Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for chicken mycoplasma synoviae antibody

Abstract

The invention belongs to the technical field of antibody detection kits, and relates to an application of chicken mycoplasma synoviae recombinant protein FBA in preparation of a chicken mycoplasma synoviae antibody indirect ELISA detection kit, which is characterized in that the amino acid sequence of the chicken mycoplasma synoviae recombinant protein FBA is as shown in SEQ ID NO.1. The indirect ELISA detection kit for the chicken mycoplasma synoviae antibody is characterized by comprising an elisa plate which takes chicken mycoplasma synoviae recombinant protein FBA as a coating antigen, wherein the amino acid sequence of the chicken mycoplasma synoviae recombinant protein FBA is shown as SEQ ID NO.1. The method is helpful for deeply developing the research on chicken mycoplasma synoviae infection and immunity, and a rapid, sensitive and good-specificity method is provided for clinical diagnosis of chicken mycoplasma synoviae infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection kits, and specifically relates to an indirect ELISA detection kit for Mycoplasma synoviae antibodies in chickens. Background Art

[0002] Mycoplasma synoviae is a pathogen that affects the growth and development of poultry. After chicken flocks are infected with this pathogen, it can cause respiratory tract lesions, arthritis, etc., resulting in slow growth and reduced egg production in chicken flocks. In addition, infected chicken flocks are more susceptible to other pathogens, such as avian influenza and Newcastle disease, causing serious harm to the poultry breeding industry. Currently, Mycoplasma synoviae has been reported in Oceania, North and South America, Africa, Europe, Asia, etc. In China, the infection rate of Mycoplasma synoviae, especially the infection rate of chicken embryos, is also becoming increasingly serious. Therefore, establishing a diagnostic method for specifically, sensitively, and rapidly detecting serum antibodies is of great significance for preventing and controlling Mycoplasma synoviae infection.

[0003] Based on clinical symptoms, pathological changes, and epidemiology, a preliminary diagnosis of Mycoplasma synoviae infection can be made, but laboratory diagnosis is still required for confirmation. Laboratory diagnosis is divided into histological diagnosis, etiological diagnosis, and serological diagnosis, and each test method has its own advantages and disadvantages. Pathogen isolation and identification is the most reliable method for diagnosing Mycoplasma synoviae, but due to the high requirements for mycoplasma culture and the difficulty of isolation, pathogen isolation and identification are less commonly used in disease diagnosis. Molecular biology diagnosis and serological detection methods are currently more commonly used in laboratories. The PCR method has good sensitivity and short detection time, but has high requirements for the operation skills of detection personnel, so it is difficult to be widely applied. Among serological diagnosis methods, ELISA has the advantages of specificity, sensitivity, simplicity, rapidity, stability, etc., has relatively low requirements for personnel operation, and is suitable for batch detection. Currently, there are already commercial ELISA antibody kits on the market that mainly use Mycoplasma synoviae VlhA as the coating antigen, but due to the recombination of Mycoplasma synoviae pseudogenes and VlhA genes, antigen variation will occur, resulting in false negative results. Therefore, it is very necessary to establish an ELISA method and kit for detecting Mycoplasma synoviae antibodies with good specificity and high sensitivity. Summary of the Invention

[0004] In order to overcome the deficiencies and drawbacks of the prior art, the purpose of the present invention is to provide an indirect ELISA detection kit for Mycoplasma synoviae antibodies, which helps to deeply carry out research on Mycoplasma synoviae infection and immunity, and provides a fast, sensitive, and specific method for the diagnosis of clinical Mycoplasma synoviae infection.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] Application of recombinant protein FBA of Mycoplasma synoviae in preparing indirect ELISA detection kit for Mycoplasma synoviae antibodies in chickens, characterized in that: the amino acid sequence of the recombinant protein FBA of Mycoplasma synoviae in chickens is as shown in SEQ ID NO.1.

[0007] An indirect ELISA detection kit for Mycoplasma synoviae antibodies in chickens, characterized in that: the kit includes an enzyme-labeled plate with recombinant protein FBA of Mycoplasma synoviae in chickens with the amino acid sequence as shown in SEQ ID NO.1 as the coating antigen. The coating antigen is the recombinant protein FBA of the adhesion-related factor of Mycoplasma synoviae in chickens, and its primer sequences are as follows:

[0008] 5’-CGCGGATCC ATGCCATTAACAAACGCAAA-3’

[0009] 5’-CCGCTCGAG AGCTTTATTTTGTGATCCAA-3’

[0010] Aldolase FBA not only serves as a membrane surface protein of Mycoplasma and is an important immunogen, but also participates in the adhesion of host cells and functions as a virulence factor. By screening the optimal reaction conditions, an indirect ELISA detection kit for Mycoplasma synoviae antibodies with good specificity and high sensitivity was established. Specificity analysis shows that FBA can be used as the coating antigen to establish an indirect ELISA detection kit for detecting Mycoplasma synoviae antibodies.

[0011] A method for using an indirect ELISA detection kit for Mycoplasma synoviae antibodies in chickens, characterized by including the following steps: Dilute the purified recombinant protein FBA to 10 μg / mL at a concentration gradient and incubate it at 37 °C for 2 h; Block it with 5% BSA at 37 °C for 1 h; Dilute the specific chicken sera at 1:400 and incubate them at 37 °C for 1 h, and each serum is repeated 3 times; Dilute the HRP-labeled rabbit anti-chicken IgG at 1:4000 and incubate it at 37 °C for 1 h; Add TMB and develop color at room temperature for 15 min, and then terminate the color development with 4 mol / L dilute sulfuric acid; Read the OD 450 absorbance value. When the OD 450 value of the sample ≥ 0.85, it is determined as positive; when the OD 450 value of the sample ≤ 0.70, it is determined as negative; if it is in the intermediate value between the two, it is judged as suspicious.

[0012] Furthermore, the optimal concentration of the blocking solution is 10 g / L BSA for blocking, and the optimal blocking condition is overnight at 4 °C.

[0013] A preparation method of recombinant protein FBA of Mycoplasma synoviae in chickens, characterized by including the following steps:

[0014] Step 1: Cultivation of Mycoplasma synoviae MS251 strain and extraction of genomic DNA

[0015] Take the MS251 strain preserved in the laboratory in the early stage for resuscitation culture. Add 200 μL of the bacterial solution to 1.8 mL of the liquid medium, and use a test tube without added bacterial solution as a negative control. Incubate in a 37 °C incubator for 2 - 3 days. At this time, the medium changes from red to orange or yellow, while there is no obvious change in the negative control. Aspirate the bacterial solution into a centrifuge tube, centrifuge at 12,000 r / min for 10 min, discard the supernatant, add 100 μL of autoclaved deionized water, resuspend the precipitate, boil for 5 min, then ice-bath on ice for 3 min and centrifuge at 12,000 r / min for 10 min. The supernatant is the DNA template of the MS251 strain and is stored at -20 °C for later use;

[0016] Step 2: Primer design for amplification of Mycoplasma synoviae FBA gene and PCR amplification

[0017] Design primers with reference to the FBA gene sequence of the MS251 strain, and add restriction enzyme sites BamHⅠ and XhoⅠ at the 5' ends of the upstream and downstream respectively, and then add protective bases to ensure effective enzyme digestion can be carried out subsequently. TGA, which encodes tryptophan in mycoplasma, is regarded as a stop codon in the E. coli expression system and cannot be correctly translated, so it needs to be mutated to TGG to be correctly translated in E. coli. Therefore, use Snap Gene 4.1.8 to design primers at the mutation site and use the method of Overlap PCR amplification to achieve point mutation. The upstream primer FBA-F introduces a BamHⅠ restriction enzyme site at the 5' end, and the downstream primer FBA-R introduces an XhoⅠ restriction enzyme site at the 5' end. The size of the target fragment amplified by this pair of primers is 864 bp in total;

[0018] Table 1 Primer sequences

[0019]

[0020]

[0021] The underlined parts are the XhoⅠ and BamHⅠ restriction enzyme sites respectively; the bolded bases are the mutation sites;

[0022] After agarose gel electrophoresis of each PCR product, perform gel recovery. Mix the recovered products in the same proportion as the template and perform the second round of PCR amplification to obtain the full-length FBA gene;

[0023] Step 3: Construction and identification of prokaryotic expression plasmid

[0024] The PCR product of the full-length FBA gene was subjected to gel recovery. The gel recovery product was ligated to the pMD19-T vector, and the ligation product was transformed into Escherichia coli competent cell DH5α at a ratio of 10:1 and sent to Shanghai Bioengineering Company for sequence identification. If the sequence was correct, the plasmid of the target gene was extracted according to the plasmid extraction kit. Then, the target gene and the plasmid of pET-28a(+) were respectively double digested, and the digestion results met the expectations.

[0025] Step 4: Induced expression of recombinant protein

[0026] The recombinant expression plasmid pET-28a(+)-FBA was transferred into the expression engineering bacterium Escherichia coli BL21(DE3) competent cells. Positive cell clones with positive PCR results were selected for sequencing. Then, the correctly sequenced bacterial solution was evenly inoculated into 200 mL of LB liquid medium with kanamycin resistance and cultured in a shaker for enrichment. When the OD value reached 0.6 - 0.8, 2 mL of 100 mmol / L IPTG was added to induce expression for 4, 6, 8, and 10 h respectively. The results showed that the protein expression level was the highest after 6 h of induction.

[0027] Step 5: Solubility analysis and purification of recombinant protein

[0028] Solubility analysis showed that the recombinant protein FBA was expressed in a soluble manner. The supernatant of the lysate was purified with a Histrap TM HP purification column, and the collected protein was detected by SDS-PAGE. The results showed that a single band appeared at about 35 kD, and the purification result was good.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides an indirect ELISA detection kit for Mycoplasma synoviae antibodies using a purified recombinant protein of Mycoplasma synoviae fructose 1,6-bisphosphate aldolase (FBA) as a coating antigen, which helps to deeply carry out the research on Mycoplasma synoviae infection and immunity, and provides a fast, sensitive, and specific method for the diagnosis of clinical Mycoplasma synoviae infection. Brief description of the drawings

[0030] Figure 1 It is a schematic diagram of the full-length amplification of the FBA gene in Example 1 of the present invention;

[0031] Figure 2It is the FBA segmented amplification diagram in Example 1 of the present invention; wherein, M: DNA2000 Maker molecular mass standard; 1-3: Amplification diagram of FBA-F1 and FBA-R2; 4-6: Amplification diagram of FBA-F2 and FBA-R; 7-9: Amplification diagram of FBA-F and FBA-R1;

[0032] Figure 3 It is the full-length amplification diagram of FBA in Example 1 of the present invention; wherein, M: DNA2000 Maker molecular mass standard;

[0033] Figure 4 It is the double digestion diagram in Example 1 of the present invention;

[0034] Figure 5 It is the screening diagram of the optimal induction conditions in Example 1 of the present invention;

[0035] Figure 6 It is the SDS-PAGE in Example 1 of the present invention; wherein, A: Preliminary expression diagram of FBA protein: 1: Precipitation after induction of pET-FBA; 2: Empty plasmid after induction; 3: Supernatant after induction of pET-FBA; B: 1: Effluent; 2-6: Elution solutions of the 1st, 2nd, 3rd, 4th, and 5th tubes respectively;

[0036] Figure 7 It is the Western blot in Example 1 of the present invention; wherein, M: Prestained protein molecular mass standard; 1: fba protein 2: Negative serum. Specific implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. Example 1 Preparation of indirect ELISA antigen for Mycoplasma synoviae antibody in chickens

[0038] 1 Materials

[0039] (1) Serum, strains and plasmids: Positive and negative sera of Mycoplasma synoviae in chickens, Newcastle disease serum of chickens, and Mycoplasma gallisepticum serum were stored at -20 °C in the Animal Medicine Laboratory of Southwest Minzu University; The clinical isolate MS251 of Mycoplasma synoviae was isolated and stored by this laboratory; The prokaryotic cloning expression vector pET-28a(+) was purchased from Takara; Competent cells DH5α and BL21(DE3) were both purchased from Saiguo Technology Co., Ltd.

[0040] (2) Main reagents: LB liquid and solid media were purchased from Hangzhou Microbial Reagent Company; IPTG inducer and His-tag nickel column were purchased from GE Company; ampicillin was purchased from Saiguo Technology Co., Ltd.; T4 DNA ligase, BamHⅠ and XhoⅠ restriction endonucleases, and Tag DNA polymerase were purchased from Takara Company; BCA protein quantification kit and plasmid extraction kit were both purchased from Tiangen Biochemical Technology Co., Ltd.

[0041] (3) Main instruments: The constant temperature incubator was purchased from Shanghai Hecheng Instrument Manufacturing Company; the high-speed refrigerated centrifuge and PCR amplifier were purchased from Thermo Fisher Scientific; the constant temperature metal bath was purchased from Hangzhou Bioer Technology Company; the cell ultrasonic disruptor was purchased from Xinzhi Biotechnology Company; the ultrapure water instrument Milli-Q was purchased from Millipore, France; the microplate reader and gel semi-dry transfer device were purchased from Bio-Rad.

[0042] 2 Methods

[0043] (1) Preparation of antigen

[0044] ① Culture of Mycoplasma synoviae MS251 strain and extraction of genomic DNA

[0045] Take the MS251 strain preserved in the laboratory in the early stage for resuscitation culture. Add 200 μL of the bacterial solution to 1.8 mL of the liquid medium, and use a test tube without added bacterial solution as a negative control. Incubate in a 37 °C incubator for 2 - 3 days. At this time, the medium changes from red to orange or yellow, while there is no obvious change in the negative control. Aspirate the bacterial solution into a centrifuge tube, centrifuge at 12000 r / min for 10 min, and discard the supernatant. Add 100 μL of autoclaved deionized water, resuspend the precipitate, boil for 5 min, then ice-bath on ice for 3 min and centrifuge at 12000 r / min for 10 min. The supernatant is the DNA template of the MS251 strain and is stored at -20 °C for later use.

[0046] ② Design of primers for amplification of Mycoplasma synoviae fba gene and PCR amplification

[0047] Primers were designed based on the FBA gene sequence of the MS251 strain, and the restriction enzyme sites BamHⅠ and XhoⅠ were added to the 5' ends of the upstream and downstream primers. Protective bases were added respectively to ensure effective enzyme digestion in the follow-up. TGA, which encodes tryptophan in mycoplasma, is recognized as a stop codon in the E. coli expression system and cannot be correctly translated. Therefore, it needs to be mutated to TGG to be correctly translated in E. coli. Thus, primers were designed at the mutation site using Snap Gene 4.1.8, and site-directed mutagenesis was achieved by the method of Overlap PCR. The upstream primer FBA-F introduced the BamHⅠ restriction enzyme site at the 5' end, and the downstream primer FBA-R introduced the XhoⅠ restriction enzyme site at the 3' end. The size of the target fragment amplified by this pair of primers was 864 bp in total.

[0048] Table 1 Primer sequences

[0049]

[0050] The underlined parts are the XhoⅠ and BamHⅠ restriction enzyme sites respectively; the bolded bases are the mutation sites.

[0051] As Figure 1 shown, the corresponding fragments were amplified and obtained using the designed primers, and the amplification results are shown in Figure 2 .

[0052] After agarose gel electrophoresis of the PCR products of each segment, gel extraction was performed. The extracted products were mixed in the same proportion as the template for the second round of PCR amplification to obtain the full-length FBA gene.

[0053] ③ Construction and identification of prokaryotic expression plasmid

[0054] The PCR product of the full-length FBA gene was subjected to gel extraction. The gel-extracted product was ligated to the pMD19-T vector, and the ligation product was transformed into the E. coli competent cell DH5α at a ratio of 10:1 and sent to Shanghai Bioengineering Company for sequence identification. If the sequence was correct, the plasmid of the target gene was extracted according to the plasmid extraction kit. Then, the target gene and the plasmid of pET-28a(+) were digested with restriction enzymes respectively, and the digestion results met the expectations.

[0055] ④ Induced expression of recombinant protein

[0056] The recombinant expression plasmid pET-28a(+)-FBA was transferred into the competent cells of the expression engineering bacterium Escherichia coli BL21(DE3). Positive cell clones with a positive PCR result were picked for sequencing. Then, the bacterial solution with correct sequencing was evenly inoculated into 200 mL of LB liquid medium with kanamycin resistance and cultured in a shaker for enrichment. When the OD value reached 0.6 - 0.8, 2 mL of 100 mmol / L IPTG was added to induce expression for 4, 6, 8, and 10 h respectively. The results showed that the protein expression level was the highest after 6 h of induction.

[0057] ⑤ Solubility analysis and purification of the recombinant protein

[0058] Solubility analysis showed that the recombinant protein FBA was expressed in a soluble form ( Figure 6 A). The supernatant of the lysate was purified with a Histrap TM HP purification column, and the collected protein was detected by SDS-PAGE. The results showed that a single band appeared at around 35 kD ( Figure 6 B), and the purification result was good.

[0059] (2) Detection of antigen immunogenicity

[0060] The purified FBA protein was loaded for SDS-PAGE electrophoresis. After transfer, it was blocked with 5% skim milk powder for 2 h. Then, it was incubated overnight with the positive serum of Mycoplasma synoviae of chickens as the primary antibody, and at the same time, the negative serum of SPF chickens was set as the control group. HRP-labeled rabbit anti-chicken IgG was used as the secondary antibody for Western blot analysis. The results showed that the purified recombinant protein could specifically bind to the positive serum of Mycoplasma synoviae of chickens, while there was no reaction with the negative one, as Figure 7 shown. It indicated that the FBA recombinant protein had good reactivity and could specifically bind to the antibody of Mycoplasma synoviae of chickens.

[0061] (3) Detection of antigen specificity

[0062] The purified recombinant protein FBA was diluted to 10 μg / mL at a concentration gradient and incubated at 37 °C for 2 h. It was blocked with 5% BSA at 37 °C for 1 h. The specific chicken sera were all diluted at 1:400 and incubated at 37 °C for 1 h. Each serum was replicated 3 times. HRP-labeled rabbit anti-chicken IgG was diluted at 1:4000 and incubated at 37 °C for 1 h. After adding TMB and developing color at room temperature for 15 min, the color development was terminated with 4 mol / L dilute sulfuric acid, and the OD was read with an enzyme-linked immunosorbent assay reader 450Absorbance value. The coated recombinant protein FBA was evaluated for specific binding with positive sera of Mycoplasma synoviae in chickens as the standard positive, SPF chicken sera as the standard negative, and positive sera of Mycoplasma gallisepticum and Newcastle disease in chickens. The results showed that the recombinant protein FBA had a strong positive reaction with the positive serum of Mycoplasma synoviae in chickens, no reaction with the negative serum, and also no reaction with the positive sera of Mycoplasma gallisepticum and Newcastle disease in chickens (as shown in Table 2).

[0063] Table 2 Specificity evaluation results

[0064]

[0065] Note: The values in the table are OD 450 readings, P is positive, N is negative.

[0066] Example 2 Establishment and condition optimization of indirect ELISA method for Mycoplasma synoviae in chickens

[0067] 1 Materials

[0068] (1) Sera: Positive and negative sera of Mycoplasma synoviae in chickens were stored at -20 °C in the Animal Medicine Laboratory of Southwest Minzu University;

[0069] (2) Main reagents: Rabbit anti-chicken IgG-HRP was purchased from Beijing Boao Sen Biotechnology Co., Ltd.; Bovine serum albumin V (BSA) was purchased from Saiguo Technology Co., Ltd.; Single-component TMB chromogenic solution was purchased from Beijing Sizhengbai Biotechnology Co., Ltd.

[0070] (3) Main instruments: Incubator was purchased from Shanghai HeCheng Instrument Manufacturing Co., Ltd.; Ultra-pure water instrument Milli-Q was purchased from Millipore Corporation, France; Microplate reader was purchased from Bio-Rad Company.

[0071] 2 Methods

[0072] (1) Determination of the optimal working concentrations of antigen and antibody

[0073] The antigen was serially diluted to 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml with buffer, and each dilution was repeated three times. After blocking with 5% BSA, the negative and positive sera of Mycoplasma synoviae in chickens were serially diluted to 1:100, 1:200, 1:400, and 1:800 with antibody diluent. After adding TMB and developing color at room temperature for 15 min, the color development was terminated with 4 mol / L dilute sulfuric acid, and the absorbance value at OD 450 was read with a microplate reader, and the P / N value was calculated. The antigen concentration when the P / N value was the largest was selected as the optimal antigen concentration, and the corresponding primary antibody dilution was the optimal primary antibody dilution. The results are shown in Table 3. The optimal antigen coating concentration determined in this experiment was 10 μg / mL, and the optimal serum dilution was 1:400.

[0074] Table 3 Determination of the Optimal Working Concentrations of Antigens and Primary Antibodies

[0075]

[0076] Note: P: Positive serum; N: Negative serum

[0077] (2) Determination of the Optimal Blocking Solution

[0078] According to the above-optimized conditions, PBS containing 5, 10, and 20 g / L BSA was used as the blocking solution, and blocking was carried out at 37°C for 2 h and overnight at 4°C respectively. The conditions corresponding to the maximum P / N value were taken as the optimal blocking solution and blocking conditions. The results showed that when the blocking solution was 10 g / L and blocking was carried out overnight at 4°C, the P / N value was the largest.

[0079] Table 4 Optimization of the Blocking Solution

[0080]

[0081]

[0082] (3) Determination of the Serum Reaction Time

[0083] After coating the plates and blocking according to the above-optimized conditions, the reaction was carried out at 37°C for 30 min, 60 min, and 90 min, and 3 replicates were made for each time point. The OD 450 values were compared, and the time corresponding to the maximum P / N value was taken as the optimal working time of the serum. The results of the serum reaction at different times are shown in Table 5, that is, the reaction time of the primary antibody serum was 60 min as the best.

[0084] Table 5 Determination of the Serum Reaction Time

[0085]

[0086] (4) Optimization of the Dilution Ratio and Action Time of the Enzyme-Labeled Secondary Antibody

[0087] According to the above-optimized conditions, the enzyme-labeled secondary antibody was diluted at 1:4000, 1:8000, and 1:12000 respectively, added to the 96-well enzyme-labeled plate, and the reaction was carried out at 37°C for 30, 45, and 60 min. The OD 450 values were compared, and the conditions corresponding to the maximum P / N value were taken as the optimal dilution multiple and optimal working time of the secondary antibody. The reaction results of HRP-labeled goat anti-chicken IgG at different dilution ratios are shown in Table 6. When the enzyme-labeled secondary antibody was diluted at 1:4000 and incubated for 45 min, it was the best.

[0088] Table 6 Determination of the Dilution Ratio of the Enzyme-Labeled Secondary Antibody

[0089]

[0090] (5) Screening of the optimal color development time

[0091] According to the above optimized conditions, the color development times of the substrate were set to 5, 10, and 15 min respectively, and the time corresponding to the maximum P / N value was taken as the optimal color development time. The results showed that the optimal color development time was 15 min (as shown in Table 7).

[0092] Table 7 Optimal color development time

[0093]

[0094] (6) Determination of the positive and negative critical values

[0095] Determination of the critical value: Twenty chicken serum samples negative for Mycoplasma synoviae were detected by the optimized indirect ELISA method, and the OD 450 mean value (X) and standard deviation (S) of all sera were calculated. Samples with an OD 450 value ≥ X + 3S were determined to be positive, samples with an OD 450 value ≤ X + 2S were determined to be negative, and those between the two were considered suspicious. From the results in Table 8, it can be seen that X = 0.41, S = 0.14, X + 3S = 0.85, X + 2S = 0.70. When the sample OD 450 value ≥ 0.85, it was determined to be positive; when the sample OD 450 value ≤ 0.70, it was determined to be negative; if it was between the two values, it was considered suspicious.

[0096] Table 8 OD values of indirect ELISA detection of 20 chicken serum samples negative for Mycoplasma synoviae 450

[0097]

[0098] (7) Repeatability test

[0099] Five positive sera and five negative sera were taken, and the ELISA plates were coated with the target protein prepared in the same batch. These 10 serum samples were detected by ELISA, with 3 replicate wells for each serum sample, and the OD 450 values were measured to calculate the within-batch coefficient of variation for each serum. At the same time, these 10 serum samples were detected using ELISA plates coated with 3 different batches, with 3 replicate wells for each serum, and the between-batch coefficient of variation was calculated. The results showed that the within-batch coefficient of variation was between 0.11% and 4.58%, and the between-batch coefficient of variation was between 1.89% and 5.26%, indicating good repeatability test results.

[0100] (8) Detection of clinical sera

[0101] A total of 200 serum samples suspected of Mycoplasma synoviae infection from Chengdu, Sichuan Province were detected using the established FBA-ELISA. The results showed that the positive rate was 85%, with a relatively high detection rate, indicating that the indirect ELISA method established based on the FBA recombinant protein had good sensitivity.

[0102] The embodiments of the present application have been described above in conjunction with the accompanying drawings. However, the present application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them fall within the protection scope of the present application.

Claims

1. Application of recombinant protein FBA of Mycoplasma synoviae in preparing an indirect ELISA detection kit for Mycoplasma synoviae antibodies in chickens, characterized in that: The amino acid sequence of the recombinant protein FBA of Mycoplasma synoviae in chickens is as shown in SEQ ID NO.

1.

2. An indirect ELISA kit for detecting antibodies against Mycoplasma synoviae in chickens, characterized in that: The kit includes an enzyme-labeled plate with the recombinant protein FBA of Mycoplasma synoviae in chickens with the amino acid sequence as shown in SEQ ID NO.1 as the coated antigen.

3. The indirect ELISA detection kit for Mycoplasma synoviae of chickens according to claim 2, wherein: The kit further includes a coating buffer, a blocking solution, a washing solution, an antibody dilution solution, a positive serum of Mycoplasma synoviae in chickens, a negative serum of Mycoplasma synoviae in chickens, an enzyme-labeled secondary antibody, a substrate chromogenic solution, and a termination solution.

4. The indirect ELISA kit for detecting Mycoplasma synoviae in chickens according to claim 3, wherein: The coating buffer is composed of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4 mixed with Tween 20, and the volume concentration of Tween 20 in the buffer is 0.05%; the blocking solution is composed of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4 and bovine serum albumin (BSA), and the mass concentration of bovine serum albumin (BSA) in the blocking solution is 5%; the antibody dilution solution is purchased from Wuhan Boster Biological Engineering Co., Ltd.; the washing solution is composed of a phosphate buffer with a concentration of 0.01 mol / L and a pH value of 7.4 mixed with Tween 20, and the volume concentration of Tween 20 in the washing solution is 0.05%; the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-chicken IgG; The substrate chromogenic solution is a single-component TMB chromogenic solution; the termination solution is dilute sulfuric acid with a concentration of 4 mol / L.

5. The indirect ELISA detection kit for Mycoplasma synoviae in chickens according to claim 4, and the usage method of the kit is as follows: (1) Coating of the enzyme-labeled plate antigen: Dilute the recombinant protein FBA of Mycoplasma synoviae in chickens with the coating buffer to 10 μg / mL, add 100 μL per well to the enzyme-labeled plate, incubate overnight at 4°C, wash the plate 3 times with the washing solution for 5 minutes each time, and pat dry; (2) Blocking of the enzyme-labeled plate: Add 200 μL of the blocking solution to each well of the coated enzyme-labeled plate, block at 37°C for 1 h, wash the plate 3 times with the washing solution for 5 minutes each time, and pat dry; (3) Adding the diluted serum: Add 100 μL per well of the test serum diluted 1:400 times with the antibody dilution solution, add 100 μL per well of the positive serum of Mycoplasma synoviae in chickens diluted 1:400 times with the antibody dilution solution as a positive control, add the negative serum of Mycoplasma synoviae in chickens diluted 1:400 times with the antibody dilution solution as a negative control, act at 37°C for 1 h, wash the plate 3 times with the washing solution for 5 minutes each time, and pat dry; (4) Adding the diluted enzyme-labeled secondary antibody: Add 100 μL per well of the enzyme-labeled secondary antibody diluted 1:4000 times with the washing solution, act at 37°C for 1 h, wash the plate 3 times with the washing solution for 5 minutes each time, and pat dry; (5) Adding the substrate for chromogenic reaction: Add 100 μL of the TMB substrate chromogenic solution to each well, and develop color at 37°C in the dark for 15 minutes; (6) Adding the termination solution to complete the detection: Add 50 μL of the termination solution to each well, measure the OD450 value on an enzyme-labeled immunosorbent assay instrument; when the OD450 value of the sample ≥ 0.85, it is determined as positive; when the OD450 value of the sample ≤ 0.70, it is determined as negative; if it is between the two intermediate values, it is judged as suspicious.

6. A preparation method of recombinant protein FBA of Mycoplasma synoviae in chickens, characterized in that, It includes the following steps: Step 1: Cultivation of Mycoplasma synoviae MS251 strain and extraction of genomic DNA Resuscitate and culture the MS251 strain preserved in the laboratory earlier. Add 200 μL of the bacterial solution to 1.8 mL of liquid medium, and use a test tube without added bacterial solution as a negative control. Incubate in a 37 °C incubator for 2 - 3 days. At this time, the medium changes from red to orange or yellow, while there is no obvious change in the negative control. Pipette the bacterial solution into a centrifuge tube, centrifuge at 12,000 r / min for 10 min, discard the supernatant, add 100 μL of autoclaved deionized water, resuspend the precipitate, boil for 5 min, then ice-bath on ice for 3 min and centrifuge at 12,000 r / min for 10 min. The supernatant is the DNA template of the MS251 strain, and it is stored at -20 °C for later use; Step 2: Design of primers for amplification of Mycoplasma synoviae FBA gene and PCR amplification Design primers with reference to the FBA gene sequence of the MS251 strain, and add restriction enzyme sites BamHⅠ and XhoⅠ to the 5' ends of the upstream and downstream respectively. Then add protective bases to ensure effective enzyme digestion can be carried out later. TGA, which encodes tryptophan in mycoplasma, is recognized as a stop codon in the Escherichia coli expression system and cannot be correctly translated. Therefore, it needs to be mutated to TGG to be correctly translated in Escherichia coli. So, use Snap Gene 4.1.8 to design primers at the mutation site and achieve site-directed mutagenesis by the method of Overlap PCR. The upstream primer FBA-F introduces a BamHⅠ restriction enzyme site at the 5' end, and the downstream primer FBA-R introduces an XhoⅠ restriction enzyme site at the 3' end. The size of the target fragment amplified by this pair of primers is 864 bp in total; Table 1 Primer sequences The underlined parts are the XhoⅠ and BamHⅠ restriction enzyme sites respectively; the bolded bases are the mutation sites; After agarose gel electrophoresis of each PCR product, perform gel extraction. Mix the extracted products in the same proportion as the template and perform a second round of PCR amplification to obtain the full-length FBA gene; Step 3: Construction and identification of prokaryotic expression plasmid Perform gel extraction on the PCR product of the full-length FBA gene. The gel-extracted product is ligated to the pMD19-T vector, and the ligation product is transformed into Escherichia coli competent cell DH5α at a ratio of 10:1 and sent to Shanghai Bioengineering Company for sequence identification. If the sequence is correct, extract the plasmid of the target gene according to the plasmid extraction kit. Then, perform double enzyme digestion on the target gene and the plasmid of pET-28a(+) respectively, and the enzyme digestion results meet the expectations; Step 4: Induced expression of recombinant protein The recombinant expression plasmid pET-28a(+)-FBA was transferred into the competent cells of the expression engineering bacterium Escherichia coli BL21(DE3). Positive cell clones with a positive PCR result were picked for sequencing. Then, the bacterial solution with correct sequencing was evenly inoculated into 200 mL of LB liquid medium with kanamycin resistance and cultured in a shaker for enrichment. When the OD value reached 0.6 - 0.8, 2 mL of 100 mmol / L IPTG was added for induction culture for 4, 6, 8, and 10 h, respectively. The results showed that the protein expression level was the highest after 6 h of induction; Step 5. Solubility analysis and purification of the recombinant protein Solubility analysis showed that the recombinant protein FBA was expressed in a soluble form and the supernatant of the lysate was purified using a Histrap TM HP purification column, and the collected protein was then detected by SDS-PAGE. The results showed a single band around 35 kD, indicating good purification results.

Images