology, so that the reactogenicity and sensitivity can be effectively balanced, and the requirement of the variable surface lipoproteins as ELISA detection antigens is met. The antigen composition of the invention is used as the coating antigen of the ELISA method, and the detection rate of the domestic mycoplasma bovis epidemic serotype is obviously higher than that of the existing imported kit.

Drawings

FIG. 1 is SDS electrophoresis and western-blot of 4979.1 protein expressed by pronucleus, wherein M is protein Marker, 1 is ultrasonication precipitation, 2 is ultrasonication supernatant, and 3 is 1.

FIG. 2 is an SDS electrophoresis chart and a western-blot chart of the 0148.1 protein expressed by the pronucleus, wherein M is a protein Marker, 1 is an ultrasonic crushing supernatant, 2 is an ultrasonic crushing precipitate, and 3 is a western-blot of 2.

FIG. 3 is an SDS electrophoresis chart and a western-blot chart of the 2700.1 protein expressed by the pronucleus, wherein M is a protein Marker, 1 is an ultrasonic crushing supernatant, 2 is an ultrasonic crushing precipitate, and 3 is a western-blot of 2.

FIG. 4 shows the results of the analysis of the reactogenicity of three antigenic proteins.

FIG. 5 shows the results of the analysis of the reactogenicity of the combination of the three antigens.

Detailed Description

Example 1 obtaining of Mycoplasma bovis-specific antigenic proteins

1.1 acquisition of complete Gene sequences of different strains of M.bovis

33 representative Mycoplasma bovis (Mycoplasma bovis) complete gene sequences were found from the National Center for Biotechnology Information (NCBI), and the complete gene sequences were obtained by sequencing of Mycoplasma bovis (isolated in the literature "diagnosis and treatment report of Mycoplasma bovis, Lishuguang, et al, Proc. zoology of domestic animals", 2017, 38 (2): 64-66 "1.4, hereinafter referred to as" Mycoplasma bovis Shandong strain ") isolated in the laboratory.

1.2 computer predictive analysis of antigenic proteins

The protein localization, transmembrane region, antigen parameters and homology alignment of different mycoplasma bovis strains are analyzed by using online biology software PSORTb, Vaxign 2Beta, BLASTp and the like to obtain the following candidate mycoplasma bovis specific antigen proteins 4979.1, 0148.1 and 2700.1.

1.3 codon optimization of candidate Mycoplasma bovis specific antigen protein and construction of expression vector

And analyzing whether the candidate mycoplasma bovis specific antigen protein contains TGA codons by using DNAstar software, modifying the TGA codons into TGG, further performing codon optimization, codon usage frequency analysis, Escherichia coli expression adaptability parameter prediction and the like by using online codon optimization software, and synthesizing and connecting the optimized gene sequence to a vector PET-32A to obtain a recombinant vector so as to express the corresponding candidate mycoplasma bovis specific antigen protein.

The sequence information of the optimized candidate mycoplasma bovis specific antigen protein is as follows:

4979.1 variable surface lipoproteins: the amino acid sequence (285aa, SEQ ID No.1) is as follows;

MKKSKFLLLTTLSPIISLPFLSASCITGEKADNKMEKDIKINENTDEKNSSETMNDKQKQNKSSIDSKMEEKADKDSSKSEEGKSMENEHADEKNSSETMNDKQKQNKSSIDSKMEEKADKDSSKSEEGKSMENEHADEKNSSETMNDKQKQDSGTNSDQKDQDSKTNGSSNEPIRPSESAQSDMQIENSEINTYLDEFNEYVKEASLLKEKKEGTEKAKELLDKNPELEKAVKTLSAFHEHLKSGLEKIKKLIEKDIKNPGSELLDTLKEYVKYRKEIQNAIKN；

4979.1 optimized gene sequence of variable surface lipoprotein (855bp, SEQ ID No.4) is as follows:

ATGAAAAAGTCAAAATTTTTGCTATTAACAACTTTGTCTCCGATCATCAGCCTTCCGTTTCTGTCGGCCAGCTGCATTACCGGTGAAAAGGCGGATAATAAAATGGAGAAAGACATTAAGATCAATGAGAACACCGATGAAAAAAACAGCTCCGAGACGATGAATGATAAGCAGAAGCAAAACAAGAGCTCGATCGACAGCAAAATGGAGGAAAAAGCTGATAAGGACAGCAGTAAGTCCGAGGAGGGCAAATCCATGGAAAACGAACATGCTGATGAGAAAAACTCCTCCGAGACCATGAACGACAAACAAAAACAAAACAAAAGCTCGATTGACAGCAAGATGGAGGAGAAAGCGGACAAGGACTCTTCCAAATCTGAAGAGGGCAAATCCATGGAAAACGAGCACGCCGACGAAAAGAACAGCTCAGAAACCATGAATGATAAGCAAAAGCAAGATAGCGGCACCAATAGCGATCAGAAGGACCAGGATAGCAAAACGAATGGTAGCAGCAACGAACCGATTCGCCCAAGCGAAAGCGCGCAGAGCGACATGCAGATCGAAAATTCTGAGATTAACACCTATCTGGATGAATTCAACGAGTACGTGAAGGAAGCAAGCCTGCTCAAAGAGAAGAAAGAGGGCACGGAAAAGGCGAAAGAGTTGCTGGACAAAAACCCGGAACTGGAAAAAGCGGTTAAAACCCTGTCTGCGTTCCACGAACATCTGAAGTCTGGTCTGGAGAAAATCAAAAAGCTGATTGAAAAGGACATCAAGAACCCGGGTTCGGAGCTGTTGGACACCTTAAAGGAGTACGTCAAGTATCGTAAAGAGATCCAGAATGCAATTAAGAAC；

0148.1 variable surface lipoproteins: the amino acid sequence (265aa, SEQ ID No.2) is as follows:

MKKSKFLLLGSVASLASIPFVAAKCGETKEEEKKKPEGDQKPGGDKNPGENKTPGENTDQGKNPGGDKNPGGDKNPGENTEPDKNPGGDKNPGENKTPEGNKTPEGNKTPEGNKTPGENKTPERNPEEGSTNNGIGWGENHLWGKDEDDLEKSKQESDEAEREGEDSEAESEKGSDTESSKKDKGSDTESSKKDKDPKTESSTKAKDPETQSGKPVVKPMVRVTENTSENSKVKKVDKYWDGVTPEDKKKAIESGKFWAEHWELE；

0148.1 optimized gene sequence of variable surface lipoprotein (795bp, SEQ ID No.5) is as follows:

ATGAAAAAGTCAAAATTTCTATTATTGGGATCTGTGGCGAGCCTGGCATCGATCCCGTTCGTGGCGGCTAAGTGCGGTGAAACCAAAGAGGAGGAGAAAAAGAAGCCGGAGGGCGACCAGAAACCGGGTGGCGACAAGAACCCGGGCGAGAACAAGACCCCGGGCGAAAACACGGATCAAGGTAAAAATCCGGGGGGCGATAAAAACCCGGGCGGTGACAAGAACCCGGGCGAGAACACCGAGCCGGATAAGAATCCGGGCGGCGATAAGAATCCGGGTGAGAACAAAACTCCGGAAGGCAACAAAACCCCTGAGGGCAACAAGACGCCAGAGGGTAATAAGACTCCGGGCGAGAACAAGACCCCGGAGCGCAATCCGGAGGAGGGTAGCACGAATAATGGTATCGGTTGGGGTGAGAACCACCTGTGGGGGAAAGACGAGGATGATCTGGAGAAAAGCAAACAAGAATCAGACGAGGCCGAACGTGAAGGTGAAGACAGCGAAGCTGAATCGGAAAAAGGTTCCGACACCGAAAGTAGCAAGAAAGACAAGGGCAGCGACACGGAATCCAGCAAGAAGGACAAGGATCCGAAAACCGAGTCTTCTACCAAAGCGAAAGACCCGGAAACCCAGTCCGGTAAACCGGTGGTTAAACCCATGGTTCGTGTTACCGAGAACACCAGCGAAAACAGCAAGGTGAAAAAAGTCGATAAGTACTGGGATGGTGTTACCCCAGAAGATAAAAAGAAGGCGATTGAAAGCGGTAAGTTTTGGGCAGAACATTGGGAATTGGAA；

2700.1 variable surface lipoproteins: the amino acid sequence (290aa, SEQ ID No.3) is as follows:

MKKSKFLILGSIASSSLLMIAASCDVKKKDVETSNSEKQDSKTNSQSNSGNQGSRPDSESNSGNQGSKINSQSNSGNQGSRPDSESNSEKQDSKTNSQSNSGNQGSRPDSESNSGNQGSKINSQSNSGNQGSRPDSESNSGNQGSKTDSESNSENQDSKTDGSRHESTTPSESTQNDIPTENYKIDDYLNRVKTYGKEANLLITLLAGAWTENYDELQKKYASLWPIVTEFFKLHEKIVSKFDEIKKEIEEFFGKPESKNNKLDNLLKQYEKSRDEIKDAIEVLKRLQKK；

2700.1 the optimized gene sequence (870bp, SEQ ID No.6) of the protein is as follows;

ATGAAAAAGTCAAAATTTTTAATACTAGGAAGCATCGCCAGCAGCTCCCTGTTAATGATTGCGGCGTCTTGCGATGTTAAAAAGAAGGACGTGGAGACAAGCAACAGCGAGAAACAGGACTCGAAGACCAACTCGCAATCTAACTCCGGTAACCAGGGCTCTCGTCCGGACTCCGAGTCCAACTCCGGCAACCAAGGTTCGAAGATCAATAGCCAGAGTAACAGCGGCAACCAAGGTTCACGTCCGGACAGCGAGTCTAATAGCGAGAAACAAGACTCCAAGACCAATTCCCAAAGCAACTCCGGTAACCAGGGTAGCCGTCCGGACTCTGAGTCCAACAGCGGTAACCAGGGTAGCAAAATTAACTCCCAGAGCAATAGCGGCAACCAGGGTAGCCGCCCAGACTCTGAGAGCAACTCCGGTAACCAGGGCTCGAAGACCGACAGCGAGTCTAACAGCGAGAACCAAGACAGCAAGACTGACGGCAGCCGCCATGAATCTACGACCCCGAGCGAAAGTACCCAGAATGATATTCCGACCGAAAATTACAAGATCGATGATTATCTGAATCGTGTTAAAACGTACGGCAAAGAGGCAAATTTGCTCATTACCCTGTTGGCTGGCGCGTGGACCGAAAATTATGATGAACTGCAGAAAAAGTATGCGAGCCTTTGGCCGATTGTGACCGAGTTCTTCAAACTGCACGAAAAGATCGTCAGCAAGTTTGATGAAATTAAAAAGGAAATCGAGGAATTTTTCGGTAAACCGGAATCTAAGAACAACAAACTGGATAATCTGTTGAAGCAATACGAAAAAAGCAGAGATGAGATCAAAGATGCAATCGAAGTTCTGAAACGTCTGCAAAAAAAG。

1.4 expression and purification of antigenic proteins

Respectively transferring the recombinant vectors in the step 1.3 into expression host bacteria BL21, selecting positive colonies, inoculating an LB culture medium containing Kan antibiotics, inducing by using IPTG, purifying by using commercial HIS labels to obtain corresponding antigen proteins, and detecting by electrophoresis until the sizes meet expectations, wherein the results are shown in figures 1-3.

Example 2 analysis of the reactogenicity of different Mycoplasma bovis-specific antigenic proteins

The purified antigen protein obtained in step 1.4 of example 1 was coated on the reaction wells of the ELISA plate according to coating concentration gradients of 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5 and 0.25. mu.g/mL, respectively.

The inactivated culture of mycoplasma bovis Shandong strain is used to immunize New Zealand rabbit to prepare positive serum, and the research of its reactogenicity is carried out, and the nonimmune New Zealand white rabbit serum is used as negative control.

Preparation of a buffer:

coating buffer (ph9.6 carbonate buffer): 1.59g of sodium carbonate, 2.93g of sodium bicarbonate, and the volume of the solution was adjusted to 1000ml using ultrapure water, and the pH was confirmed to be 9.6 by a pH meter, and the solution was filtered through a 0.44 μm membrane for use.

Washing buffer solution: weighing 8.0g of sodium chloride, 0.2g of potassium dihydrogen phosphate, 6.29g of disodium hydrogen phosphate dodecahydrate and ultrapure water, fixing the volume to 1000ml, adding 0.5ml of Tween-20, adjusting the pH value to 7.2, filtering by using a 0.44 mu m membrane, and storing for later use.

Sealing liquid: bovine Serum Albumin (BSA) was prepared at 0.8% in a washing buffer, and the resulting mixture was subjected to filtration sterilization to prepare a blocking solution.

Sample diluent: same as the confining liquid.

Positive serum: the purified protein is prepared by immunizing New Zealand white rabbits.

Negative control: healthy New Zealand white rabbit serum without immune antigen protein.

Enzyme-labeled secondary antibody: the HRP-labeled staphylococcal protein A is diluted by a blocking solution at a ratio of 1:5000 when in use.

Stopping liquid: 1M sulfuric acid.

Experimental procedure

1. The 3 proteins for purification of example 1 were diluted to 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, and 0.25. mu.g/ml using coating buffer, and added to ELISA reaction plates at 100. mu.l/well overnight at 4 ℃.

2. Spin-drying to remove coating buffer, adding 150 μ l washing buffer into each well, shaking for 2min, and spin-drying; the washing was repeated 3 times.

3. Blocking buffer was added at 200. mu.l per well and blocked for 1 hour.

4. Washing buffer solution is used, 200 mu l of washing buffer solution is used for each hole, shaking is carried out for 2min, and drying is carried out; the washing was repeated 3 times.

5. The prepared positive serum (diluted 1:100 using blocking buffer) was added in 100. mu.l per well, set in 8 replicates, and reacted at 37 ℃ for 30 min.

6. Washing buffer solution is used, 200 mu l of washing buffer solution is used for each hole, shaking is carried out for 2min, and drying is carried out; the washing was repeated 3 times.

7. Add enzyme-labeled secondary antibody diluted with blocking buffer 1:5000, 100. mu.l per well, and react at 37 ℃ for 30 min.

8. Washing buffer solution is used, 200 mu l of washing buffer solution is used for each hole, shaking is carried out for 2min, and drying is carried out; the washing was repeated 3 times.

9. Adding TMB color development solution, each well is 100 μ l, and reacting for 15min at room temperature in dark.

10. Stop solution was added in an amount of 50. mu.l per well, and the OD450 value was measured using a microplate reader.

As a result: the OD450 values for different specific coating antigens at different coating concentrations are shown in table 1, table 2 and table 3. Plots were made using GraphPAD prism based on coating antigen concentration and OD450 averages as shown in figure 4.

Reactogenicity of antigenic proteins of Table 1, 4979.1

Table 2, 0148.1 reactogenicity of antigenic proteins

Table 3, 2700.1 reactogenicity of antigenic proteins

The results showed that by plotting a horizontal line with an OD450 ═ 1.0 in fig. 4, the coating concentrations of the corresponding three antigenic proteins 4979.1, 0148.1, 2700.1 were 2, 8, 20 μ g/ml, respectively.

Example 3 analysis of reaction specificity of Mycoplasma bovis-specific antigen protein

The procedure of example 2 was followed except that: 4979.1, 0148.1 and 2700.1 proteins were coated on ELISA plates using 2. mu.g/ml, 8. mu.g/ml and 20. mu.g/ml, respectively, as coating antigens; the specificity of the rabbit-derived positive serum of the protein-prepared mycoplasma bovis is determined by replacing the positive serum of the goat subspecies of the mycoplasma bovis, the mycoplasma alcaligenes, the mycoplasma filiform subspecies, or the mycoplasma filiform subspecies.

The positive serum of the mycoplasma lactis, the mycoplasma alcaligenes, the mycoplasma filiform subspecies goat subspecies or the mycoplasma filiform subspecies is prepared by the following steps:

mycoplasma agalactiae (CVCC344), Mycoplasma alcaligenes (YX), Mycoplasma filiformis subspecies caprine (CVCC3009), or Mycoplasma filiformis subspecies filiformis (CVCC378) were all sourced from the open-backed laboratory at the State department of veterinary and livestock research institute, Binshou, Shandong province. The 4 mycoplasma inactivated cultures were used to immunize new zealand white rabbits, respectively, to prepare corresponding positive serum antibodies.

As a result: as shown in Table 4, the OD450 values in the measurement of the specificity of the ELISA detection method using the positive sera of M.lactis, M.alcaligenes, M.filiformis subspecies goat, M.filiformis, and M.filiformis subspecies were all 0.182 or less, and the OD450 of the positive sera of M.bovis proteins was 0.958-1.158, indicating that the 3 candidate antigen proteins in example 2 did not cross-react with the positive sera of M.lactis, M.alcaligenes, M.filiformis subspecies goat, and M.filiformis, i.e., they were able to specifically detect the positive sera of M.bovis.

TABLE 4 results of specificity detection of different envelope antigens

Example 4 combination and optimization of different Mycoplasma bovis-specific antigenic proteins

Determination of combination ratio of specific antigen proteins of different mycoplasma bovis

According to the comparison of the OD450 value reactogenicity of each antigen protein in example 2, 4989.1, 0148.1 and 2700.0 have coating concentrations of about 2, 8 and 20. mu.g/ml at an OD450 value of 1, so that the mixed coating mass ratios of 4979.1 to 0148.1, 4979.1 to 2700.1, 0148.1 to 2700.1 antigen proteins are set to 1:4, 1:10 and 2:5, respectively.

Second, screening of optimal coating concentration of combinations of mycoplasma bovis-specific antigen proteins

The total coating concentration of antigen proteins mixed two by two in step one is set as a gradient of 20, 10, 5 and 2.5. mu.g/ml, coating and ELISA detection are carried out according to the method of example 2, and the optimal coating concentration of the antigen combination is determined. The results are shown in tables 5, 6, 7 and 5.

TABLE 5, 4979.1 and 0148.1 test results of antigen proteins mixed at 1:4

TABLE 6, 4979.1 test results of 1:10 mixing of antigen protein 2700.1

TABLE 7, 0148.1 test results of 2:5 mixtures of antigen proteins with 2700.1

The results show that by plotting a horizontal line with an OD450 ═ 1.0 in fig. 5, the optimal coating concentrations for the three combinations 4979.1 and 0148.1, 4979.1 and 2700.1, 0148.1 and 2700.1 were 5, 13, 20 μ g/ml, respectively. Wherein, the combination curve of 4979.1 and 0148.1 is reasonable, and the optimal concentration is the lowest, which is 5 mug/ml, thereby being convenient for reducing the subsequent cost.

Example 5 Blind sample detection

1. ELISA plates were coated using the optimum coating antigen concentrations of 4979.1 and 0148.1 determined in example 4, 5. mu.g/ml, mixed at a ratio of 1: 4.

2. 30 negative clinical specimens were screened using a commercial Mycoplasma bovis antibody detection kit (manufactured by Bovinheck, Canada, catalog number 201012) for determination of cut-off values.

3. Three replicates were performed after 1:100 dilutions of each negative clinical sample (serum) from step 2, and OD450 values were determined according to the ELISA protocol in example 2, and the cut-off was calculated as OD450 mean (X) +3 × standard deviation (S). And when the average value of the OD450 of the sample to be detected is less than the critical value, judging the result to be negative, and when the average value of the OD450 of the sample to be detected is more than or equal to the critical value, judging the result to be positive.

4. Using a commercial mycoplasma bovis antibody detection kit (bovinheck, ca, catalog No. 201012) as a control, the ELISA plate of step 1 was used to perform the ELISA procedures of example 2 on the clinical 301 remaining samples to be tested (bovine serum), respectively, and the results are shown in table 8.

TABLE 8 comparison of the results of the blind tests

Reagent kit	Number of positive samples	Number of negative samples	Rate of positive detection
				Control	147	154	48.8％
Step
	1	172	129	57.14％

The results in table 8 show that the positive detection rate of mycoplasma bovis antibodies using the ELISA plate of step 1 is significantly higher than the control commercial kit.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

SEQUENCE LISTING

<110> Shandong province Binzhou animal husbandry veterinary research institute

<120> antigen composition for detecting mycoplasma bovis antibody, kit and application thereof

<130> JN-CNP202125

<160> 6

<170> PatentIn version 3.5

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Met Lys Lys Ser Lys Phe Leu Ile Leu Gly Ser Ile Ala Ser Ser Ser

1 5 10 15

Leu Leu Met Ile Ala Ala Ser Cys Asp Val Lys Lys Lys Asp Val Glu

20 25 30

Thr Ser Asn Ser Glu Lys Gln Asp Ser Lys Thr Asn Ser Gln Ser Asn

35 40 45

Ser Gly Asn Gln Gly Ser Arg Pro Asp Ser Glu Ser Asn Ser Gly Asn

50 55 60

Gln Gly Ser Lys Ile Asn Ser Gln Ser Asn Ser Gly Asn Gln Gly Ser

65 70 75 80

Arg Pro Asp Ser Glu Ser Asn Ser Glu Lys Gln Asp Ser Lys Thr Asn

85 90 95

Ser Gln Ser Asn Ser Gly Asn Gln Gly Ser Arg Pro Asp Ser Glu Ser

100 105 110

Asn Ser Gly Asn Gln Gly Ser Lys Ile Asn Ser Gln Ser Asn Ser Gly

115 120 125

Asn Gln Gly Ser Arg Pro Asp Ser Glu Ser Asn Ser Gly Asn Gln Gly

130 135 140

Ser Lys Thr Asp Ser Glu Ser Asn Ser Glu Asn Gln Asp Ser Lys Thr

145 150 155 160

Asp Gly Ser Arg His Glu Ser Thr Thr Pro Ser Glu Ser Thr Gln Asn

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Trp Pro Ile Val Thr Glu Phe Phe Lys Leu His Glu Lys Ile Val Ser

225 230 235 240

Lys Phe Asp Glu Ile Lys Lys Glu Ile Glu Glu Phe Phe Gly Lys Pro

245 250 255

Glu Ser Lys Asn Asn Lys Leu Asp Asn Leu Leu Lys Gln Tyr Glu Lys

260 265 270

Ser Arg Asp Glu Ile Lys Asp Ala Ile Glu Val Leu Lys Arg Leu Gln

275 280 285

Lys Lys

290

<210> 4

<211> 855

<212> DNA

<213> Artificial sequence

<400> 4

atgaaaaagt caaaattttt gctattaaca actttgtctc cgatcatcag ccttccgttt 60

ctgtcggcca gctgcattac cggtgaaaag gcggataata aaatggagaa agacattaag 120

atcaatgaga acaccgatga aaaaaacagc tccgagacga tgaatgataa gcagaagcaa 180

aacaagagct cgatcgacag caaaatggag gaaaaagctg ataaggacag cagtaagtcc 240

gaggagggca aatccatgga aaacgaacat gctgatgaga aaaactcctc cgagaccatg 300

aacgacaaac aaaaacaaaa caaaagctcg attgacagca agatggagga gaaagcggac 360

aaggactctt ccaaatctga agagggcaaa tccatggaaa acgagcacgc cgacgaaaag 420

aacagctcag aaaccatgaa tgataagcaa aagcaagata gcggcaccaa tagcgatcag 480

aaggaccagg atagcaaaac gaatggtagc agcaacgaac cgattcgccc aagcgaaagc 540

gcgcagagcg acatgcagat cgaaaattct gagattaaca cctatctgga tgaattcaac 600

gagtacgtga aggaagcaag cctgctcaaa gagaagaaag agggcacgga aaaggcgaaa 660

gagttgctgg acaaaaaccc ggaactggaa aaagcggtta aaaccctgtc tgcgttccac 720

gaacatctga agtctggtct ggagaaaatc aaaaagctga ttgaaaagga catcaagaac 780

ccgggttcgg agctgttgga caccttaaag gagtacgtca agtatcgtaa agagatccag 840

aatgcaatta agaac 855

<210> 5

<211> 795

<212> DNA

<213> Artificial sequence

<400> 5

atgaaaaagt caaaatttct attattggga tctgtggcga gcctggcatc gatcccgttc 60

gtggcggcta agtgcggtga aaccaaagag gaggagaaaa agaagccgga gggcgaccag 120

aaaccgggtg gcgacaagaa cccgggcgag aacaagaccc cgggcgaaaa cacggatcaa 180

ggtaaaaatc cggggggcga taaaaacccg ggcggtgaca agaacccggg cgagaacacc 240

gagccggata agaatccggg cggcgataag aatccgggtg agaacaaaac tccggaaggc 300

aacaaaaccc ctgagggcaa caagacgcca gagggtaata agactccggg cgagaacaag 360

accccggagc gcaatccgga ggagggtagc acgaataatg gtatcggttg gggtgagaac 420

cacctgtggg ggaaagacga ggatgatctg gagaaaagca aacaagaatc agacgaggcc 480

gaacgtgaag gtgaagacag cgaagctgaa tcggaaaaag gttccgacac cgaaagtagc 540

aagaaagaca agggcagcga cacggaatcc agcaagaagg acaaggatcc gaaaaccgag 600

tcttctacca aagcgaaaga cccggaaacc cagtccggta aaccggtggt taaacccatg 660

gttcgtgtta ccgagaacac cagcgaaaac agcaaggtga aaaaagtcga taagtactgg 720

gatggtgtta ccccagaaga taaaaagaag gcgattgaaa gcggtaagtt ttgggcagaa 780

cattgggaat tggaa 795

<210> 6

<211> 870

<212> DNA

<213> Artificial sequence

<400> 6

atgaaaaagt caaaattttt aatactagga agcatcgcca gcagctccct gttaatgatt 60

gcggcgtctt gcgatgttaa aaagaaggac gtggagacaa gcaacagcga gaaacaggac 120

tcgaagacca actcgcaatc taactccggt aaccagggct ctcgtccgga ctccgagtcc 180

aactccggca accaaggttc gaagatcaat agccagagta acagcggcaa ccaaggttca 240

cgtccggaca gcgagtctaa tagcgagaaa caagactcca agaccaattc ccaaagcaac 300

tccggtaacc agggtagccg tccggactct gagtccaaca gcggtaacca gggtagcaaa 360

attaactccc agagcaatag cggcaaccag ggtagccgcc cagactctga gagcaactcc 420

ggtaaccagg gctcgaagac cgacagcgag tctaacagcg agaaccaaga cagcaagact 480

gacggcagcc gccatgaatc tacgaccccg agcgaaagta cccagaatga tattccgacc 540

gaaaattaca agatcgatga ttatctgaat cgtgttaaaa cgtacggcaa agaggcaaat 600

ttgctcatta ccctgttggc tggcgcgtgg accgaaaatt atgatgaact gcagaaaaag 660

tatgcgagcc tttggccgat tgtgaccgag ttcttcaaac tgcacgaaaa gatcgtcagc 720

aagtttgatg aaattaaaaa ggaaatcgag gaatttttcg gtaaaccgga atctaagaac 780

aacaaactgg ataatctgtt gaagcaatac gaaaaaagca gagatgagat caaagatgca 840

atcgaagttc tgaaacgtct gcaaaaaaag 870

Claims

1. An antigen composition for specifically detecting mycoplasma bovis antibodies, wherein the antigen composition comprises a first antigen, and the amino acid sequence of the first antigen comprises a sequence shown in SEQ ID No.1, or a sequence which has more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID No.1 and has the same or similar function with the sequence shown in SEQ ID No. 1.

2. The antigenic composition of claim 1 which further comprises a second antigen having an amino acid sequence comprising the sequence of SEQ ID No.2 or a sequence which is more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the sequence of SEQ ID No.2 and which is functionally identical or similar to the sequence of SEQ ID No. 2;

preferably, the mass ratio of the first antigen to the second antigen in the antigen composition is 1: (1-20), preferably, 1: (1-10), more preferably, 1: (2-7), more preferably, 1: (3-5).

3. An antigen for specifically detecting mycoplasma bovis antibodies, wherein said antigen is the first antigen of claim 1.

4. A polynucleotide capable of encoding an antigenic composition of said mycoplasma bovis antibody of claim 1 or 2;

preferably, the polynucleotide sequence encoding said first antigen comprises the sequence shown in SEQ ID No.3, or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID No.3, or the complement of the above,

preferably, the polynucleotide sequence encoding the second antigen comprises the sequence shown in SEQ ID No.4, or a sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequence shown in SEQ ID No.4, or a complementary sequence of the above sequences.

5. An expression cassette, recombinant vector, recombinant bacterium or recombinant cell comprising the polynucleotide of claim 4.

6. Use of the antigenic composition of claim 1 or 2, the antigen of claim 3, the polynucleotide of claim 4, or the expression cassette, recombinant vector, recombinant bacterium or recombinant cell of claim 5 in the preparation of a Mycoplasma bovis detection product,

preferably, the method for detecting mycoplasma bovis comprises ELISA method,

preferably, the target of mycoplasma bovis detection is mycoplasma bovis antibody, which comprises polyclonal antibody or monoclonal antibody, more preferably, the polyclonal antibody is derived from body fluid of animal, more preferably, blood, more preferably, serum.

7. An ELISA kit for specifically detecting Mycoplasma bovis antibodies, said kit comprising a coating antigen, said coating antigen being the antigen composition of claim 1 or 2 or the antigen of claim 3;

preferably, when the coating antigen is the antigen of claim 3, the first antigen is coated at a concentration of 0.5-3.5 μ g/ml, preferably, 1.5-2.5 μ g/ml, more preferably, 2.0 μ g/ml;

when the coating antigen is the antigen composition of claim 2,

8. The kit according to claim 7, further comprising a solid support for immobilizing the coated antigen, and/or an enzyme-labeled secondary antibody, and/or a coating buffer, and/or a sample diluent, and/or a blocking solution, and/or a positive control, and/or a negative control, and/or a developing solution, and/or a stop solution.

9. Use of the antigen composition of claim 1 or 2, the antigen of claim 3, the polynucleotide of claim 4, the expression cassette, recombinant vector, recombinant bacterium or recombinant cell of claim 5, or the kit of claim 7 or 8 for the specific detection of Mycoplasma bovis,

preferably, the method for detecting mycoplasma bovis comprises ELISA method,

10. A method for specifically detecting Mycoplasma bovis antibodies, comprising the step of contacting the antigen composition of claim 1 or 2 or the antigen of claim 3 with antibodies in a test sample,

preferably, the sample to be tested is derived from a body fluid, more preferably, blood, more preferably, serum of an animal;

preferably, the method of detection comprises ELISA.