Application of streptococcus suis virulence protein in pathogen detection and attenuated strain preparation

Abstract

The invention discloses application of streptococcus suis virulence protein in pathogen detection and attenuated strain preparation, and belongs to the technical field of biological products. The invention finds that the B9H0105210 protein is related to the pathogenicity of streptococcus suis and is a novel streptococcus suis virulence protein for the first time. The B9H0105210 protein in the streptococcus suis type 2 SC19 strain is inactivated or the coding gene of the B9H0105210 protein is deleted, so that the pathogenicity of the streptococcus suis type 2 SC19 strain can be obviously reduced, and the streptococcus suis type 2 SC19 strain can be used as an attenuated live vaccine for preventing and treating streptococcus suis infection. The polyclonal antibody prepared by taking the B9H0105210 protein as the antigen provides an important tool for researching the pathogenic mechanism of streptococcus suis. By utilizing the principle of antigen-antibody specific recognition, the method can sensitively, quickly and economically detect the illness condition of the livestock group, and is suitable for detecting a large number of samples.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and particularly relates to the application of a Streptococcus suis virulence protein in pathogen detection and preparation of attenuated strains. Background Art

[0002] Streptococcus suis is an important zoonotic pathogen, which can cause diseases such as septicemia, meningitis, and endocarditis in pigs; human infection with Streptococcus suis can cause streptococcal toxic shock syndrome, which can be fatal in severe cases. Streptococcus suis not only causes significant economic losses to the pig industry, but also poses a serious threat to public health safety.

[0003] Studying the virulence factors of Streptococcus suis and further exploring its pathogenic mechanism are the basis for preventing and controlling Streptococcus suis. When Streptococcus suis invades the host, it not only needs the host to provide various substances required for maintaining the life activities of the bacteria, but also has to cope with the pressure from the host immune system. In order to survive in the complex and changeable host environment, Streptococcus suis often regulates its multiple virulence genes in the most effective way, so as to achieve colonization, diffusion, escape, and long-term survival in the host. Currently reported factors related to the virulence of Streptococcus suis mainly include: capsular polysaccharide, hemolysin, muramidase-released protein, extracellular factor, secreted nuclease SsnA, adenosine synthase Ssads, C5a peptidase ScpA, IgA1 protease, and H factor-binding protein Fhb, etc. Therefore, deeply exploring new virulence proteins of Streptococcus suis not only helps to understand the pathogenicity of Streptococcus suis, but also provides potential targets for developing new prevention and control strategies. Summary of the Invention

[0004] Aiming at the above-mentioned existing technologies, the purpose of the present invention is to provide the application of a Streptococcus suis virulence protein in pathogen detection and preparation of attenuated strains. The present invention discovers through research that the B9H01_05210 protein is related to the pathogenicity of Streptococcus suis and is a new Streptococcus suis virulence protein; based on the B9H01_05210 protein, specific detection of Streptococcus suis pathogens and preparation of attenuated strains can be achieved.

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

[0006] In the first aspect of the present invention, there is provided the application of the B9H01_05210 protein as a Streptococcus suis virulence protein in the following (1) or (2):

[0007] (1) Preparation of attenuated Streptococcus suis strains;

[0008] (2) Preparation of a vaccine for preventing or treating Streptococcus suis infection;

[0009] The amino acid sequence of the B9H01_05210 protein is shown in SEQ ID NO.1; specifically as follows:

[0010] MIYLDNAATTALSPTALQRMMEVAQENYGNPSSIHQSGRKANQCLRQSRQEIAQILSASPEQIIFTSGGSEADTLAIQGYALAHQSKGKHLITTAIEHHAVLHTMQYLEDRFGFEVTYIQPVNQVITAQQILDALRPDTILVSVMFANNETGQLLPIKEIGELLADHQAVFHVDAVQAIGKIAISPSDYGIDLLAASAHKFHGPKGMGFLYSKVHKFDSLIHGGKQEQQHRAGTENLPAIAAMATALTEQYESLDTHHQHVSELRQHIIEGLSGLDYYLNQAGPHLPHVLNIGFLGKLNEQLLMQLDLAGIAVSSGSACTAGVVQNSHVLEAMYGKDSHRLKESIRISLSEINTKEEIDTLIIELKKILGG。

[0011] In the above application, by inactivating the B9H01_05210 protein in Streptococcus suis, the toxicity of Streptococcus suis can be reduced, and a Streptococcus suis attenuated strain can be prepared.

[0012] Furthermore, based on the prepared Streptococcus suis attenuated strain, it can be developed into a vaccine for preventing and treating Streptococcus suis infection.

[0013] As a preferred embodiment, the vaccine is an attenuated live vaccine.

[0014] In the second aspect of the present invention, there is provided the use of the coding gene of the B9H01_05210 protein as a virulence gene of Streptococcus suis in the following (1) or (2):

[0015] (1) Preparing a Streptococcus suis attenuated strain;

[0016] (2) Preparing a vaccine for preventing or treating Streptococcus suis infection;

[0017] The coding gene of the B9H01_05210 protein is a DNA molecule shown in the following i) or ii):

[0018] i) A DNA molecule whose nucleotide sequence is shown in SEQ ID NO.2;

[0019] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 other than i).

[0020] In the above application, genetic engineering means such as homologous recombination and CRISPR / Cas gene editing technology can be used to knockout the coding gene of B9H01_05210 protein in Streptococcus suis, so as to reduce the pathogenicity of Streptococcus suis, thereby constructing a Streptococcus suis attenuated strain.

[0021] In the third aspect of the present invention, there is provided the use of B9H01_05210 protein as an antigen in the following (1) or (2):

[0022] (1) Preparation of polyclonal antibodies;

[0023] (2) Preparation of products for detecting Streptococcus suis;

[0024] The amino acid sequence of the B9H01_05210 protein is shown in SEQ ID NO.1.

[0025] In the above application, animals can be immunized with B9H01_05210 protein as an antigen to prepare polyclonal antibodies; the prepared polyclonal antibodies can effectively detect Streptococcus suis immunogens. In the research and prevention and control of Streptococcus suis, the preparation of polyclonal antibodies is of great significance. Polyclonal antibodies can be used to develop rapid and sensitive immunological diagnostic methods, such as ELISA, immunofluorescence and immunochromatographic test strips, etc., to provide support for the early detection and typing of Streptococcus suis. In addition, polyclonal antibodies can also be used to study the antigenic characteristics, immune escape mechanisms and host immune responses of Streptococcus suis, providing a theoretical basis for vaccine design and immune strategies. At the same time, polyclonal antibodies can be used as candidate drugs for passive immunotherapy to directly neutralize pathogens or toxins, providing an emergency treatment method for patients or animals infected with Streptococcus suis. In vaccine research and development, polyclonal antibodies can also be used for vaccine potency evaluation and antigen screening to help optimize the vaccine formulation and improve the protective effect of the vaccine. In summary, the preparation of polyclonal antibodies against Streptococcus suis not only provides an important tool for the diagnosis and treatment of diseases, but also lays a solid foundation for in-depth research on the pathogenic mechanism and prevention and control strategies of Streptococcus suis.

[0026] In the fourth aspect of the present invention, there is provided a polyclonal antibody based on B9H01_05210 protein, and the polyclonal antibody is prepared by the following method:

[0027] Using the B9H01_05210 protein shown in SEQ ID NO.1 as an antigen to immunize animals, immunize 2-4 times to obtain immune serum; purify the immune serum to prepare polyclonal antibodies.

[0028] Preferably, the B9H01_05210 protein is prepared by the following method:

[0029] The coding gene of the B9H01_05210 protein was ligated with a prokaryotic expression vector to obtain a ligation product; the ligation product was transformed into Escherichia coli competent cells, and positive clones were screened to prepare a recombinant plasmid; the recombinant plasmid was transformed into BL21(DE3) competent cells to obtain positive monoclonal colonies; the positive monoclonal colonies were induced to culture and purified to prepare the B9H01_05210 protein.

[0030] More preferably, IPTG with a final concentration of 20 mM was added for induction culture; the temperature of induction culture was 35 °C, and the time of induction culture was 4 - 6 h.

[0031] More preferably, the purification was specifically as follows: the bacterial liquid after induction culture was collected, and the thalli were collected by centrifugation; the thalli were resuspended in a lysis buffer, and the thalli were broken to obtain a cell lysate; the cell lysate was subjected to affinity chromatography.

[0032] Preferably, the immunized animal was a New Zealand white rabbit.

[0033] In the fifth aspect of the present invention, there is provided the use of the above polyclonal antibody in the preparation of a product for detecting Streptococcus suis.

[0034] In the above use, the Streptococcus suis is Streptococcus suis serotype 2 strain SC19.

[0035] Using the polyclonal antibody prepared by the present invention, specific detection of Streptococcus suis serotype 2 strain SC19 can be achieved.

[0036] The beneficial effects of the present invention:

[0037] (1) The present invention for the first time studies and discovers that the B9H01_05210 protein is related to the pathogenicity of Streptococcus suis and is a new virulence protein of Streptococcus suis. Inactivating the B9H01_05210 protein in Streptococcus suis serotype 2 strain SC19 or deleting the coding gene of the B9H01_05210 protein can significantly reduce the pathogenicity of Streptococcus suis serotype 2 strain SC19 and can be used as a live attenuated vaccine for the prevention and treatment of Streptococcus suis infection.

[0038] (2) The polyclonal antibody prepared using the B9H01_05210 protein as an antigen provides an important tool for studying the pathogenic mechanism of Streptococcus suis. Based on the principle of specific recognition between antigen and antibody, it can sensitively, quickly, and economically detect the disease status of livestock herds, and is suitable for the detection of a large number of samples. This antibody can be detected by various serological methods such as protein hybridization and ELISA, and can also be used to make test strips through loading, with intuitive result judgment. By using this specific antibody, it can be clinically used for the rapid diagnosis of Streptococcus suis infection, helping doctors formulate treatment plans in a timely manner. In addition, polyclonal antibodies can also be used for passive immunotherapy. By injecting them into infected pigs, it helps their immune systems quickly recognize and eliminate pathogens, thereby alleviating symptoms and accelerating recovery. Through experiments such as immunoblotting and immunofluorescence staining using this antibody, researchers can deeply analyze the function and mechanism of action of the B9H01_05210 protein during the process of bacterial infection. In addition, this antibody can also be used to screen and identify host proteins and molecules related to Streptococcus suis infection, providing a theoretical basis for the development of new antibacterial strategies and vaccines. Through in-depth research on the B9H01_05210 protein and its antibody, scientific researchers can better understand the pathogenic process of Streptococcus suis and lay a foundation for future anti-infection research. Description of the Drawings

[0039] Figure 1 : Amplification results of the upstream and downstream homologous arms of the target gene B9H01_05210; among them, lane 1 is the upstream homologous arm; lane 2 is the downstream homologous arm.

[0040] Figure 2 : Identification results of the B9H01_05210 gene deletion recombinant plasmid pSET4s-ΔB9H01_05210.

[0041] Figure 3 : Identification results of the PCR-verified gene deletion strain SC19ΔB9H01_05210, where M 1 : Marker of DL5000; M 2 : Marker of DL2000; 1: SC19; 2 - 16: PCR identification of suspected strains to screen out the target strains; 6, 8, 11, 16: successfully constructed SC19ΔB9H01_05210.

[0042] Figure 4 : Results of the colony count determination of wild strain SC19 and gene deletion strain SC19ΔB9H01_05210 in mouse tissues and organs.

[0043] Figure 5 : Virulence experiment (LD 50)。

[0044] Figure 6 : Immunoprotective effect of the attenuated vaccine of the gene deletion strain SC19ΔB9H01_05210 against the infection of Streptococcus suis type 2 strain SC19 in mice.

[0045] Figure 7 : Immunoprotective effect of the attenuated vaccine of the gene deletion strain SC19ΔB9H01_05210 against the infection of Streptococcus suis type 2 strain P1 / 7 in mice.

[0046] Figure 8 : Agarose gel electrophoresis results of the cloning product of the coding gene of protein B9H01_05210; among them, lane 1 is the PCR amplification result.

[0047] Figure 9 : Double digestion identification results of the recombinant expression vector construction; among them, lane 1: plasmid digestion identification result; 2: plasmid empty vector control; 3: target gene amplification product control.

[0048] Figure 10 : Identification results of the transformation of BL21 Escherichia coli; among them, lanes 1-3: single colony cultures selected on the Amp-resistant plate.

[0049] Figure 11 : GST-B9H01_05210 protein purification process; among them, 1: supernatant of ultrasonicated bacterial solution; 2: flow-through solution; 3-7: PBS washing solution for impurities; 8-10: elution solution.

[0050] Figure 12 : Western Blot detection of protein purification results.

[0051] Figure 13 : Results of detecting the titer of polyclonal antibodies by ELISA method.

[0052] Figure 14 : Western blot identification results of polyclonal antibodies against immunogenic proteins; lane 1 is 1 μg of immunogenic protein, lane 2 is 5 μg of immunogenic protein, and lane 3 is 10 μg of immunogenic protein.

[0053] Figure 15 : Western blot identification results of polyclonal antibodies against the bacterial lysate of Streptococcus suis. Specific implementation manners

[0054] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0055] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below in combination with specific embodiments.

[0056] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specific conditions are carried out according to conventional test methods or the operation manuals recommended by the suppliers. Among them:

[0057] The GenBank accession number of the wild-type strain SC19 of Streptococcus suis type 2 is MNPY00000000.1. Streptococcus suis P1 / 7 is described in the literature "Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States." Veterinary Microbiology 156(3-4):290-296.

[0058] Example 1: Construction of a gene-deleted strain of Streptococcus suis type 2

[0059] 1. Construction of the recombinant vector pSET4sΔB9H01_05210 lacking the B9H01_05210 gene

[0060] Using the genome of the wild-type strain SC19 of Streptococcus suis type 2 as a template, the upstream and downstream homologous arm fragments on both sides of the B9H01_05210 gene (the nucleotide sequence is shown in SEQ ID NO.2) were amplified with the primers Up-B9H01_05210-F / Up-B9H01_05210-R and Down-B9H01_05210-F / down-B9H01_05210-R in Table 1 respectively, and the upstream and downstream homologous arms of the B9H01_05210 gene were ligated and recovered.

[0061] Table 1: Primers for constructing gene-deleted strains

[0062]

[0063] Reaction system: 2×Taq Master PCR Mix 25 μL, DNA template 1 μL, Forward primer (20 μM) 1 μL, Reverse primer (20 μM) 1 μL, ddH 2 O was supplemented to 50 μL.

[0064] Reaction procedure: 95°C for 5 min, 94°C for 1 min, 55°C for 30 s, 72°C for 1 min, 30 cycles, 72°C for 10 min. Recover and purify the PCR products according to the instructions of the PCR product purification kit from BIOMIGA. Take 2 μL for detection. The results are as Figure 1 shown, and the upstream and downstream homologous arms of the target length are successfully amplified.

[0065] Extract the pSET4s plasmid according to the instructions of the plasmid extraction kit. Perform double digestion on the pSET4s plasmid with EcoRI and BamHⅠ. Recover the double-digested product of pSET4s, and then ligate the amplified upstream and downstream homologous arm fragments of the B9H01_05210 gene into the double-digested pSET4s plasmid; Take 10 μL of the ligated product and add it to 100 μL of Escherichia coli competent cells, mix well, place on ice for 30 min, treat at 42°C for 90 s, and ice bath for 2 min. Add 900 μL of LB medium and incubate at 30°C for 10 min for resuscitation. Shake in a 30°C shaker for 90 min, take 100 μL of the bacterial solution and spread it evenly on a plate containing antibiotics, and culture overnight in a 30°C incubator. Pick several colonies on the plate for colony PCR amplification, and detect the products by gel electrophoresis. The electrophoresis results are as Figure 2 shown. At the same time, send the plasmid to Sangon Biotech for sequencing.

[0066] The above results show that: The recombinant vector pSET4sΔB9H01_05210 lacking the B9H01_05210 gene was successfully constructed in this invention.

[0067] 2. Construction of Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210)

[0068] Electrotransform the recombinant vector pSET4sΔB9H01_05210 into SC19 competent cells. The electrotransformation conditions are:

[0069] Voltage 2500V, resistance 500 ohms, time 5 ms. Immediately add THB medium after the electric shock, mix well and transfer to an EP tube; after preheating at 30 °C for 10 min, culture in a shaker at 37 °C for 2 - 3 h; take 100 μL of the cultured bacterial liquid and spread it on a THB plate containing 50 μg / mL spectinomycin, pick the grown single colonies and place them in THB liquid medium (containing 50 μg / mL spectinomycin), culture overnight at 37 °C; dilute the bacterial liquid and spread it on a THB solid medium (containing 50 μg / mL spectinomycin), culture at 37 °C for 12 h; select the grown single colonies, place them in THB liquid medium (without spectinomycin), culture overnight at 30 °C, transfer to a new THB liquid medium (without spectinomycin) at a ratio of 1:5 and continue to culture at 30 °C, transfer and culture 3 times; after continuous culture, dilute the bacterial liquid in gradient and spread it on a THB solid medium (without spectinomycin), culture overnight at 37 °C; pick the single colonies on the above solid medium and streak them on THB solid media containing 50 μg / mL spectinomycin and without spectinomycin respectively, culture at 37 °C for 12 h; select the colonies that do not grow on the THB solid medium containing 50 μg / mL spectinomycin but grow on the THB solid medium without spectinomycin, transfer them to a new THB liquid medium (without spectinomycin) and culture overnight at 37 °C for identification.

[0070] The primers used for PCR identification of the deletion strain are as follows:

[0071] B9H01_05210 - secondary exchange - F: TTCTAAATGCACCGATTGCG;

[0072] B9H01_05210 - secondary exchange - R: ATGGCTGCATCCTTATTCTCA.

[0073] Reaction system: 2×Taq Master PCR Mix 10 μL, DNA template 1 μL, forward primer (20 μM) 1 μL, reverse primer (20 μM) 1 μL, ddH 2 O 7 μL.

[0074] Reaction procedure: pre - denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 90 s, cycle 35 times, then extension at 72 °C for 10 min, cool at 16 °C.

[0075] The results of PCR identification are as Figure 3 shown. The results indicate that the fragment lengths of the suspected strains in lanes 6, 8, 11, and 16 are significantly shorter than that of the wild - type SC19 in lane 1, so they are the target - fragment deletion strains.

[0076] The strain with the deletion of the target fragment identified by PCR was subjected to genome resequencing (Sangon Biotech). The results of genome resequencing (Sangon Biotech) showed that the B9H01_05210 gene of the strain with the deletion of the target fragment identified by PCR was deleted.

[0077] The above results proved that the gene-deleted strain of Streptococcus suis type 2 (SC19ΔB9H01_05210) was successfully constructed in this invention.

[0078] Example 2: Colony counting experiment of the gene-deleted strain of Streptococcus suis type 2 (SC19ΔB9H01_05210)

[0079] The wild-type strain SC19 and the gene-deleted strain of Streptococcus suis type 2 (SC19ΔB9H01_05210) were respectively inoculated into THB liquid medium, cultured in a shaker at 37 °C and 220 rpm, transferred after 12 h, and used to challenge mice at a dose of 2.5×10 8 CFU. After 24 h, the tissues and organs of the mice were collected in a sterile environment and homogenized. Use a 100 μL micropipette to aspirate 100 μL of the 1:10 sample homogenate, slowly inject it along the tube wall into a sterile test tube containing 900 μL of diluent, and shake the test tube to mix the sample homogenate. Select the sample homogenate with an appropriate dilution. When performing a 10-fold serial dilution, aspirate 10 μL of the sample and drop it into the sterile THB solid medium. Each dilution was repeated 3 times. At the same time, 10 μL of the blank diluent was respectively aspirated as a blank control. After the diluent solidified, turn the solid medium over and culture it at 36 °C ± 1 °C for 48 h ± 2 h. After the culture was completed, take out the medium, observe it with the naked eye, record the dilution factor and the corresponding number of colonies, and the colony count was expressed in colony-forming units (CFU).

[0080] The results were as Figure 4 shown. The bacterial colonization amount of the gene-deleted strain SC19ΔB9H01_05210 in tissues and organs was significantly lower than that of the wild strain SC19.

[0081] Example 3: Virulence experiment of the gene-deleted strain of Streptococcus suis type 2 (SC19ΔB9H01_05210) (LD 50 )

[0082] Fifty female mice aged 18 - 20 g with basically the same physiological status were randomly divided into 10 groups, with 5 mice in each group. Among them: 5 groups were respectively intraperitoneally injected with the wild strain SC19 at doses of 5×10 8 , 2.5×10 8 , 1×10 8 , 5×10 7 , 2.5×10 7 CFU; the other 5 groups were respectively intraperitoneally injected with doses of 5×108 , 2.5×10 8 , 1×10 8 , 5×10 7 , 2.5×10 7 CFU of the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210).

[0083] After inoculation, observe for 5 days, record the status of the mice every 8 hours, record the number of dead and surviving mice within 5 days, and calculate the LD 50 .

[0084] The results are as Figure 5 shown. The results show that: the LD 50 of the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210) is 1×10 8 CFU, and the LD 50 of the wild-type SC19 is 2×10 7 CFU, indicating that its virulence is lower than that of the wild-type SC19.

[0085] Example 4: Immunoprotective experiment of the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210)

[0086] Randomly divide 20 female mice weighing 18 - 20 g into 4 groups, with 5 mice in each group; among them: groups 1 - 2 serve as the immunization groups and are injected intramuscularly with a dose of 2.5×10 8 CFU of the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210); groups 3 - 4 serve as the PBS control groups and are injected intramuscularly with an equal volume of PBS.

[0087] 14 days after immunization, challenge group 1 of the immunization groups and group 3 of the PBS control groups with an equal dose (5×10 8 CFU) of the wild-type Streptococcus suis strain SC19; challenge group 2 of the immunization groups and group 4 of the PBS control groups with an equal dose (5×10 8 CFU) of the Streptococcus suis P1 / 7.

[0088] Observe for 7 days after challenge, record the status of the mice every day, count the number of surviving mice, and calculate the survival rate of the mice.

[0089] The results are as Figures 6-7As shown, the survival rate of mice immunized with the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210) was significantly increased under the challenge of P1 / 7 and SC19, indicating that the Streptococcus suis type 2 gene deletion strain (SC19ΔB9H01_05210) has protective and cross-immunization effects.

[0090] Example 5: Preparation of B9H01_05210 Protein

[0091] 1. Amplification of target gene fragment:

[0092] Using the genome of the Streptococcus suis type 2 wild-type strain SC19 as a template, the B9H01_05210 gene fragment with the nucleotide sequence shown in SEQ ID NO.2 was amplified by PCR.

[0093] Set the PCR reaction program: pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, for a total of 30 cycles; finally, final extension at 72°C for 10 minutes.

[0094] Detection of PCR products: Weigh 0.8g of agarose, add 100mL of 1×TAE buffer, heat and dissolve, cool to 50 - 60°C, add 5μL of nucleic acid dye and mix well, pour into the gel mold and insert the comb, and wait for the gel to solidify. Take 5μL of PCR product and mix it with 1μL of 6× loading buffer for loading, and at the same time add DNA molecular weight standard. Electrophoresis was carried out at 120V for 30 - 40 minutes in 1×TAE buffer, observed and photographed under the gel imaging system to confirm whether the target gene fragment was amplified.

[0095] The results are as Figure 8 shown, and the gene fragment of the target length was amplified.

[0096] 2. Construction of recombinant plasmid:

[0097] A pair of restriction endonucleases (BamH I and Xho I) were used to double-digest the pGEX-4T-1 vector and the amplified target gene fragment. The target gene fragment after digestion was ligated to the linearized vector using T4 DNA ligase. The ligation reaction system includes: linearized vector: 60ng; PCR fragment: 180ng; T4 DNA ligase: 1μL; 10× ligation buffer: 2μL; sterile distilled water: made up to 20μL; incubated at 12°C for 2 hours to ensure the ligation efficiency.

[0098] Transform the ligation product into DH5α competent cells. Spread the transformed bacterial solution on an LB agar plate containing Ampicillin (50 mg / L) and incubate it upside down at 37 °C for 15 hours. Pick a single colony, extract the plasmid and perform double digestion to verify whether the correct target fragment is inserted into the recombinant plasmid.

[0099] The results are as Figure 9 shown, and the correct target fragment is inserted into the recombinant plasmid.

[0100] Extract the plasmid from the positive clone and send it to a sequencing company for sequencing verification. The sequence of the inserted fragment is as shown in SEQ ID NO.2, with the correct sequence and correct orientation. That is, the recombinant plasmid is successfully constructed.

[0101] 3. Protein expression and purification:

[0102] Transform the constructed recombinant plasmid into BL21(DE3) competent cells. Spread the transformed bacterial solution on an LB agar plate containing Ampicillin. Incubate it upside down at 37 °C for 15 hours until the colonies are clearly visible. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing the resistance. Culture it on a shaker at 37 °C and 200 rpm for 8 hours. Extract plasmid DNA and perform colony PCR identification: Take 6 μL of the bacterial solution and perform PCR amplification using specific primers, and detect by electrophoresis whether the target fragment is amplified.

[0103] The results are as Figure 10 shown, and the target fragment is amplified.

[0104] Pick the positive clone containing the recombinant plasmid and inoculate it into 10 mL of LB liquid medium containing Ampicillin resistance. Culture it on a shaker at 37 °C and 200 rpm until OD600 ≈ 0.6 - 0.8. At this time, the bacteria are in the logarithmic growth phase and are suitable for expression. Add 20 mM IPTG (isopropyl-β-D-thiogalactoside) to the medium to induce the gene expression driven by the T7 promoter; induction conditions: 35 °C, 180 rpm, induce for 5 h.

[0105] Collect 200 ml of the induced bacterial solution, centrifuge and concentrate the bacteria, resuspend the bacterial pellet in 15 mL of lysis buffer (PBS), and lyse the bacteria using an ultrasonic crusher or French Press on ice. Incubate the GST affinity agarose beads with the bacterial lysate at 4 °C for 2 hours, then add the mixture to the chromatography column. After waiting for the flow-through to flow out, add 10 column volumes of PBS for washing impurities. After three times, add the elution buffer to elute the protein from the agarose beads, and collect the purified protein solution. Monitor the protein purification process by SDS PAGE; detect the purified protein by Western Blot.

[0106] The results are as Figures 11-12 shown, and the results indicate that the B9H01_05210 protein was successfully prepared by the present invention.

[0107] Example 6: Preparation of Polyclonal Antibody Based on B9H01_05210 Protein

[0108] 1. Preparation of polyclonal antibody:

[0109] Select healthy, adult New Zealand white rabbits, weighing about 2.5 kg. Conduct a preliminary health check on the animals to ensure no diseases and infections. After a one-week stable adaptation, collect 2 ml of blood from the marginal ear vein, separate the serum and store it in a -80°C refrigerator as negative serum.

[0110] Use the B9H01_05210 protein prepared in Example 5 as an antigen to immunize the above-mentioned New Zealand white rabbits. At the first immunization, mix the B9H01_05210 protein with an equal volume of complete Freund's adjuvant and emulsify it into an emulsion; for subsequent booster immunizations, use IFA to mix with the antigen. The immunization protocol is as follows:

[0111] (1) First immunization: Immunize the rabbits by subcutaneous multi-point injection with the prepared antigen-adjuvant emulsion. The total dose of protein for each rabbit is 800 μg, and it is injected at 4 sites.

[0112] (2) Booster immunization: After the first immunization, conduct a booster immunization once every week. Each time, mix 400 μg of protein with IFA and inject it in the same way as the first time.

[0113] Determine the number and interval of booster immunizations according to the monitoring results of antibody titers. In this experiment, 1 booster immunization was carried out.

[0114] (3) Final immunization and blood collection:

[0115] At week after the last booster immunization, conduct the final immunization by injecting the rabbits with a mixture of 400 μg of protein and IFA. Seven days after the final immunization, collect blood from the rabbit heart to obtain high-titer antiserum, which is the polyclonal antibody.

[0116] 2. Detection of polyclonal antibody titer:

[0117] (1) Serum separation: Place the collected blood in a sterile anticoagulant tube and let it stand for 2 hours to allow the blood to clot. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and collect the supernatant as serum. Transfer the serum to a new sterile centrifuge tube and store it at -20°C or -80°C for later use.

[0118] (2) Coating of ELISA plate: Select a flat bottom 96-well ELISA plate, dilute the B9H01_05210 protein to 200 μg / mL with carbonate buffer (pH 9.6), add 100 μL to each well, and coat overnight at 4°C.

[0119] (3) Blocking non-specific binding sites: Wash the ELISA plate three times with PBST (PBS containing 0.05% Tween-20). Add 5% skim milk powder, 100 μL to each well, and block at 37°C for 2 hours.

[0120] (4) Adding the serum sample to be tested: Remove the blocking solution and wash three times. Dilute the serum sample in a series of gradients (such as 1:1000, 1:2000, 1:4000, etc.), add 100 μL of the diluted serum to each well, and incubate at 37°C for 2 hours.

[0121] (5) Adding the secondary antibody and developing color: Wash three times, add the horseradish peroxidase (HRP)-labeled anti-rabbit IgG secondary antibody, dilute to an appropriate concentration, 100 μL to each well, and incubate at 37°C for 1 hour. After washing again, add the TMB substrate solution and develop color in the dark for 15 minutes. Add the stop solution (1M H 2 SO 4 ), and terminate the reaction.

[0122] (6) OD value measurement: Use an enzyme-linked immunosorbent assay reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0123] (7) Titer calculation: Calculate the titer of the antibody according to the OD value, usually determined by the relationship between the sample dilution and the OD value. Select the highest dilution with an OD value greater than twice the OD value of the negative serum as the antibody titer.

[0124] The results are as Figure 13 shown, and the titer of the polyclonal antibody serum is approximately 1:16000.

[0125] Example 7: Identification of antigen by Western blot of polyclonal antibody

[0126] 1. Western blot identification of the immunogenic protein by polyclonal antibody:

[0127] (1) SDS-PAGE electrophoresis: Prepare a 12% SDS-PAGE gel, mix the protein sample with the loading buffer, and boil at 100°C for 5 minutes. Load the sample, add protein samples containing 1 μg, 5 μg, and 10 μg of B9H01_05210 protein to three wells in sequence, and add a prestained protein Marker at the same time. Electrophoresis conditions: Constant voltage electrophoresis at 80 V until the sample enters the separating gel, and then constant voltage electrophoresis at 120 V until the bromophenol blue reaches the bottom of the gel.

[0128] (2) Membrane transfer: Transfer the proteins in the gel onto a PVDF membrane or nitrocellulose membrane using the wet transfer method or semi-dry transfer method. Membrane transfer conditions: Semi-dry transfer, 20 v, 30 minutes.

[0129] (3) Blocking: Place the membrane in 5% non-fat milk powder and block at room temperature for 1 hour to block non-specific binding.

[0130] (4) Primary antibody incubation: Incubate the blocked membrane with the polyclonal antibody prepared in Example 6 (1:1000) at room temperature for 2 hours. Wash three times with PBST (PBS containing 0.05% Tween-20), 5 minutes each time.

[0131] (5) Secondary antibody incubation: Add HRP-labeled anti-rabbit IgG secondary antibody (1:5000 - 1:10000) and incubate at room temperature for 1 hour. Wash three times with PBST, 5 minutes each time.

[0132] (6) Color development: Use an ECL chemiluminescence kit, mix the membrane with the substrate, and react in the dark for 1 - 2 minutes. Detect the signal using a chemiluminescence imaging system and record the results.

[0133] The results are as Figure 14 shown. By comparing with the prestained Marker, confirm that the molecular weight is correct. Specific bands are shown and there are no non-specific bands.

[0134] 2. Western blot identification of polyclonal antibody against Streptococcus suis bacterial lysate:

[0135] (1) Strain culture and protein expression: Inoculate strain SC19 and the Streptococcus suis serotype 2 gene deletion strain (SC19ΔB9H01_05210) constructed in Example 1 into LB liquid medium and culture in a shaker at 37 °C and 200 rpm until OD600 ≈ 0.6 - 0.8.

[0136] (2) Bacterial cell collection and lysis: Centrifuge to collect the bacterial cells, resuspend them in PBS buffer, and add proteinase k. Use an ultrasonic cell disruptor to lyse the bacterial cells, and after complete lysis, centrifuge to take the supernatant.

[0137] (3) Electrophoresis: Load the supernatant and precipitate onto a 12% SDS-PAGE gel, 10 μg per well. Electrophoresis conditions: 80 V to the separating gel, then 120 V until the bromophenol blue reaches the bottom.

[0138] (4) Transfer the proteins in the gel to a PVDF membrane using the semi-dry transfer method at 15 V for 30 minutes. Blocking and antibody incubation. Block the membrane with 5% skim milk powder for 1 hour at room temperature. Wash three times with PBST, 5 minutes each time. Add the diluted polyclonal antibody (1:2000 - 1:10000) and incubate overnight at 4°C. After washing three times, add the HRP-labeled secondary antibody (1:5000 - 1:10000) and incubate for 1 hour at room temperature. Wash three times again.

[0139] (5) Development and detection: Use ECL reagent for development and expose to film or chemiluminescence imaging system.

[0140] The results are as Figure 15 shown. This polyclonal antibody has specific binding to the B9H01_05210 protein in the bacterial lysate, showing specific bands.

[0141] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of B9H01_05210 protein as a virulence protein of Streptococcus suis in the following (1) or (2): (1) preparing an attenuated strain of Streptococcus suis; (2) Preparation of vaccines for preventing or treating Streptococcus suis infection; The amino acid sequence of the B9H01_05210 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The vaccine is a live attenuated vaccine.

3. Use of the gene encoding the B9H01_05210 protein as a virulence gene of Streptococcus suis in the following (1) or (2): (1) preparing an attenuated strain of Streptococcus suis; (2) Preparation of vaccines for preventing or treating Streptococcus suis infection.

4. The use according to claim 3, characterized in that: The coding gene of the B9H01_05210 protein is a DNA molecule as shown in i) or ii) below: i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 except i).

5. Use of B9H01_05210 protein as an antigen in the following (1) or (2): (1) preparing polyclonal antibodies; (2) preparing products for detecting Streptococcus suis; The amino acid sequence of the B9H01_05210 protein is shown in SEQ ID NO.

1.

6. A polyclonal antibody based on B9H01_05210 protein, characterized in that: The polyclonal antibody is prepared by the following method: The B9H01_05210 protein shown in SEQ ID NO.1 is used as an antigen to immunize animals for 2-4 times to obtain immune serum; the immune serum is purified to prepare polyclonal antibodies.

7. The polyclonal antibody according to claim 6, characterized in that The B9H01_05210 protein was prepared by the following method: The coding gene of the B9H01_05210 protein and the prokaryotic expression vector are connected to obtain a connection product; the connection product is transformed into Escherichia coli competent cells, positive clones are screened, and a recombinant plasmid is prepared; the recombinant plasmid is transformed into BL21 (DE3) competent cells to obtain positive monoclonal colonies; the positive monoclonal colonies are induced and cultured, and purified to prepare the B9H01_05210 protein.

8. The polyclonal antibody according to claim 7, characterized in that IPTG was added at a final concentration of 20 mM for induction culture; the induction culture temperature was 35° C., and the induction culture time was 4-6 h.

9. The polyclonal antibody according to claim 7, characterized in that The purification specifically comprises: collecting the bacterial solution after induction culture, collecting the bacterial bodies by centrifugation; resuspending the bacterial bodies in a lysis buffer, breaking the bacterial bodies to obtain a cell lysate; and subjecting the cell lysate to affinity chromatography.

10. Use of the polyclonal antibody according to any one of claims 6 to 9 in the preparation of a product for detecting Streptococcus suis.

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