CN113604492B

CN113604492B - Fusion gene, fusion protein, preparation method and mycoplasma bovis subunit vaccine

Info

Publication number: CN113604492B
Application number: CN202111060347.1A
Authority: CN
Inventors: 方鹏飞; 孔迪; 曹文龙; 滕小锘; 张大鹤
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2023-07-07
Anticipated expiration: 2041-09-10
Also published as: CN113604492A

Abstract

The application discloses a fusion gene, a fusion protein, a preparation method and a mycoplasma bovis subunit vaccine. The fusion protein is expressed from the first sequence or the second sequence. The nucleotide sequence of the first sequence is shown as SEQ ID NO:1 and the nucleotide sequence of the second sequence is as set forth in SEQ ID NO:1 has at least 95% similarity. Vaccines include fusion proteins and adjuvants. The fusion protein has higher antigenicity, immunogenicity and expression level than natural protein, stronger immunogenicity, high animal safety, and can be prepared by large-scale serum-free suspension culture by using a bioreactor, so that large-scale batch production can be realized, the batch is stable, the quality control is easy, and the production cost of vaccines can be greatly reduced.

Description

Fusion gene, fusion protein, preparation method and mycoplasma bovis subunit vaccine

Technical Field

The application relates to the technical field of subunit vaccines, in particular to a fusion gene, a fusion protein, a preparation method and a mycoplasma bovis subunit vaccine.

Background

Mycoplasma bovis (M.bovis) is a causative agent against cattle, it belongs to the Protophyte kingdom, the phylum Thick-walled bacteria, the class of mollicutes, the order of Mycoplasma, the family of Mycoplasma, the genus of Mycoplasma, the size between bacteria and viruses, the thallus structure is simple, and no cell wall exists. After Mycoplasma bovis infects cattle, the infected cattle can have symptoms such as body temperature rise, mental depression, asthma, clear flow or purulent nasal juice at the beginning of the disease, and then have symptoms such as diarrhea, emaciation, coarse and disordered fur, and can cause Mycoplasma bovis related diseases such as bovine pneumonia, mammitis, arthritis, tympanitis, and the like (Mycoplasma bovis-associated disease, mbad).

The mycoplasma bovis spread is wide in spread, and can be spread through the respiratory tract spread, body fluid spread (such as milk, genital secretion and the like) or vertical spread (Vertical transmission) and the like, so that healthy cattle groups are easy to be infected by mycoplasma bovis, especially cows and beef cattle are highly susceptible to the mycoplasma bovis, and the sick cattle need to be treated in time so as to prevent the spread of infection.

At present, mycoplasma bovis is mainly treated by antibiotics and whole-cell vaccines. However, because mycoplasma bovis does not have a cell wall, it is insensitive to antibiotics acting against the cell wall and is prone to develop resistance thereto. Whole cell vaccines include inactivated vaccines and partially attenuated live vaccines. The whole cell vaccine needs to culture mycoplasma bovis, the culture cost is high, and the antigen of mycoplasma bovis is easy to change along with the change of culture conditions, so that the antigen is different, the production of the whole cell vaccine is unstable, the protection capability of the whole cell vaccine is limited, the protection time is short, the side effect is large, and the possibility of virulence reversion exists.

Disclosure of Invention

The application provides a fusion gene, a fusion protein, a preparation method and a mycoplasma bovis subunit vaccine, which are not antibiotic vaccines and whole cell vaccines, can improve the defects of the antibiotic vaccines and the whole cell vaccines, and have high specificity and sensitivity to the treatment of mycoplasma bovis.

In order to overcome the defects, the application adopts the following technical scheme:

the application provides a fusion gene, the nucleotide sequence of which is selected from any one of the following sequences:

(1) A first sequence, the nucleotide sequence of which is shown as SEQ ID NO:1 is shown in the specification;

(2) A second sequence, the nucleotide sequence of which is identical to the nucleotide sequence shown in SEQ ID NO:1 has at least 95% similarity.

In some embodiments, the second sequence has one or more nucleotide substitutions, deletions, or insertions relative to the first sequence.

The present application provides a plasmid vector (pUC-M.bovis-Fu plasmid vector) comprising: a pUC plasmid; and a fusion gene inserted into the pUC plasmid.

Wherein, the pUC plasmid is selected from any one of pUC17 plasmid, pUC18 plasmid and pUC19 plasmid.

The present application provides a transfer vector (pF-m.bovis-Fu transfer vector) comprising: baculovirus transfer plasmids; and a fusion gene inserted into the baculovirus transfer plasmid.

Wherein, the baculovirus transfer plasmid is selected from any one of pFastBac 1, pVL1393 and pFastBac dual.

The present application provides a recombinant plasmid (Bacmid-m.bovis-Fu recombinant plasmid) comprising: bacmid vector; and a fusion gene inserted into the Bacmid of the baculovirus vector.

The present application provides a transfected cell formed by transfecting a host cell with a recombinant plasmid under the influence of a transfection system. The host cell may be selected from any one of Sf9 cells, high Five cells, or Sf21 cells.

The present application provides a fusion protein formed by expressing a fusion gene from transfected cells; the nucleotide sequence of the fusion gene is selected from any one of the following sequences:

The application provides a fusion protein, the amino acid sequence of which is selected from any one of the following sequences:

(1) And a third sequence, the amino acid sequence of which is shown as SEQ ID NO:2 is shown in the figure;

(2) And a fourth sequence, the amino acid sequence of which is identical to the amino acid sequence shown in SEQ ID NO:2 has at least 95% similarity.

The application provides a preparation method of fusion protein, which comprises the following steps:

(1) Preparing a fusion gene; the nucleotide sequence of the fusion gene is selected from a first sequence or a second sequence, and the nucleotide sequence of the first sequence is shown as SEQ ID NO:1 and the nucleotide sequence of the second sequence is as set forth in SEQ ID NO:1 has at least 95% similarity;

(2) Cloning the fusion gene into a baculovirus transfer plasmid to obtain a transfer vector (pF-M.bovis-Fu transfer vector);

(3) Transforming DH10Bac strain with transfer vector, screening recombinant strain, extracting recombinant plasmid containing fusion gene (Bacmid-M.bovis-Fu recombinant plasmid), and transferring host cell to obtain transfected cell containing recombinant baculovirus gene (rBac-M.bovis-Fu); the recombinant baculovirus gene contains the fusion gene;

(4) And collecting cell cultures of transfected cells, and purifying to obtain the fusion protein.

The application provides application of the fusion protein in preparing an antibody of mycoplasma bovis.

The application provides a subunit vaccine which contains the fusion protein and pharmaceutically acceptable adjuvant.

Wherein the concentration of the fusion protein in the subunit vaccine may be 100.+ -.10. Mu.g/ml.

The adjuvant can be selected from any one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, MONTANIDE ISA 51 VG, liquid paraffin, squalane, saponin, vegetable oil, and cytokine.

Due to the adoption of the technical scheme, the application has the following technical effects:

First, the present application employs direct concatenation of a truncated GK and a truncated GPDH to obtain a fusion gene, and expresses the fusion gene to obtain a fusion protein. The test shows that the fusion protein has higher antigenicity and immunogenicity than natural protein, higher expression level than natural protein, higher immunogenicity and high animal safety.

In addition, the fusion protein can be prepared by large-scale serum-free suspension culture by using a bioreactor, can realize large-scale batch production, has stable batch-to-batch and easy quality control, can greatly reduce the production cost of vaccines, and does not have the phenomenon of virulence reversion.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a gel electrophoresis chart of PCR amplification products of pUC-M.bovis-Fu plasmid vector.

FIG. 2 is a gel electrophoresis chart of colony PCR after culturing in LB liquid medium.

FIG. 3 is a schematic representation of the sequence composition of pF-M.bovis-Fu transfer vector.

FIG. 4 is a SDS-PAGE protein band of each cell culture.

FIG. 5 is a graph showing the Western Blot detection results of SDS-PAGE products of FIG. 4.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.

The present application will be described in detail below. The following description of the embodiments is not intended to limit the preferred embodiments.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the scope of the present application.

Unless otherwise indicated, all reagents and materials referred to herein are commercially available or may be prepared by one of ordinary skill in the art in accordance with common general knowledge.

[ fusion Gene ]

The application provides a fusion gene, which is called M.bovis-Fu gene for short. Fu means Fusion (Fusion). The fusion gene can be expressed as a fusion protein for preparing mycoplasma bovis subunit vaccine. The nucleotide sequence of the fusion gene may be selected from any one of the following sequences:

Wherein the first sequence is 1293 nucleotides in total, and comprises a stop codon in addition to the truncated sequence of Glycerol Kinase (GK) gene and Glycerol-3-phosphate dehydrogenase (Glycerol-3-phosphate Dehydrogenase, GPDH) gene on the surface of cell membrane of Mycoplasma bovis. The truncated sequences of the two genes are directly connected, but not connected through a nucleotide connecting segment (such as a nucleotide sequence with a connecting function), so that the expressed fusion protein has double enzymatic activity and stability, has double activity of glycerol kinase and glycerol-3-phosphate dehydrogenase, and cannot influence antigen activity because of being cut into two monomer enzymes by unknown enzymes.

The full-length sequence of the Glycerol Kinase (GK) gene of mycoplasma bovis has 1506 nucleotides, and the full-length sequence is shown in SEQ ID NO:3, the truncated sequence is 619 th nucleotide to 1371 st nucleotide of the full-length sequence. The truncated sequence is a conserved sequence of glycerol kinase.

The full-length sequence of the glycerol-3-phosphate dehydrogenase (GPDH) gene of mycoplasma bovis has 996 nucleotides, and the full-length sequence is shown as SEQ ID NO:4, the truncated sequence is from nucleotide 208 to nucleotide 744 of the full length sequence. The truncated sequence is a conserved sequence of glycerol-3-phosphate dehydrogenase.

The conserved sequences of the two genes have sequence stability in different mycoplasma bovis strains, and after the fusion genes are formed by series splicing, the fusion proteins formed by the expression of the fusion genes can realize stable immune activity, so that the mycoplasma bovis subunit vaccine can be prepared. If the subunit vaccine is produced by using the full-length sequences of the two genes, the expressed fusion protein is easy to form a polymer and can partially or completely cover the active center of the enzyme, so that the immune activity of the fusion protein is lower than that of the fusion protein formed by the conserved sequences, and the whole activity of the mycoplasma bovis subunit vaccine is difficult to be exerted.

In addition, the protein expression quantity of the fusion gene formed by adopting the conserved sequences of the two genes is higher than that of the fusion gene formed by the full-length sequences of the two genes, and the fusion gene has better immune activity after being prepared into subunit vaccine.

In addition to using the truncated original nucleotide sequences of both genes to make a fusion gene having a first sequence, a second sequence having at least 95% similarity to the first sequence can be used to make the fusion gene. The second sequence may have at least 96% similarity to the first sequence, may have at least 97% similarity, may have at least 98% similarity, or may have at least 99% similarity. Similarity is defined as the ratio of the number of nucleotides in the second sequence that are identical to the first sequence to the total number of nucleotides in the first sequence. The second sequence has one or more nucleotide substitutions, deletions or insertions relative to the first sequence, the number of nucleotides being substituted, deleted or inserted being such that the second sequence has at least 95% similarity to the first sequence. If the similarity is too low, the fusion protein formed by the second sequence will have poor immunological activity. The substitution may be performed by substitution of guanine (G), cytosine (C), thymine (T) and adenine (A) in pairs. For example, thymine (T) at a certain position is replaced with guanine (G), and the rest is the same.

[ vector containing fusion Gene ]

The present application provides a series of vectors containing the above fusion genes, each of which has its own function, as described in detail below:

1. pUC-M.bovis-Fu plasmid vector:

pUC-M.bovis-Fu plasmid vectors include the fusion gene (M.bovis-Fu gene) described above and pUC17 vectors. The pUC-M.bovis-Fu plasmid vector was used for PCR amplification of the fusion gene. The nucleotide sequence of the upstream primer M.bovis-Fu-F adopted in the amplification is shown as SEQ ID NO:5, specifically 5'-ATAGGATCCATGCCGCGCAGCATTCTGCCGGAAATTAAA-3'; the nucleotide sequence of the downstream primer M.bovis-Fu-R is shown in SEQ ID NO:6, in particular 5'-ATAAAGCTTTTAGCCAATCGCGCTCAGTTCCAGCGG-3'.

The construction method of pUC-M.bovis-Fu plasmid vector comprises: the M.bovis-Fu gene was cloned into pUC17 vector according to the molecular cloning method.

2. pF-M.bovis-Fu transfer vector

pF-M.bovis-Fu transfer vectors include fusion genes (M.bovis-Fu gene) and pFastBac 1 plasmids. The pF-M.bovis-Fu transfer vector was used to transfer the fusion gene into DH10Bac competent cells.

The construction method of the pF-M.bovis-Fu transfer vector comprises the following steps: the pFastBac 1 plasmid and the M.bovis-Fu gene were digested with BamHI and HindIII enzymes at 37℃respectively, and then the digested pFastBac 1 plasmid and the digested M.bovis-Fu were ligated using T4 DNA ligase, transformed and amplified with DH 5. Alpha. Competent cells, and screened for the correct pF-M.bovis-Fu transfer vector.

3. Bacmid-M.bovis-Fu recombinant plasmid

The Bacmid-M.bovis-Fu recombinant plasmids include fusion genes (M.bovis-Fu genes) and baculovirus plasmids (Baculovirus plasmid, abbreviated as Bacmid). A baculovirus plasmid is a plasmid with a baculovirus genome that can shuttle between bacteria and insect cells, and therefore, the Bacmid-M.bovis-Fu recombinant plasmid can also be referred to as a Bacmid-M.bovis-Fu shuttle plasmid.

The construction method of the Bacmid-M.bovis-Fu recombinant plasmid comprises the following steps: pF-M.bovis-Fu transfer vector was added to DH10Bac competent cells for transformation, and the transformants were screened for Bacmid-M.bovis-Fu recombinant plasmid.

In the above method, DH10Bac competent cells contained baculovirus plasmid (Bacmid) and helper plasmid (The helper plasmid). After the pF-M.bovis-Fu transfer vector (containing the fusion gene) enters DH10Bac competent cells, the baculovirus plasmid (Bacmid) and the pF-M.bovis-Fu transfer vector are recombined under the action of the helper plasmid to generate a Bacmid-M.bovis-Fu recombinant plasmid, namely the transformation process.

In the above method, the screening may be performed by selecting a monoclonal method, specifically including:

(1) Coating DH10Bac bacterial liquid containing Bacmid-M.bovis-Fu recombinant plasmid on LB solid medium containing gentamicin, kanamycin, tetracycline, 5-bromo-4-chloro-3-indole-beta-D-galactoside (also known as X-gal) and isopropyl-beta-D-thiogalactoside (also known as IPTG) for culture;

(2) Selecting positive white colony, and culturing on LB solid medium containing gentamicin, kanamycin, tetracycline, 5-bromo-4-chloro-3-indole-beta-D-galactoside (also known as X-gal) and isopropyl-beta-D-thiogalactoside (also known as IPTG) by streaking;

(3) Then selecting positive single colony, inoculating on LB liquid medium containing gentamicin, kanamycin, tetracycline, 5-bromo-4-chloro-3-indole-beta-D-galactoside (also known as X-gal) and isopropyl-beta-D-thiogalactoside (also known as IPTG) for culture;

(4) And finally, extracting the Bacmid-M.bovis-Fu recombinant plasmid in the culture solution.

[ cell containing fusion Gene, recombinant baculovirus Gene and fusion protein ]

The application provides a cell containing a fusion gene, a recombinant baculovirus gene containing the fusion gene and a fusion protein formed by expressing the fusion gene by the cell.

Wherein, the cell containing the fusion gene is obtained by transfecting host cells with a Bacmid-M.bovis-Fu recombinant plasmid under the action of a transfection system.

Transfection systems include Cellfectin transfection reagents and the like. Host cells include Sf9 cells, high Five cells, or Sf21 cells. sf9 cells, also known as spodoptera frugiperda cells (Spodoptera frugiperda cell), belong to insect cells and can be used to produce the recombinant baculovirus gene rBac-m. r represents recombination (recombinant), bac is the abbreviation of Bacmid, and M.bovis-Fu represents the fusion gene of the application.

The Bacmid-m.bovis-Fu recombinant plasmid is capable of shuttling between bacteria and insect cells, and when entered into insect cell Sf9, is capable of recombination to produce the recombinant baculovirus gene rBac-m.bovis-Fu. The recombinant baculovirus gene rBac-M.bovis-Fu contains a baculovirus recombinant gene and a fusion gene (M.bovis-Fu).

The above-described cells containing the fusion gene are capable of expressing the fusion gene to form a fusion protein. When the nucleotide sequence of the fusion gene corresponding to the fusion protein is the first sequence, the theoretical molecular weight of the fusion protein expressed by the recombinant baculovirus gene rBac-M.bovis-Fu is 47kDa. If the nucleotide sequence of the fusion gene corresponding to the fusion protein is the second sequence, the theoretical molecular weight of the fusion protein expressed by the recombinant baculovirus gene rBac-M.bovis-Fu is converted according to the difference of the second sequence.

The fusion proteins can be used to prepare mycoplasma bovis subunit vaccines. The amino acid sequence of the fusion protein may be selected from any one of the following sequences:

Wherein the third sequence is formed by concatenating a truncated amino acid sequence of glycerol kinase on the surface of a cell membrane of mycoplasma bovis and a truncated amino acid sequence of glycerol-3-phosphate dehydrogenase. The two amino acid sequences are directly linked, rather than via an amino acid linker fragment (e.g., an amino acid sequence that serves as a linker). Because the fusion protein is formed by directly connecting a first amino acid sequence with glycerol kinase activity and a second amino acid sequence with glycerol-3-phosphate dehydrogenase activity in series, and the glycerol kinase and the glycerol-3-phosphate dehydrogenase are key enzymes for generating energy by using glycerol as a carbon source for mycoplasma bovis, the direct connection of the amino acid sequences with the two enzyme activities enables the active center positions of the two enzymes to be closer, and has stronger immune activity and better stability compared with a whole-cell vaccine.

The fusion protein can be expressed by a first sequence or a second sequence, or can be expressed by the first sequence or the second sequence, and then the expressed sequence is subjected to amino acid substitution, insertion, truncation or extension and other processing by adopting protein engineering. But it should be ensured that the amino acid sequence after processing has at least 95% similarity to the amino acid sequence originally expressed.

In addition, in addition to using the original amino acid sequences of the two enzymes to make a fusion protein having a third sequence, a fourth sequence having at least 95% similarity to the third sequence may be used to make the fusion protein. The fourth sequence may have at least 96% similarity to the third sequence, may have at least 97% similarity to the third sequence, may have at least 98% similarity to the third sequence, or may have at least 99% similarity to the fourth sequence. Similarity is defined as the ratio of the number of amino acids in the fourth sequence that are identical to the third sequence to the total number of amino acids in the third sequence. The fourth sequence has one or more amino acid substitutions, deletions or insertions relative to the third sequence, the number of nucleotides being substituted, deleted or inserted being such that the fourth sequence has at least 95% similarity to the third sequence. If the similarity is too low, the fusion protein characterized by the fourth sequence has poor immunological activity and it is difficult to prepare subunit vaccines with the desired immunological activity. Substitution or insertion is required to ensure substitution or insertion of the identical amino acid, but substitution or insertion of the specific amino acid cannot be adopted, otherwise the active center of the enzyme is affected, and thus the immunocompetence is affected.

Illustratively, the acidic amino acids may be replaced with each other, the basic amino acids may be replaced with each other, and the neutral amino acids may be replaced with each other, but the acidic amino acids and the basic amino acids may not be replaced with each other so as not to affect the catalytic function of the enzyme. For another example, the amino acid near the insertion site is an acidic amino acid, and the amino acid at the insertion site should also be an amino acid identical thereto, i.e., an acidic amino acid. Insertion of basic amino acids or neutral amino acids is the same.

The mycoplasma bovis subunit vaccine is prepared by extracting key protein components of pathogenic immunogens of mycoplasma bovis, and compared with the whole protein of the pathogenic immunogens, the key protein components have more stable amino acid sequences and have stronger and better specific immune activity. The mycoplasma bovis subunit vaccine contains no nucleic acid, and can induce the host to produce humoral immunity after being injected into the host, so that the number of antibodies produced in the host is more than that of the whole cell vaccine, and the specificity of the antibodies is stronger than that of the whole cell vaccine. Key protein components include glycerol kinase and glycerol-3-phosphate dehydrogenase of mycoplasma bovis. The full-length amino acid sequence of the glycerol kinase of mycoplasma bovis is shown in SEQ ID NO:7, the full-length amino acid sequence of the glycerol-3-phosphate dehydrogenase is shown as SEQ ID NO: shown at 8. In comparison with the two sequences, the third sequence of the present application is formed by concatenating the truncated amino acid sequence of glycerol kinase on the surface of the cell membrane of Mycoplasma bovis and the truncated amino acid sequence of glycerol-3-phosphate dehydrogenase.

The inventors found in the study that the mechanism of mycoplasma bovis infection in healthy cattle is closely related to the way mycoplasma bovis metabolizes carbon using a carbon source. Lipid metabolism in animals produces glycerol, the primary carbon source for mycoplasma bovis. Specific glycerol kinase and glycerol-3-phosphate dehydrogenase exist on the surface of cell membranes of mycoplasma bovis of different species. Glycerol kinase is capable of catalyzing glycerol to produce glycerol 3-phosphate (also known as glycerol-3-phosphate), and glycerol-3-phosphate dehydrogenase is capable of catalyzing glycerol 3-phosphate with the aid of coenzyme I (nicotinamide adenine dinucleotide) and producing dihydroxyacetone phosphate (Dihydroxacetone Phosphate, DHAP) and nicotinamide adenine dinucleotide in a reduced state (Nicotinamide Adenine Dinucleotide, NADH). The reduced nicotinamide adenine dinucleotide is capable of reducing dihydroxyacetone phosphate in the glycolytic pathway and releasing energy for ATP synthesis, thereby providing energy and substances required for metabolism for mycoplasma bovis metabolism. Since glycerol kinase and glycerol-3-phosphate dehydrogenase are specifically present on the cell membrane of mycoplasma bovis, the released energy can promote the binding of other functional proteins on the cell membrane surface of mycoplasma bovis to host cells (such as respiratory tract mucosal cells, genital tract mucosal cells, etc. of cattle), and thus glycerol kinase and glycerol-3-phosphate dehydrogenase are involved in adhesion between mycoplasma bovis and host cells, and have immunogenicity with a certain specificity and sensitivity, can be used for preparing mycoplasma bovis subunit vaccines.

After the mycoplasma bovis subunit vaccine is inoculated into animals such as cattle, the animals such as cattle can generate humoral immunity, namely antibodies against glycerol kinase and glycerol-3-phosphate dehydrogenase are generated in body fluid. After the living mycoplasma bovis enters the cattle body, the antibody in the cattle body fluid can be combined with glycerol kinase and glycerol-3-phosphate dehydrogenase on the surface of the mycoplasma bovis, so that the mycoplasma bovis cannot utilize glycerol as a carbon source to generate energy, and the cell of the cattle cannot be well adhered, so that the mycoplasma bovis cannot survive in the animal body, the animal cannot be infected by the mycoplasma bovis, and the vaccine effect is achieved.

Subunit vaccine

The present application provides a subunit vaccine comprising the fusion protein described above and an adjuvant.

Wherein the concentration of the fusion protein in the subunit vaccine is 100+/-10 mug/ml.

The adjuvant comprises one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, MONTANIDE ISA 51 VG, liquid paraffin, squalane, saponin, vegetable oil, and cytokine, preferably MONTANIDE ISA 206 VG. The immunogenicity of Subunit vaccine (subnit vaccinee) is greatly improved after the Subunit vaccine is matched with the adjuvant.

The application adopts a baculovirus insect cell expression system to express mycoplasma bovis M.bovis-Fu fusion protein, and prepares a genetic engineering subunit vaccine (subnit vaccinee) through the fusion protein. The subunit vaccine disclosed by the application can block the glycerol metabolic pathway of mycoplasma bovis by simultaneously inhibiting the enzyme activities of GPDH and GK on the cell membrane surface of mycoplasma bovis, so that the pathogenicity of mycoplasma bovis in animals is influenced. Through various experimental detection, the subunit vaccine can generate stronger humoral immunity in an animal body, the immunized animal can resist virulent attack, and the bioreactor can be used for carrying out amplified production, so that the production cost of the vaccine is greatly reduced.

The present application is further illustrated below with reference to examples.

EXAMPLE construction of pF-M.bovis-Fu transfer vector

The present example provides a method for constructing pF-M.bovis-Fu transfer vector, comprising the steps of:

1. constructing a pUC-M.bovis-Fu plasmid vector, and amplifying and purifying the M.bovis-Fu fusion gene by using the pUC-M.bovis-Fu plasmid vector to obtain a purified M.bovis-Fu fusion gene; the method specifically comprises the following steps:

(1-1) an M.bovis-Fu fusion gene (sequence shown as SEQ ID NO: 1) was synthesized in Nanjing Jinsri Biotechnology Co., ltd.) and cloned into pUC17 vector to obtain pUC-M.bovis-Fu plasmid vector.

(1-2) PCR amplification was performed using pUC-M.bovis-Fu plasmid as a template, M.bovis-Fu-F as an upstream primer, and M.bovis-Fu-R as a downstream primer. The gene sequence of M.bovis-Fu-F is shown as SEQ ID NO. 5, and the gene sequence of M.bovis-Fu-R is shown as SEQ ID NO. 6. The PCR amplification system is shown in Table 1.

TABLE 1 PCR amplification System of the genes of bovis-Fu

The amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 94℃for 45 seconds, annealing at 54℃for 45 seconds, elongation at 72℃for 1 minute, 35 cycles; extension was carried out at 72℃for 10 minutes.

In order to verify whether the target gene obtained after amplification is an M.bovis-Fu fusion gene, gel electrophoresis was performed on the PCR product obtained after amplification. As shown in FIG. 1, the gel electrophoresis pattern has 3 lanes, lane 1 is the lane where the target gene is located, lane 2 is the blank, and lane 3 (M) is the lane of the standard (Marker). Lane 1 shows a target band at the 1.3kbp position, the molecular weight corresponding to the theoretical molecular weight of the M.bovis-Fu fusion gene, indicating successful amplification of the target gene.

(1-3) recovering and purifying by using a gel recovery and purification kit to obtain the purified M.bovis-Fu fusion gene.

2. The pFastBac 1 plasmid and the M.bovis-Fu gene were digested with BamHI and HindIII enzymes, respectively, and then the digested pFastBac 1 plasmid and the digested M.bovis-Fu were ligated using T4 DNA ligase, transformed and amplified with DH 5. Alpha. Competent cells, and pF-M.bovis-Fu transfer vector with the correct sequence was selected. The method specifically comprises the following steps:

(2-1) the pFastBac 1 plasmid was digested with the cleavage reaction system shown in Table 2, and the PCR-amplified product of the M.bovis-Fu gene (i.e., the purified M.bovis-Fu fusion gene obtained in step 1) was digested with the cleavage reaction system shown in Table 2. Each cleavage reaction was double digested with BamHI and HindIII at 37℃for 3 hours.

TABLE 2 pFastBac 1 plasmid cleavage reaction System

Table 3M bovis-Fu Gene cleavage reaction System

(2-2) subjecting the two digested products to gel electrophoresis, respectively, and purifying and recovering the digested pFastBac 1 plasmid and the digested M.bovis-Fu gene fragment, respectively, using a gel recovery and purification kit.

(2-3), the digested pFastBac 1 plasmid and the digested M.bovis-Fu gene fragment (also known as the M.bovis-Fu gene cleavage product) were ligated overnight using T4 DNA ligase in a water bath at 16 ℃. The connection system is shown in Table 4.

TABLE 4M plasmid ligation System for the genes bovis-Fu and pFastBac 1

(2-4) 10. Mu.l of the ligation product obtained in the step (2-3) was added to 100. Mu.l of DH 5. Alpha. Competent cells, mixed well, heat-shocked at 42℃for 90 seconds, ice-bathed for 2 minutes, and then 900. Mu.l of LB medium without Amp was added thereto, followed by culturing at 37℃for 1 hour. 1.0mL of the bacterial liquid was concentrated to 100. Mu.L by centrifugation, and the concentrated solution was spread on LB solid medium containing Amp, and incubated at 37℃for 16 hours.

(2-5), single colonies on a flat-plate LB solid medium were picked up and inoculated with LB liquid medium, respectively, cultured at 37℃for 2 hours, and colony PCR was performed using the bacterial liquid as a template and M.bovis-Fu-F and M.bovis-Fu-R as primers. Gel electrophoresis is carried out on colony PCR products to verify the size of the target gene. As shown in FIG. 2, the gel electrophoresis pattern had 8 lanes, and the colony PCR products corresponding to the 6 colonies (i.e., 6 replicates) picked in lanes 1 to 6 each had a band around 1.3kbp, indicating that these 6 samples were positive samples. Lane 7 is a blank control lane, lane 8 (lane M) is a standard (Marker) lane for displaying whether the remaining lanes are present with a corresponding molecular weight.

(2-6), sequencing bacterial liquid with positive colony PCR identification result (namely bacterial liquid corresponding to a sample with a band around 1.3 kbp) by a sequencing company, and selecting bacterial liquid corresponding to a sample with correct sequencing for preservation. The bacterial liquid with correct sequence contains pF-M.bovis-Fu transfer vector with target gene (M.bovis-Fu fusion gene), and the sequence composition is shown in figure 3.

EXAMPLE two construction of Bacmid-M.bovis-Fu recombinant plasmid (containing baculovirus genome)

The embodiment provides a Bacmid-M.bovis-Fu recombinant plasmid, and the construction method comprises the following steps:

1. adding pF-M.bovis-Fu transfer vector into DH10Bac competent cells for transformation to obtain a transformant; the method specifically comprises the following steps:

mu.l of pF-M.bovis-Fu plasmid in example 1 was added to 100. Mu.l of DH10Bac competent cells, mixed well, ice-bathed for 30 minutes, heat-shocked in a water bath at 42℃for 90 seconds, ice-bathed for 2 minutes, and 900. Mu.l of LB liquid medium without Amp was added to culture at 37℃for 5 hours. After diluting 100. Mu.l of the bacterial liquid 81-fold, 100. Mu.l of the diluted bacterial liquid was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37℃for 48 hours to obtain a transformant.

2. Screening the transformant to obtain a Bacmid-M.bovis-Fu recombinant plasmid, which specifically comprises the following steps:

white colonies with large areas are picked up by using an inoculating needle, streaked on LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, cultured for 48 hours at 37 ℃, then single colonies (monoclonal) are picked up and inoculated on LB liquid culture medium containing gentamicin, kanamycin and tetracycline for culturing, strains are preserved, plasmids are extracted, and the Bacmid-M.bovis-Fu recombinant plasmids are obtained.

Example III obtaining recombinant baculovirus Gene

The embodiment provides a method for obtaining a recombinant baculovirus gene, which comprises the following steps:

1. 0.8X10 cells were seeded in six well plates ⁶ The confluence of Sf9 cells was 50-70%. The following complexes were prepared for each well: mu.l Cellfectin transfection reagent diluted with 100. Mu.l transfection medium T1Briefly vortexing; mu.g of the recombinant Bacmid-M.bovis-Fu plasmid obtained in example II was diluted with 100. Mu.l of the transfection medium T1, and the diluted transfection reagent and the Bacmid-M.bovis-Fu plasmid were mixed and gently blown to prepare a transfection mixture.

2. After Sf9 cells are attached, the transfection complex is added, the mixture is incubated for 5 hours at 27 ℃, the supernatant is removed, 2mLSF-SFM fresh culture medium is added, the culture is carried out for 4 to 5 days at 27 ℃ and the supernatant is harvested, and the recombinant baculovirus gene rBac-M.bovis-Fu is obtained.

After transfection of Sf9 cells with the recombinant baculovirus gene rBac-m.bovis-Fu, sf9 cells express the recombinant baculovirus gene rBac-m.bovis-Fu to form a fusion protein, which is contained in the cell culture of transfected Sf9 cells.

The virus content of the harvested F1 generation recombinant baculovirus is detected by an indirect immunofluorescence method, and the virus content of rBac-M.bovis-Fu F1 generation virus is 1.83 multiplied by 10 ⁸ TCID ₅₀ . Amplifying recombinant baculovirus rBac-M.bovis-Fu as seed virus for later use.

In addition, recombinant baculoviruses expressing the control group shown in the following table 5 were constructed according to the above example method. In Table 5, the sequence shown in SEQ ID NO. 4 is the full-length nucleotide sequence of GDPH, and the expressed protein is the full-length amino acid sequence of GDPH. The sequence shown in SEQ ID NO. 3 is the full-length nucleotide sequence of GK, and the expressed protein is the full-length amino acid sequence of GK. The full length amino acid sequence of GDPH and the full length amino acid sequence of GK served as controls for the fusion proteins of the present application.

Table 5 group set for control group

EXAMPLE four SDS-PAGE detection

The present example provides a method for detecting cell cultures of transfected sf9 cells, which is mainly aimed at detecting whether the cell cultures of transfected cells contain fusion proteins expressed by the gene of interest.

The method of this example was carried out by performing SDS-PAGE on the cell cultures of Sf9 cells obtained in example III and containing the recombinant baculovirus gene (rBac-M.bovis-Fu), sf9 cells of control group 1 containing the GPDH full-length gene, and Sf9 cells of control group 2 containing the GK full-length gene, while using Sf9 cells infected with empty baculovirus as a negative control. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. Mu.l of 5×loading buffer was added, the mixture was subjected to boiling water bath for 5 minutes, centrifugation was carried out at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% concentration gel) electrophoresis, and after electrophoresis, the gel was stained and decolorized to observe a target band.

As shown in FIG. 4, the protein band pattern of this example has a total of 5 lanes. The band shown in lane 1, in which the target band appears at a molecular weight of about 57kDa, was obtained from the cell culture of control group 2, and indicated that the cell culture of control group 2 contained GK protein (having the full-length amino acid sequence). The band shown in lane 2, in which the target band appears at a molecular weight of about 37kDa, was obtained from the cell culture of control group 1, and indicated that the cell culture of control group 1 contained GDPH protein (with the full-length amino acid sequence). The band indicated in lane 3, in which the target band appears at a molecular weight of about 47kDa, was obtained from cell cultures from the experimental group, indicating that the cell cultures from the rBac-M.bovis-Fu experimental group contain a fusion protein expressed by the M.bovis-Fu fusion gene, which is made up of a truncated GK protein in tandem with a truncated GDPH protein. Lane 4 is a negative control, and no target bands appear at 37kDa, 47kDa and 57kDa, suggesting that the negative control group does not express the three proteins. Lane 5 is a Marker lane for displaying the relevant molecular weight. The above results demonstrate that the fusion protein is correctly expressed in Sf9 cells.

Example five Western Blot detection

The embodiment provides a method for carrying out Western Blot detection on a product after SDS-PAGE electrophoresis in the fourth embodiment, which specifically comprises the following steps: the product after SDS-PAGE electrophoresis of example four was transferred onto nitrocellulose membrane (NC membrane), blocked with 5% nonfat milk powder for 2 hours, incubated for 2 hours with rabbit anti-M.bovis positive serum, rinsed, incubated for 2 hours with HRP-labeled goat anti-rabbit polyclonal antibody as secondary antibody, rinsed, then added dropwise with enhanced chemiluminescent fluorogenic substrate, photographed using a chemiluminescent imager, and the results of FIG. 5 were obtained.

As shown in FIG. 5, the protein band pattern of this example has a total of 5 lanes. The band shown in lane 1, in which the target band appears at a molecular weight of about 57kDa, was obtained from the cell culture of control group 2, and indicated that the cell culture of control group 2 contained GK protein (having the full-length amino acid sequence). The band shown in lane 2, in which the target band appears at a molecular weight of about 37kDa, was obtained from the cell culture of control group 1, and indicated that the cell culture of control group 1 contained GDPH protein (with the full-length amino acid sequence). The band indicated in lane 3, in which the target band appears at a molecular weight of about 47kDa, was obtained from cell cultures from the experimental group, indicating that the cell cultures from the rBac-M.bovis-Fu experimental group contain a fusion protein expressed by the M.bovis-Fu fusion gene, which is made up of a truncated GK protein in tandem with a truncated GDPH protein. Lane 4 is a negative control, and no target bands appear at 37kDa, 47kDa and 57kDa, suggesting that the negative control group does not express the three proteins. Lane 5 is a Marker lane for displaying the relevant molecular weight.

In the above results, the recombinant baculovirus expression samples all had the target bands, and the negative control had no target bands, indicating that the target fusion protein was expressed correctly in Sf9 cells.

Example six insect cell bioreactor serum-free suspension culture

Example three laboratory pilot cultures of transfected Sf9 cells were performed and experiments in examples four and five demonstrated that the pilot cultures contained the target fusion protein, indicating that the pilot results gave the correct expression of the target fusion protein. In order to further expand the expression level of the fusion protein of interest, it is necessary to use a bioreactor for the amplification culture in order to provide conditions and parameters for industrial applications.

The present example provides a method for the scale-up culture of transfected Sf9 cells using a bioreactor comprising the steps of:

1. aseptically culturing Sf9 insect cells in 1000mL shake flask for 3-4 days until the concentration reaches 3-5×10 ⁶ Inoculating Sf9 insect cells into 5L bioreactor at a concentration of 3-8X10 when cell/mL and activity is greater than 95% ⁵ cell/mL. When the concentration of Sf9 insect cells reaches 3-55×10 ⁶ At cell/mL, sf9 insect cells are inoculated into a 50L bioreactor, and the Sf9 insect cells grow to a concentration of 3-55 multiplied by 10 ⁶ Inoculating the cell/mL into a 500L bioreactor, and continuing cell culture;

2. when the cell concentration of the insect cells in the Sf9 bioreactor of 500L reaches 2 to 85 multiplied by 10 ⁶ And (3) inoculating rBac-M.bovis-Fu recombinant baculovirus for transfection in the cell/mL, wherein the culture condition of the bioreactor is that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80%, and the stirring speed is 100-180rpm. In consideration of the optimum conditions for cell culture, it is preferable that the pH is 6.2, the temperature at the cell culture stage is set at 27℃and the dissolved oxygen is 50%, and the stirring speed is 100-180rpm.

3. After further culturing for 5-9 days after transfection, BEI at one thousandth of the final concentration is added, and after 48 hours of action at 37 ℃, na at two thousandth of the final concentration is added ₂ S ₂ O ₃ Inactivation was terminated. The cell culture supernatant was harvested by centrifugation or hollow fiber filtration and stored at 2-8deg.C (i.e., M.bovis-Fu protein stock).

For the verification comparison experiments, protein stocks expressing control group 1 and control group 2 may also be prepared in the same manner.

In this embodiment, since the volume of the bioreactor is relatively large, and it is difficult to achieve complete and precise control of various experimental parameters in different areas, the experimental parameters are suitably represented by range values.

EXAMPLE seven protein purification

The M.bovis-Fu protein stock solution obtained in example six contains many impurities in addition to the M.bovis-Fu fusion protein, and therefore, purification is required.

This example provides a method for purifying the M.bovis-Fu protein stock solution obtained in example six, comprising the steps of:

ultrasonically crushing one of the M.bovis-Fu protein stock solutions obtained in the sixth embodiment, centrifuging for 30 minutes at 12000r/min, collecting supernatant, filtering with a 0.22 μm filter membrane, removing impurities, and concentrating for 10 times by using an ultrafiltration tube with a molecular weight cutoff of 10kDa to obtain purified target protein.

The purified target protein was quantified using BCA total protein, and then the purity of the target protein was determined in combination with gray-scale scanning, resulting in a concentration of 830. Mu.g/mL of M.bovis-Fu fusion protein with a purity of 98%. This demonstrates that the use of the bioreactor of example six successfully resulted in a highly pure M.bovis-Fu fusion protein.

Example eight subunit vaccine preparation

The embodiment provides a preparation method of subunit vaccine. The method comprises the following steps:

the purified M.bovis-Fu fusion protein liquid obtained in the seventh embodiment is diluted according to a certain proportion, and then added into MONTANIDE ISA 206 VG adjuvant, so that the concentration of the fusion protein in the final emulsified vaccine is 100 mug/ml, and the vaccine is preserved at 4 ℃ after quality inspection is qualified.

For the verification comparison experiment, the protein stock solutions of the control group 1 and the control group 2 can be mixed in a ratio of 1:1 to prepare a vaccine according to the method, and the vaccine is taken as the control group A and is stored at 4 ℃.

Thus, the vaccine of the experimental group contains fusion proteins, which are formed by concatenating truncated GK protein and truncated GDPH protein. The vaccine of the control A group is formed by mixing protein stock solutions of the control 1 and the control 2 in equal proportion, so the control A group contains two proteins, including GK protein with full-length amino acid sequence and GDPH protein with full-length amino acid sequence, and the two proteins are not connected in series, but exist in the vaccine independently.

The relevant properties of the subunit vaccine of the present application were verified by various immunological experiments as follows.

Test example 1 vaccine safety test

The purpose of this test example is to verify the biosafety of the vaccine of this application, the method of verification comprising the steps of:

adult healthy Balb/c mice were selected 10, each about 25 g. The cells were randomly divided into 2 groups of 5 cells each. One group is an immunized group, each mouse is intramuscular injected with 1.0mL of mycoplasma bovis subunit vaccine of the invention, one group is a negative control group, and each mouse is injected with the same amount of physiological saline in the same way. The body temperature of the mice was measured and the mental state of the mice was observed for 7 consecutive days.

The results show that all mice have no symptoms of fever, inappetence, coarse fur, listlessness and the like, and have no death condition. The vaccine of the invention is proved to meet the safety requirement. As mentioned above, if an animal is infected with Mycoplasma bovis, the animal may develop symptoms such as elevated body temperature, mental depression, asthma, clear flow or purulent nasal juice, which may be an indication of whether the animal is infected with Mycoplasma bovis.

Test example 2 antibody level detection test

The purpose of this test example was to infect healthy cattle using the vaccine of this application to detect whether the vaccine of this application could produce antibodies to healthy cattle, thereby validating the immune activity of the vaccine of this application.

The test example inspection method comprises the following steps:

15 healthy calves with ages of 10-15 days are randomly divided into 3 groups, and 5 calves are selected from each group. Group 1 is an immunization group, and each calf was injected with 3mL of subunit vaccine (containing fusion protein) via neck muscle. Group 2 is control group a, and each calf was injected with 3mL of the common vaccine (containing an equal proportion of the mixture of full length GK protein and full length GDPH protein) in the neck muscle. Group 3 is a blank control group, and each calf is injected with the same amount of physiological saline in the same way. Secondary immunization (secondary immunization) was performed at the same dose 10 days after the primary immunization (primary immunization). Blood is collected through jugular vein and serum is separated before first, 10 days after first and 14 days after second, and antibody detection is carried out by using mycoplasma bovis antibody ELISA detection kit. The results are shown in Table 6.

TABLE 6 antibody level detection results

Note that: when the S/P value of the sample to be detected is more than or equal to 0.418, judging that the sample to be detected is positive; and when the S/P value of the sample to be detected is less than 0.418, judging that the sample to be detected is negative. S/P value= (sample-n)/(active-n).

As can be seen from table 6, after the systematic errors of the blank control group are removed, the immune group generates more than 2 times of the antibody level of the control group a after the first and second immunity, which indicates that the vaccine of the present application has good immune activity, and the immune activity is far higher than the mixture of the full-length GK protein and the full-length GDPH protein.

Test example 3 complement sterilization test

In test example 2, the relevant vaccine was injected into healthy cattle, and then, humoral immunity of healthy cattle was induced, that is, antibodies were produced in blood. The aim of this test was to examine the in vitro sterilizing capacity of antibodies in cattle against mycoplasma. In order to enhance the bactericidal effect of the antibody on mycoplasma bovis, complement was added. Complement is a commercial product derived from the freeze-dried complement product of the national veterinary drug institute.

The specific method of the test example comprises the following steps:

the bovine serum and the bovine negative serum before immunization were taken 14 days after the second immunization of each group, and complement sterilization was performed. Positive, complement and blank groups were set simultaneously, and the reaction systems of each group are shown in table 7. After the reaction, the solutions of each group are diluted by ten times ratio and respectively taken 10 ⁴ 、10 ⁵ 、10 ⁶ 100. Mu.L of the diluted suspension was plated on mycoplasma bovis solid medium, 3 media were plated for each dilution, colony counts were performed after 5-7 days of incubation, and the test was repeated 3 times. And the sterilization rate is calculated according to the following formula: sterilization rate (%) = [ |test group CFU-negative group cfu|/complement group CFU]100% and the specific test results are shown in Table 7.

TABLE 7 complement sterilization test reaction System and results

In the test example, mycoplasma bovis PG45 is used as a test strain. The experimental results are described below:

blank group PBS liquid was added to mycoplasma bovis suspension as a blank control.

Complement group additionally added complement in mycoplasma bovis suspension, the average value of the cultured colony is approximately equal to that of the blank group, which shows that complement itself has no effect of killing mycoplasma bovis basically.

Negative group pre-priming bovine serum was added to mycoplasma bovis suspension. Because the pre-immune serum is not subjected to humoral immunity, and does not contain antibodies against mycoplasma bovis, the pre-immune serum has no effect of killing mycoplasma bovis basically, so that the average value of the cultured colonies is approximately equal to that of a blank group and a complement group.

Control group A added bovine serum 14 days after the second immunization to the mycoplasma bovis suspension. The vaccine of the control A group which adopts the equal ratio mixture of the full-length GK protein and the full-length GDPH protein enables healthy cattle to generate humoral immunity, the sterilization average value of the vaccine is 46.69%, which indicates that the vaccine plays a certain role in sterilization, but the vaccine has low immunocompetence and is difficult to be applied commercially.

The immune group of the invention adds bovine serum 14 days after the secondary immunization into mycoplasma bovis suspension. The immune group adopts vaccine containing fusion protein to make healthy cattle produce humoral immunity, and the average sterilization value is 85.54%, so that the sterilization effect of positive group vaccine is basically achieved. The experimental results show that: the mycoplasma bovis subunit vaccine can generate specific antibodies after stimulating humoral immunity of healthy cattle, so that the mycoplasma bovis subunit vaccine has good immune activity.

Test example 4 adhesion inhibition test

After mycoplasma bovis enters the animal body, glycerol Kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) on the cell membrane provide energy for its adhesion to the animal cell surface using glycerol as a carbon source. The antibody produced after humoral immunity can be specifically combined with Glycerol Kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH), so that the glycerol is difficult to provide energy, and mycoplasma bovis is difficult to adhere to the cell membrane of animal cells, and the infectious capacity of mycoplasma bovis is reduced. The aim of this test example is to investigate the inhibition of the adhesion capacity of serum after humoral immunity to mycoplasma bovis, comprising the following steps:

1. adding mycoplasma bovis PG45 suspension into a 1.5mL centrifuge tube according to the infection amount of 500MOI, adding bovine serum of the inactivated immune group calf of the invention after 14 days after the double-immunity treatment, so that the ratio of the serum to the total volume is 1:100, and carrying out water bath at 37 ℃ for 1h. Control a, positive, negative and blank groups were set up at the same time.

2. After centrifugation, washing, EBL cells that had been pretreated were infected by resuspension with 1mL of pre-warmed serum-free DMEM, allowed to act for 2h at 37℃and after 5 washes with sterile PBS digested with 200. Mu.L trypsin. After complete digestion, complete nutrient solution was added to terminate digestion under a microscope.

3. After collecting cells, centrifuging and washing, adding 1mL of preheated mycoplasma bovis culture medium, and standing at room temperature for 10min. The solutions of each group are diluted by ten times ratio and respectively taken to be 10 ⁴ 、10 ⁵ 、10 ⁶ 100. Mu.L of the diluted suspension was plated on mycoplasma bovis solid medium, 3 media were plated for each dilution, colony counts were performed after 5-7 days of incubation, and the test was repeated 3 times. And the adhesion inhibition rate was calculated according to the following formula: adhesion inhibition (%) = [ |test group CFU-negative group cfu|/blank group CFU]100% and the test results are shown in Table 8.

TABLE 8 adhesion inhibition test results

From the results in Table 8, the adhesion inhibition rate of the Mycoplasma bovis by the immunized group of the invention is far higher than that of the control A group, which shows that the fusion protein of the invention can inhibit the adhesion of Mycoplasma bovis, thereby reducing the strong toxicity of Mycoplasma bovis.

The foregoing has outlined rather broadly the more detailed description of the present application, wherein specific examples have been provided to illustrate the principles and embodiments of the present application, the description of the examples being provided solely to assist in the understanding of the method of the present application and the core concepts thereof; meanwhile, those skilled in the art will have variations in the specific embodiments and application scope in light of the ideas of the present application, and the present description should not be construed as limiting the present application in view of the above.

Sequence listing

<110> Suzhou Shino Biotechnology Co., ltd

<120> fusion gene, fusion protein, preparation method and mycoplasma bovis subunit vaccine

<130> SUP210103CN

<141> 2021-09-10

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ccgagcttta ccggcctggg cagcccgtat tgggatagct atagccgcgg cgcgattttt 480

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Val Thr Phe Pro His Leu Phe Ser Arg Asp Asn Ala Ser Glu Ile Arg

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Ile Thr Ser Ser Ile Gly Asp Gln Gln Ser Ala Leu Phe Gly Gln Leu

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Cys Leu Glu Lys Gly Gln Ser Lys Ile Thr Tyr Gly Thr Gly Cys Phe

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Ile Leu Thr Asn Thr Gly Ser Gln Ile Val Lys Ser Asn His Gly Leu

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Leu Thr Thr Ile Ala Tyr Ser Phe Lys Asp Lys Ile Leu Tyr Ala Leu

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Glu Gly Ser Val Met Ile Ala Gly Ala Ala Val Gln Trp Leu Arg Asp

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Asn Leu Lys Ile Val Tyr Ser Ala Leu Glu Thr Glu Trp Tyr Ala Gly

115 120 125

Gln Val Asn Asp Asp Arg Arg Val Tyr Val Val Pro Ser Phe Thr Gly

130 135 140

Leu Gly Ser Pro Tyr Trp Asp Ser Tyr Ser Arg Gly Ala Ile Phe Gly

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Leu Asp Arg Gly Thr Lys Arg Glu His Ile Val Arg Ala Thr Leu Glu

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Ala Ile Ala Tyr Gln Ala Asn Asp Val Val Ser Ala Met Gly Lys Asp

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Met Asn Asp Pro Ile Lys Ile Phe Lys Val Asp Gly Gly Ala Ser Asn

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Asn Lys Phe Met Met Gln Phe Gln Ser Asn Ile Ser Gln Ser Lys Val

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

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Ala Gly Leu Ala Thr Gly Tyr Trp Lys Ser Ala Leu Ser Glu Leu Asp

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Phe Met Ile Leu Ala Val Pro Ser Ser Ala Ile Asp Ser Val Leu Gly

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Lys Ile Gly Asp Val Leu Gly Thr Gln Lys Ile Lys Val Ile Asn Val

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Ala Lys Gly Ile Asp Ser Lys Thr Lys Lys Phe Phe Ser Asp Val Leu

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Val Glu Lys Phe Ser Ser Asn Ile Glu Gln Tyr Cys Ser Ile Leu Gly

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Pro Ser Phe Ala Thr Glu Val Phe Glu Asn Ala Leu Thr Met Ile Asn

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Val Val Gly Pro Asn Glu Gln Phe Leu Thr Glu Val Ser Gln Thr Phe

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Asn Asn Lys Tyr Phe Arg Leu Val Val Asn Ser Asp Glu Lys Gly Ser

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

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

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

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Ser Asn Asn Glu Leu Pro Leu Glu Leu Ser Ala Ile Gly

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ctgaaaattg tgtatagcgc gctggaaacc gaatggtatg cgggccaggt gaacgatgat 1020

cgccgcgtgt atgtggtgcc gagctttacc ggcctgggca gcccgtattg ggatagctat 1080

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attaacagcg gcattaacag caaatatttt ggcaaccgcc gctttaacaa cccggaaaac 180

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gtgccgagca gcgcgattga tagcgtgctg ggcaaaattg gcgatgtgct gggcacccag 300

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gatgtgctgg tggaaaaatt tagcagcaac attgaacagt attgcagcat tctgggcccg 420

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gaacagtttc tgaccgaagt gagccagacc tttaacaaca aatattttcg cctggtggtg 540

aacagcgatg aaaaaggcag cgaactgttt gcggcgctga aaaacgtgct ggcgattggc 600

attggcgcga ttacctatat gcatccgtat aaaaacaccg aaagcgcgct gctggcggcg 660

ggcgcgaaag aaatttatca gatttataaa gaactgctga aaaccagcaa caacgaactg 720

ccgctggaac tgagcgcgat tggcgatatt tttctgacct gcagcagcct gaaaagccgc 780

aactttctgt ttggcaccca gattgcggaa aaaggcctga aaaccgtgct ggaagaaaac 840

accaaaaccg tggaaggcta tcataacgcg aaaattctgg aagaaattct gaacgataac 900

cagagcatta acgcgccgtt tctgcgcagc attattgatg tgctgtatca taacaaagat 960

gtgcataaac tgaccgattt tattgaaaaa tataac 996

<210> 5

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 5

ataggatcca tgccgcgcag cattctgccg gaaattaaa 39

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 6

ataaagcttt tagccaatcg cgctcagttc cagcgg 36

<210> 7

<211> 502

<212> PRT

<213> Mycoplasma bovis

<400> 7

Met Glu Lys Tyr Ile Leu Thr Leu Asp Glu Gly Thr Thr Ser Ala Arg

1 5 10 15

Thr Leu Ile Val Asn Lys Lys Gly Glu Ile Ile Ala Val Glu Gln Ala

20 25 30

Glu Phe Thr Gln His Phe Pro Lys Glu Gly Trp Val Glu His Asp Ala

35 40 45

Ile Glu Ile Trp Asn Thr Gln Arg Ser Thr Leu Val Gln Val Leu Asn

50 55 60

Lys Ser Lys Ile Ser Pro Glu Gln Ile Asp Ser Ile Gly Ile Thr Asn

65 70 75 80

Gln Arg Glu Thr Val Val Ile Trp Asn Lys Glu Thr Gly Leu Pro Ile

85 90 95

Tyr Asn Ala Ile Val Trp Gln Asp Gln Arg Thr Ala Glu Tyr Cys Gln

100 105 110

Ser Phe Asn Lys Glu Gln Leu Glu Leu Val Lys Glu Lys Thr Gly Leu

115 120 125

Ile Ile Asn Pro Tyr Phe Ser Gly Thr Lys Val Lys Trp Ile Leu Asp

130 135 140

Asn Val Pro Asn Ala Arg Glu Leu Ala Ala Glu Gly Lys Leu Met Phe

145 150 155 160

Gly Thr Ile Asn Thr Trp Leu Ile Tyr Arg Leu Thr Gly Gly Lys Val

165 170 175

Phe Ala Thr Asp His Thr Asn Ala Gln Arg Thr Leu Leu Tyr Asn Ile

180 185 190

His Thr Asn Asp Trp Asp Asn Asp Leu Leu Glu Leu Phe Asn Ile Pro

195 200 205

Arg Ser Ile Leu Pro Glu Ile Lys Ser Cys Ser Glu Asp Tyr Gly Val

210 215 220

Thr Phe Pro His Leu Phe Ser Arg Asp Asn Ala Ser Glu Ile Arg Ile

225 230 235 240

Thr Ser Ser Ile Gly Asp Gln Gln Ser Ala Leu Phe Gly Gln Leu Cys

245 250 255

Leu Glu Lys Gly Gln Ser Lys Ile Thr Tyr Gly Thr Gly Cys Phe Ile

260 265 270

Leu Thr Asn Thr Gly Ser Gln Ile Val Lys Ser Asn His Gly Leu Leu

275 280 285

Thr Thr Ile Ala Tyr Ser Phe Lys Asp Lys Ile Leu Tyr Ala Leu Glu

290 295 300

Gly Ser Val Met Ile Ala Gly Ala Ala Val Gln Trp Leu Arg Asp Asn

305 310 315 320

Leu Lys Ile Val Tyr Ser Ala Leu Glu Thr Glu Trp Tyr Ala Gly Gln

325 330 335

Val Asn Asp Asp Arg Arg Val Tyr Val Val Pro Ser Phe Thr Gly Leu

340 345 350

Gly Ser Pro Tyr Trp Asp Ser Tyr Ser Arg Gly Ala Ile Phe Gly Leu

355 360 365

Asp Arg Gly Thr Lys Arg Glu His Ile Val Arg Ala Thr Leu Glu Ala

370 375 380

Ile Ala Tyr Gln Ala Asn Asp Val Val Ser Ala Met Gly Lys Asp Met

385 390 395 400

Asn Asp Pro Ile Lys Ile Phe Lys Val Asp Gly Gly Ala Ser Asn Asn

405 410 415

Lys Phe Met Met Gln Phe Gln Ser Asn Ile Ser Gln Ser Lys Val Ile

420 425 430

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

435 440 445

Gly Leu Ala Thr Gly Tyr Trp Lys Ser Ile Asp Glu Ile Lys Asp Thr

450 455 460

Tyr Lys Val His Phe Glu Ile Thr Pro Glu Ile Ser Ala Thr Glu Ala

465 470 475 480

Asn Lys Leu Val Lys Gly Trp Asn Val Ala Val Lys Arg Thr Phe Asn

485 490 495

Trp Thr Lys Asp Ile Glu

500

<210> 8

<211> 332

<212> PRT

<213> Mycoplasma bovis

<400> 8

Met Thr Lys Lys Ile Thr Ile Ile Gly Thr Gly Ala Trp Ala Ser Gly

1 5 10 15

Leu Ala Ser Val Leu Ser Tyr Asn Asn His Lys Ile Lys Met Trp Gly

20 25 30

Ile Asp Gln Lys Glu Ile Ser Asp Ile Asn Ser Gly Ile Asn Ser Lys

35 40 45

Tyr Phe Gly Asn Arg Arg Phe Asn Asn Pro Glu Asn Ile Lys Ala Thr

50 55 60

Leu Asp Leu Lys Asp Ala Leu Ser Glu Leu Asp Phe Met Ile Leu Ala

65 70 75 80

Val Pro Ser Ser Ala Ile Asp Ser Val Leu Gly Lys Ile Gly Asp Val

85 90 95

Leu Gly Thr Gln Lys Ile Lys Val Ile Asn Val Ala Lys Gly Ile Asp

100 105 110

Ser Lys Thr Lys Lys Phe Phe Ser Asp Val Leu Val Glu Lys Phe Ser

115 120 125

Ser Asn Ile Glu Gln Tyr Cys Ser Ile Leu Gly Pro Ser Phe Ala Thr

130 135 140

Glu Val Phe Glu Asn Ala Leu Thr Met Ile Asn Val Val Gly Pro Asn

145 150 155 160

Glu Gln Phe Leu Thr Glu Val Ser Gln Thr Phe Asn Asn Lys Tyr Phe

165 170 175

Arg Leu Val Val Asn Ser Asp Glu Lys Gly Ser Glu Leu Phe Ala Ala

180 185 190

Leu Lys Asn Val Leu Ala Ile Gly Ile Gly Ala Ile Thr Tyr Met His

195 200 205

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

210 215 220

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

225 230 235 240

Pro Leu Glu Leu Ser Ala Ile Gly Asp Ile Phe Leu Thr Cys Ser Ser

245 250 255

Leu Lys Ser Arg Asn Phe Leu Phe Gly Thr Gln Ile Ala Glu Lys Gly

260 265 270

Leu Lys Thr Val Leu Glu Glu Asn Thr Lys Thr Val Glu Gly Tyr His

275 280 285

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

290 295 300

Ala Pro Phe Leu Arg Ser Ile Ile Asp Val Leu Tyr His Asn Lys Asp

305 310 315 320

Val His Lys Leu Thr Asp Phe Ile Glu Lys Tyr Asn

325 330

Claims

1. A fusion gene, which is characterized in that the nucleotide sequence of the fusion gene is shown as a first sequence, and the nucleotide sequence of the first sequence is shown as SEQ ID NO: 1.

2. A plasmid vector, wherein the plasmid vector comprises: a pUC plasmid; and the fusion gene according to claim 1 inserted into the pUC plasmid.

3. The plasmid vector according to claim 2, wherein said pUC plasmid is selected from any one of pUC17 plasmid, pUC18 plasmid and pUC19 plasmid.

4. A transfer vector, the transfer vector comprising: baculovirus transfer plasmids; and the fusion gene of claim 1 inserted into the baculovirus transfer plasmid.

5. The transfer vector of claim 4, wherein the baculovirus transfer plasmid is selected from any one of pFastBac 1, pVL1393 and pFastBac dual.

6. A recombinant plasmid, characterized in that it comprises: bacmid vector; and the fusion gene according to claim 1 inserted into the Bacmid of the baculovirus vector.

7. A transfected cell, wherein the transfected cell is formed by transfecting a host cell with the recombinant plasmid of claim 6;

the host cell is selected from any one of Sf9 cells, high Five cells or Sf21 cells.

8. A fusion protein formed by expressing a fusion gene from the transfected cell of claim 7; the nucleotide sequence of the fusion gene is shown as a first sequence, and the nucleotide sequence of the first sequence is shown as SEQ ID NO: 1.

9. A fusion protein, wherein the amino acid sequence of the fusion protein is shown as a third sequence, and the amino acid sequence of the third sequence is shown as SEQ ID NO: 2.

10. A method for producing the fusion protein according to claim 8 or 9, comprising the steps of:

preparing a fusion gene; the nucleotide sequence of the fusion gene is shown as a first sequence, and the nucleotide sequence of the first sequence is shown as SEQ ID NO:1 is shown in the specification;

cloning the fusion gene into baculovirus transfer plasmid to obtain transfer vector;

transforming DH10Bac strain by using the transfer vector, screening out recombinant strain, extracting recombinant plasmid containing the fusion gene, and transferring the host cell to obtain transfected cell;

And collecting a cell culture of the transfected cells, and purifying to obtain the fusion protein.

11. Use of a fusion protein according to claim 8 or 9 for the preparation of antibodies to mycoplasma bovis.

12. A subunit vaccine comprising the fusion protein of claim 8 or 9 and an adjuvant;

the concentration of the fusion protein in the subunit vaccine is 100+/-10 mug/ml;

the adjuvant is selected from any one or more of MONTANIDE ISA 206VG, MONTANIDE ISA 201VG, MONTANIDE ISA 51VG, liquid paraffin, squalane, saponin, vegetable oil and cytokine.