CN113754744A

CN113754744A - Mycoplasma bovis protein SBP-2 and application thereof

Info

Publication number: CN113754744A
Application number: CN202111109156.XA
Authority: CN
Inventors: 郭亚男; 王建东; 谢建亮; 何生虎; 梁小军; 李勇
Original assignee: Institute Of Animal Science Ningxia Academy Of Agricultural And Forestry Sciences
Current assignee: Institute Of Animal Science Ningxia Academy Of Agricultural And Forestry Sciences
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2021-12-07
Anticipated expiration: 2041-09-22
Also published as: CN113754744B

Abstract

The application provides a mycoplasma bovis protein and application thereof. The application provides mycoplasma bovis protein (SBP-2) in mycoplasma bovis lipid membrane protein family, a gene for coding the protein is P48 gene, and a nucleotide sequence of the gene is shown as a sequence table in SEQ ID NO. 1. The amino acid sequence of the mycoplasma bovis protein SBP-2 is shown in SEQ ID NO. 2. The method adopts a prokaryotic expression technology to construct pET-28a-P48 recombinant plasmid and introduce BL21 escherichia coli, then screens IPTG high-efficiency expression conditions, and utilizes a nickel column affinity chromatography technology to purify SBP-2 protein. The result shows that SBP-2 is soluble membrane protein and the protein size is 51 KDa. Western blot results show that the prokaryotic expression SBP-2 protein has good reactogenicity. The application provides a mycoplasma bovis vaccine which can stimulate calves to generate obvious specific immune response and achieve high antibody level.

Description

Mycoplasma bovis protein SBP-2 and application thereof

Technical Field

The application relates to the technical field of recombinant proteins, in particular to mycoplasma bovis SBP-2 and application thereof.

Background

Mycoplasma bovis (Mycoplasma bovis) is a member of the class mollicutes, the order Mycoplasmatales, the family Mycoplasmataceae, and the genus Mycoplasma, and is one of the important pathogenic pathogens of cattle. Mycoplasma bovis has a simple structure, no cell wall, no cell nucleus, only ribose, and can pass through a 0.22 μm filter membrane. The gene sequence of Mycoplasma bovis is about 1080kbp, has a low G + C content, and is about 27.8% -32.9%. The mechanisms of virulence factors and pathogenicity of mycoplasma bovis remain largely unknown, but the high frequency altered expression capacity of the mycoplasma bovis membrane surface protein (Vsp) family plays an important role in mycoplasma bovis pathogenicity. In the mycoplasma bovis genome, a plurality of membrane protein genes (at least 13 genes encode genes capable of expressing variable surface lipoproteins) with an adhesion function can be expressed. Several membrane proteins (Vsp) have been identified in bovine mycoplasma, such as VspA, VspB, VspC, VspF, VspO and VspL. These genes operate through various mechanisms, including DNA transposition and intrachromosomal recombination within the Vsp locus, altering the antigenic properties of their surface components, can act to enhance colonization, attachment and evasion of the host immune defense system, and are highly immunogenic and capable of inducing mucosal immunity.

The protein expressed by the mycoplasma bovis P48 gene is a conserved protein, has specificity, and is one of membrane proteins of the main immunogenicity of the mycoplasma bovis. The protein is a main protein for causing immune regulation after mycoplasma bovis infection, and has close relation with the pathogenic mechanism of mycoplasma bovis infection. Antibodies to this protein have also been found in animals tested for infection with or naturally infected with mycoplasma bovis pathogens. However, no commercial vaccine exists in China, the immune effect of the inactivated vaccine is not obvious at home, and no membrane protein vaccine can be used for preventing the infection of mycoplasma bovis pathogen.

Disclosure of Invention

The application provides mycoplasma bovis SBP-2 and application thereof, which are used for solving the problems that no commercial vaccine exists at home, the immune effect of foreign inactivated vaccine is not obvious, and no membrane protein vaccine can be used for preventing infection of mycoplasma bovis pathogen.

In a first aspect, the application provides mycoplasma bovis protein SBP-2, wherein a gene for coding the mycoplasma bovis protein is a P48 gene, and a nucleotide sequence of the mycoplasma bovis protein is shown as a sequence table in SEQ ID NO 1; wherein the nucleotide is obtained by optimizing TGA base expressing tryptophan in Mycoplasma bovis P48 gene to TGG.

Preferably, the amino acid sequence of the mycoplasma bovis protein SBP-2 is shown in SEQ ID NO. 2.

In a second aspect, the present application provides a recombinant plasmid, which is the pET-28a-P48 plasmid comprising the Mycoplasma bovis protein SBP-2 described above.

In a third aspect, the present application provides an escherichia coli comprising the above recombinant plasmid, classified under the names: escherichia coli (Escherichia coli) preserved in China general microbiological culture Collection center (CGMCC) with the preservation date of 2021, 8 months and 9 days and the preservation number of CGMCC NO: 23133.

In a fourth aspect, the application provides the use of the mycoplasma bovis protein SBP-2 in the manufacture of a medicament for preventing a disease caused by mycoplasma bovis.

In a fifth aspect, the application provides an application of mycoplasma bovis protein SBP-2 in preparing mycoplasma bovis vaccines.

In a sixth aspect, the present application provides a mycoplasma bovis vaccine, comprising the SBP-2 protein and an immunological adjuvant.

The application provides a mycoplasma bovis protein and application thereof. The application provides mycoplasma bovis SBP-2, a gene for coding the protein is P48 gene, and the nucleotide sequence of the protein is shown as SEQ ID NO. 1 in a sequence table. The amino acid sequence of the mycoplasma bovis SBP-2 is shown in SEQ ID NO 2. The method adopts a prokaryotic expression technology to construct pET-28a-P48 recombinant plasmid and introduce BL21 escherichia coli, then screens IPTG high-efficiency expression conditions, and utilizes a nickel column affinity chromatography technology to purify SBP-2 protein. The result shows that SBP-2 is soluble membrane protein and the protein size is 51 KDa. Western blot results show that the prokaryotic expression SBP-2 protein has good reactogenicity.

The application provides an application of the SBP-2 protein in preparation of mycoplasma bovis vaccines. The application provides a mycoplasma bovis vaccine, which comprises the SBP-2 protein and an immunologic adjuvant. In order to investigate whether the SBP-2 recombinant protein vaccine plays a role in preventing mycoplasma bovis diseases, the SBP-2 recombinant protein vaccine is prepared, mycoplasma bovis inactivated vaccines are used as a reference, then calf immunity test research is carried out, infection test is carried out on the 15 th day after immunity is finished, and pathological histological observation is carried out 28 days after infection. As a result, it was found that: the antibody level of the NC group calf is always kept in a stable negative state; after the protein vaccine is injected for one week, the antibody level of the protein vaccine group calf is increased to be positive, and then a stable positive state is maintained; after the inactivated vaccine group is immunized for 1 time, the antibody level of the mycoplasma bovis begins to rise, can reach positive on day 15, reaches the maximum value at week 3, and then is stable. The IgA content of the protein vaccine group at week 2 after the first immunization was significantly different from that of the NC group (P < 0.05). IgM levels were very significantly different from NC groups at week 2 after the first immunization and at week 1 after infection (P < 0.01). IgG levels were very significantly different from NC groups 1 week after the first immunization and 1 week after infection (P < 0.01). Pathological dissection of calves shows that: the lung of the protein vaccine group has slight pathological changes; the PC group cattle have serious pathological changes; no pathological changes can be seen by naked eyes in the inactivated vaccine group and the NC group. The SBP-2 protein vaccine can stimulate calves to generate obvious specific immune response and reach a high antibody level; the SBP-2 protein vaccine stimulates the humoral immune system of calves, and further shows that the SBP-2 protein as the conserved surface lipoprotein of mycoplasma bovis can generate antibodies aiming at the mycoplasma bovis membrane protein when used as a vaccine for animal immunization and can reduce the invasion of mycoplasma bovis pathogeny to a certain extent.

Drawings

In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram of a pET-28a (+) plasmid provided in the examples herein;

FIG. 2 is a map of the plasmid pET-28 a-P48;

FIG. 3 is the induced expression diagram under the conditions of 0.6mmol/L and 0.8mmol/L in the present application example;

FIG. 4 is the induction expression diagram under the conditions of 1.0mmol/L and 1.2mmol/L in the present application example;

FIG. 5 is a graph showing the results of protein purification in examples of the present application;

FIG. 6 is a diagram showing the result of Western blot in the example of the present application;

FIG. 7 is a graph of the body temperature changes before and after immunization and infection of calves in the examples of the present application;

FIG. 8 is a graph showing the change in specific antibodies in examples of the present application;

FIG. 9 is a graph showing the results of a pathological anatomy examination of a protein vaccine group in the examples of the present application; a, pleural effusion; substantial flesh change of lung; c normal kidney; normal liver; e, congestion of lung and parenchymal flesh change; f, normal larynx; g normal trachea;

FIG. 10 is a diagram of pathological anatomy of a PC group according to an embodiment of the present application; a, lung tissue adhesion; b, bleeding and congestion of the lung; c, the lung tissues generate offwhite cheese-like or purulent necrotic lesions with substantial flesh change and different sizes; d, bleeding inside lung tissues and having suppurative and caseous necrotic foci;

FIG. 11 is a graph showing the results of a pathological anatomy examination of an inactivated vaccine group in the example of the present application;

FIG. 12 is a sectional view of tracheal tissue of a protein vaccine group in an example of the present application; a, the structure of the trachea is normal; b, the hyaline cartilage structure is normal;

FIG. 13 is a lung tissue section of a protein vaccine group according to an embodiment of the present application; bronchial epithelium hyperplasia, inflammatory exudate and cellulitis nodules 100 ×; emphysema-alveolar compensatory dilation: alveoli expand, alveolar walls become thin, and some alveoli have been broken and combined into large sacs 100 ×; c bronchial epithelium was severely hyperplastic 100 ×; d terminal bronchial bleeding with inflammatory exudate 400 ×; e, emphysema: alveolar dilatation 400 ×; f, peeling off epithelial cells of the alveolar septa, and breaking the alveolar septa by 400 x; g, alveolar collapse and thickening by 400 times; h substantial flesh change and bleeding;

FIG. 14 is a diagram of the lungs of a PC group according to an embodiment of the present application;

FIG. 15 is a diagram of the lungs of a PC group according to an embodiment of the present invention;

FIG. 16 is a sectional view of an inactivated vaccine group airway tissue according to an embodiment of the present invention;

FIG. 17 is a sectional view of a lung tissue of an inactivated vaccine group according to an example of the present application;

FIG. 18 is a graph showing the variation of IgA, IgM and IgG contents in examples of the present application.

Detailed Description

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.

In a third aspect, the application provides an escherichia coli, which contains the recombinant plasmid, and is preserved in China general microbiological culture collection center with the preservation date of 2021, 8 and 9 days and the preservation number of CGMCC NO: 23133.

The following examples are provided to illustrate the acquisition of the Mycoplasma bovis SBP-2 protein and the immunization of SBP-2 protein vaccines.

Examples

S1, construction, expression and purification of mycoplasma bovis P48 gene engineering recombinant SBP-2 protein

Construction of S11 and P48 gene cloning plasmid

DNA and amino acid sequence analysis was performed by BioEdit software, and primer design was performed by Primer5.0 software.

S111, calling P48 gene and extracting target gene

Synthesizing a primer sequence of the P48 protein gene, carrying out PCR amplification for 35 cycles (wherein, a PCR amplification system is shown in a table 1-1), then sucking 5 mu L of an amplification product, directly carrying out agarose gel electrophoresis, and observing the reaction.

TABLE 1-1 PCR amplification System

The amplified target gene was subjected to agarose gel electrophoresis and the target gene was recovered.

S112, enzyme digestion

The objective gene was amplified by PCR using the EcolI and BamHI restriction enzymes and plasmid double digestion was carried out using pET-28a (+) cleavage sites at position 192 and position 198 as shown in FIG. 1.

50 mu L of plasmid digestion system (wherein, the plasmid digestion system is shown in the table 1-2) is prepared in a centrifuge tube, and the plasmid digestion is completed at 37 ℃ for 0.5 h.

TABLE 1-2 plasmid cleavage System

After plasmid digestion, 20. mu.L of a target DNA digestion system (wherein the target DNA digestion system is shown in tables 1-3) is prepared in a centrifuge tube, and double digestion is completed at 37 ℃ for 0.5 h.

TABLE 1-3 DNA double restriction enzyme System for purposes

S113, recovery of DNA fragment

And (3) recovering the Kit and the DNA fragment after completing double enzyme digestion, wherein the steps are as follows:

the gel containing the P48 target gene is cut off, put into a centrifuge tube, added with the S1 sol solution, heated in a water bath for dissolution (about 10-15 minutes), and mixed well. Then, one third volume of isopropanol was added, mixed well and water-bathed for 1 minute.

Adding the product into a nucleic acid adsorption column, centrifuging at 2000rpm for 1min, and discarding the liquid in the centrifuge tube.

Add 450. mu.L of washing solution to the adsorption column, after standing for 1min, centrifuge at 12000rpm for 30 s. The upper separation column is left, and the liquid in the centrifuge tube is discarded.

And (5) repeatedly cleaning.

Centrifuge at 2000rpm for 1min, and then leave the upper column and place in a new centrifuge tube. Add 30. mu.L of eluent, wait for 1-3 minutes, and centrifuge to collect the liquid (1min, 12000 rpm).

The liquid in the centrifuge tube is P48 DNA. The PCR product is amplified to obtain a product with the fragment size of about 1400, which is consistent with the base sequence number of the P48 protein gene.

After recovering linearized plasmid DNA by ethanol precipitation, the DNA content was determined by OD30, and recombinant plasmid was constructed by T4 DNA ligase at a relative concentration ratio of 1:3 between the vector and the target fragment.

10 mu L of recombinant DNA construction system (wherein, the recombinant DNA construction reaction system is shown in tables 1-4) is prepared in a 0.5mL centrifuge tube, and then the pET-28a-P48 recombinant plasmid is obtained after 12 hours of connection at 14-16 ℃.

TABLE 1-4 construction of the reaction System for recombinant DNA

S114, ligation reaction

Coli competent cells, comprising the steps of:

the ligation product 5. mu.L and competent cell 40. mu.L were mixed well and ice-cooled for 30 min.

Heat shock at 42 ℃ for 90s and rapid ice bath for 120 s.

400. mu.L of LB liquid medium (containing kanamycin and Kan) was added thereto, and shaking culture was carried out at 37 ℃ for 50 min.

Spreading 200 μ L of culture solution, and culturing at 37 deg.C for 10-14 hr.

S115, transformed cell and culture

Coli competent cells transformed with plasmid DNA comprising the following steps:

100 μ L of competent cells were placed on ice, 10 μ L of ligation product was added after lysis, and ice-cooled for 30 min.

Heat shock at 42 ℃ for 90s and ice bath for 120 s.

Adding 800 μ L LB culture medium (without antibiotics), shaking at 37 deg.C and 180r/min for 40-50min, and recovering cells.

Centrifuging the resuscitative bacteria liquid at 8000r/min for 2min at normal temperature.

The supernatant was aspirated at 700. mu.L and the cells were blown into a cell suspension.

100 mu L of cell suspension is taken and coated on an LB plate, the plate is rightly placed for about 30min until liquid is absorbed, then the plate is inverted, and the culture is carried out for 12h-16h at 37 ℃ until bacterial colonies appear.

Resistant selection plates were cultured and the transformed strains were inoculated on resistant solid medium. The culture was carried out at 37 ℃ for 24 hours by inversion. And (4) observing whether colonies appear, and if the colonies appear, selecting a single colony for amplification culture.

The liquid culture comprises the following steps:

when colonies are on the resistant selection plate, single colonies are selected and added to 15mL of liquid LB medium to which resistance is added.

Sealing, and shake culturing at 37 deg.C for 12 hr.

And (5) observing whether the bacterial growth state appears or not, and if the liquid is turbid, indicating that the Escherichia coli starts to grow normally.

S116, enzyme digestion identification

And (4) carrying out small-amount plasmid extraction on the escherichia coli successfully cultured in the final liquid culture, and carrying out corresponding enzyme digestion for identification.

Preparing 20 μ L enzyme digestion system (wherein, the recombinant plasmid enzyme digestion system is shown in table 1-5) in a 0.5mL centrifuge tube, reacting for 30min at 37 ℃, and then judging whether the enzyme digestion is correct through agarose electrophoresis so as to judge whether the cultured escherichia coli contains specific plasmids. As can be seen from FIG. 2, the plasmid extracted after amplification in liquid medium contains two gene fragments after enzyme digestion, the largest fragment is equal to the size of the enzyme-digested empty plasmid above 4500bp, and the small fragment is equal to the target gene fragment of P48 protein around 1500 bp. Wherein 1 and 2 in FIG. 2 represent electrophoresis results after double digestion of the recombinant plasmid pET-28 a-P48; 3. 4 represents the result of electrophoresis after double digestion of empty plasmid pET-28 a.

TABLE 1-5 recombinant plasmid restriction systems

S12, expression, purification and identification of recombinant protein in genetic engineering

S121, small expression:

the E.coli transformed with the expression plasmid was added to 2mL of LB medium supplemented with Kan, and cultured overnight at 37 ℃ with shaking in an incubator.

Subsequently, 200. mu.L of the overnight-cultured bacteria were aspirated, added to 2mL of LB medium without resistance, and cultured in an incubator at 37 ℃ for about 2 hours with shaking, and the OD600 value was measured to approximately 0.8.

IPTG (final concentration of 0.5mmol/L) was added for induction, and the mixture was cultured in an incubator at 37 ℃ for 4 hours with shaking.

1mL of the bacterial solution was collected and centrifuged to obtain cells.

Using 50 mu L H₂The cells were suspended in O, and 50. mu.L of 2X protein electrophoresis buffer was added thereto, followed by boiling for 2 minutes. Followed by protein SDS-PAGE electrophoresis to identify whether protein was expressed. As can be seen from fig. 3 and 4: after IPTG is added, the final concentration of the bacterial liquid is 0.6mmol/L, 0.8mmol/L, 1mmol/L and 1.2mmol/L, and the ratio of the 1mmol/L and 1.2mmol/L bacterial liquid to the 0.6mmol/L and 0.8 m/L bacterial liquid in the induction processThe protein expressed by mol/L bacterial liquid has high content; the content of mycoprotein of 1mmol/L and 1.2mmol/L is not greatly different, and the content of expressed bacteria liquid of 1mmol/L is hardly different between 4h and 5 h. Therefore, the final concentration of IPTG is 1mmol/L, and the expression time of 4h is the optimal expression condition.

S122, mass expression of engineering bacteria:

1) single colonies were picked and cultured with shaking in 2mL LB medium (containing Kan antibiotics) to OD600 of about 0.5.

2) According to the following steps: the ratio of 1000 was transferred to the scale-up culture.

3) Culturing until OD600 reaches about 1.5, adding IPTG to 0.4mM, and inducing at 37 deg.C for 4 h.

4) Collecting thallus, centrifuging at 8000r/min at 4 deg.C for 10 min.

5) The supernatant was discarded. The pellet was resuspended in 40mL of lysate.

6) Performing ice bath, and adopting an ultrasonic crusher or a high-pressure homogenizer to crush the cells. The ultrasound conditions were as follows: working time is 15 min; pulse: beating for 2s and stopping for 5 s; output power: 70 percent.

7) Centrifuging at 12000r/min for 10 min. The supernatant and pellet were stored and then analyzed by SDS-PAGE.

S123, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

1) And mounting the glass-made rubber plate on a rubber frame, and checking the sealing condition of the gel plate by using deionized water.

2) 5mL of 10% separation gel was prepared. The following ingredients were added in sequence:

1.9mL of deionized water; 30% Acr-Bis (29:1), 1.7 mL; 1.5mol/L Tris-HCl (pH 8.8)1.3 mL; 10% SDS, 0.05 mL; 0.05mL of 10% ammonium persulfate; TEMED, 0.002 mL.

3) After the mixture is mixed lightly, the separation glue is slowly added into a glue making groove along the edge of the glass plate until the distance between the separation glue and the comb teeth is about 1cm, and a space for concentrating the glue is reserved.

4) 1mL of deionized water was added as a cover layer using a 1mL micropipette gun, and left to stand for about 30min to allow the gel to completely coagulate.

5) After gel coagulation, the deionized water in the cover layer was poured off and 2mL of 5% concentrated gum was added. The following ingredients were added in sequence: 1.4mL of deionized water; 30% Acr-Bis (29:1), 0.33 mL; 1mol/L Tris-HCl (pH 6.8), 0.25 mL; 10% SDS, 0.02 mL; 0.02mL of 10% ammonium persulfate; TEMED, 0.002 mL.

6) After mixing gently, slowly adding the concentrated gel on the separation gel along the edge of the glass plate, inserting a 1mm comb, and standing for about 30min to completely polymerize the gel.

7) And (3) putting the foot preparation frame into an electrophoresis tank, adding about 200mL of 1 xTris-Gly electrophoresis buffer solution to completely soak the gel, slightly pulling out the comb, slightly washing the sample adding hole by using a trace liquid transfer gun to remove unpolymerized acrylamide and gel fragments, and then straightening the gel teeth by using a fine needle for later use.

8) Sample application: mu.L of the above expressed soluble protein and inclusion body protein were taken in PCR tubes, 5. mu.L of 2 XSDS loading buffer was added to each tube, after heating in boiling water at 100 ℃ for 3min, 10. mu.L of sample was added to the loading well with a micropipette, and protein MARKER was added to the adjacent lanes.

9) Electrophoresis: the initial voltage was 80V for 40 minutes, followed by increasing the voltage to 120V and electrophoresis time 1.5 hours.

10) The electrophoresis was stopped when the bromophenol blue front reached the bottom of the electrophoresis chamber, and the glass plate containing the gel was removed. The gel containing the SBP-2 protein was removed from the glass plate and the concentrated gel was cut off.

11) The cut separation gel was placed in a large petri dish, Coomassie brilliant blue was poured in for staining, and the petri dish with Coomassie brilliant blue was placed on a shaker and slowly shaken for staining for 4 h.

12) Pouring out the staining solution after staining, then immersing the gel with a destaining solution for destaining, replacing the eluents at intervals of 20min, 40min and 60min respectively, and then destaining overnight until the background blue fades away, at which time clear protein bands can be seen.

S124, high-efficiency induction expression of recombinant protein

1) BL21 E.coli containing the recombinant plasmid was inoculated on an LB plate containing 50. mu.g/mL Kan, and cultured at 37 ℃ for 12 hours.

2) The grown colonies were picked up with a sterile pipette tip and inoculated into 2mL of LB liquid medium (containing 50. mu.g/mL Kan), cultured with shaking at 37 ℃ and 220r/min overnight to give a seed solution.

3) 1500 μ L of seed solution was added to 50mL of LB liquid medium (containing 50 μ g/mLKan), 5 samples, and cultured with shaking for 3h (OD 600. apprxeq.0.8).

4) 1.0mL of the bacterial culture was taken in a 1.5mL centrifuge tube as a sample before IPTG induction (0h), and IPTG (19.14. mu.L of 50mg/mL (210mmol/L)) was added to the remaining 49mL of culture to a final concentration of 0.6mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and continued to incubate at 37 ℃ at 220r/min with shaking.

5) At 1h, 2h, 3h, 4h, and 5h, respectively, 1mL of bacterial culture was removed from the culture broth in a 1.5mL centrifuge tube.

6) Centrifuging the culture taken out for 0h, 1h, 2h, 3h, 4h and 5h at room temperature 8000r/min for 10min, and discarding the supernatant.

7) To each pellet, cell lysate and protease inhibitor were added at 200W for 10s (10 s apart) and treated 20 times. Then, the mixture is centrifuged at 10000r/min for 10min, and the supernatant is transferred to a new centrifuge tube (soluble recombinant protein) with the volume of 1.5mL, and is stored at 4 ℃ (stored at 20 ℃) for standby.

S125, nickel affinity chromatographic column purification of large-scale expression protein

1) After the SBP-2 protein is induced and expressed according to the optimal conditions, the cultured bacterial liquid is poured into a centrifugal barrel, the bacteria are collected by centrifugation at 5000rpm for 30min, the supernatant is removed, and then 50mL of solution A (20mM Tris, 500mM NaCl, 10mM imidazole, 5% glycerol, pH 8.2) is used for every 600mL of bacterial liquid to resuspend and mix the bacteria uniformly.

2) And (3) carrying out ultrasonic treatment on the thalli in an ice-water mixture, wherein the power is 200W, and the ultrasonic crushing time is 20 min.

3) The ultrasonically disrupted cells were centrifuged at 12000rpm for 10min, and then the supernatant was separated and stored at 4 ℃.

4) An appropriate amount of nickel column packing (for specific binding to the His-tag containing recombinant protein) was placed in the gravity column, and after settling down, the upper pad was lightly covered, and 10 bed volumes of the column were passed through with pure water, followed by 10 bed volumes of the column with solution a.

5) Loading: the supernatant after ultrasonication is passed through a column, and 100mL of sample is loaded, wherein the flow rate is not too fast.

6) Rebalancing: solution a was passed through the column for 25 bed volumes.

7) Impurity washing: 25 bed volumes were washed with eluent (pH 7.9Tris-HCl 20 mmol/L; imidazole 10 mmol/L; sodium chloride 0.5 mol/L).

8) And (3) elution: elution was performed with 15mL of solution B, and the tube was carefully collected. Adding solution B (pH 7.9Tris-HCl 20 mmol/L; imidazole 500 mmol/L; sodium chloride 0.5mol/L), collecting eluted protein in different tubes; after washing the column with 10 column volumes of Binding Buffer and 5 column volumes of deionized water in this order, the column was equilibrated with 3 column volumes of 20% ethanol at 4 ℃.

As can be seen from FIG. 5, the purified protein was found to be around 55kD protein Marker after purification by Ni column purification kit.

S126、Western blot

1) Transfer electrophoresis

The fusion protein is subjected to SDS-PAGE, the gel of the part to be transferred is cut out in order, and the gel is soaked in an electrotransformation buffer. Cut 6 pieces of filter paper (area slightly larger than gel block) of the same size, and soak the filter paper together with two sponges in the buffer solution.

Soaking a piece of PVDF membrane with the same size as the filter paper in a methanol solution for 10s, and then soaking the PVDF membrane in a transfer buffer solution for 3 min;

and thirdly, electrically converting the sandwich sequence as follows: positive (white)/sponge/3 layers of filter paper/nitrocellulose membrane/GE gel/3 layers of filter paper/sponge/negative (black);

2) electric conversion: placing the sandwich clip into an electrophoresis tank pre-filled with an electrotransformation membrane buffer solution, and keeping constant current for 75V for 1 h;

3) ddH for PVDF membrane after membrane conversion₂Washing with O, incubating in sealing solution for 30min, washing with washing solution for 5min for 3 times;

4) adding primary antibody (mycoplasma bovis positive serum, diluting to 1:100 with blocking solution), incubating for 1h, washing membrane for 3 times, each time for 5 min;

5) adding 1:1000 diluted secondary antibody (HRP-labeled mouse anti-bovine IgG serum), incubating for 1h, washing the membrane for 3 times, each time for 5 min;

6) fitting for mixingPreparing AEC color developing solution, slowly shaking to develop color at room temperature in dark place, and when the color depth of the specific blotting strip reaches the requirement (about 10min), using ddH₂The reaction was terminated by O rinsing.

As can be seen from the Western blot result, that is, FIG. 6, the SBP-2 protein can generate specific immunoblotting reaction with the polyclonal antibody of the mouse anti-SBP-2 protein, and a corresponding band appears around 55 KDa. Western blot shows that SBP-2 protein has good reactogenicity.

S13, analysis

In this example, the construction and prokaryotic expression of expression vector was successfully performed by using DNA recombination technology. In the process of constructing the recombinant plasmid, a 10-microliter system is selected, when the physical quantity ratio of the target DNA to the vector plasmid is 6:2, the expression plasmid can be successfully constructed, and a positive expression strain can be successfully obtained after the BL21 expression strain is introduced. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) shows that when prokaryotic expression is carried out by introducing a pET-28a-P48 expression vector into BL21 escherichia coli, 4 hours is the optimal expression time under the induction of 1MM IPTG inducer when the cell density OD600 reaches about 0.8, and the generated SBP-2 protein is soluble protein. Western blot results show that the prokaryotic expression SBP-2 protein has good reactogenicity.

S2, SBP-2 protein immune vaccine preparation and immune effect

S21, test material and test pretreatment

S211, strain and test animal

Mycoplasma bovis Ningxia-1 strain isolated from calf lung, preserved by Ningxia university college of agriculture; 12 animals of 2 months old Hostan bulls without mycoplasma infection were purchased from a Hostan Dairy farm in Zhongning.

Primary reagent

S212, design and synthesis of primers

The reference Mycoplasma bovis HB0801 gene complete sequence is respectively designed as follows: 16S rRNA universal primer and uvrC specific primer, which are synthesized by Shanghai Productivity Limited company. The primers are as follows:

s22, preparation of SBP-2 recombinant protein vaccine and preparation of mycoplasma bovis inactivated vaccine

S221, preparation of recombinant protein vaccine

The SBP-2 protein after prokaryotic expression and purification is used for measuring the protein content by a D30 nucleic acid protein analyzer, and ddH is used₂O to 1.0mg/mL, and stored at-20 ℃ until use.

Preparation of protein vaccine for first immunization: taking 7.5mL of the protein with the concentration of 1.0mg/mL for standby preservation in a 50mL centrifuge tube, and then adding an equal amount of Freund's complete adjuvant (7.5mL) into the centrifuge tube; then soaked in ice box for ultrasonic emulsification. The emulsification conditions are shown in Table 2-1.

TABLE 2-1 emulsification conditions

Preparation of protein vaccine for the second immunization: taking 7.5mL of the protein with the concentration of 1.0mg/mL for standby preservation in a 50mL centrifuge tube, and then adding an equal amount of Freund's incomplete adjuvant (7.5mL) into the centrifuge tube; then soaked in ice box for ultrasonic emulsification. The emulsification conditions were the same as the first emulsification conditions.

S222, preparation of mycoplasma bovis inactivated vaccine

Mycoplasma bovis isolate Ningxia-1 strain was isolated at a ratio of 1:100 percent of the culture medium is inoculated into the liquid culture medium of mycoplasma bovis Thiaucurt's with 5 percent of CO₂Culturing at 37 deg.C for 3 days to obtain first generation bacterial liquid, inoculating to liquid culture medium at a ratio of 1:100, and adding 5% CO₂Culturing at 37 deg.C for 3d to obtain second generation, inoculating to liquid culture medium at a ratio of 1:10, and adding 5% CO₂Culturing at 37 deg.C for 3d to obtain third generation; then measuring the concentration (CCU/mL) of the third generation bacterial liquid and adding a formaldehyde solution to make the formaldehyde in the bacterial liquid endInactivating at 37 deg.C with concentration of 0.4%, extracting inactivated bacteria liquid after 2d, inoculating to Thiaucorn's solid culture medium, and detecting.

And (3) centrifuging 16000r/min for 30min the inactivated bacterial liquid without viable bacteria after inactivation, washing the obtained precipitate for 3 times by using a physiological saline buffer solution, and then diluting the precipitate to the original concentration by using the physiological saline. And (3) mixing the washed inactivated bacterial liquid and a white oil adjuvant in an equal volume, and ultrasonically emulsifying at an interval of 2s for 10min at 100W.

S23, animal immunity test and infection test

Before the test, a nose swab of the Holstein bull calf is collected to separate and identify the mycoplasma bovis, a blood sample is collected to separate serum, and the mycoplasma bovis antibody is detected by using a mycoplasma bovis antigen detection kit. Selecting 12 male calves of 2 months old with negative serum detection and without mycoplasma pathogen detection as test objects, and feeding at a certain beef cattle farm in Yongning county of Yinchuan Ningxia in Ningxia.

The 12 negative calves were divided into 4 groups of 3. Respectively a negative control group (NC), a positive control group (PC), a protein vaccine group and an inactivated vaccine group. Each cow of the protein vaccine group is injected with 5mL of recombinant protein vaccine with the protein content of 2.5mg 1 time and 2 times at two-week interval; each cattle of the inactivated vaccine group was injected intramuscularly with 5mL of 1X 10 at two-week intervals¹⁰The Ningxia-1 inactivated vaccine of CCU/mL is used for 1 time and 2 times in total; the PC group was inoculated 3 days before the start of the experiment with 5mL of 1X 10 solution by intramuscular injection¹⁰The Ningxia-1 strain of Mycoplasma bovis at CCU/mL dose was infected for 3 consecutive infections. NC group was immunized with 5mL of physiological saline at the time of intramuscular injection in other groups.

On day 15 after the 2 nd vaccine immunization, both the protein vaccine group and the inactivated vaccine group were injected intramuscularly with 5mL of 1X 10¹⁰The infection test is carried out on the Ningxia-1 strain of mycoplasma bovis at CCU/mL dose, 3d is infected continuously, and the NC group is injected with Thiauucort's liquid culture medium at the same dose through muscle and continuously observes for 28 days.

Measuring the body temperature 7: 00-8: 00 every day for 0-15 days, and measuring the body temperature once every other day after 15 days until the infection test is finished; serum was collected 1 time a week before day 0 (first immunization), and after day 0, blood was collected 1 time and serum was separated every 7 days for 4 groups of test cattle until 4 weeks after the end of infection. On day 28 post-infection, pathological dissection was performed on PC, NC and test groups of cattle, and lung, trachea, larynx, heart and kidney tissues were collected from the cattle fixed in 10% formalin solution.

Starting after infection of animals, nasal swabs were collected 1 time per week for 3 groups of test cattle and diluted with PBS. Inoculating the nasal swab in Thiaucorn's liquid medium and solid medium CO₂Incubator 5% CO₂Culturing at 37 ℃, and observing colony morphology after 2d of microscopic examination; filtering the bacterial liquid by using a 0.22 mu m bacterial filter, inoculating the bacterial liquid in a liquid culture medium for culture, extracting culture DNA after culturing for 2d, and identifying mycoplasma by using a 16S rRNA primer and identifying mycoplasma bovis and mycoplasma agalactiae by using a uvrC primer.

Pathological dissection of cattle is carried out 28 days after animal infection test, and joint fluid, pleural effusion, pericardial fluid and lung tissue of cattle are collected. Sterilizing the surface of the collected lung tissue by flame, cutting a small piece of the deep lung tissue, and then inoculating the small piece of deep lung tissue into a mycoplasma bovis Thiaucort's liquid screening culture medium and a mycoplasma bovis Thiaucort's solid screening culture medium under an aseptic operation condition. Placing the solid culture medium in 5% CO₂CO at 37 deg.C₂After the culture box is cultured for 2 days, the colony morphology is observed under a microscope; and (3) culturing the liquid culture medium in a constant-temperature shaking table at 37 ℃ for 2 days, observing the color change condition of the culture medium, filtering the yellow culture solution by using a 0.22um sterilizing filter membrane, repeatedly culturing the filtrate, and extracting DNA (deoxyribonucleic acid) to perform mycoplasma bovis molecular biological identification.

The collected tissues were soaked in 10% formalin solution, fixed for 7 days, embedded in paraffin, HE-stained, and observed under an optical microscope.

Serological tests are carried out on the collected serum according to the specifications of the Mycoplasma bovis ELISA antibody detection Kit, the Bovine IgM ELISA Kit, the Bovine IgG ELISA Kit and the Bovine IgA ELISA Kit in Belgium, negative and positive detection and content standard curve calculation are carried out on the obtained data according to the specifications, and the numerical value is converted into the content.

S24, result analysis

As can be seen from FIG. 7, during the test period, the temperature of the NC group was maintained at 39.10-39.50 ℃ and was within the normal body temperature of the cattle; the body temperature of the PC group is higher than 39.50 ℃ from day 14 and shows a rising trend, the highest body temperature is 40.40 ℃, the most general temperature difference with the NC group is 1.15 ℃, and the difference is significant (P is less than 0.05); the body temperature of the protein vaccine group before immunization is not different from that of the NC group, but the body temperature of the calf starts to rise about 1 week after mycoplasma bovis pathogen infection, and finally the body temperature is close to the maximum body temperature of the PC group, and the difference is obvious compared with the NC group (P is less than 0.05); the body temperature change of the inactivated vaccine group at the 1 st vaccination is not obvious, the body temperature is slightly increased after the 2 nd vaccination, the highest body temperature is 39.60 ℃, the difference with the NC group is not obvious (P is more than 0.05), and the highest body temperature is 39.6 ℃ after the infection test is not obvious (P is more than 0.05) compared with the NC group.

As can be seen from FIG. 8, the specific antibody detection before the test was negative in the protein vaccine group, NC group and PC group, and the difference between the groups was not significant (P > 0.05); after the protein vaccine is injected for one week, the antibody level of the protein vaccine group calf is increased to be positive, and then a stable positive state is maintained; the PC group shows positive antibody level increase one week after the virus infection, then reduces after three weeks, and then is in a more stable positive state; the detection of the mycoplasma bovis antibodies in the NC group is negative in the whole test stage, the antibody level is stable, and the difference is not significant (P > 0.05); the NC group is very different from the protein vaccine group and the PC group (P < 0.01); the antibody level of the body of the protein vaccine after the cattle are immunized is not obviously different from that of the positive group (P > 0.05). The inactivated vaccine group starts to rise in antibody level 1 week after inoculation of the mycoplasma bovis inactivated vaccine, the difference between the antibody level and the antibody level is not significant (P is more than 0.05) at week 0, the mycoplasma bovis antibody at week 2 is positive and continuously rises, the difference between the antibody level and the antibody level is significant (P is less than 0.01) at week 0, the antibody level reaches the maximum value at week 3, and the antibody level of the mycoplasma bovis is in a stable state from week 4 to week 6; after week 6 infection with mycoplasma bovis pathogen, mycoplasma bovis antibody levels continued to rise, and then were in a steady state.

Pathological anatomical examination of protein vaccine group calves is shown in fig. 9: pathological changes of kidney, liver, larynx and trachea are not visible, a large amount of yellowish effusion is in the chest cavity, bleeding and blood stasis of the lung are caused, the lung is adhered to the chest cavity, and obvious substantive pathological changes are caused. As can be seen from FIG. 10, the results of the autopsy of the PC group revealed: lung tissue adhesion, lung bleeding and congestion, substantial fleshing of lung tissue, grey cheese-like or suppurative necrotic foci with different sizes, internal hemorrhage of lung tissue with suppurative and cheese-like necrotic foci, and no pathological changes of other tissues can be seen with naked eyes. As can be seen from FIG. 11, the recombinant examination of the inactivated vaccine revealed that: the lung, larynx, heart, liver, trachea, kidney and joint tissues have no macroscopic lesions.

As can be seen from FIG. 12, the protein vaccine group calf has normal tracheal tissue structure, and in the lung tissue, bronchiole epithelial hyperplasia, inflammatory exudate, cellulitis nodule, hemorrhage, and inflammatory exudate; emphysema-alveolar compensatory dilation: alveoli dilate, alveolar walls become thin, and some alveoli break up to form large sacs; epithelial cell detachment at alveolar septum, alveolar septal rupture, alveolar collapse and thickening, substantial hyperplasia of alveolar, and hemorrhage. As can be seen from FIGS. 13 to 15, the bronchioles of the PC group were narrowed, the folds were increased, a small amount of exfoliated epithelial cells were present in the lumen, the alveoli were collapsed, the alveolar septa were thickened, the tissue was proliferated, and a small amount of cells were exfoliated in the alveolar lumen. As can be seen from FIGS. 16 to 17, the inactivated vaccine group showed no significant histopathological changes in lung and trachea tissues.

Wherein, in FIG. 13, A is bronchial epithelial hyperplasia, inflammatory exudate and cellulitis nodule 100X; b is emphysema-alveolar compensatory dilation: alveoli expand, alveolar walls become thin, and some alveoli have been broken and combined into large sacs 100 ×; c is severe hyperplasia of bronchial epithelium 100X; d is terminal bronchial bleeding with inflammatory exudate 400 ×; e is emphysema: alveolar dilatation 400 ×; f is alveolar septal epithelial cell shedding and alveolar septal rupture of 400 x; g is alveolar collapse and thickening of 400X; h indicates substantial flesh change and bleeding.

In fig. 14, the lungs of the PC group are 100 × and 400 ×, respectively, and it can be seen that the lungs of the PC group: bronchiectasis, increased plica, and a few exfoliated epithelial cells in the lumen; fig. 15 shows the PC group lungs 100 x and 400 x, respectively, and it can be seen that: the alveolus collapses, the alveolar septum thickens, and a small amount of cells fall off in the alveolar cavity; FIG. 16 shows inactivated vaccine group trachea tissue sections 100X and 400X, respectively; FIG. 17 shows inactivated vaccine lung tissue sections 100X and 400X, respectively.

As can be seen from fig. 18, the IgA content started to increase 1 week after the 1 st immunization in the protein vaccine group, reached the maximum at 2 weeks, then started to decrease, and then was maintained in a steady state; the whole IgA content of the protein vaccine group is slightly higher than that of the IgA of the NC group, the IgA content of 2 weeks after the first immunization is remarkably different from that of the NC group (P is less than 0.05), and the NC group always maintains a stable state.

As can be seen from fig. 18, IgG levels began to rise 1 week after 1 st immunization in the protein vaccine group, reaching a maximum at 2 weeks; IgG content is obviously increased after 1 week of mycoplasma bovis pathogen infection, IgG content is very different from that of the NC group (P is less than 0.01) at 2 weeks after the first immunization and 1 week after infection, and the NC group always maintains a relatively stable state.

As can be seen from fig. 18, IgM reached a maximum in the protein vaccine group at week 2 after the 1 st immunization; the IgM content is obviously increased after 1 week of mycoplasma bovis pathogen infection, the difference between the IgM content 1 week after the first immunization and 1 week after infection is extremely obvious compared with that of the NC group (P is less than 0.01), and the NC group always maintains a relatively stable state.

The isolation and identification of mycoplasma are carried out on nasal swabs of an inactivated vaccine group, a protein vaccine group NC group and a PC group collected weekly after infection, and the findings are as follows: the PC group separates and identifies mycoplasma bovis pathogen from collected pathological materials from 1 week after infection; the mycoplasma bovis pathogen is not separated and identified in the whole test period of the NC group; the inactivated vaccine group can be used for separating and identifying mycoplasma bovis pathogen 1 week after infection, the mycoplasma bovis pathogen is not separated and identified after 3 weeks, and the protein vaccine group can be used for separating the mycoplasma bovis pathogen starting 1 week after infection.

For PC group calves, 5mL of 1X 10 was injected into the calves through 3 consecutive intramuscular injections¹⁰Infection with a CCU/mL dose of Mycoplasma bovis Ningxia-1 strain resulted in the finding that: the PC group shows clinical symptoms such as body temperature rise, dyspnea, cough and the like clinically; body temperature started at 14d and was higher than 39.50 ℃ and showed a tendency to increase, with a maximum body temperature of 40.40 ℃ and a maximum temperature difference of 1.15 ℃ from the NC group (P is significant) (the temperature difference is<0.05), can be collected during the whole test processSeparating mycoplasma bovis from the collected nasal swabs; the PC group shows positive antibody level increase one week after the virus infection, then reduces after three weeks, and then is in a more stable positive state; the case autopsy finds that: lung tissue adhesion, lung bleeding and blood stasis, lung tissue parenchyma change and grey cheese-like or suppurative necrotic foci with different sizes, lung tissue internal bleeding with suppurative and cheese-like necrotic foci, and no macroscopic pathological changes of other tissues; the histological observation result shows that bronchiole is reduced, the plica is increased, epithelial cells are slightly exfoliated in the lumen, the alveoli are collapsed, the alveolar septa are thickened, the tissue is proliferated, and a small amount of cells are exfoliated in the alveolar lumen. The results are consistent with the clinical symptoms and pathological histological changes caused by mycoplasma bovis infecting cattle, so that the following results can be obtained: 3 consecutive injections through muscle of 5mL 1X 10¹⁰A CCU/mL dose of a Mycoplasma bovis strain successfully replicates the Mycoplasma bovis case model.

The specific antibodies of the protein vaccine group, the NC group and the PC group are detected to be negative before the test, the difference between the groups is not significant (P is more than 0.05), the statistical significance is achieved, and the test animals selected in the test meet the test conditions. After the protein vaccine is injected for one week, the antibody level of the protein vaccine group calf is increased to be positive, and then a stable positive state is maintained; the PC group shows positive antibody level increase one week after the virus infection, then reduces after three weeks, and then is in a more stable positive state; the NC group is negative in mycoplasma bovis antibody detection in the whole test stage, the antibody level is stable, the difference is not significant (P >0.05), and the difference is very significant (P <0.01) compared with the protein vaccine group and the PC group. The SBP-2 protein vaccine can stimulate the calf to generate obvious specific immune response and reach high antibody level.

The IgA content of the protein vaccine group at 2 weeks after the first immunization is remarkably different from that of the NC group (P <0.05), and the NC group always maintains a relatively stable state. IgG content was significantly different (P <0.01) from the NC group at 2 weeks after the first immunization and 1 week after infection, and the NC group was maintained in a stable state. The protein vaccine group reached a maximum at week 2 after the 1 st immunization; the IgM content is obviously increased after 1 week of mycoplasma bovis pathogen infection, the difference between the IgM content 1 week after the first immunization and 1 week after infection is extremely obvious compared with that of the NC group (P is less than 0.01), and the NC group always maintains a relatively stable state. The SBP-2 protein vaccine stimulates the humoral immune system of the calf, and further the SBP-2 protein is used as the conserved surface lipoprotein of the mycoplasma bovis to prepare the protein vaccine which can generate antibodies aiming at the mycoplasma bovis membrane protein when the protein vaccine is used for animal immunization.

Pathological dissection of protein vaccine group calves shows that: pathological changes of kidney, liver, larynx and trachea are not visible, a large amount of yellowish effusion is in the chest cavity, bleeding and blood stasis of the lung are caused, the lung is adhered to the chest cavity, and obvious substantive pathological changes are caused. The PC group autopsy finds that: lung tissue adhesion, lung bleeding and blood stasis, lung tissue parenchyma change and grey cheese-like or suppurative necrotic foci with different sizes, lung tissue internal bleeding with suppurative and cheese-like necrotic foci, and no macroscopic pathological changes of other tissues; in histological analysis, the protein vaccine group calf has normal tracheal tissue structure, and in lung tissue, bronchiole epithelial hyperplasia, inflammatory exudate, cellulitis nodule, hemorrhage, and inflammatory exudate; emphysema-alveolar compensatory dilation: alveoli dilate, alveolar walls become thin, and some alveoli break up to form large sacs; the alveolar septal epithelial cells are exfoliated, the alveolar septal is broken, the alveoli are collapsed and thickened, the alveoli are substantially proliferated, and the bleeding is similar to the pathological changes of the tissues of the PC group. The mycoplasma bovis pathogene is mainly used for causing the lung organs of calves to generate substantial pathological changes after intramuscular injection, and other organ tissues are normal; the lung tissue change of the protein vaccine group is relatively lighter than that of the PC group, which indicates that the SBP-2 protein can reduce the infection of mycoplasma bovis pathogen to a certain extent, but can not completely block the pathogenicity of the pathogen. Therefore, the SBP-2 protein is one of the main pathogenic proteins of mycoplasma bovis pathogen invading the body, and can be used as one of protein vaccine candidate proteins.

2 passages 5mL of 1X 10¹⁰Intramuscular injection of CCU/mL inactivated vaccine found: after the inactivated mycoplasma bovis vaccine is immunized for 1 time, the antibody level of mycoplasma bovis begins to rise, can reach positive on day 15, reaches the maximum value at week 3, and then is stable. 4 th week advanceInfection, antibody content decreased, and then plateaus. The body temperature of the infected inactivated vaccine group calf is in the normal body temperature range, and the difference with the NC group is not significant (P)>0.05), no respiratory symptoms such as cough; pathological anatomy of lung, larynx, heart, liver, trachea, kidney and joint tissues has no macroscopic pathological changes; the lung was not seen in the histological analysis and the trachea produced histological lesions. The research shows that the inactivated vaccine of mycoplasma bovis developed by the experiment can prevent the invasion of mycoplasma bovis bacteria.

Sequence listing

<110> Ningxia academy of agriculture and forestry, animal science institute (Ningxia grass and animal engineering research center)

<120> mycoplasma bovis protein SBP-2 and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1419

<212> DNA

<213> Mycoplasma bovis (Mycoplasma bovis)

<400> 1

gaattcatga aaaaaaataa attctacctg ttcctgggtg ccgccccggt cctgtcggtc 60

ccgctggtcg ctgcaagctg tggtgacaaa tactttaaag aaaccgaagt tgatggtgtc 120

aaaacggtga ccacgctgtc acatattgtc tcgcgtaaag gtctgaaact gcgcgatggt 180

ctgaccgtgg acaacgcacc gcgtgcagcc tttattacgg atgaaggttc cgttcatgac 240

gaaagcttca atcagtctgg ctgggaagcc gtccacaaaa tcagttatga actgggtctg 300

gataaagcac aagtttccgg caacaaaaat ctgcgcaaca aagtctacga accgaaaaaa 360

ggtgaactgg cgagctctta taaaaatgcc attgacagtt cctttcgtta catcgttctg 420

tgcggtttca cccacaaagc agctctgtat ggcctggaac cggaatacat taagaaaatt 480

aaagataaca acatcgtgtt catcaccgtt gatttcgaca ttcagcaaga tgcatctacg 540

ggcgaaccgg cggccaaagc ttttgtggac aaaatcggcc agggtcgtct gattccggtt 600

atcttcgata ccaaacaagc agcttatatt gcgggtcgcg cactggctga ttacttcagc 660

aaaatctaca aagacaaccc ggaaaaacgt accattggtg cattcggcgg tatcccgtgg 720

ccggcagtgt ctgattttat tgctggcacg ttccagggta ttatcgactg gaataaagaa 780

catccggaag cgaaaaccaa aagtctgaac aatacgatcg aactgaaaac cagttttacg 840

tccggtgaac cggtggcagt tgcagcaatt aacagcgtca tcaaagcaac cgcatcatat 900

ccggtggcag gctcgctgtc atcggatacg gccaaagaaa tcaaaaaact gggtgacaaa 960

aacaaattca tcatcggcgt tgatgcagac cagaaaaatg ctctgaaagg tcaccgtatt 1020

ttcacctcag tgatgaaact gatcggccag gcggtttata acgtcctggc cgatctgtac 1080

tcgcagggtg aaaatagcct gtctctgcaa ccgggctttg aaattggcaa gaaaaacggc 1140

gaagcgaaag tgttcggcta tggtgaaaat ggcgcaagca aatacgtcgg tgtggctacc 1200

agtggcctgc tggattccaa aaacgacgaa attgcaaata aagctctgga agaagcaacc 1260

aaatattacg aaagcaaaaa agcggaaatc cagaaaacgc tgtctggtca actggaagaa 1320

gcgaaaaaag ccctgggcac caaatggccg gatcaaccgg cggaccaatt cggcaaaatg 1380

attaactggc tggcaaaaga aacgcaaaaa taaggatcc 1419

<210> 2

<211> 468

<212> PRT

<213> Mycoplasma bovis (Mycoplasma bovis)

<400> 2

Met Lys Lys Asn Lys Phe Tyr Leu Phe Leu Gly Ala Ala Pro Val Leu

1 5 10 15

Ser Val Pro Leu Val Ala Ala Ser Cys Gly Asp Lys Tyr Phe Lys Glu

20 25 30

Thr Glu Val Asp Gly Val Lys Thr Val Thr Thr Leu Ser His Ile Val

35 40 45

Ser Arg Lys Gly Leu Lys Leu Arg Asp Gly Leu Thr Val Asp Asn Ala

50 55 60

Pro Arg Ala Ala Phe Ile Thr Asp Glu Gly Ser Val His Asp Glu Ser

65 70 75 80

Phe Asn Gln Ser Gly Trp Glu Ala Val His Lys Ile Ser Tyr Glu Leu

85 90 95

Gly Leu Asp Lys Ala Gln Val Ser Gly Asn Lys Asn Leu Arg Asn Lys

100 105 110

Val Tyr Glu Pro Lys Lys Gly Glu Leu Ala Ser Ser Tyr Lys Asn Ala

115 120 125

Ile Asp Ser Ser Phe Arg Tyr Ile Val Leu Cys Gly Phe Thr His Lys

130 135 140

Ala Ala Leu Tyr Gly Leu Glu Pro Glu Tyr Ile Lys Lys Ile Lys Asp

145 150 155 160

Asn Asn Ile Val Phe Ile Thr Val Asp Phe Asp Ile Gln Gln Asp Ala

165 170 175

Ser Thr Gly Glu Pro Ala Ala Lys Ala Phe Val Asp Lys Ile Gly Gln

180 185 190

Gly Arg Leu Ile Pro Val Ile Phe Asp Thr Lys Gln Ala Ala Tyr Ile

195 200 205

Ala Gly Arg Ala Leu Ala Asp Tyr Phe Ser Lys Ile Tyr Lys Asp Asn

210 215 220

Pro Glu Lys Arg Thr Ile Gly Ala Phe Gly Gly Ile Pro Trp Pro Ala

225 230 235 240

Val Ser Asp Phe Ile Ala Gly Thr Phe Gln Gly Ile Ile Asp Trp Asn

245 250 255

Lys Glu His Pro Glu Ala Lys Thr Lys Ser Leu Asn Asn Thr Ile Glu

260 265 270

Leu Lys Thr Ser Phe Thr Ser Gly Glu Pro Val Ala Val Ala Ala Ile

275 280 285

Asn Ser Val Ile Lys Ala Thr Ala Ser Tyr Pro Val Ala Gly Ser Leu

290 295 300

Ser Ser Asp Thr Ala Lys Glu Ile Lys Lys Leu Gly Asp Lys Asn Lys

305 310 315 320

Phe Ile Ile Gly Val Asp Ala Asp Gln Lys Asn Ala Leu Lys Gly His

325 330 335

Arg Ile Phe Thr Ser Val Met Lys Leu Ile Gly Gln Ala Val Tyr Asn

340 345 350

Val Leu Ala Asp Leu Tyr Ser Gln Gly Glu Asn Ser Leu Ser Leu Gln

355 360 365

Pro Gly Phe Glu Ile Gly Lys Lys Asn Gly Glu Ala Lys Val Phe Gly

370 375 380

Tyr Gly Glu Asn Gly Ala Ser Lys Tyr Val Gly Val Ala Thr Ser Gly

385 390 395 400

Leu Leu Asp Ser Lys Asn Asp Glu Ile Ala Asn Lys Ala Leu Glu Glu

405 410 415

Ala Thr Lys Tyr Tyr Glu Ser Lys Lys Ala Glu Ile Gln Lys Thr Leu

420 425 430

Ser Gly Gln Leu Glu Glu Ala Lys Lys Ala Leu Gly Thr Lys Trp Pro

435 440 445

Asp Gln Pro Ala Asp Gln Phe Gly Lys Met Ile Asn Trp Leu Ala Lys

450 455 460

Glu Thr Gln Lys

465

Claims

1. The mycoplasma bovis protein SBP-2 is characterized in that a gene for coding the mycoplasma bovis protein is a P48 gene, and the nucleotide sequence of the mycoplasma bovis protein is shown as a sequence table in SEQ ID NO 1; wherein the nucleotide is obtained by optimizing TGA base expressing tryptophan in Mycoplasma bovis P48 gene to TGG.

2. The mycoplasma bovis protein SBP-2 according to claim 1, wherein the amino acid sequence of said mycoplasma bovis protein SBP-2 is represented by SEQ ID NO. 2.

3. A recombinant plasmid, wherein said plasmid is the pET-28a-P48 plasmid comprising mycoplasma bovis SBP-2 according to claim 1.

4. Escherichia coli comprising the recombinant plasmid according to claim 3, which is deposited at the CGMCC with the preservation number of CGMCC NO. 23133 at 2021, 8 and 9 days.

5. Application of mycoplasma bovis protein SBP-2 in preparation of medicines for preventing diseases caused by mycoplasma bovis is provided.

6. An application of mycoplasma bovis protein SBP-2 in preparing mycoplasma bovis vaccine is provided.

7. A mycoplasma bovis vaccine, comprising SBP-2 protein of claim 1 and an immunoadjuvant.