A multi-epitope protein of Mycoplasma synoviae in chickens and its application

Abstract

The present invention provides a multi-epitope protein of Mycoplasma synoviae of chickens and its application. The amino acid sequence of the multi-epitope protein of Mycoplasma synoviae of chickens is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the multi-epitope protein of Mycoplasma synoviae of chickens is shown in SEQ ID NO.2. After purification and identification, the multi-epitope protein of Mycoplasma synoviae of chickens was subjected to an immunization challenge protection test using SPF chicks, which initially showed good immunoprotective effects. This study lays a foundation for the research and development of genetic engineering vaccines against Mycoplasma synoviae of chickens.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a multi-antigenic epitope protein of Mycoplasma synoviae in chickens, and its preparation and application. Background Art

[0002] Mycoplasma synoviae (MS) causes swollen joints, synovial bursa and tendon inflammation, and enlargement of parenchymal organs in diseased chickens. Although the mortality rate is within 10%, it is difficult to eradicate after the chicken flock is infected. It can be transmitted horizontally and vertically, and often occurs in mixed infections. Due to the long-term nature of MS infection and its easy replication and survival in the host, MS is often difficult to control, causing huge economic losses to the poultry industry. Currently in the market, the method of antibody detection mainly relies on imported ELISA commercial kits, but the imported ELISA commercial kits are expensive and not suitable for large-scale detection in chicken farms to purify the chicken flock. Therefore, it is of great significance to develop a more economical MS antibody ELISA detection kit to replace imported products for the monitoring of MS infection in China.

[0003] Currently, the treatment of Mycoplasma synoviae disease mainly uses sensitive antibiotics, mainly tylosin, spectinomycin, spiramycin and their macrolide drugs, etc. With the long-term abuse of drugs by people, the drug resistance of mycoplasma to macrolides and other antibiotic drugs is increasing continuously. And the common vaccines on the market are the attenuated vaccine strain MS-H and the inactivated oil emulsion vaccine of MS, which are relatively expensive. It is of great significance to develop a more economical MS vaccine to replace imported products for the prevention and control of MS in China. Summary of the Invention

[0004] The purpose of the present invention is to provide a multi-antigenic epitope protein of Mycoplasma synoviae in chickens, and its preparation and application.

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

[0006] A multi-antigenic epitope protein of Mycoplasma synoviae in chickens, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] A gene encoding the above multi-antigenic epitope protein of Mycoplasma synoviae in chickens, whose nucleotide sequence is shown in SEQ ID NO.2.

[0008] A recombinant expression vector containing the above gene encoding the multi-antigenic epitope protein of Mycoplasma synoviae in chickens, and its starting vector is pET-32a(+).

[0009] A recombinant expression bacterium containing the above recombinant expression vector, and its starting strain is Escherichia coli.

[0010] Use of the above-mentioned multi-epitope protein of Mycoplasma synoviae in chickens for preparing a Mycoplasma synoviae genetic engineering vaccine.

[0011] In this study, bioinformatics software and related websites were used to analyze the dominant linear epitopes of MS structural proteins to obtain epitope sequences. The antigenic sites were analyzed by Protean software and named MS-eLPD. Then, the amino acid sequence of the multi-epitope peptide MS-eLPD was deduced into a nucleotide sequence through genetic codons, and the dominant codons of Escherichia coli were selected as much as possible. The nucleotide sequence of the antigenic epitope peptide was sent to a biological company for gene synthesis, and the target gene and plasmid were ligated by a ligase. The recombinant prokaryotic expression plasmid of MS-eLPD was transformed into DH5α competent cells, and the plasmid was extracted using a plasmid extraction kit. The recombinant plasmid was used as a template, and the recombinant plasmid was double-digested with EcoRΙ and Hind III and subjected to nucleic acid electrophoresis. The recombinant plasmid identified as positive by enzyme digestion was sent for sequencing. The correctly constructed recombinant prokaryotic expression plasmid was named pET-32a(+)-MS-eLPD. The pET-32a(+)-MS-eLPD recombinant plasmid was transformed into BL21. After overnight culture, single isolated colonies were picked and cultured continuously in LB liquid medium, and IPTG was used to induce protein expression.

[0012] After a large amount of induction expression and purification of MS-eLPD, SPF chicks were immunized twice, and challenged with the pathogenic MS isolated and identified. The experimental chickens in the challenge control group developed the disease, while the immunized group obtained protection.

[0013] The advantages of the present invention are as follows:

[0014] The common vaccines on the current market are two types: the live attenuated vaccine MS-H strain and the inactivated oil emulsion vaccine of MS. Although the inactivated vaccine can block the vertical transmission of MS, increase the humoral antibody, and temporarily relieve the symptoms during infection, it cannot completely eliminate the wild virus in the chicken body and prevent the invasion of wild virus; the live attenuated vaccine MS-H strain can induce mucosal immunity in the body. Research shows that Mycoplasma does not contain lipopolysaccharide and peptidoglycan on its surface, but there are a large number of lipoproteins on its surface. Mycoplasma lipoproteins can participate in the adhesion to host cells and can affect the host immune system. Lipoproteins stimulate cells to produce pro-inflammatory factors and cytokines, causing damage to host cells. At the same time, Mycoplasma lipoproteins can escape the surveillance of the host immune system, enabling them to survive in host cells for a long time and promoting and assisting the development of other diseases. With the continuous development of bioinformatics technology, bioinformatics has been widely applied. At present, bioinformatics analysis technology is widely used to predict the structure of proteins, which plays an important role in the research of epitope vaccines.

[0015] Multi-epitope vaccines are prepared by concatenating multiple nucleic acid sequence fragments of epitopes and recombining them into a vector using recombinant DNA technology. The multi-epitope genetic engineering vaccine antigens can be used to prepare multivalent vaccines with multiple protective capabilities against multiple serotypes of a single pathogen, or as broad-spectrum vaccines for preventing multiple diseases. A multi-epitope vaccine refers to a vaccine that simultaneously carries multiple target antigens and auxiliary epitopes. It is prepared based on the amino acid sequences of the target antigen epitopes and represents a new design concept of genetic engineering vaccines developed in recent years, representing the research direction and development trend of a new generation of vaccines. Multi-epitope vaccines have many advantages that traditional genetic engineering vaccines do not have: multi-epitope vaccines can be efficiently presented with antigens; they can effectively cope with the mutations of pathogenic microorganism antigens and many adverse factors existing in immune responses; they have unique advantages in cellular immunity, etc.

[0016] With the long-term abuse of drugs by people, the drug resistance of mycoplasma to macrolides and other antibiotic drugs has increased, making subsequent treatment more difficult. And there are many defects in the commonly available vaccines on the market. In the research of vaccines, the research of genetic engineering vaccines is not common at home and abroad. With the in-depth research on multi-epitope vaccines, it provides convenience for the research of mycoplasma genetic engineering vaccines. Brief Description of the Drawings

[0017] Figure 1A : Prediction diagram of protein secondary structure. Figure 1B : Prediction diagram of protein tertiary structure.

[0018] Figure 2 : Restriction enzyme digestion electrophoresis identification of recombinant plasmid pET-32a(+)-MS-eLPD.

[0019] Figure 3 : SDS-PAGE analysis of recombinant protein pET-32a(+)-MS-eLPD.

[0020] Figure 4 : Western blot analysis.

[0021] Figure 5 : Postmortem changes of chicks infected with MS-NP.

[0022] Figure 6 : Respiratory tract pathological sections of chicks infected with MS-NP.

[0023] Figure 7 : Spleen pathological sections of chicks infected with MS-NP. Detailed Embodiments

[0024] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0025] Example 1

[0026] 1 Materials and Methods

[0027] 1.1 Materials

[0028] 1.1.1 Strains, strains, expression vectors and experimental animals

[0029] The prokaryotic expression plasmid pET-32a(+) was preserved in the inventor's laboratory; competent cells of Escherichia coli DH5α and BL21(DE3) were both purchased from TransGen Biotech Co., Ltd. in Beijing; SPF chicken embryos were purchased from Fujian Sunner Development Co., Ltd.; Mycoplasma synoviae MS-NP strain was isolated and identified by the inventor's laboratory. Sequencing found that the MS-NP strain was in the same branch as Mycoplasma synoviae HN01 strain, 5-9 strain and JX01 strain isolated in China, and was distantly related to the attenuated live vaccine MS-H strain in Australia.

[0030] 1.1.2 Main reagents

[0031] Ni-NTA Resin, substrate for TMB ELISA and T4 DNA ligase were all purchased from TransGen Biotech Co., Ltd. in Beijing; endotoxin-free plasmid midiprep kit was purchased from CW Biotech Co., Ltd. in Beijing; restriction enzymes QuickCut EcoRΙ and QuickCut HindIII were purchased from Takara Bio Inc. in Dalian; isopropyl-β-D-thiogalactoside (IPTG), ampicillin (Amp) and imidazole were all purchased from Solarbio Science & Technology Co., Ltd. in Beijing; SDS-PAGE Gel Kit was purchased from CW Biotech Co., Ltd. in Beijing; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; Mycoplasma synoviae antibody detection kit was purchased from IDEXX BioResearch Co., Ltd. in Beijing; bovine serum albumin (BSA) was purchased from Yeasen Biotech Co., Ltd. in Shanghai; 96-well ELISA plates were purchased from CORNING; rabbit anti-chicken IgG-HRP was purchased from Proteintech; CTB mucosal immune adjuvant was purchased from Absin Bioscience Inc. in Shanghai; MS-H commercial attenuated vaccine was donated by Fujian Sunner Development Co., Ltd. Other conventional reagents such as sodium carbonate, sodium bicarbonate, sodium chloride, potassium chloride, disodium hydrogen phosphate, Tween-20, etc. were preserved in this laboratory.

[0032] 1.2 Construction of MS-eLPD prokaryotic expression plasmid

[0033] 1.2.1 Prediction and screening of B / T cell antigenic epitopes of Mycoplasma synoviae

[0034] The ABCpred website and SYFPEITHI website were used to predict antigen epitopes, and the DNAStar software Protean program was used to analyze and predict the B cell epitopes (antigenicity, hydrophilicity, surface possibility, flexibility, etc.) related parameters of Mycoplasma synoviae DLD, LP78, VLHA, LDH, ENO and other proteins.

[0035] Log in to the IEBD website (http: / / tools.immuneepitope.org / main / ) and the SYFPEITHI database website (http: / / www.syfpeithi.de / bin / mhcserver.dll / ), respectively input the amino acid sequences of potential dominant epitopes predicted by the above analysis, and evaluate the T cell sites with strong correlation (correlation>85%) in each fragment.

[0036]

[0037] 1.2.2 Design and synthesis of MS-eLPD nucleotide sequence

[0038] The antigenic epitopes were arranged in series through 4 glycine residues (GGGG) from the selected antigenic epitopes to obtain the expected multi-epitope peptide sequence, and the antigenic site of the multi-epitope peptide was analyzed by Protean software, and the multi-epitope peptide was named MS-eLPD. The amino acid sequence (SEQ ID NO.1) of the multi-epitope peptide MS-eLPD was derived as a nucleotide sequence by genetic codons, and the dominant codons of Escherichia coli were selected as much as possible. At the same time, EcoR Ι and Hind III restriction sites were added at both ends of the sequence to obtain a total of 1113 bases (SEQ ID NO.2). The designed multi-epitope peptide nucleotide sequence was sent to a biological company for gene synthesis.

[0039] 1.2.3 Construction and identification of MS-eLPD prokaryotic expression plasmid

[0040] The target gene shown in SEQ ID NO.2 and the pET-32a(+) plasmid were ligated by T4 DNA ligase. The reaction system and conditions are shown in the following table to obtain the recombinant plasmid pET-32a(+)-MS-eLPD. The ligation product was transformed into Escherichia coli (E.coli) competent cells DH5α, reacted on ice for 30 min; heat shocked at 42 °C for 90 s, placed on ice for 2 min after heat shock, and then 1 mL of LB liquid medium without Amp was added and cultured at 25 °C for 45 min; 100 μL of the bacterial solution was evenly spread on a solid LB medium plate containing Amp (1:1000) and incubated upside down in a 37 °C incubator overnight. The next day, single colonies were randomly selected and cultured in LB liquid medium containing Amp (1:1000). After 3 h, the bacterial solution was taken as a template, and PCR reaction was carried out using T7 universal primers (F: 5'-TAATACGACTCACTATAGGG-3'; R: 5'-GCTAGTTATTGCTCAGCGG-3'), and agarose gel electrophoresis was performed.

[0041]

[0042] 1.3 Prokaryotic expression of MS-eLPD and SDS-PAGE analysis

[0043] 1.3.1 Induced expression of the recombinant bacteria of the multi-epitope fusion gene

[0044] The correctly identified recombinant plasmid pET-32a(+)-MS-eLPD was transferred into E.coli BL21(DE3) competent cells. The next day, a single colony was picked and cultured overnight at 37 °C with shaking. 200 μL of the bacterial solution was inoculated into 20 ml of liquid LB medium containing Amp (1:1000) resistance and cultured until the OD 600nm reached 0.6 - 0.8, 20 μL of 1.0 mmol / L IPTG was added and induced at 25 °C for 2 h, 4 h, 6 h, 8 h, and 10 h. 1 ml of the bacterial solution was collected each time. At the same time, the empty vector and the recombinant bacteria without IPTG induction were set as negative controls. Centrifuge at 12000 r / min for 1 min, and take the supernatant and precipitate for SDS-PAGE electrophoresis analysis to determine the relative expression levels of soluble protein and inclusion body of MS-eLPD in E.coli.

[0045] 1.3.2 Purification of the recombinant protein of the multi-epitope fusion gene

[0046] According to the instructions of the ProteinIso Ni-NTA Resin kit, the target protein was purified. Elution was carried out with imidazole at concentrations of 20 mM, 50 mM, 80 mM, 100 mM, and 200 mM respectively. After elution, the elution effluent was taken for SDS-PAGE analysis to determine the concentration of the eluent.

[0047] 1.3.3 Dialysis and Concentration of Multi-Epitope Fusion Gene Recombinant Protein

[0048] Since the MS-eLPD solution eluted during purification contains a high concentration of imidazole, which will affect subsequent experiments, the purpose of this step is to dialyze the purified MS-eLPD recombinant protein to remove imidazole. Gradient dialysis is carried out, dialyzing successively in solutions containing 50 mM, 25 mM, 10 mM, and 0 mM imidazole, with each dialysis lasting 3 h.

[0049] After dialysis, PEG 20000 is used for concentration: Add PEG 20000 powder to cover the surface of the dialysis bag. After about 1 h, the renatured protein can be concentrated several times. The purified, dialyzed, and concentrated MS-eLPD recombinant protein is subjected to SDS-PAGE detection. The specific operations are as follows:

[0050] (1) Install the glass plates of the vertical electrophoresis tank device;

[0051] (2) Preparation of polyacrylamide gel separating gel: Referring to the instructions of the Beyotime kit, prepare 5 mL of 10% separating gel; after mixing, add it between the glass plates, and seal the top layer with 1 mL of absolute ethanol, taking care to make the liquid surface flat. Let it act at room temperature for 30 - 45 min;

[0052] (3) Preparation of polyacrylamide gel stacking gel: Prepare 2 mL of 5% stacking gel mixture; Pour off the water on the separating gel and blot it dry, then add the above mixture. Insert the comb between the glass plates. At room temperature, the polyacrylamide gel stacking gel takes 15 - 30 min to polymerize;

[0053] (4) Sample treatment and loading: Add an equal volume of 2×SDS loading buffer to the sample solutions prepared in the above experiments, lyse them in a 100 °C water bath for 10 min, centrifuge at 12000 r / min for 1 - 2 min at room temperature, and take 10 μL of the supernatant for SDS electrophoresis analysis;

[0054] (5) Electrophoresis: Add Tris-glycine electrophoresis buffer. The voltage of the stacking gel is 80 v, and the voltage of the separating gel is 120 v. Electrophoresis is carried out at 4 - 8 °C until the bromophenol blue reaches the lower end of the electrophoresis tank and then stops;

[0055] (6) Staining and decolorization: Take out the electrophoresed polyacrylamide gel from the glass plates, stain it with Coomassie Brilliant Blue staining solution, stain at room temperature for 20 - 30 min, and then put it into the decolorizing solution for decolorization until the protein bands are clear;

[0056] (7) Gel imaging and preservation: Use a gel imaging system to image and preserve the decolorized polyacrylamide gel.

[0057] 1.3.4 Determination of protein concentration and Western blot identification

[0058] According to the instructions of the Easy II Protein Quantitative Kit (BCA), calculate the protein concentration to be measured based on the readings of the microplate reader.

[0059] Using clinical chicken MS positive serum as the primary antibody and horseradish peroxidase-labeled goat anti-chicken IgG as the secondary antibody, perform Western blot analysis. Refer to the test methods in "Common Experimental Methods in Immunology" edited by Zhu Liping et al. The specific operations are as follows:

[0060] (1) Preparation of protein samples: Based on the above expression results, prepare a protein sample solution for immunoblot analysis;

[0061] (2) SDS-PAGE electrophoresis: Prepare a gel plate and perform electrophoresis;

[0062] (3) Transfer electrophoresis: After electrophoresis, take out the gel and rinse it 3 times with PBST. Immerse 2 cut Bio-Rad Mini Trans-Blot Filter Papers and 1 nitrocellulose membrane (NC membrane) similar in size to the gel in the transfer buffer for 10 min in advance, and assemble them in the order of cathode plate, sponge pad, filter paper, separating gel, NC membrane, filter paper, sponge pad. Fill with transfer buffer, keep a constant current of 100 mA, and perform electrophoresis for 2 - 3 h;

[0063] (4) Blocking: After electrophoresis, disassemble the device and take out the NC membrane. Rinse it several times with PBST, add an appropriate volume of 5% skim milk, and block it at room temperature for 4 - 6 h;

[0064] (5) Action of the primary antibody: Pour out the blocking solution, add 1×TBST washing solution and gently wash 3 times, 5 min each time. Pour out the washing solution, add the MS positive serum diluted 1:1000 until it completely covers, and gently shake it with a rotary shaker for 2 - 3 h;

[0065] (6) Action of the secondary antibody: Wash the NC membrane and add HRP-labeled goat anti-chicken IgG diluted 1:3000, and incubate it at room temperature for 2 h. After incubation, wash the NC membrane.

[0066] (7) Color development: Use the freshly prepared ECL color development solution to develop the color of the NC membrane. Imaging and preservation: Use a gel imaging system to image and save the developed membrane.

[0067] 2 Animal experiments

[0068] 2.1 Preparation of the infected bacterial solution

[0069] Rejuvenate the cryopreserved MS-NP strain isolated and identified in the laboratory, and perform large-scale culture using a liquid medium. Incubate it in an incubator at 37°C. After the pH value of its medium drops and the color changes from red to yellow, perform CCU determination, and dilute it to 10 9 CCU / mL for standby.

[0070] 2.2 Chick immunization program

[0071] Purchase 56 one-day-old SPF chicks from Sunner Development Co., Ltd. After raising them for one week, randomly select 7 chicks' venous blood as the pre-experiment negative control. Randomly divide all chicks into 7 groups, with 8 chicks in each group. Set up a blank control group Ba, a challenged group Bb, and a commercial vaccine group Bc; according to the difference in the immunization route, set up a subcutaneous injection group Aa, an eye-drop and nasal-drop group Ab, a muscle injection group Ac, and an adjuvant injection control group Ad. The immunization method is as follows: At 7 days old, emulsify the purified MS-eLPD recombinant protein solution and Freund's complete adjuvant at a volume ratio of 1:1 (the final solubility of the MS-eLPD recombinant protein is 1 mg / mL), and immunize according to the following immunization program. At 14 days old, randomly select 3 chicks' wing venous blood from each group and separate the serum. At 21 days old, first draw venous blood from each group of chicks and then boost immunize once with the same dose as the first immunization. At 28 days old, randomly select 3 chicks' wing venous blood from each group and separate the serum. At 31 days old, perform challenge. Except for the Ba group, the chicks in the other 6 groups are challenged with the MS-NP bacterial solution by nasal drop. Use a 1 ml needleless syringe to drip 0.2×10 9 CCU / mL of the MS-NP bacterial solution into the nasal cavity of each chick. Observe the morbidity and mortality of the chicken flock after challenge, count its mortality rate, and continuously observe for 10 days. At 38 days old, randomly select 3 chicks' wing venous blood from each group and separate the serum. At 41 days old, weigh the chickens and then perform euthanasia, observe the pathological changes of tissues such as the laryngeal trachea, lungs, and air sacs of the chickens in different experimental groups, and make detailed records. Finally, refer to the literature for lesion grade scoring. Dissect and collect samples such as the trachea and spleen for further detection and analysis.

[0072]

[0073]

[0074] 2.4 Clinical symptom observation

[0075] Observe the clinical symptoms of each group of chickens daily, and make detailed records of the onset time and clinical symptom characteristics.

[0076] 2.5 Histopathological examination

[0077] Observe the pathological changes of the trachea and spleen tissues. The preparation process of the pathological tissue sections is as follows:

[0078] Collect the trachea and spleen of experimental animals and place them in neutral formaldehyde (final concentration 4% formic acid PBS solution). After fixing at room temperature for 24 hours, trim the blocks. Trim the tissue blocks into 0.5×0.5×0.5 cm 3 size, and then place them in fresh formaldehyde solution and fix at room temperature for 24 hours. Continuously rinse the fixed tissue samples with running water for 24 hours to wash away the fixative; dehydrate using the ethanol gradient step-up dehydration method. Gradient alcohol dehydration: 75%, 1.5 hours; 85%, 1 hour; 95%, 1 hour; 100%, 1 hour. Clear with xylene: Xylene (Ⅰ) for 15 minutes, Xylene (Ⅱ) for 10 minutes. Embed the tissue in wax. Place the well-cleared tissue into the melted wax (Ⅰ) and impregnate for 1 hour; then transfer it to wax (Ⅱ) and impregnate for another 1 hour. After the wax block is fixed, trim it to be slightly larger than the tissue block, and then section, with a thickness generally of 4 - 5 μm.

[0079] Section staining: Extend the sections in water at 38 - 42 °C, then place them on glass slides, label, and dry. Remove wax with xylene: Xylene (Ⅰ) for 5 - 10 minutes, Xylene (Ⅱ) for 5 - 10 minutes. Rehydrate with gradient alcohol: 100% alcohol for 5 minutes; 95% alcohol for 3 minutes; 85% alcohol for 3 minutes; 75% alcohol for 2 minutes; deionized water for 2 minutes. Stain: Hematoxylin for 2 minutes; 1% eosin ethanol for 1 minute. Repeat the gradient alcohol dehydration and xylene clearing again. Mount with neutral gum. Observe and photograph under a microscope.

[0080] 2.6 ELISA detection of serum antibodies

[0081] Coat the ELISA plate with the prokaryotic-expressed purified antigen MS-eLPD (0.2 μg / mL), 100 μL / well; detect antibodies using the previously established indirect ELISA method. The specific steps are as follows: Dilute the chicken sera collected at each time point (7d, 14d, 21d, 28d, 38d) according to the detection results in 2.6. Add the diluted serum samples of each group to the enzyme-labeled wells on the same enzyme-labeled plate, with each sample repeated in 3 wells. At the same time, set up negative serum control and positive serum control. Use the corresponding OD 450nm value as the reference value for antibody level.

[0082] 2.8 Re-isolation and identification of MS in experimental animals

[0083] Sterilely collect the throat swabs of experimental animals for re-isolation and identification of Mycoplasma gallisepticum.

[0084] 3 Results

[0085] 3.1 Protein structure prediction

[0086] Use the Expasy online prediction website for analysis, excluding regions that are not easily formed into epitopes such as helices and folds. The secondary prediction results are asFigure 1A As shown, the tertiary structure is as Figure 1B shown.

[0087] 3.2 Identification Results of Double Enzyme Digestion of Recombinant Plasmid

[0088] After ligating the target gene and pET-32a(+) plasmid with T4 DNA ligase, the ligation product was transformed into DH5α cloning strain. As Figure 2 shown, two clear target bands were visible after double enzyme digestion verification of the plasmid of the cloning strain, indicating the successful construction of the recombinant plasmid. The recombinant plasmid was named pET-32a(+)-MS-eLPD. The recombinant plasmid with correct double enzyme digestion verification was sent to Fujian Sangon Biotech Co., Ltd. for sequencing, and the sequencing result was correct.

[0089] 3.3 Results of Prokaryotic Expression and SDS-PAGE Analysis

[0090] The results showed that no obvious target band was seen in the uninduced group of the recombinant plasmid (lane 1); an obvious specific band of about 54KD (lane 2) was visible in the supernatant after sonication of the induced group of the recombinant plasmid, and obvious bands were visible in the supernatant after sonication (lane 3); a small amount of protein was visible in the precipitate (lane 4). The results showed that the recombinant plasmid pET-32a(+)-MS-eLPD could express an MS-eLPD fusion protein of about 54KD size after induction with IPTG, and the MS-eLPD fusion protein mainly existed in the form of soluble protein ( Figure 3 ). The target protein was purified with a nickel affinity chromatography column and then identified by SDS-PAGE. The results showed that the target protein could be eluted with 100 mM imidazole eluent. The purified target protein was concentrated, and the OD 562nm value was 3.568. It was calculated that its concentration was 49.14 μg / ml.

[0091] 3.4 Identification of the Target Protein by Western Blot

[0092] The purified recombinant protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. Western blot detection was performed using clinical chicken positive serum as the primary antibody. The results showed ( Figure 4 ) that the target protein had good immunoreactivity with the serum antibody in the Western blot method.

[0093] 3.5 Analysis of Immunization and Challenge Protection Effect

[0094] 3.5.1 Safety Evaluation

[0095] No obvious stress reactions or discomfort reactions at the inoculation sites were observed in the chicks immunized with MS-eLPD+ adjuvant within 24 hours. Their diet and water intake were normal. After dissection two weeks after immunization, no tumors or other conditions were found at the immunization sites or throughout the body, indicating that the recombinant protein MS-eLPD prepared in this experiment had no obvious irritation to chickens, and there was no discomfort at the inoculation sites or throughout the body, and its safety was good.

[0096] 3.5.2 Clinical symptoms and pathological changes of chicks During the entire experimental period, there were no abnormal changes in the clinical symptoms and growth and development of the chickens in the control group. The chickens in the experimental group were smaller in size compared with those in the control group. As the infection time passed, the feathers of the chickens in the infected group were disheveled and dull, they were listless, their feed intake decreased, and some chickens showed mild respiratory symptoms. After dissection, ( Figure 5 ), there were no abnormal changes in the blank control group. A small number of chickens in the experimental group had mild sternal cysts, and there was a small amount of fluid after dissection; different numbers of chickens in different groups of the experimental group had severe air sac inflammation, with an increase in follicles. According to reference

[90] , the thoracic air sacs or abdominal air sacs were scored based on the severity of air sac inflammation: 0 indicated no visible macroscopic air sac damage; 1 indicated visible lymph follicle lesions or mild turbidity of the air sac; 2 indicated mild thickening of the air sac, usually with small caseous exudates aggregating; 3 indicated obvious thickening of the air sac, decreased transparency, and a large amount of caseous exudates in a single air sac. The scores of each group are shown in the following table.

[0097] Group Number of lesions Score Group Aa 2 / 8 1 Group Ab 1 / 8 1 Group Ac 0 / 8 1 Group Ad 4 / 8 2 Group Ba 0 / 8 0 Group Bb 5 / 8 1-2 Group Bc 2 / 8 1

[0098] 3.5.3 Pathological section analysis

[0099] Sections were randomly selected from the three groups for observation (n = 3). Group A was the PBS control group (only Ba group), Group B was the immunized group (Aa group, Ab group, Ac group, Bc group), and Group C was the challenged group (Ad group, Bb group).

[0100] As Figure 6 shown, the basic structure of the trachea in Group A was clear and intact; in Group B, the tracheal mucosal epithelial cells gradually became columnar, and a small amount of lymphocytes infiltrated and partially exfoliated; in Group C, the cilia on the surface of the tracheal epithelium were arranged disorderly and a large amount exfoliated; a large number of irregular mucous glands were formed in the mucosal layer, as well as a large amount of lymphocyte infiltration and widened gaps.

[0101] As Figure 7 shown, there were no obvious histopathological changes in the spleens of the chicks in the control group (Group A), the number of lymphocytes in the spleens of the chicks in Group B increased, a small amount of lymphocytes infiltrated, and the gaps widened; in the spleens of the chicks in Group C, there were necrosis phenomena such as karyorrhexis and karyolysis of lymphocytes; the cell structure was blurred, the boundary between the red pulp and the white pulp was unclear, and some cell nuclei disappeared in the lymphocytes, showing diffuse necrosis.

[0102] 3.5.4 ELISA detection of antibody levels

[0103] The chicken sera collected at each time point (7d, 14d, 21d, 28d, 38d) were used to detect the antibody titers of chicken sera by the established indirect ELISA method, and the changing trends of the antibody levels in the chicken sera of different immunization groups were observed. Using the purified MS-eLPD protein as the antigen, the protein coating concentration was 0.5 μg / mL, 100 μL per well, and a 96-well ELISA plate was coated. The test positive serum and the test negative serum were diluted 1:100. After incubating the primary antibody (30 min) and the secondary antibody (60 min), the average value of the readings of the positive serum and the negative serum measured 3 times was calculated, and the ratio of the positive serum to the negative serum (P / N value) was calculated.

[0104] The results are shown in the following table. The antibody level in the blank group (Ba group) was always very low, lower than the negative-positive critical value (the threshold was 0.2785). In the commercial inactivated vaccine immunization group (Bc group), after the first immunization, the antibody level was lower than the negative-positive critical value. After the second immunization, there was a significant change in the antibody titer (p < 0.001). After the bacterial challenge, the serum antibody level increased rapidly, far greater than the negative-positive critical value. In the immunization groups (Aa group, Ab group), the antibody level gradually increased after the first immunization and was higher than the negative-positive critical value. After the second immunization, the antibody level continued to increase. After the bacterial challenge, the antibody level increased slightly and then remained roughly stable. The final antibody level in the Aa group was significantly higher than that in the Bc group, and the difference in the final antibody level between the Ab group and the Bc group was not significant. In the Ac group, the antibody level remained at a relatively low level after the primary immunization, increased significantly after the second immunization and was higher than the negative-positive critical value. After the bacterial challenge, the antibody level increased slightly again but was lower than that in the Bc group. At the same time, the antibody levels in the adjuvant control group (Ad group) and the bacterial challenge group (Bb group) were always very low before the bacterial challenge, lower than the negative-positive critical value. After the bacterial challenge (31d), there was a significant change in the antibody level, and it was higher than the negative-positive critical value.

[0105]

Claims

1. A gene encoding a multi-epitope protein of Mycoplasma synoviae of chickens, characterized in that: The nucleotide sequence is as shown in SEQ ID NO.

2.

2. A recombinant expression vector containing the gene described in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that: Its starting vector is pET-32a(+).

4. A recombinant expression bacterium containing the recombinant expression vector described in claim 2.

5. The recombinant expression bacterium according to claim 4, wherein: Its starting strain is Escherichia coli.

6. Use of the Mycoplasma synoviae multi-epitope protein encoded by the gene described in claim 1 in the preparation of a Mycoplasma synoviae genetic engineering vaccine.

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