Recombinant Mycoplasma synoviae antigen protein, its coding gene, preparation method and application

Abstract

The present invention discloses a recombinant Mycoplasma synoviae antigen protein, its coding gene, preparation method and application. The present invention respectively expresses and purifies rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145 recombinant proteins by using a prokaryotic expression system. Furthermore, the polyclonal antiserum prepared by immunizing BALB / c mice has a significant growth inhibitory effect on Mycoplasma synoviae strains WVU1853, Ningxia / 2017-1, and Shandong / 2017-2. The neutralizing antibody titer can reach up to 1:256 at most, and it has the potential to be developed into a Mycoplasma synoviae subunit vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a recombinant Mycoplasma synoviae antigen protein, its encoding gene, a preparation method and applications thereof. Background Art

[0002] Mycoplasma synoviae (MS) in chickens can cause synovitis, air sacculitis, Eggshell apex abnormality (EAA), etc., which can lead to problems such as reduced feed return rate, growth retardation, increased culling rate and reduced egg production, resulting in huge economic losses. Mycoplasma synoviae can also present mixed infections with other pathogens, leading to more severe clinical symptoms and higher mortality.

[0003] Controlling Mycoplasma synoviae infection mainly includes: purification of breeding flocks, drug prevention and treatment, and vaccination. Mycoplasma synoviae can be transmitted horizontally and vertically. The purification of breeding flocks has high economic costs and great difficulties. The abuse of antibiotics can significantly enhance its drug resistance and increase food safety risks. Currently, the vaccine products used for preventing and controlling Mycoplasma synoviae mainly include attenuated live vaccines and inactivated vaccines, and there is no commercialized Mycoplasma synoviae subunit vaccine.

[0004] Neutralizing antigens are a class of antigens that can induce neutralizing antibodies against pathogens and play important roles in inducing immune protection, immune evaluation and vaccine design. There is currently no relevant report on Mycoplasma synoviae proteins with neutralizing activity. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a recombinant Mycoplasma synoviae antigen protein with immune neutralizing and protective activity.

[0006] Another technical problem to be solved by the present invention is to provide a nucleic acid or gene encoding the recombinant Mycoplasma synoviae antigen protein.

[0007] Another technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector or a recombinant bacterium containing the nucleic acid or gene.

[0008] Another technical problem to be solved by the present invention is to provide a preparation method for the recombinant Mycoplasma synoviae antigen protein.

[0009] Another technical problem to be solved by the present invention is to provide the applications of the recombinant Mycoplasma synoviae antigen protein, the nucleic acid or gene, the expression cassette, the recombinant vector and the recombinant bacterium in the preparation of drugs, polyclonal antibodies or vaccines for treating or preventing Mycoplasma synoviae infection.

[0010] Another technical problem to be solved by the present invention is to provide a polyclonal antibody.

[0011] The last technical problem to be solved by the present invention is to provide a vaccine.

[0012] Technical solution: To solve the above technical problems, the present invention provides a recombinant Mycoplasma synoviae antigen protein, which is a protein selected from the following (a), (b), or (c):

[0013] (a) A protein having an amino acid sequence as shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38;

[0014] (b) A protein derived from (a) in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of (a) and having the activity of Mycoplasma synoviae antigen protein;

[0015] (c) A protein having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

[0016] The content of the present invention further includes a nucleic acid or gene encoding the recombinant Mycoplasma synoviae antigen protein, and the nucleic acid or gene is a gene or nucleic acid selected from the following (i), (ii), or (iii):

[0017] (i) A gene or nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11;

[0018] (ii) A gene or nucleic acid in which one or several bases are substituted, deleted, or added in the nucleotide sequence of (i) and having the activity of encoding Mycoplasma synoviae antigen protein;

[0019] (iii) A gene or nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0020] Among them, preferably, the recombinant Mycoplasma synoviae antigen protein includes rRS02775, rRS01455, rRS01745, rRS01040, rRS01750 or rRS02145, and their corresponding amino acid sequences are shown as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12 respectively.

[0021] The content of the present invention also includes an expression cassette, a recombinant vector or a recombinant bacterium, which contains the nucleic acid or gene described above.

[0022] Among them, the recombinant vector is obtained by inserting the nucleic acid or gene into a vector. Preferably, the nucleic acid or gene is inserted between the T7 terminator sequence and the lac operator sequence of the vector.

[0023] Among them, the vector includes but is not limited to prokaryotic expression vectors.

[0024] Preferably, the prokaryotic expression vector includes but is not limited to Escherichia coli expression vectors, and the Escherichia coli expression vectors include but are not limited to pET-32a.

[0025] Among them, the nucleic acid or gene contains the gene encoding Mycoplasma synoviae RS02775 protein, and the nucleic acid or gene sequence is the sequence shown as SEQ ID NO:1 obtained by codon optimization;

[0026] Among them, the nucleic acid or gene contains the gene encoding Mycoplasma synoviae RS01455 protein, and the nucleic acid or gene sequence is the sequence shown as SEQ ID NO:3 obtained by codon optimization;

[0027] Among them, the nucleic acid or gene contains the gene encoding Mycoplasma synoviae RS01745 protein, and the nucleic acid or gene sequence is the sequence shown as SEQ ID NO:5 obtained by codon optimization;

[0028] Among them, the nucleic acid or gene contains the gene encoding Mycoplasma synoviae RS01040 protein, and the nucleic acid or gene sequence is the sequence shown as SEQ ID NO:7 obtained by codon optimization;

[0029] Among them, the nucleic acid or gene contains the gene encoding Mycoplasma synoviae RS01750 protein, and the nucleic acid or gene sequence is the sequence shown as SEQ ID NO:9 obtained by codon optimization;

[0030] Among them, the nucleic acid or gene contains a gene encoding the Mycoplasma synoviae RS02145 protein, and the nucleic acid or gene sequence is the sequence shown in SEQ ID NO:11 obtained by codon optimization.

[0031] Preferably, the 3′ end of the gene encoding the Mycoplasma synoviae RS02775 protein is linked to a 6×His tag peptide sequence, and the 5′ end is successively linked to an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is the pET-32a-RS02775 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0032] Preferably, the 3′ end of the gene encoding the Mycoplasma synoviae RS01455 protein is linked to a 6×His tag peptide sequence, and the 5′ end is successively linked to an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is the pET-32a-RS01455 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0033] Preferably, the 3′ end of the gene encoding the Mycoplasma synoviae RS01745 protein is linked to a 6×His tag peptide sequence, and the 5′ end is successively linked to an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is the pET-32a-RS01745 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0034] Preferably, the 3′ end of the gene encoding the Mycoplasma synoviae RS01040 protein is linked to a 6×His tag peptide sequence, and the 5′ end is successively linked to an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is the pET-32a-RS01040 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0035] Preferably, the 3′ end of the gene encoding the Mycoplasma synoviae RS01750 protein is linked to a 6×His tag peptide sequence, and the 5′ end is successively linked to an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is the pET-32a-RS01750 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0036] Preferably, the 3′ end of the protein-encoding gene of Mycoplasma synoviae RS02145 is ligated with a 6×His tag peptide sequence, and the 5′ end is successively ligated with an S-Tag tag peptide sequence, a 6×His tag peptide sequence, and a TrxA tag peptide sequence. The recombinant expression vector is a pET-32a-RS02145 vector with the target gene fragment inserted between the T7 terminator sequence and the lac operator sequence.

[0037] The present invention also includes a method for preparing the recombinant Mycoplasma synoviae antigen protein, comprising the following steps:

[0038] 1) Obtaining the nucleic acid or gene described above;

[0039] 2) Introducing the nucleic acid or gene obtained in step 1) into a vector to obtain a recombinant vector;

[0040] 3) Introducing the recombinant vector into a host bacterium for induced expression to obtain the recombinant Mycoplasma synoviae antigen protein.

[0041] The steps for obtaining nucleic acids or genes in step 1) include: referring to the RS02775 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 591671 - 593977 of CP083748.1, the RS01455 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 203259 - 205574 of CP034544.1, the RS01745 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 370710 - 372185 of CP083748.1, the RS01040 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 311556 - 314174 of CP163310.1, the RS01750 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 147506 - 149740 of CP163310.1, and the RS02145 gene sequence of Mycoplasma synoviae in chickens shown by the nucleotide sequence at positions 445670 - 447094 of CP083748.1. According to the codon bias of Escherichia coli, the sequences of RS02775, RS01455, RS01745, RS01040, RS01750, and RS02145 were point - mutated by overlap PCR. The stop codon TGA in the sequences was mutated to TGG. The mutated sequence of RS02775 is as shown in SEQ ID NO:1, the mutated sequence of RS01455 is as shown in SEQ ID NO:3, the mutated sequence of RS01745 is as shown in SEQ ID NO:5, the mutated sequence of RS01040 is as shown in SEQ ID NO:7, the mutated sequence of RS01750 is as shown in SEQ ID NO:9, and the mutated sequence of RS02145 is as shown in SEQ ID NO:11.

[0042] The specific steps in step 2) include: respectively digesting and ligating the optimized target genes in step 1) to the pET - 32a vector, transforming the ligation products into Trans - T1 competent cells, coating them on LB plates containing ampicillin resistance, culturing at 37°C for 12 - 16 h, picking single colonies and inoculating them into LB medium respectively, identifying the recombinant plasmids by PCR and sequencing. The recombinant plasmids are named pET - 32a - RS02775, pET - 32a - RS01455, pET - 32a - RS01745, pET - 32a - RS01040, pET - 32a - RS01750, or pET - 32a - RS02145.

[0043] The specific steps in step 3) include: transforming the constructed recombinant plasmid into Escherichia coli BL21(DE3) respectively, identifying positive strains by PCR, and inducing the positive strains to express rRS02775, rRS01455, rRS01745, rRS01040, rRS01750 or rRS02145 recombinant proteins by ITPG.

[0044] The present invention also includes a purification method for recombinant Mycoplasma synoviae antigen proteins, and the corresponding purified recombinant proteins are obtained by purifying rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145 recombinant proteins by affinity chromatography.

[0045] The present invention also includes the application of the recombinant Mycoplasma synoviae antigen proteins, the nucleic acids or genes, the expression cassettes, recombinant vectors or recombinant bacteria in the preparation of drugs, polyclonal antibodies or vaccines for treating or preventing Mycoplasma synoviae infection.

[0046] The present invention also includes a polyclonal antibody, and the polyclonal antibody is obtained by immunizing an animal with the recombinant Mycoplasma synoviae antigen protein.

[0047] Preferably, the polyclonal antibody is a neutralizing protective antibody, and the neutralizing protective antibody can induce the body to produce neutralizing protective antibodies against Mycoplasma synoviae WVU1853 strain, Ningxia / 2017-1 strain, Shandong / 2017-2 strain after mixing the purified rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145 recombinant proteins with Freund's adjuvant respectively and uniformly.

[0048] The present invention also includes a vaccine, and the vaccine includes the recombinant Mycoplasma synoviae antigen protein.

[0049] Among them, the vaccine also includes an adjuvant.

[0050] Preferably, the recombinant Mycoplasma synoviae antigen proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145 with immune neutralizing and protective activities provided by the present invention can be used to prepare Mycoplasma synoviae subunit vaccines.

[0051] Specifically, the purified recombinant Mycoplasma synoviae antigen proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 with immunoneutralizing and protective activities were mixed and formulated so that each milliliter volume contained 111.1 μg of each of the rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 recombinant proteins. An appropriate aluminum hydroxide adjuvant was added so that the volume ratio of the adjuvant phase to the protein phase was 1:3, and the final concentration of each protein in the vaccine was 83.3 μg / ml.

[0052] Mechanism of the invention: The present invention provides six antigen proteins of recombinant Mycoplasma synoviae with immunoneutralizing and protective effects, namely the Mycoplasma synoviae RS02775, RS01455, RS01745, RS01040, RS01750, and RS02145 proteins screened by the reverse vaccinology method. Subsequently, the rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 recombinant proteins were expressed through a prokaryotic expression system. The polyclonal antibodies induced by the recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 have a neutralizing effect on multiple strains of Mycoplasma synoviae.

[0053] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The polyclonal antisera prepared by immunizing with the rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 recombinant proteins of the present invention have a significant growth inhibitory effect on the Mycoplasma synoviae WVU1853 strain, Ningxia / 2017-1 strain, and Shandong / 2017-2 strain. The neutralizing antibody titer can reach above 1:256, and it has the potential to be developed into a subunit vaccine against Mycoplasma synoviae, providing a candidate antigen for the development of a subunit vaccine against Mycoplasma synoviae, which is of great significance for the prevention and control of Mycoplasma synoviae. Description of the drawings

[0054] Figure 1 It is the gel electrophoresis result of the overlap PCR amplification products of the RS02775, RS01455, RS01745, RS01040, RS01750, and RS02145 genes;

[0055] Figure 2Gel electrophoresis results of PCR amplification products of colony samples transformed with genes RS02775, RS01455, RS01745, RS01040, RS01750, and RS02145;

[0056] Figure 3 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS02775;

[0057] Figure 4 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS01455;

[0058] Figure 5 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS01745;

[0059] Figure 6 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS01040;

[0060] Figure 7 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS01750;

[0061] Figure 8 Map of the constructed recombinant prokaryotic expression vector pET-32a-RS02145;

[0062] Figure 9 SDS-PAGE identification results of the recombinant rRS02775 protein expressed in E. coli;

[0063] Figure 10 SDS-PAGE identification results of the recombinant rRS01455 protein expressed in E. coli;

[0064] Figure 11 SDS-PAGE identification results of the recombinant rRS01745 protein expressed in E. coli;

[0065] Figure 12 SDS-PAGE identification results of the recombinant rRS01040 protein expressed in E. coli;

[0066] Figure 13 SDS-PAGE identification results of the recombinant rRS01750 protein expressed in E. coli;

[0067] Figure 14 SDS-PAGE identification results of the recombinant rRS02145 protein expressed in E. coli;

[0068] Figure 15Identification results of purified recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145;

[0069] Figure 16 Western blot identification results of polyclonal antiserum against recombinant protein rRS02775;

[0070] Figure 17 Western blot identification results of polyclonal antiserum against recombinant protein rRS01455;

[0071] Figure 18 Western blot identification results of polyclonal antiserum against recombinant protein rRS01745;

[0072] Figure 19 Western blot identification results of polyclonal antiserum against recombinant protein rRS01040;

[0073] Figure 20 Western blot identification results of polyclonal antiserum against recombinant protein rRS01750;

[0074] Figure 21 Western blot identification results of polyclonal antiserum against recombinant protein rRS02145;

[0075] Figure 22 Results of chicken footpad and joint lesion scores after immunization with a single protein

[0076] Figure 23 Results of chicken footpad and joint lesion scores after using the subunit vaccine of the present invention

[0077] Figure 24 Comparison results of chicken footpad and tarsometatarsal joint lesions in each group after using the subunit vaccine of the present invention. Detailed implementation mode

[0078] The implementation scheme of the present invention will be described in detail and comprehensively in combination with specific embodiments below. Obviously, the described embodiments are only used to illustrate a part of the embodiments of the present invention, and should not be regarded as a limitation on the scope of the present invention. In the embodiments, the specific conditions not specifically described are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not indicating the manufacturer are all commercially available conventional products.

[0079] Example 1 Construction and identification of expression vector pET-32a-RS02775

[0080] 1. Amplification and recovery of the RS02775 gene: Referring to the nucleotide sequence at positions 591671 - 593977 of Mycoplasma synoviae strain 5 - 9 (GenBank No. CP083748.1), according to the codon preference of Escherichia coli, the RS02775 gene sequence was mutated by the overlap PCR method. The mutated gene sequence is shown as SEQ ID NO:1. The amino acid sequence translated from SEQ ID NO:1 is 100% identical to the amino acid sequence translated from the nucleotide sequence at positions 591671 - 593977 of CP083748.1, and both are the amino acid sequence shown as SEQ ID NO:33. Using the optimized RS02775 gene sequence as a template, and RS02775F and RS02775R as upstream and downstream primers (the primer sequences of RS02775F and RS02775R are shown as SEQ ID NO:13 and SEQ ID NO:14), PCR amplification was carried out according to the instructions of Nanjing Novoprotein Biotechnology Co., Ltd.'s 2×Phanta Max Master Mix (DyePlus). The amplification system and reaction program are shown in Table 1:

[0081] Table 1 RS02775 gene amplification system and reaction program

[0082]

[0083] The PCR amplification product was detected by 1% agarose gel electrophoresis. The size of the target gene is as Figure 1 shown, and a target band appeared at the 2325bp position, indicating that the target gene amplification was successful. It was recovered and purified using a gel recovery and purification kit.

[0084] 2. Digestion and recovery of the pET - 32a vector: The extracted pET - 32a plasmid was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis and then recovered and purified using a gel recovery and purification kit.

[0085] 3. Digestion and recovery of the RS02775 gene fragment: The recovered and purified RS02775 gene fragment was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis and then recovered and purified using a gel recovery and purification kit.

[0086] 4. Ligation: The purified and recovered RS02775 gene fragment was ligated to the pET - 32a plasmid fragment using T4 DNA ligase.

[0087] 5. Transformation: Take 5 μL of the ligation product and add it to 50 μL of BL21(DE3) competent cells. Gently flick to mix evenly, incubate on ice for 30 min, perform heat shock in a 42 °C water bath for 45 sec, then quickly place it on ice and let it stand for 2 min. Add 500 μL of LB liquid medium without resistance and culture at 37 °C for 1 h. Concentrate the bacterial solution to 100 μL and spread it on LB solid medium with ampicillin resistance, and culture at 37 °C for 14 h.

[0088] 6. Colony PCR and sequencing identification: Pick single colonies on the plate and inoculate them into LB liquid medium respectively, and culture at 37 °C for 4 h. Use the bacterial solution as a template, and use S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) as primers for bacterial solution PCR identification. Perform gel electrophoresis on the PCR products to verify the band size. Samples with a 2475 bp band are positive samples as Figure 2 shown. Send the positive strains to Anhui General Biotech Co., Ltd. for sequencing, and select the plasmids with correct sequencing and the corresponding bacterial solutions for preservation. The schematic diagram of the vector map of the correctly constructed recombinant vector pET-32a-RS02775 is as Figure 3 shown, the amino acid sequence of the recombinant protein it expresses is as shown in SEQ ID NO:2, and the corresponding gene sequence is as shown in SEQ ID NO:27.

[0089] Example 2 Construction and identification of expression vector pET-32a-RS01455

[0090] 1. Amplification and recovery of RS01455 gene Refer to the nucleotide sequence at positions 203259 - 205574 of Mycoplasma synoviae HN01 strain (GenBank No.CP034544.1). According to the codon preference of Escherichia coli, the RS01455 gene sequence was mutated by the overlap PCR method. The mutated gene sequence is as shown in SEQ ID NO:3. The amino acid sequence translated from SEQ ID NO:3 has 100% identity with the amino acid sequence translated from the nucleotide sequence at positions 203259 - 205574 of CP034544.1, and both are the amino acid sequence shown in SEQ ID NO:34. Using the optimized RS01455 gene sequence as a template, RS01455F and RS01455R as upstream and downstream primers (the primer sequences of RS01455F and RS01455R are shown in SEQ ID NO:15 and SEQ ID NO:16 respectively), perform PCR amplification according to the instructions of Nanjing Novoprotein Biotechnology Co., Ltd.'s 2×Phanta Max Master Mix (Dye Plus). The amplification system and reaction procedure are shown in Table 2:

[0091] Table 2 RS01455 Gene Amplification System and Reaction Program

[0092]

[0093] The PCR amplification products were detected by 1% agarose gel electrophoresis. The size of the target gene was as Figure 1 shown, and a target band appeared at the 2334 bp position, indicating that the target gene was successfully amplified. The gel recovery and purification kit was used for recovery and purification.

[0094] 2. Digestion and Recovery of pET-32a Vector: The extracted pET-32a plasmid was digested with BamH I and Sal I restriction endonucleases. The digested products were subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0095] 3. Digestion and Recovery of RS01455 Gene Fragment: The recovered and purified RS01455 gene fragment was digested with BamH I and Sal I restriction endonucleases. The digested products were subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0096] 4. Ligation: The purified and recovered RS01455 gene fragment was ligated to the pET-32a plasmid fragment using T4 DNA ligase.

[0097] 5. Transformation: 5 μL of the ligation product was added to 50 μL of BL21(DE3) competent cells, gently flicked to mix, incubated on ice for 30 min, heat-shocked in a 42°C water bath for 45 sec, and then quickly placed on ice and allowed to stand for 2 min. 500 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37°C for 1 h. The bacterial solution was concentrated to 100 μL and spread on an LB solid medium containing ampicillin resistance, and cultured at 37°C for 14 h.

[0098] 6. Colony PCR and Sequencing Identification: Single colonies on the plate were picked and inoculated into LB liquid medium, and cultured at 37°C for 4 h. Using the bacterial solution as a template, S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) were used as primers for colony PCR identification. The PCR products were subjected to gel electrophoresis to verify the band size. Samples with a 2505 bp band were positive samples as Figure 2 shown. The positive strains were sent to Anhui General Biology Co., Ltd. for sequencing, and the plasmids with correct sequencing and the corresponding bacterial solutions were selected for preservation. The schematic diagram of the constructed correct recombinant vector pET-32a-RS01455 vector map was as Figure 4 shown. The amino acid sequence of the expressed recombinant protein was as shown in SEQ ID NO:4, and the corresponding gene sequence was as shown in SEQ ID NO:28.

[0099] Construction and Identification of Expression Vector pET-32a-RS01745 in Example 3

[0100] 1. Amplification and Recovery of RS01745 Gene: Referring to the nucleotide sequence at positions 370710 - 372185 of Mycoplasma synoviae strain 5 - 9 (GenBank No. CP083748.1), according to the codon preference of Escherichia coli, the RS01745 gene sequence was mutated by the overlap PCR method. The mutated gene sequence is shown in SEQ ID NO:5. The amino acid sequence translated from SEQ ID NO:5 is 100% identical to the amino acid sequence translated from the nucleotide sequence at positions 370710 - 372185 of CP083748.1, and both are the amino acid sequence shown in SEQ ID NO:35. Using the optimized RS01745 gene sequence as a template and RS01745 F and RS01745 R as upstream and downstream primers (the primer sequences of RS01745 F and RS01745 R are shown in SEQ ID NO:17 and SEQ ID NO:18), PCR amplification was carried out according to the instructions of 2×Phanta Max Master Mix (Dye Plus) from Nanjing Novoprotein Scientific Inc. The amplification system and reaction program are shown in Table 3:

[0101] Table 3 RS01745 Gene Amplification System and Reaction Program

[0102]

[0103] The PCR amplification product was detected by 1% agarose gel electrophoresis. The size of the target gene is as Figure 1 shown, and a target band appeared at the 1494bp position, indicating that the target gene was successfully amplified. It was recovered and purified using a gel recovery purification kit.

[0104] 2. Digestion and Recovery of pET-32a Vector: The extracted pET-32a plasmid was digested with BamH I and Xho I restriction endonucleases. The digestion products were subjected to gel electrophoresis and then recovered and purified using a gel recovery purification kit.

[0105] 3. Digestion and Recovery of RS01745 Gene Fragment: The recovered and purified RS01745 gene fragment was digested with BamH I and Xho I restriction endonucleases. The digestion products were subjected to gel electrophoresis and then recovered and purified using a gel recovery purification kit.

[0106] 4. Ligation: The purified and recovered RS01745 gene fragment was ligated to the pET-32a plasmid fragment using T4 DNA ligase.

[0107] 5. Transformation: Take 5 μL of the ligation product and add it to 50 μL of BL21(DE3) competent cells. Gently flick to mix evenly, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 sec, then quickly place on ice and let stand for 2 min. Add 500 μL of LB liquid medium without resistance and culture at 37°C for 1 h. Concentrate the bacterial solution to 100 μL and spread it on LB solid medium with ampicillin resistance, then culture at 37°C for 14 h.

[0108] 6. Colony PCR and sequencing identification: Pick single colonies on the plate and inoculate them into LB liquid medium respectively, then culture at 37°C for 4 h. Use the bacterial solution as a template, and S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) as primers for colony PCR identification. Perform gel electrophoresis on the PCR products to verify the band size. Samples with a 1644 bp band are positive samples as Figure 2 shown. Send the positive strains to Anhui General Biotech Co., Ltd. for sequencing, and select the plasmids with correct sequencing and the corresponding bacterial solutions for preservation. The schematic diagram of the vector map of the correctly constructed recombinant vector pET-32a-RS01745 is as Figure 5 shown. The amino acid sequence of the recombinant protein it expresses is as shown in SEQ ID NO:6, and the corresponding gene sequence is as shown in SEQ ID NO:29.

[0109] Example 4 Construction and identification of expression vector pET-32a-RS01040

[0110] 1. Amplification and recovery of RS01040 gene: Refer to the nucleotide sequence at positions 311556 - 314174 of Mycoplasma synoviae strain HB01 (GenBank No.CP163310.1). According to the codon preference of Escherichia coli, the RS01040 gene sequence was mutated by the overlap PCR method. The mutated gene sequence is as shown in SEQ ID NO:7. The amino acid sequence translated from SEQ ID NO:7 is 100% identical to the amino acid sequence translated from the nucleotide sequence at positions 311556 - 314174 of CP163310.1, and both are the amino acid sequence shown in SEQ ID NO:36. Using the optimized RS01040 gene sequence as a template, RS01040F and RS01040R as upstream and downstream primers (the primer sequences of RS01040F and RS01040R are shown in SEQ ID NO:19 and SEQ ID NO:20 respectively), perform PCR amplification according to the instructions of Nanjing Novoprotein Biotechnology Co., Ltd.'s 2×Phanta Max Master Mix (Dye Plus). The amplification system and reaction program are shown in Table 4:

[0111] Table 4 Gene amplification system and reaction program of RS01040

[0112]

[0113]

[0114] The PCR amplification products were detected by 1% agarose gel electrophoresis. The size of the target gene was as Figure 1 shown. A target band appeared at the 2637bp position, indicating successful amplification of the target gene. The gel recovery and purification kit was used for recovery and purification.

[0115] 2. Digestion and recovery of pET-32a vector: The extracted pET-32a plasmid was digested with BamH I and Xho I restriction endonucleases. The digested products were subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0116] 3. Digestion and recovery of RS01040 gene fragment: The recovered and purified RS01040 gene fragment was digested with BamH I and Xho I restriction endonucleases. The digested products were subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0117] 4. Ligation: The purified and recovered RS01040 gene fragment was ligated with the pET-32a plasmid fragment using T4 DNA ligase.

[0118] 5. Transformation: Take 5 μL of the ligation product and add it to 50 μL of BL21(DE3) competent cells. Gently flick to mix, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 sec, then quickly place on ice and let stand for 2 min. Add 500 μL of antibiotic-free LB liquid medium and culture at 37°C for 1 h. Concentrate the bacterial solution to 100 μL and spread it on an LB solid medium with ampicillin resistance, and culture at 37°C for 14 h.

[0119] 6. Colony PCR and sequencing identification: Pick single colonies on the plate and inoculate them into LB liquid medium respectively, and culture at 37°C for 4 h. Using the bacterial solution as a template and S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) as primers for colony PCR identification. The PCR products were subjected to gel electrophoresis to verify the band size. Samples with a 2787bp band were positive samples as Figure 2 shown. The positive strains were sent to Anhui General Biology Co., Ltd. for sequencing, and the plasmids with correct sequencing and the corresponding bacterial solutions were selected for preservation. The schematic diagram of the constructed correct recombinant vector pET-32a-RS01040 vector map is as Figure 6As shown, the amino acid sequence of the recombinant protein it expresses is as shown in SEQ ID NO:8, and the corresponding gene sequence is as shown in SEQ ID NO:30.

[0120] Example 5 Construction and Identification of Expression Vector pET-32a-RS01750

[0121] 1. Amplification and Recovery of RS01750 Gene: Referring to the nucleotide sequence at positions 147506 - 149740 of Mycoplasma synoviae strain HB01 (GenBank No. CP163310.1), according to the codon preference of Escherichia coli, the RS01750 gene sequence was mutated by overlap PCR method. The mutated gene sequence is as shown in SEQ ID NO:9. The amino acid sequence translated from SEQ ID NO:9 has 100% identity with the amino acid sequence translated from the nucleotide sequence at positions 147506 - 149740 of CP163310.1, and both are the amino acid sequence shown in SEQ ID NO:37. Using the optimized RS01750 gene sequence as a template, RS01750F and RS01750R as upstream and downstream primers (the primer sequences of RS01750F and RS01750R are as shown in SEQ ID NO:21 and SEQ ID NO:22), PCR amplification was carried out according to the instructions of 2×Phanta Max Master Mix (Dye Plus) of Nanjing Novoprotein Scientific Co., Ltd. The amplification system and reaction program are shown in Table 5:

[0122] Table 5 RS01750 Gene Amplification System and Reaction Program

[0123]

[0124] The PCR amplification product was detected by 1% agarose gel electrophoresis. The size of the target gene is as Figure 1 shown, and a target band appeared at the 2253bp position, indicating that the target gene was successfully amplified. It was recovered and purified using a gel recovery and purification kit.

[0125] 2. Digestion and Recovery of pET-32a Vector: The extracted pET-32a plasmid was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis and then recovered and purified using a gel recovery and purification kit.

[0126] 3. Digestion and Recovery of RS01750 Gene Fragment: The recovered and purified RS01750 gene fragment was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis and then recovered and purified using a gel recovery and purification kit.

[0127] 4. Ligation: Use T4 DNA ligase to ligate the purified and recovered RS01750 gene fragment with the pET-32a plasmid fragment.

[0128] 5. Transformation: Take 5 μL of the ligation product and add it to 50 μL of BL21(DE3) competent cells. Flick gently to mix, incubate on ice for 30 min, heat shock in a 42 °C water bath for 45 sec, then quickly place on ice and let stand for 2 min. Add 500 μL of antibiotic-free LB liquid medium and culture at 37 °C for 1 h. Concentrate the bacterial solution to 100 μL and spread it on an LB solid medium with ampicillin resistance, and culture at 37 °C for 14 h.

[0129] 6. Colony PCR and sequencing identification: Pick single colonies on the plate and inoculate them into LB liquid medium respectively, and culture at 37 °C for 4 h. Use the bacterial solution as a template, and S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) as primers for colony PCR identification. Perform gel electrophoresis on the PCR products to verify the band size. Samples with a 2403 bp band are positive samples as Figure 2 shown. Send the positive strains to Anhui General Biology Co., Ltd. for sequencing, and select the plasmids with correct sequencing and the corresponding bacterial solutions for preservation. The schematic diagram of the vector map of the correctly constructed recombinant vector pET-32a-RS01750 is as Figure 7 shown. The amino acid sequence of the recombinant protein it expresses is shown in SEQ ID NO:10, and the corresponding gene sequence is shown in SEQ ID NO:31.

[0130] Example 6 Construction and Identification of Expression Vector pET-32a-RS02145

[0131] 1. Amplification and recovery of the RS02145 gene: Referring to the nucleotide sequence at positions 445670 - 447094 of Mycoplasma synoviae strain 5 - 9 (GenBank No. CP083748.1), according to the codon preference of Escherichia coli, the RS02145 gene sequence was optimized by the overlap PCR method. The optimized gene sequence is shown as SEQ ID NO: 11. The amino acid sequence translated from SEQ ID NO: 11 has 100% identity with the amino acid sequence translated from the nucleotide sequence at positions 445670 - 447094 of CP083748.1, and both are the amino acid sequence shown as SEQ ID NO: 38. Using the optimized RS02145 gene sequence as a template, and RS02145F and RS02145R as upstream and downstream primers (the primer sequences of RS02145F and RS02145R are shown as SEQ ID NO: 23 and SEQ ID NO: 24), PCR amplification was carried out according to the instructions of 2×Phanta Max Master Mix (Dye Plus) of Nanjing Novoprotein Scientific Inc. The amplification system and reaction program are shown in Table 6:

[0132] Table 6 RS02145 gene amplification system and reaction program

[0133]

[0134] The PCR amplification product was detected by 1% agarose gel electrophoresis. The size of the target gene is as Figure 1 shown, and a target band appeared at the 1443bp position, indicating that the target gene amplification was successful. The gel recovery and purification kit was used for recovery and purification.

[0135] 2. Digestion and recovery of the pET - 32a vector: The extracted pET - 32a plasmid was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0136] 3. Digestion and recovery of the RS02145 gene fragment: The recovered and purified RS02145 gene fragment was digested with BamH I and Xho I restriction endonucleases. The digested product was subjected to gel electrophoresis, and the gel recovery and purification kit was used for recovery and purification.

[0137] 4. Ligation: The purified and recovered RS02145 gene fragment was ligated with the pET - 32a plasmid fragment using T4 DNA ligase.

[0138] 5. Transformation: Take 5 μL of the ligation product and add it to 50 μL of BL21(DE3) competent cells. Gently flick to mix evenly, place on ice bath for 30 min, heat shock in a 42 °C water bath for 45 sec, then quickly place on ice and let stand for 2 min. Add 500 μL of antibiotic-free LB liquid medium and culture at 37 °C for 1 h. Concentrate the bacterial solution to 100 μL and spread it on LB solid medium with ampicillin resistance, and culture at 37 °C for 14 h.

[0139] 6. Colony PCR and sequencing identification: Pick single colonies on the plate and inoculate them into LB liquid medium respectively, and culture at 37 °C for 4 h. Use the bacterial solution as a template, and S.tag and T7ter (the sequences are shown in SEQ ID NO:25 and SEQ ID NO:26 respectively) as primers for colony PCR identification. Perform gel electrophoresis on the PCR products to verify the band sizes. Samples with a 1593 bp band are positive samples as Figure 2 shown. Send the positive strains to Anhui General Biotech Co., Ltd. for sequencing, and select the plasmids with correct sequencing and the corresponding bacterial solutions for preservation. The schematic diagram of the vector map of the correctly constructed recombinant vector pET-32a-RS02145 is as Figure 8 shown, the amino acid sequence of the recombinant protein it expresses is as shown in SEQ ID NO:12, and the corresponding gene sequence is as shown in SEQ ID NO:32.

[0140] Example 7 Expression and purification of recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145

[0141] 1. Verification of the expression of target proteins: Pick 6 correctly constructed positive strains from Examples 1 - 6 into 5 mL of LB liquid medium respectively, and culture at 37 °C for 4 - 6 h until OD600 reaches 0.4. Add IPTG with a final concentration of 1 mmol / L and induce at 37 °C for 6 h. Concentrate the induced bacterial solution by 10 times and ultrasonically lyse for 10 min, then centrifuge at 12000 r / min for 5 min to separate the supernatant of the lysate. Perform SDS-PAGE vertical electrophoresis on the separated supernatant and precipitate of the lysate. As Figure 9 shown, the rRS02775 recombinant protein can be expressed in the precipitate, and there is a band at a position with a molecular weight of about 104 kDa, which is consistent with the expectation; as Figure 10 shown, the rRS01455 recombinant protein can be expressed in the supernatant, and there is a band at a position with a molecular weight of about 104 kDa, which is consistent with the expectation; as Figure 11 shown, the rRS01745 recombinant protein is expressed in both the supernatant and the precipitate, and there is a band at a position with a molecular weight of about 76 kDa, which is consistent with the expectation; as Figure 12As shown, the rRS01040 recombinant protein can be expressed in the supernatant, and there is a band at a molecular weight of approximately 119 kDa, which is consistent with the expectation; as Figure 13 As shown, the rRS01750 recombinant protein can be expressed in the supernatant, and there is a band at a molecular weight of approximately 100 kDa, which is consistent with the expectation; as Figure 14 As shown, the rRS02145 recombinant protein can be expressed in the supernatant, and there is a band at a molecular weight of approximately 70 kDa, which is consistent with the expectation.

[0142] 2. Large-scale protein expression: Streak the six positive strains constructed in Examples 1 to 6 on a plate for resuscitation. Pick single colonies into 5 mL of LB liquid medium respectively and culture at 37 °C until the logarithmic growth phase. Transfer the fresh bacterial liquid to 500 mL of LB liquid medium and culture at 37 °C for 4 - 6 h until the OD600 is 0.4. The induction conditions are as described in the previous item. Use a centrifuge to collect the bacterial cells at 6000 r / min for 5 min.

[0143] 3. Protein purification: Resuspend the six kinds of collected bacterial cells evenly with 50 mL of buffer respectively, then break the bacterial cells by ultrasonic treatment, purify them with GenScript Ni-NTA affinity chromatography medium, collect the six kinds of purified protein solutions, and analyze the purification effect of the target protein by SDS-PAGE electrophoresis, as Figure 15 shown. Sterilize the six kinds of purified protein solutions by filtration through a 0.22 μm filter membrane and store them at 4 °C.

[0144] Preparation of polyclonal antibody sera against recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 in Example 8

[0145] 1. Immunization: Immunize 6-week-old BALB / c mice with the purified recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 as antigens respectively. The immunization method is subcutaneous multi-point injection. When immunizing for the first time, mix the purified protein solution with an equal volume of Freund's complete adjuvant, and the immunization dose is 100 μg of recombinant protein per mouse. Immunize again two weeks after the first immunization, with an immunization dose of 50 μg per mouse. Immunize for the third time two weeks after the second immunization, with an immunization dose of 100 μg per mouse. The adjuvants for the second and third immunizations are Freund's incomplete adjuvant.

[0146] 2. Serum collection: One week after the third immunization, collect blood by cardiac puncture. Place the blood at 37 °C for 2 h, and serum separation can be seen. Centrifuge at 5000 r / min for 10 min to collect the supernatant, obtain the polyclonal antibody serum, and store it at -20 °C.

[0147] 3. Antibody titer detection: The indirect ELISA method was used to detect the polyclonal antibody titers of recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 in the prepared antiserum. The purified recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 were respectively diluted with coating buffer (0.05 mol / L sodium carbonate - sodium bicarbonate buffer, pH 9.6) to a final concentration of 2 μg / mL as antigens, and the two-fold serially diluted polyclonal antiserum was used as the primary antibody. The serum of non-immunized BALB / c mice was used as the negative control, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. Color development was performed by the TMB method. After adding the stop solution to terminate the reaction, the A450 value of each well was detected using an enzyme-linked immunosorbent assay (ELISA) reader. When the ratio of the A450 value of the well to be tested to the A450 value of the negative control well was ≥ 2.1, it was judged as positive, and the maximum dilution factor at which it could be judged as positive was used as the antibody titer of the serum to be tested. The results showed that the titers of the antisera against recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 prepared by the indirect ELISA method were 1:5120000, 1:2560000, 1:5120000, 1:10240000, 1:10240000, and 1:2560000, respectively.

[0148] Example 9 Identification of the polyclonal antibody sera of recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 by Western blot

[0149] Whole cell proteins of Mycoplasma synoviae Ningxia / 2017-1 strain and the tag proteins induced, expressed, and purified after transforming BL21(DE3) Escherichia coli with the pET-32a empty vector were used as samples for SDS electrophoresis. The products after electrophoresis were transferred onto a nitrocellulose membrane (NC membrane). Western blot identification was performed using the 500-fold diluted solutions of the six antisera prepared in Example 8 as the primary antibodies and HRP-labeled goat anti-mouse IgG as the secondary antibody. The identification results of the polyclonal antibody serum of recombinant protein rRS02775 are as Figure 16 shown. The prepared polyclonal antibody could specifically bind to Mycoplasma synoviae, and there was a band at a molecular weight of approximately 84 kDa, which was consistent with the expectation. The identification results of the polyclonal antibody serum of recombinant protein rRS01455 are as Figure 17 shown. The prepared polyclonal antibody could bind to Mycoplasma synoviae, and there was a band at a molecular weight of approximately 84 kDa, which was consistent with the expectation. The identification results of the polyclonal antibody serum of recombinant protein rRS01745 are asFigure 18 As shown, the prepared polyclonal antibody can specifically bind to Mycoplasma synoviae of chickens, and there is a band at a molecular weight of approximately 56 Da, which is consistent with the expectation. The identification results of the polyclonal antibody serum of recombinant protein rRS01040 are as Figure 19 As shown, the prepared polyclonal antibody can specifically bind to Mycoplasma synoviae of chickens, and there is a band at a molecular weight of approximately 99 kDa, which is consistent with the expectation. The identification results of the polyclonal antibody serum of recombinant protein rRS01750 are as Figure 20 As shown, the prepared polyclonal antibody can specifically bind to Mycoplasma synoviae of chickens, and there is a band at a molecular weight of approximately 80 kDa, which is consistent with the expectation. The identification results of the polyclonal antibody serum of recombinant protein rRS02145 are as Figure 21 As shown, the prepared polyclonal antibody can specifically bind to Mycoplasma synoviae of chickens, and there is a band at a molecular weight of approximately 50 kDa, which is consistent with the expectation.

[0150] Example 10 Determination of the neutralization titers of specific polyclonal antisera of recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145

[0151] Add 100 μL of the diluted polyclonal antiserum prepared in Example 9 to each well in the first column of a 96-well round-bottom deep-well plate. The dilution factor is 16-fold, and the diluent is Mycoplasma synoviae liquid medium (Mycoplasm Broth Base (mycoplasma basal medium) 25.5 g / L, glucose 3.3 g / L, coenzyme 100 mg / L, arginine 400 mg / L, cysteine 100 mg / L, phenol red 0.02%, porcine serum 15%, pH 7.8). Subsequently, perform a 2-fold serial dilution for each column, and set 8 dilution degrees for each serum. Add 200 μL of Mycoplasma synoviae liquid medium to each well in the ninth column as a negative control, and add 100 μL of Mycoplasma synoviae liquid medium to the tenth column as a positive control. Add 10 CCU to each well in the first to eighth columns and the tenth column 4Mycoplasma synoviae bacterial solution at a concentration of CCU / ml. Cover it with the matching silicone lid to complete the sealing, and incubate it in a 37°C biochemical incubator until the color change of the positive control is completed. Take the serum dilution gradient of the critical well with no color change as the neutralization titer. The results showed that the specific polyclonal sera of recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145 had neutralizing effects on Mycoplasma synoviae strains WVU1853, Ningxia / 2017-1, and Shandong / 2017-2 (ZHANG X, CHEN Y, XIE D, et al. Multi-locus sequence typing analysis of Mycoplasma synoviae isolates reveals unique sequence types in China[J]. Veterinary microbiology, 2021, 259(109101.)). The neutralization titers are shown in Table 7:

[0152] Table 7 Neutralization titers of polyclonal sera of recombinant proteins

[0153]

[0154] Example 11 Immunization challenge protection test of subunit vaccine prepared from a single protein

[0155] 1. Preparation of subunit vaccine: Dilute the 6 purified recombinant proteins in Step 4 of Example 7 with normal saline to a protein concentration of 555.6 μg / mL, and then uniformly mix the diluted protein solution with aluminum hydroxide gel adjuvant in a ratio of 3:1 to prepare the subunit vaccine. The aluminum hydroxide gel adjuvant is purchased from Thermo Fisher Scientific, product number: 77161.

[0156] 2. Immunization: Immunize 10 21-day-old SPF chickens with the prepared subunit vaccine by intramuscular injection in the chest, with an immunization dose of 0.12 mL per chicken. Another 10 chickens are set as the challenge control, and 10 chickens are set as the blank control. Twenty-one days after immunization, a second immunization is carried out with the same dose. Twenty-one days after the second immunization, the experimental chickens in each immunized group and the challenge control group are challenged by injecting 0.1 ml of the bacterial solution with a viable bacteria count of about 7 CCU / mL into the footpad. Observe the morbidity of the experimental chickens within 21 days.

[0157] ​Statistical results of immune protection efficacy: Paw pad lesions were scored according to the following criteria: No obvious swelling lesion of the paw pad was scored 0; Mild swelling lesion and thickening of the paw pad were scored 1; Obvious swelling of the paw pad, and tissue hyperplasia was visible upon autopsy, scored 2; Obvious swelling of the paw pad, mild deformation of the foot, obvious tissue hyperplasia and granuloma were visible upon autopsy, and there was a small amount of purulent exudate, scored 3; Severe swelling of the paw pad, severe deformation of the foot, obvious tissue hyperplasia was visible upon autopsy, a large number of granulomas appeared, and there was a large amount of purulent exudate, scored 4. Joint lesions were scored according to the following criteria: No obvious swelling lesion of the joint was scored 0; Mild swelling lesion and exudation of the joint were scored 1; Obvious swelling of the joint, and tissue hyperplasia was visible upon autopsy, scored 2; Obvious swelling of the joint, mild deformation of the foot, obvious tissue hyperplasia and granuloma were visible upon autopsy, and there was a small amount of purulent exudate, scored 3; Severe swelling and deformation of the joint, obvious tissue hyperplasia was visible upon autopsy, a large number of granulomas appeared, and there was a large amount of purulent exudate, scored 4. The results are as Figure 22 shown. The average lesion scores of joints and paw pads in each immunized group were lower than those in the challenge control group. One-way ANOVA was used to perform a significant analysis of the data. The joint lesion scores of the rRS02775 immunized group, rRS01455 immunized group, rRS01745 immunized group, rRS01040 immunized group, and rRS01750 immunized group were significantly lower (P<0.05) than those in the challenge control group. The paw pad lesion scores of the rRS02775 immunized group and rRS01750 immunized group were significantly lower (P<0.05) than those in the challenge control group. The incidence rates of each experimental group are shown in Table 8. The incidence rates of paw pad swelling and tarsal joint synovitis in each protein immunized group were lower than those in the challenge control group. The results indicate that after immunization with recombinant proteins rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, and rRS02145, the incidence rates of chicken paw swelling and tarsal joint synovitis can be reduced, and at the same time, the lesion degree can be significantly alleviated, showing good immune protection effects.

[0158] Table 8 Incidence rates of each group in the immune protection efficacy test of subunit vaccines prepared with single proteins

[0159]

[0160] Example 12 Immune challenge protection test of subunit vaccines prepared with multi-protein combinations

[0161] 1. Preparation of subunit vaccine: The 6 purified recombinant proteins in step 4 of Example 7 were mixed and diluted with physiological saline to a total protein concentration of 666.6 μg / mL, with the concentration of each recombinant protein being 111.1 μg / mL. Then, the mixed protein solution was uniformly mixed with aluminum hydroxide gel adjuvant in a ratio of 3:1 to prepare a subunit vaccine. The aluminum hydroxide gel adjuvant was purchased from Thermo Fisher Scientific, product number: 77161.

[0162] 2. Immunization: Fifty 21-day-old SPF chickens were randomly divided into 5 groups of 10 chickens each. Subunit vaccine group: Immunized with the prepared subunit vaccine by intramuscular injection in the chest, with an immunization dose of 0.12 ml per chicken. Twenty-one days after the first immunization, a second immunization was carried out with the same dose. Inactivated Mycoplasma synoviae vaccine group: Immunized with the inactivated Mycoplasma synoviae vaccine for chickens (YBF-MS1 strain) (purchased from Qingdao Bioengineering Co., Ltd.) by intramuscular injection in the chest, with an immunization dose of 0.5 ml per chicken. Twenty-one days after the first immunization, a second immunization was carried out with the same dose. MS-H temperature-sensitive live vaccine group: Immunized with the MS-H temperature-sensitive live vaccine (purchased from Australian Bioresources Company), with an immunization dose of 30 μL per chicken. The challenge control group and the blank control group were not immunized. At 63 days of age of the experimental chickens, except for the blank control group, each group of experimental chickens was challenged by injecting 0.1 ml of a bacterial suspension with a viable bacterial count of approximately 10 7 CCU / mL into the footpad. The morbidity of the experimental chickens was observed within 21 days.

[0163] 3. Statistical analysis of the results of immune protection efficacy: The scoring criteria for the lesions of the footpad and joints were the same as those in Example 11. The results are as Figure 23 shown. The average lesion scores of the joints and footpads in each immunized group were lower than those in the challenge control group. One-way ANOVA was used to perform a significant analysis of the data. The differences in the lesion scores of the footpad and joints between the subunit vaccine immunized group and the challenge control group were extremely significant (P < 0.01). The differences in the lesion scores of the footpad and joints between the inactivated vaccine immunized group and the challenge control group were not significant. The difference in the footpad lesion score between the MS-H immunized group and the challenge control group was extremely significant (P < 0.01), and the difference in the joint lesion score between the MS-H immunized group and the challenge control group was significant (P < 0.05). The morbidity rates of each experimental group are shown in Table 9. The morbidity rates of footpad swelling and tarsal joint synovitis in the subunit vaccine immunized group were both 2 / 10. The morbidity rates of footpad swelling and tarsal joint synovitis in the MS-H live vaccine immunized group were both 3 / 10. The morbidity rate of footpad swelling in the inactivated vaccine immunized group was 5 / 10, and the morbidity rate of tarsal joint synovitis was 3 / 10.

[0164] Table 9 Morbidity rates of each group in the immune protection efficacy test of the subunit vaccine prepared from the multi-protein combination

[0165]

[0166] It can be seen that Figure 24 after immunization with this subunit vaccine, the degree of footpad lesions in chickens and the morbidity rate of tarsal joint synovitis can be significantly reduced, and it has a good immune protection effect.

Claims

1. Use of a recombinant chicken synoviae Mycoplasma antigen protein composition in the preparation of a medicament for treating or preventing chicken synoviae Mycoplasma infection, wherein the amino acid sequences of each protein in the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO:

38.

2. Use of a nucleic acid or gene encoding a recombinant chicken synoviae Mycoplasma antigen protein composition in the preparation of a medicament for treating or preventing chicken synoviae Mycoplasma infection, wherein the nucleotide sequences of the nucleic acids or genes of each protein of the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:

11.

3. Use of an expression cassette containing a nucleic acid or gene encoding a recombinant chicken synoviae Mycoplasma antigen protein composition in the preparation of a medicament for treating or preventing chicken synoviae Mycoplasma infection, wherein the nucleotide sequences of the nucleic acids or genes of each protein of the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:

11.

4. Use of a recombinant vector containing a nucleic acid or gene encoding a recombinant chicken synoviae Mycoplasma antigen protein composition in the preparation of a medicament for treating or preventing chicken synoviae Mycoplasma infection, wherein the nucleotide sequences of the nucleic acids or genes of each protein of the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:

11.

5. The use according to claim 4, characterized in that: The recombinant vector is obtained by inserting the nucleic acid or gene between the T7 terminator sequence and the lac operator sequence of the vector.

6. Use of a recombinant bacterium containing a nucleic acid or gene encoding a recombinant chicken synoviae Mycoplasma antigen protein composition in the preparation of a medicament for treating or preventing chicken synoviae Mycoplasma infection, wherein the nucleotide sequences of the nucleic acids or genes of each protein of the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:

11.

7. The use according to any one of claims 1 to 6, characterized in that: The medicament includes a polyclonal antibody or a vaccine.

8. A vaccine, characterized in that The vaccine comprises a recombinant chicken synoviae Mycoplasma antigen protein composition, and the amino acid sequences of each protein in the recombinant chicken synoviae Mycoplasma antigen protein composition are shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:

38.

9. The vaccine according to claim 8, characterized in that The vaccine also includes an adjuvant.

Images