CN113527511A - Fusion protein, preparation method, application, expression system and vaccine thereof - Google Patents

Abstract

The invention provides a fusion protein, and a preparation method, application, an expression system and a vaccine thereof, and relates to the technical field of biology. The invention screens out the dominant antigen epitopes of PCV2 and PCV3Cap protein, which has good antigenicity and easy high expression, then fuses each dominant epitope with PEA partial functional fragment, and simultaneously adds carboxyl terminal sequence at the C end of the fusion protein to enhance immunogenicity and increase the solubility of the fusion protein. The obtained fusion protein has the advantages of targeting positioning, good antigenicity and high expression level. The fusion protein is used as an antigen to prepare vaccines, and has the advantages of good safety, stable titer, no toxic or side effect and activation of cellular immune response.

Description

Fusion protein, preparation method, application, expression system and vaccine thereof

The application is a divisional application with application date of 09 and 19 in 2018, application number of 201811104817.8 and invented name of fusion protein and a preparation method, application, an expression system and a vaccine thereof.

Technical Field

The invention relates to the technical field of biology, in particular to a fusion protein, and a preparation method, application, an expression system and a vaccine thereof.

Background

Porcine Circovirus (PCV) belongs to the family circoviridae, single-stranded, circular DNA viruses. The major classifications are the nonpathogenic porcine circovirus type (PCV1) and the pathogenic porcine circovirus type (PCV 2). It is known that Cap protein is structural protein of PCV, and has good immunogenicity. In 2016, 10 months, r.palinski at Kansas state university, usa and t.g.phan at san francisco university, california, et al, all reported a new PCV genotype, namely PCV 3. The virus is separated from the sick sow or piglet, and PCV2 is detected as negative. Genomic sequence analysis shows that the PCV3 genome comprises 2000 bases, has a similar genomic structure with PCVl and PCV2, and mainly encodes cap and rep genes. Porcine circovirus type 3 is detected in domestic pig farm cases, a large number of viruses are found in aborted sows and piglets suffering from dermatitis and nephropathy, and the porcine circovirus type is infected and attacked at the same time, so that great harm is caused.

PCV related diseases (PCVAD) include postwelingmultisystemic wasting syndrome (PMWS), Porcine Dermatitis Nephropathic Syndrome (PDNS), respiratory diseases (respiratory diseases), reproductive disorders (reproductive failure) and intestinal diseases (enteropathy), among others. In particular to the main Pathogeny of Multisystemic Wasting Syndrome (PMWS) of weaned pigs, which causes the immune suppression of swinery and causes great economic loss to the pig industry.

Porcine circovirus infection causes lymphocyte depletion and an imbalance in lymphocyte subpopulation ratios, resulting in immunosuppression. PCV is mainly proliferated in lymphoid tissues and infection may cause lymphocyte depletion, lymphocyte apoptosis. Dendritic Cells (DCs) play an important role in both innate and adaptive immunity in the body. Vincent et al found that 80% of dendritic cells can interact with PCV, which can exist and maintain infectivity in dendritic cells for a long time, but does not cause pathological changes and death of the dendritic cells, so that the PCV is a new way to escape degradation of DCs and also provides a new mechanism for viruses to escape immunity.

After PCV infection, the antibody generating capacity of lymphocyte is reduced, the cell immunity is reduced, the phagocytosis and elimination capacity of alveolar macrophage to pathogen is also reduced, the activity of inhibitory T cell is enhanced, the disease resistance of organism is reduced, other pathogens are easy to invade, secondary infection of porcine reproductive and respiratory syndrome, mycoplasma and the like can be caused, and thus, multiple vaccination failures are caused.

The porcine circovirus whole virus inactivated vaccine and the subunit vaccine play a certain role in humoral immunity of organisms, but for porcine circovirus which is a pathogen mainly based on cellular immunity, how to eliminate the virus through the cellular immunity is the direction for fundamentally preventing diseases.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is a first object of the present invention to provide a fusion protein to alleviate at least one of the technical problems of the prior art.

It is another object of the present invention to provide a method for preparing the above fusion protein, which can realize the expression of the above fusion protein of high quality in a large amount.

Another object of the present invention is to provide a protein expression system capable of expressing the above fusion protein, which can directly and rapidly obtain the above fusion protein in large quantities.

Another object of the present invention is to provide the use of the above fusion protein.

The invention also aims to provide a vaccine containing the fusion protein, which has good immunogenicity and high titer and solves the technical problems of low safety, poor or unstable efficacy, high production cost, low protection rate and the like of the vaccine in the prior art.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a fusion protein which comprises a pseudomonas exotoxin A structural domain section, a porcine circovirus type 2 Cap protein dominant antigen epitope section, a porcine circovirus type 3Cap protein dominant antigen epitope section and a carboxyl terminal part;

wherein the pseudomonas exotoxin A domain comprises a pseudomonas exotoxin A domain I and a pseudomonas exotoxin A domain II.

Further, the pseudomonas exotoxin a domain segment is expressed by a nucleotide sequence shown by SEQ ID No.1, the porcine circovirus type 2 Cap protein dominant epitope segment is expressed by a nucleotide sequence shown by SEQ ID No.2, the porcine circovirus type 3Cap protein dominant epitope segment is expressed by a nucleotide sequence shown by SEQ ID No.3, and the carboxyl end portion is expressed by a nucleotide sequence comprising a nucleotide sequence shown by SEQ ID No. 4;

preferably, the carboxy-terminal portion is expressed by the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

Further, the sequence of the pseudomonas exotoxin A domain segment, the porcine circovirus type 2 Cap protein dominant epitope segment, the porcine circovirus type 3Cap protein dominant epitope segment and the carboxyl terminal portion is PEA-PCV2 Cap-PCV3 Cap-carboxyl terminal, and the segments are connected through flexible amino acids;

preferably, the flexible amino acid has an amino acid sequence shown as SEQ ID NO. 7.

Further, a complement protein C3d is also included between the pseudomonas exotoxin A domain segment and the porcine circovirus type 2 Cap protein dominant epitope segment; c3d recognizes non-self antigens, promotes the uptake and presentation of antigens by antigen-presenting cells, promotes the shift of immune response patterns, changes of antibody types, increases the level of neutralizing antibodies, and is a bridge connecting innate immunity and specific immunity.

Preferably, the complement protein C3d is expressed by the nucleotide sequence shown in SEQ ID NO. 8.

The invention also provides a preparation method of the fusion protein, and a gene for coding the fusion protein is expressed in a host.

Further, expressing a gene encoding the fusion protein using a mammalian expression system;

preferably, the gene encoding the fusion protein is expressed using a CHO cell expression system;

preferably, an expression vector containing a gene encoding the fusion protein is provided, the expression vector is introduced into CHO cells, and then the CHO cells are screened to obtain a CHO cell strain which stably expresses the fusion protein, and the CHO cell strain is expressed to obtain the fusion protein;

preferably, the screening comprises pressure screening and monoclonal screening;

preferably, the production method further comprises the step of acclimatizing the CHO cell stably expressing the fusion protein to suspension culture.

The invention also provides a protein expression system capable of expressing the fusion protein.

The invention also provides the application of the fusion protein or the fusion protein prepared by the preparation method in any one of the following (A) to (D):

A) preparing a porcine circovirus vaccine;

B) preparing antibodies of porcine circovirus;

C) preparing a kit for detecting porcine circovirus;

D) preparing diagnostic antigen of porcine circovirus.

In addition, the invention also provides a vaccine containing the fusion protein.

Further, the concentration of the fusion protein in the vaccine is 10-100 mug/ml, preferably 20-80 mug/ml, and more preferably 50 mug/ml;

preferably, the vaccine further comprises adjuvants including at least one of vaccine adjuvants, stabilizers and antibiotics;

preferably, the vaccine adjuvant comprises aluminium hydroxide gel, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or ISA201RVG, preferably ISA201RVG is used.

The fusion protein provided by the invention comprises a pseudomonas exotoxin A structural domain segment, a porcine circovirus type 2 Cap protein dominant antigen epitope segment, a porcine circovirus type 3Cap protein dominant antigen epitope segment and a carboxyl terminal part. The invention screens out dominant epitopes without PCV2 and PCV3Cap protein, which has good antigenicity and easy high expression, then fuses each dominant epitope with PEA partial functional fragment, and adds carboxyl terminal sequence and enhances immunogenicity at the C end of the fusion protein, thereby increasing the solubility of the fusion protein. The obtained fusion protein has the advantages of good antigenicity and high expression level. The fusion protein is used as an antigen to prepare vaccines, and has the advantages of good safety, stable titer and no toxic or side effect.

The invention provides a preparation method of the fusion protein, which expresses a gene sequence for coding the fusion protein in a host and has the advantages of simple preparation method, low cost, powerful vaccine function and the like.

The invention provides a protein expression system capable of expressing the fusion protein, the expression system can be but is not limited to a recombinant vector, a recombinant cell and other systems containing gene sequences for expressing the fusion protein, and a large amount of the fusion protein can be simply, conveniently and quickly purified and obtained by utilizing the protein expression system.

The vaccine containing the fusion protein provided by the invention does not relate to strong toxicity, has no pathogenicity, has good safety, does not cause adverse reaction, does not generate irrelevant antibodies, has high titer and good stability, can generate higher immunogenicity after clinical use, reduces the production cost of the vaccine, and has high protection rate.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a plasmid map of pET-PA-C3d-Cap (PCV2) provided in example 1 of the present invention.

FIG. 2 is a plasmid map of pET-PA-C3d-Cap (PCV3) provided in example 1 of the present invention.

FIG. 3 is a plasmid map of pET-PA-C3d-Cap (PCV2/PCV3) provided in example 1 of the present invention.

FIG. 4 shows the result of enzyme digestion identification of the pET-PA-C3d-Cap-K recombinant positive plasmid provided in example 1 of the present invention.

Fig. 5 shows the immunohistochemical detection results of the vaccine provided in example 3 of the present invention after immune challenge.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

The invention provides a fusion protein which comprises a pseudomonas exotoxin A structural domain section, a porcine circovirus type 2 Cap protein dominant antigen epitope section, a porcine circovirus type 3Cap protein dominant antigen epitope section and a carboxyl terminal part;

wherein the pseudomonas exotoxin A domain comprises a pseudomonas exotoxin A domain I and a pseudomonas exotoxin A domain II.

The predominant epitope of Cap protein of PCV2 and PCV3 is directionally introduced into a specific position in dendritic cells through the special functions of binding of a Pseudomonas Exotoxin A (PEA) carrier and a cell surface receptor and transmembrane transport, and the predominant epitope is displayed on the cell surface in the form of endogenous antigen after presentation to mediate cellular immune response.

In one embodiment, the pseudomonas exotoxin a domain i is divided into a pseudomonas exotoxin a domain ia and a pseudomonas exotoxin a domain ib. Wherein deletion of most of the amino acids of the A domain Ib of the Pseudomonas exotoxin does not affect the PEA activity.

The carboxy terminal portion of the present invention is an endoplasmic reticulum receptor binding sequence. The term "endoplasmic reticulum receptor binding sequence" is used to refer to a group of polypeptide molecules having a similar carboxy terminus to that of the endoplasmic reticulum membrane of a cell and further having the ability to retain the protein in the lumen of the endoplasmic reticulum for caseination, so that the fusion antigen is closer to a peptide fragment of the foreign protein. Typically, the carboxy terminus is between 4 and 13 residues in length.

The invention screens out dominant epitopes without PCV2 and PCV3Cap protein, which has good antigenicity and easy high expression, then fuses each dominant epitope with PEA partial functional fragment, and adds carboxyl terminal sequence and enhances immunogenicity at the C end of the fusion protein, thereby increasing the solubility of the fusion protein. The obtained fusion protein has the advantages of good antigenicity and high expression level. The fusion protein is used as an antigen to prepare vaccines, and has the advantages of good safety, stable titer and no toxic or side effect.

In some preferred embodiments, the pseudomonas exotoxin a domain segment is expressed by the nucleotide sequence set forth in SEQ ID No.1, the porcine circovirus type 2 Cap protein dominant epitope segment is expressed by the nucleotide sequence set forth in SEQ ID No.2, the porcine circovirus type 3Cap protein dominant epitope segment is expressed by the nucleotide sequence set forth in SEQ ID No.3, and the carboxy-terminal portion is expressed by the nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID No. 4.

Preferably, the carboxy-terminal portion is expressed by the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

The pseudomonas exotoxin A domain segment is expressed by the nucleotide sequence shown in SEQ ID NO.1 to obtain an amino acid sequence shown in SEQ ID NO. 9; the dominant antigen epitope segment of the porcine circovirus type 2 Cap protein is expressed by a nucleotide sequence shown by SEQ ID NO.2 to obtain an amino acid sequence shown by SEQ ID NO. 10; the dominant antigen epitope segment of the porcine circovirus type 3Cap protein is expressed by a nucleotide sequence shown by SEQ ID NO.3 to obtain an amino acid sequence shown by SEQ ID NO. 11; the carboxyl end parts are expressed by the nucleotide sequences shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 respectively to obtain the amino acid sequences shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 respectively.

In some preferred embodiments, the pseudomonas exotoxin a domain segment, the porcine circovirus type 2 Cap protein dominant epitope segment, the porcine circovirus type 3Cap protein dominant epitope segment, and the carboxy-terminal portion are arranged in the order PEA-PCV2 Cap-PCV3 Cap-carboxy-terminal, linked by flexible amino acids.

The pseudomonas exotoxin A domain segment, the porcine circovirus type 2 Cap protein dominant antigen epitope segment, the porcine circovirus type 3Cap protein dominant antigen epitope segment and the carboxyl terminal part are connected by flexible amino acids, so that the purpose of protein recombination is achieved, and the mutual influence formed by respective high-grade structures of the segments is avoided.

Preferably, the flexible amino acid has an amino acid sequence shown as SEQ ID NO. 7.

In some preferred embodiments, complement protein C3d is further included between the pseudomonas exotoxin a domain segment and the porcine circovirus type 2 Cap protein dominant epitope segment.

The complement protein C3d molecule is a novel adjuvant, and has promotion effect in influenza virus gene immunity. The fusion protein provided by the invention is connected with complement protein C3d, and has important effects on improving the antibody titer of the vaccine and activating the cellular immune response of an organism.

Preferably, the complement protein C3d is expressed by the nucleotide sequence shown in SEQ ID NO. 8.

The complement protein C3d is expressed by the nucleotide sequence shown in SEQ ID NO.8 to obtain the amino acid sequence shown in SEQ ID NO. 15.

The gene coding the fusion protein is expressed in a host.

The method has the advantages of simple preparation method, low cost, powerful vaccine function and the like.

The host in the present invention may be, but is not limited to, an E.coli expression system, a yeast expression system, an insect expression system, a plant expression system, or a mammalian expression system. Since the structure and biological properties of the protein expressed by mammalian cells are closer to those of the natural protein after translation processing, the present invention preferably expresses the gene encoding the above fusion protein using a mammalian expression system.

In a preferred embodiment of the invention, the gene encoding the fusion protein is expressed using a mammalian expression system. Since the structure and biological properties of the protein expressed by mammalian cells are closer to those of the natural protein after translation processing, the present invention preferably expresses the gene encoding the above fusion protein using a mammalian expression system.

In a more preferred embodiment of the present invention, the gene encoding the above fusion protein is expressed using a CHO cell expression system. The CHO cell is Chinese hamster ovary (Chinese hamster ovary) cell, and has the following advantages:

(1) has accurate post-translational folding and modifying functions, and the expressed protein is most similar to natural protein molecules in the aspects of molecular structure, physical and chemical properties and biological functions.

(2) Has the extracellular secretion function of the product, and is convenient for separating and purifying downstream products.

(3) Has the high-efficiency amplification and expression capacity of the recombinant gene.

(4) Has the characteristics of adherent growth and higher shear stress and osmotic pressure resistance. Suspension culture can also be carried out, and the expression level is higher.

(5) CHO belongs to fibroblast, secretes little endogenous protein of the CHO, and is beneficial to the separation of exogenous protein.

In some embodiments of the invention, an expression vector comprising a gene encoding the fusion protein is provided, the expression vector is introduced into CHO cells, and the CHO cells are then screened for CHO cell lines stably expressing the fusion protein, and the CHO cell lines are expressed to obtain the fusion protein. The method is simple and convenient, is easy to operate, can successfully screen out the eukaryotic cell strain with stable and high expression fusion protein, provides a feasible technical scheme for the subsequent utilization of the obtained eukaryotic cell strain for various researches including application of vaccine preparation and the like, and lays a good foundation.

In some embodiments of the invention, the screening comprises pressure screening and monoclonal screening. In the present invention, G418 can be selected for pressure screening.

In a preferred embodiment of the present invention, the production method further comprises the step of acclimatizing the CHO cell stably expressing the fusion protein to suspension culture. The CHO grows in an adherent way under the general condition, so that the growth of the cell amount and the expression of the fusion protein are limited, the problem can be effectively solved by acclimatizing the adherent cells into a cell strain which can be cultured in a suspension way, the expression amount of the fusion protein is increased, and the vaccine cost is reduced.

In one embodiment, the method for preparing the fusion protein may comprise:

(1) artificially synthesizing a PEA gene and a porcine circovirus dominant antigen epitope Cap protein gene, selecting flexible amino acids to connect the gene segments, inserting a carboxyl terminal into the 3' end of the gene segment, and then respectively connecting the gene segment and the gene segment to a cloning vector to obtain a cloning vector pMD18-T containing the PEA gene, the porcine circovirus dominant antigen epitope Cap protein gene and the carboxyl terminal; (2) carrying out enzyme digestion on a cloning vector containing PEA, porcine circovirus dominant antigen epitope Cap protein genes and a carboxyl terminal to construct an expression vector simultaneously containing PEA, porcine circovirus dominant antigen epitope Cap protein genes and the carboxyl terminal, namely an expression vector pET32a of porcine circovirus fusion protein; introducing an expression vector of the porcine circovirus fusion protein into a receptor bacterium BL21 for induction expression; (3) and (3) transfecting CHO cells with positive plasmids, screening by G418 under pressure to obtain CHO cells expressing the fusion protein, filtering supernate by a 0.45um filter, and purifying the fusion protein by using a Ni + column to obtain the porcine circovirus fusion protein.

Wherein, typical double restriction enzyme cutting sites can be EcoR I and Hind III; the main components of the culture medium can be glucose, trypsin, yeast extract powder, casein, vitamin B1, NaCl and MgSO₄。

The PET-32a vector is used as an expression vector, the formed fusion protein has a small label, and the purified protein obtained without cutting is close to a natural structure, so that high immunogenicity is achieved.

A protein expression system capable of expressing the fusion protein. The protein expression system can be, but is not limited to, a recombinant vector, a recombinant cell and other systems containing gene sequences for expressing the fusion protein, and a large amount of the fusion protein can be obtained by simply, conveniently and quickly purifying the protein expression system.

The fusion protein or the fusion protein prepared by the preparation method is applied to at least one of the following (A) to (D):

A) preparing a porcine circovirus vaccine;

B) preparing antibodies of porcine circovirus;

C) preparing a kit for detecting porcine circovirus;

D) preparing diagnostic antigen of porcine circovirus.

The fusion protein provided by the invention has better immunogenicity, so that the fusion protein can be used for preparing vaccines, and can generate higher antibody titer after immunizing animals. The antibody of the porcine circovirus prepared by using the fusion protein and the fusion protein can be applied to the preparation of various detection reagents and kits for detecting the porcine circovirus or the antibody. For example, an ELISA kit containing the porcine circovirus antibody is used for detecting the porcine circovirus, or a colloidal gold immunochromatographic test strip containing the fusion protein is used for detecting the content of the porcine circovirus antibody in a sample to be detected.

Specifically, the fusion protein or the fusion protein prepared by the preparation method can be used for preparing products for preventing diseases caused by PCV2 and/or PCV3 infection;

preferably, the disease caused by PCV2 and/or PCV3 infection comprises postweaning multisystemic wasting syndrome or porcine dermatitis nephrotic syndrome.

A vaccine containing the fusion protein. The vaccine has good safety, does not cause adverse reaction, does not produce irrelevant antibodies, has high titer and good stability, and reduces the production cost of the porcine circovirus vaccine. The vaccine can generate higher antibody titer after immunizing animals, so that the animals can obtain better protection effect.

In some preferred embodiments, the concentration of the fusion protein in the vaccine is 10-100. mu.g/ml, and may be, for example, but not limited to, 10. mu.g/ml, 20. mu.g/ml, 30. mu.g/ml, 40. mu.g/ml, 50. mu.g/ml, 60. mu.g/ml, 70. mu.g/ml, 80. mu.g/ml, 90. mu.g/ml or 100. mu.g/ml, preferably 20-80. mu.g/ml, more preferably 50. mu.g/ml.

Preferably, the vaccine further comprises adjuvants including at least one of vaccine adjuvants, stabilizers and antibiotics;

preferably, the vaccine adjuvant comprises aluminium hydroxide gel, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or ISA201RVG, preferably ISA201RVG is used.

In a specific embodiment, a method for preparing a vaccine comprising the above fusion protein comprises:

and (3) performing sterile filtration on the prepared fusion protein, performing concentration measurement, setting and diluting the protein fusion protein according to concentration dose, and then mixing and emulsifying the protein fusion protein with an adjuvant to obtain the porcine circovirus vaccine.

In order to facilitate a further understanding of the present invention, the technical solutions of the present invention will now be described in detail with reference to the preferred embodiments.

Unless otherwise specified, all instruments or reagents used in the examples of the present invention are commercially available. The test methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The main reagents and medicine sources of the invention are as follows:

both the pET-32a expression vector and CHO cells were purchased from Invitrogen; m2 medium, fetal bovine serum, and trypsin were purchased from Hyclone; EcoRI, XhoI, T4 DNA ligase, RNase and the like, all purchased from NEB. Primary antibody was purchased from kino JBT, korea; horse radish peroxidase-labeled goat anti-mouse serum purchased from Sigma company; aluminum hydroxide gel, Freund's complete adjuvant, Freund's incomplete adjuvant were purchased from Sigma; white oil adjuvant or ISA201RVG from sibirak corporation;

tris, Urea, Yeast Extract, Tryptone were purchased from OXOID. IPTG is a product of Promega, and ampicillin (Amp), kanamycin (Kan), agar powder, DAB, SDS, TEMED, agarose, low-melting agarose, ammonium persulfate, acrylamide and the like are purchased from Huamei bioengineering company. Absolute ethyl alcohol, phenol, chloroform, Tween-20, citric acid and the like are analytically pure; NaCl, MgSO₄、NaHCO₃、Na₂CO₃、KH₂PO₄、Na₂HPO₄KC1, glucose, trypsin, yeast extract powder, casein peptone and vitamin B1 were all purchased from Xinjiang Baoxin bioengineering Co.

EXAMPLE 1 expression, identification and purification of the fusion protein PA-C3d-Cap-K

According to the gene sequence of Pseudomonas exotoxin A (see SEQ ID NO.1, accession number: CP007224.1, see SEQ ID NO.1), the gene sequence of porcine complement C3d (see SEQ ID NO.8), the gene sequence of Cap protein of PCV2-ADDLPP 10069 strain (see SEQ ID NO.2) and the gene sequence of Cap protein of PCV3-US/MN strain (NCBI accession number: KX898030.1) (see SEQ ID NO.3) reported by NCBI, the Pseudomonas Exotoxin A (PEA) and the domain I and II proteins, porcine complement C3d, porcine circovirus Cap protein dominant epitope and carboxyl polypeptide containing SEQ ID NO.4 select flexible amino acid (SEQ ID NO.7) are finally designed to be connected in series according to a certain combination to express the fusion protein with good immunogenicity.

1.1 construction and identification of PEA Domain I and II and Cap Gene cloning vector

According to the selected PEA structural domain I and II gene sequences, the porcine complement C3d sequence and the Cap gene sequence, after being optimized by CHO cell codons, the fusion protein gene sequence is entrusted to Shanghai's chemical company for artificial synthesis and is inserted into pMD18-T, and the obtained recombinant plasmids are respectively named as pMD18-PA-Cap-K and pMD18-PA-C3d-Cap-K cloning vectors.

1.2 construction and identification of pET-PA-Cap-K expression vector

The pMD18-PA-Cap-K cloning vector and the pET-32a expression vector are respectively subjected to double enzyme digestion by EcoR I and Hind III, after enzyme digestion products are electrophoresed, PA-Cap-K and pET-32a gene fragments are recovered by a DNA recovery kit, and the PA-Cap-K and pET-32a enzyme slice segment genes are connected overnight by a DNA connection kit to construct the pET-PA-Cap-K expression vector. The expression vector was introduced into BL21 competent cells and cultured overnight using LB medium. Extracting culture bacteria plasmid by using a plasmid extraction kit, carrying out double enzyme digestion identification by using EcoRI and HindIII, sending the identified positive plasmid to a gene company for sequencing analysis, wherein the sequencing result shows that: pET-PA-Cap-K is constructed correctly.

1.3 construction and identification of pET-PA-C3d-Cap-K expression vector

The pMD18-PA-C3d-Cap-K cloning vector and the pET-32a expression vector are subjected to double enzyme digestion by using EcoR I and Hind III respectively, after enzyme digestion products are subjected to electrophoresis, a DNA recovery kit is used for recovering PA-C3d-Cap-K and pET-32a gene fragments, a DNA ligation kit is used for carrying out gene ligation on the PA-C3d-Cap-K and pET-32a enzyme fragment overnight, and the pET-PA-C3d-Cap-K expression vector is constructed. The expression vector was introduced into BL21 competent cells and cultured overnight using LB medium. Extracting culture bacteria plasmid by using a plasmid extraction kit, carrying out double enzyme digestion identification by using EcoRI and HindIII, and displaying the identification result: after the digestion, a vector fragment of about 6000bp and a target band of about 2500bp appear, as shown in FIG. 4, wherein lane 1 is pET-PA-C3d-Cap (PCV2), lane 2 is pET-PA-C3d-Cap (PCV3), and lane 3 is pET-PA-C3d-Cap (PCV2/PCV 3). The positive plasmid is sent to a gene company for sequencing analysis, and the sequencing result shows that: pET-PA-C3d-Cap-K was constructed correctly.

Wherein, the constructed pET-PA-C3d-Cap-K plasmid comprises a pET-PA-C3d-Cap (PCV2) plasmid map shown in figure 1, a pET-PA-C3d-Cap (PCV3) plasmid map shown in figure 2 and a pET-PA-C3d-Cap (PCV2/PCV3) plasmid map shown in figure 3.

1.4 establishment and screening of expression cells

1.4.1 transfection

Preparation of plasmids: extracting the recombinant plasmid by using an extraction kit (according to the kit instruction), and centrifuging the obtained plasmid at 12000rpm for 10min for later use; collection of CHO cells: centrifuging suspension cultured CHO cells, washing the cells once by using a PBS solution, and centrifuging and collecting the cells;

electrotransformation reaction system (200 uL): 3X 10⁶CHO cells, 20ug plasmid; and (3) electrotransformation conditions and culture: adding an electrotransformation reaction system containing cells and plasmids into an electric shocking cup, shocking three times under the conditions of 1500V and 10ms, transferring the cells after shocking into two 10cm plates containing 10mL of adherent culture medium, and carrying out electroporation at 37 ℃ and 5% CO₂The culture was carried out for 1 day.

1.4.2 Positive clone selection

Drug screening: changing the liquid of the electric shock cell culture plate cultured for 1 day, adding G418 with the final concentration of 1.8mg/mL after 2 days of culture, and changing a new adherent culture medium for 7 days after 2 days of culture;

positive clone selection and detection: cultivation methodCulturing for 7 days, picking the cells to be cultured in 96-well plate, culturing in adherent culture medium at 37 deg.C and 5% CO₂And after 7 days of culture, adding 100 mu L of suspension culture medium M2 to perform expression culture for 2 days, using an empty plate culture medium for dot blot detection, transferring the high-expression clone into a 24-well plate, using an adherent culture medium to perform culture for 2 days, then changing the culture medium into M2 culture, using the culture medium for western blot (Westernblot, WB) detection, and finally obtaining the high-expression clone according to an experimental result.

1.4.3 Shake flask culture of recombinant Positive clonal cells

Adherent culture: the CHO cells cultured in T75 flask were digested with trypsin, and then suspended and allowed to stand at 37 ℃ in 10mL of M2 medium with 5% CO₂Culturing to a monolayer, and continuously subculturing;

suspension culture in 500mL Erlenmeyer flask: digesting CHO cells cultured in a T75 square bottle by using pancreatin, adding 10mL of serum-free M2 medium, suspending and standing for 2 days, centrifugally collecting the cells, transferring the cells to a shake flask at the rotation speed of 100rpm, the temperature of 37 ℃ and the CO content of 5 percent₂Culturing under the condition; the suspension cultured recombinant CHO cells were then cultured at 1X 10⁶The cells/mL of the culture medium were inoculated into a 500mL Erlenmeyer flask containing 100mLM2 medium at a final concentration, and vitamin K was added to the medium at a final concentration of 1mg/L at 100rpm, 37 ℃ and 5% CO₂Samples were taken daily to determine cell density, fusion protein concentration and glucose concentration in the culture medium.

5L bioreactor fermentation culture: recombinant CHO cells cultured in suspension at 1X 10⁶Inoculating the final concentration of the protein/mL into a bioreactor filled with a 3LM2 culture medium, generally amplifying the protein by 5-8 times, expressing the antigen when the protein is amplified to a specific volume, culturing at 37 ℃ to 5 days, reducing the temperature to 32 ℃, adjusting the pH to 7.5 +/-0.1, culturing at a proper rotating speed, adding 10% of the initial working volume of Efficientfeed on 4 days and 9 days, detecting the glucose concentration every day, and supplementing the glucose to 3-4 g/L when the glucose concentration is lower than 2.5 g/L. When the cell viability is lower than 75%, the supernatant is harvested to be the required antigen.

By optimizing the culture condition and the feeding time of the cell strain, the feeding amount finally obtains the expression of the fusion protein of 1.0 mg/ml.

1.4.4 expression and identification of fusion proteins

Protein gel electrophoresis: SDS-PAGE gel is prepared from the upper 5% concentrated gel and the lower 10% separated gel by electrophoresis at 400mA and 100V for 10min, and then at 400mA and 150V for 1 h.

Immunoblotting: transferring a sample into a PDVF membrane in a membrane transferring buffer solution after SDS-PAGE gel separation, wherein the membrane transferring condition is 100V voltage and 400mA current for 1h, the PDVF membrane is subjected to the action of a Cap protein monoclonal antibody for 4h through sealing and incubation, adding HRP (horse radish peroxidase) labeled goat-anti-mouse secondary antibody diluent, incubating for 2h at room temperature, washing for three times through PBST (para-phenylenedicarboxymethane-polyacrylamide gel electrophoresis), each time for 10min, and finally performing color development detection by using a DAB color development solution.

1.4.5 purification of fusion proteins

The expressed fusion protein sample was centrifuged at 8000rpm for 10min, and after collecting the supernatant, Ni was used⁺The affinity column was purified, and the purified protein was dialyzed against physiological saline and identified by SDS-PAGE. The identification result shows that: after SDS-PAGE electrophoresis of the purified product, the target protein fragment is consistent with the expected size. The content of the fusion protein is 0.8mg/mL, and the fusion protein is frozen and stored at the temperature of minus 80 ℃.

Example 2 preparation of porcine circovirus vaccine

Diluting the fusion protein prepared in the example 1 by using a PBS (phosphate buffer solution), mixing the diluted fusion protein solution with SEPPIC ISA201R VG adjuvant according to the mass fraction of 50%, stirring at the rotating speed of 8000r/min for 10min, adding 0.01% (volume ratio) of thimerosal solution before stopping stirring to ensure that the final concentration of the thimerosal solution is not more than one ten thousandth, fully oscillating and uniformly mixing, performing sterile inspection, viscosity measurement and stability measurement according to the requirement of the annex of the current Chinese veterinary drug dictionary, and placing at 4 ℃ for later use after the sterility inspection, the viscosity measurement and the stability measurement are qualified.

Example 3 application of porcine circovirus vaccine group

Different components of the porcine circovirus subunit vaccine were prepared by the methods provided in examples 1 and 2, and piglets were immunized in groups according to different components (PCV2 antibody titer was not higher than 1:64), as shown in table 1. Blood is collected after immunization, PCV antibody titer is carried out by applying an indirect immunofluorescence assay (IFA), the change rule of the antibody level is evaluated, and secondary immunization is carried out 14 days after the first immunization. 28 days after the first immunization, the vaccine efficacy was evaluated by challenge.

TABLE 1 grouping of piglet Immunity porcine circovirus vaccine compositions

After vaccine immunization, the level of PCV Cap protein antibody is significantly different compared with that of a control group, the antibody water average of each immunization group is positive, and the average titer of PCV2 antibody is not lower than 1: 1024; the PCV2 antibody titer levels were comparable for each immunization group. Among them, the PCV2 antibody titer produced by the fusion protein of the invention is equivalent to that of a single protein group, but the PCV3 antibody titer is obviously higher than that of the single protein group (second group), which indicates that the fusion protein of the invention has an unexpected effect on PCV3 virus immunization. None of the control groups turned positive, and the average titer of PCV2 antibody was not higher than 1:64, and the specific test results are shown in Table 2.

TABLE 2 PCV antibody IFA detection results 28 days after immunization of porcine circovirus vaccine compositions

After antigens expressed in different forms are prepared into vaccines, the immunity efficacy test of the vaccines is carried out. The result of the challenge test shows that the viremia of the vaccine immunity group, the weight of the pig and the antigen-resistant result (obtained according to the score of the immunohistochemical detection result) in the tissue cell are shown in the table 3, and the specific immunohistochemical detection result is shown in the table 5.

TABLE 3 porcine circovirus vaccine composition PCV2 efficacy test results

Note: the PCV2 pathogenic judgment standard refers to the quality standard of porcine circovirus type II baculovirus vector inactivated vaccine (CP08) strain of Wuhanzhongbo biological product GmbH, and the specific content of the pathogenic pig judgment standard is as follows:

(1) the piglet is judged to have poor development and clinical symptoms of PCV2 infection if the relative weight gain rate of the piglet is more than or equal to 5 percent;

(2) after the toxin is attacked, PCV2 antigen in the blood is detected as positive reaction;

(3) PCV2 antigen was positive in the dissected pig tissues 28 days after challenge.

If the number of the 3 patients meets the above 2, the disease is judged to be a disease.

Wherein, the relative weight gain rate of the piglets is calculated in the following way:

the vaccine immunity challenge protection standard is as follows: the experimental pig 4/5 of the vaccine immunity group is protected, and the vaccine is judged to be qualified; the control group 5/5 attacked the disease.

As can be seen from Table 3, after 28 days after immunization, PCV2 seed virus infection, PCV2 evaluation results in serum neutralization tissues of all piglets after challenge of immunization group piglets are negative;

the relative weight gain rate of each immune group is not higher than 5 percent, which indicates that the piglets grow normally. As can be seen from the average daily gain analysis of the experimental pigs in each group, the experimental pigs in the third group are-2.458 percent and are obviously superior to the other two immune groups; the fusion protein vaccine has an obvious effect of generating antibodies after immunizing experimental pigs compared with a single protein group, can better resist the damage of viruses to organisms and reduce the influence on the weight.

As can be seen from the specific tissue Immunohistochemistry (IHC) detection result in fig. 5, the virus antigens in the lymph nodes of the experimental pigs in each immunization group are significantly less than those in the control group, wherein the staining of the cells in the lymph node detection of the experimental pigs in the third group is blue, no positive antigen is detected, the cells are uniformly distributed, and no cell shedding is seen, which indicates that the virus does not cause serious damage to the tissues and organs, while the positive antigens are detected in different degrees in a single protein group, the number of the cells is significantly less, the uniformity is poor, and a certain cell shedding exists.

Example 4 vaccine construction experiments

Porcine circovirus subunit vaccines with different sequences were prepared using the methods provided in examples 1 and 2, and the specific sequence cases are shown in table 4.

TABLE 4 different porcine circovirus subunit vaccine antigen nucleotide sequences

Group of	Sequence of
		First group	NO.16
Second group	NO.17
		Third group	NO.18

The vaccine compositions in the different groups in table 5 below were prepared and verified according to the methods of examples 1-2, and the antibody titer, antigen stability, and soluble protein content after immunization were measured, and the results are shown in tables 5, 6, and 7, respectively.

TABLE 5 post-immunization antibody detection of porcine circovirus vaccine of each combination structure of piglet immunization

TABLE 6 porcine circovirus vaccine immune post-antibody detection of piglet immunization respective combinatorial structures

TABLE 7 analysis of the stability after purification of each combinatorial protein

According to the detection results, after the vaccine immune experimental pig is prepared by combining various forms, the antibody level generation conditions have certain differences. The combination of the present invention has good effects in terms of antibody production, antigen preservation, expression level and stability.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Tiankang biological products Ltd

<120> fusion protein, preparation method, application, expression system and vaccine thereof

<160> 18

<170> PatentIn version 3.5

<210> 1

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<212> DNA

<213> Artificial sequence

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atgcacctga taccccattg gatccccctg gtcgccagcc tcggcctgct cgccggcggc 60

tcgtccgcgt ccgccgccga ggaagccttc gacctctgga acgatgcgcc aaggcctgcg 120

tgctcgacct caaggacggc gtgcgttcca gccgcatgag cgtcgacccg gccatcgccg 180

acaccaacgg ccagggcgtg ctgcactact ccatggtcct ggagggcggc aacgacgcgc 240

tcaagctggc catcgacaac gccctcagca tcaccagcga cggcctgacc atccgcctcg 300

aaggcggcgt cgagccgaac aagccggtgc gctacagcta cacgcgccag gcgcgcggca 360

gttggtcgct gaactggctg gtaccgatcg gccacgagaa gccctcgaac atcaaggtgt 420

tcatccacga actgaacgcc ggcaaccagc tcagccacat gtcgccgatc tacaccatcg 480

agatgggcga cgagttgctg gcgaagctgg cgcgcgatgc caccttcttc gtcagggcgc 540

acgagagcaa cgagatgcag ccgacgctcg ccatcagcca tgccggggtc agcgtggtca 600

tggcccaggc ccagccgcgc cgggaaaagc gctggagcga atgggccagc ggcaaggtgt 660

tgtgcctgct cgacccgctg gacggggtct acaactacct cgcccagcag cgctgcaacc 720

tcgacgatac ctgggaaggc aagatctacc gggtgctcgc cggcaacccg gcgaagcatg 780

acctggacat caagcccacg gtcatcagtc atcgcctgca cttccccgag ggcggcagcc 840

tggccgcgct gaccgcgcac caggcctgcc acctgccgct ggagactttc acccgtcatc 900

gccagccgcg cggctgggaa caactggagc agtgcggcta tccggtgcag cggctggtcg 960

ccctctacct ggcggcgcga ctgtcatgga accaggtcga ccaggtgatc cgcaacgccc 1020

tggccagccc cggcagcggc ggcgacctgg gcgaagcgat ccgcgagcag ccggagcagg 1080

cccgtctggc cctgaccctg gccgccgccg agagcgagcg cttcgtccgg cagggcaccg 1140

gcaacgacga ggccggcgcc gccaact 1167

<210> 2

<211> 279

<212> DNA

<213> Artificial sequence

<400> 2

gtagacatga tgagattcaa tattaatgac tttcttcccc caggacctcc cagccccagc 60

cagggtgaca ggggagtggg ctccagtgct gttattctag atgataactt tgtaacacct 120

cccagcccca gctccactat tgattacttc caaccaaaca acaaaagaaa tcagctgcct 180

cccagcccca gcgtagacca cgtaggcctc ggcactgcgt tcgaaaatag tatataccct 240

cccagcccca gcaatcttaa agacccccca cttaaccct 279

<210> 3

<211> 309

<212> DNA

<213> Artificial sequence

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agaagaaaac tattcattag gaggcctccc agccccagca tgaacgtcat ttccgttgga 60

acccctccca gccccagcag ctttgaatat tataagatac taaagatgaa agttacactc 120

agccctgtaa tttctccggc tcagcctccc agccccagca gccgttactt cacccccaaa 180

ccaattctgg cgggacctcc cagccccagc cacccaggac aaagcctctt ctttttctcc 240

agacctccca gcccagccga ccccaccgtt caatggggag cactgctttg gagcatttat 300

gtcccagaa 309

<210> 4

<211> 15

<212> DNA

<213> Artificial sequence

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agggaagacc taaag 15

<210> 5

<211> 39

<212> DNA

<213> Artificial sequence

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aaggacgaac taagggaaga cctaaagaag gacgaacta 39

<210> 6

<211> 42

<212> DNA

<213> Artificial sequence

<400> 6

agggaagacc taaagaggga agacctaaag aaggacgaac ta 42

<210> 7

<211> 5

<212> PRT

<213> Artificial sequence

<400> 7

Pro Pro Ser Pro Ser

1 5

<210> 8

<211> 945

<212> DNA

<213> Artificial sequence

<400> 8

accccctccg gctgtgggga gcagaacatg atcggcatga cgcccacagt catcgctgtg 60

cactacctgg acagcaccga acaatgggag aagttcggcc tggagaagag gcaggaagcc 120

ttggagctca tcaagaaggg gtacacccag caactggcct tcagacaaaa gaactcagcc 180

tttgccgcct tccaggaccg gctgtccagc accctgctga cagcctatgt ggtcaaggtc 240

ttcgctatgg cagccaacct catcgccatc gactcccagg tcctctgtgg ggccgtcaaa 300

tggctgatcc tggagaagca gaagcctgat ggagtcttcg aggagaatgg gcccgtgata 360

caccaagaaa tgattggtgg cttcaagaac actgaggaga aagacgtgtc cctgacagcc 420

tttgttctca tcgcgctgca ggaggctaaa gacatctgtg aaccacaggt caatagcctg 480

ttgcgcagca tcaataaggc aagagacttc ctcgcagact actacctaga attaaaaaga 540

ccatatactg tggccattgc tggttatgcc ctggctctat ctgacaagct ggatgagccc 600

ttcctcaaca aacttctgag cacagccaaa gaaaggaacc gctgggagga acctggccag 660

aagctccaca atgtggaggc cacatcctac gccctcttgg ctctgctggt agtcaaagac 720

tttgactctg tccctcctat tgtgcgctgg ctcaatgagc agagatacta cggaggtggc 780

tatggatcta cccaggccac tttcatggtg ttccaagcct tggcccaata ccagaaggat 840

gtccctgatc acaaggatct gaacctggat gtgtccatcc acctgcccag ccgcagcgct 900

ccagtcaggc atcgtatcct ctgggaatct gctagccttc tgcgg 945

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Ala Pro Asp Thr Pro Leu Asp Pro Pro Gly Arg Gln Pro Arg Pro Ala

1 5 10 15

Arg Arg Arg Leu Val Arg Val Arg Arg Arg Gly Ser Leu Arg Pro Leu

20 25 30

Glu Arg Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg

35 40 45

Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gln

50 55 60

Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu

65 70 75 80

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

85 90 95

Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser

100 105 110

Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro

115 120 125

Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu

130 135 140

Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu

145 150 155 160

Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe

165 170 175

Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser

180 185 190

His Ala Gly Val Ser Val Val Met Ala Gln Ala Gln Pro Arg Arg Glu

195 200 205

Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp

210 215 220

Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu

225 230 235 240

Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro

245 250 255

Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu

260 265 270

His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala

275 280 285

Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly

290 295 300

Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala

305 310 315 320

Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile

325 330 335

Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala

340 345 350

Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala

355 360 365

Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala

370 375 380

Gly Ala Ala Asn

385

<210> 10

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<212> PRT

<213> Artificial sequence

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Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Pro

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Pro Ser Pro Ser Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile

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Leu Asp Asp Asn Phe Val Thr Pro Pro Ser Pro Ser Ser Thr Ile Asp

35 40 45

Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Pro Pro Ser Pro Ser

50 55 60

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

65 70 75 80

Pro Ser Pro Ser Asn Leu Lys Asp Pro Pro Leu Asn Pro

85 90

<210> 11

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<212> PRT

<213> Artificial sequence

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Arg Arg Lys Leu Phe Ile Arg Arg Pro Pro Ser Pro Ser Met Asn Val

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Ile Ser Val Gly Thr Pro Pro Ser Pro Ser Ser Phe Glu Tyr Tyr Lys

20 25 30

Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala Gln

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Pro Pro Ser Pro Ser Ser Arg Tyr Phe Thr Pro Lys Pro Ile Leu Ala

50 55 60

Gly Pro Pro Ser Pro Ser His Pro Gly Gln Ser Leu Phe Phe Phe Ser

65 70 75 80

Arg Pro Pro Ser Pro Ser Arg Pro His Arg Ser Met Gly Ser Thr Ala

85 90 95

Leu Glu His Leu Cys Pro Arg

100

<210> 12

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<212> PRT

<213> Artificial sequence

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Arg Glu Asp Leu Lys

1 5

<210> 13

<211> 13

<212> PRT

<213> Artificial sequence

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Lys Asp Glu Leu Arg Glu Asp Leu Lys Lys Asp Glu Leu

1 5 10

<210> 14

<211> 14

<212> PRT

<213> Artificial sequence

<400> 14

Arg Glu Asp Leu Lys Arg Glu Asp Leu Lys Lys Asp Glu Leu

1 5 10

<210> 15

<211> 315

<212> PRT

<213> Artificial sequence

<400> 15

Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr

1 5 10 15

Val Ile Ala Val His Tyr Leu Asp Ser Thr Glu Gln Trp Glu Lys Phe

20 25 30

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

35 40 45

Thr Gln Gln Leu Ala Phe Arg Gln Lys Asn Ser Ala Phe Ala Ala Phe

50 55 60

Gln Asp Arg Leu Ser Ser Thr Leu Leu Thr Ala Tyr Val Val Lys Val

65 70 75 80

Phe Ala Met Ala Ala Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys

85 90 95

Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val

100 105 110

Phe Glu Glu Asn Gly Pro Val Ile His Gln Glu Met Ile Gly Gly Phe

115 120 125

Lys Asn Thr Glu Glu Lys Asp Val Ser Leu Thr Ala Phe Val Leu Ile

130 135 140

Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln Val Asn Ser Leu

145 150 155 160

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

165 170 175

Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala

180 185 190

Leu Ser Asp Lys Leu Asp Glu Pro Phe Leu Asn Lys Leu Leu Ser Thr

195 200 205

Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln Lys Leu His Asn

210 215 220

Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu Val Val Lys Asp

225 230 235 240

Phe Asp Ser Val Pro Pro Ile Val Arg Trp Leu Asn Glu Gln Arg Tyr

245 250 255

Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln

260 265 270

Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His Lys Asp Leu Asn

275 280 285

Leu Asp Val Ser Ile His Leu Pro Ser Arg Ser Ala Pro Val Arg His

290 295 300

Arg Ile Leu Trp Glu Ser Ala Ser Leu Leu Arg

305 310 315

<210> 16

<211> 2717

<212> DNA

<213> Artificial sequence

<400> 16

atgcacctga taccccattg gatccccctg gtcgccagcc tcggcctgct cgccggcggc 60

tcgtccgcgt ccgccgccga ggaagccttc gacctctgga acgatgcgcc aaggcctgcg 120

tgctcgacct caaggacggc gtgcgttcca gccgcatgag cgtcgacccg gccatcgccg 180

acaccaacgg ccagggcgtg ctgcactact ccatggtcct ggagggcggc aacgacgcgc 240

tcaagctggc catcgacaac gccctcagca tcaccagcga cggcctgacc atccgcctcg 300

aaggcggcgt cgagccgaac aagccggtgc gctacagcta cacgcgccag gcgcgcggca 360

gttggtcgct gaactggctg gtaccgatcg gccacgagaa gccctcgaac atcaaggtgt 420

tcatccacga actgaacgcc ggcaaccagc tcagccacat gtcgccgatc tacaccatcg 480

agatgggcga cgagttgctg gcgaagctgg cgcgcgatgc caccttcttc gtcagggcgc 540

acgagagcaa cgagatgcag ccgacgctcg ccatcagcca tgccggggtc agcgtggtca 600

tggcccaggc ccagccgcgc cgggaaaagc gctggagcga atgggccagc ggcaaggtgt 660

tgtgcctgct cgacccgctg gacggggtct acaactacct cgcccagcag cgctgcaacc 720

tcgacgatac ctgggaaggc aagatctacc gggtgctcgc cggcaacccg gcgaagcatg 780

acctggacat caagcccacg gtcatcagtc atcgcctgca cttccccgag ggcggcagcc 840

tggccgcgct gaccgcgcac caggcctgcc acctgccgct ggagactttc acccgtcatc 900

gccagccgcg cggctgggaa caactggagc agtgcggcta tccggtgcag cggctggtcg 960

ccctctacct ggcggcgcga ctgtcatgga accaggtcga ccaggtgatc cgcaacgccc 1020

tggccagccc cggcagcggc ggcgacctgg gcgaagcgat ccgcgagcag ccggagcagg 1080

cccgtctggc cctgaccctg gccgccgccg agagcgagcg cttcgtccgg cagggcaccg 1140

gcaacgacga ggccggcgcc gccaactacc ccctccggct gtggggagca gaacatgatc 1200

ggcatgacgc ccacagtcat cgctgtgcac tacctggaca gcaccgaaca atgggagaag 1260

ttcggcctgg agaagaggca ggaagccttg gagctcatca agaaggggta cacccagcaa 1320

ctggccttca gacaaaagaa ctcagccttt gccgccttcc aggaccggct gtccagcacc 1380

ctgctgacag cctatgtggt caaggtcttc gctatggcag ccaacctcat cgccatcgac 1440

tcccaggtcc tctgtggggc cgtcaaatgg ctgatcctgg agaagcagaa gcctgatgga 1500

gtcttcgagg agaatgggcc cgtgatacac caagaaatga ttggtggctt caagaacact 1560

gaggagaaag acgtgtccct gacagccttt gttctcatcg cgctgcagga ggctaaagac 1620

atctgtgaac cacaggtcaa tagcctgttg cgcagcatca ataaggcaag agacttcctc 1680

gcagactact acctagaatt aaaaagacca tatactgtgg ccattgctgg ttatgccctg 1740

gctctatctg acaagctgga tgagcccttc ctcaacaaac ttctgagcac agccaaagaa 1800

aggaaccgct gggaggaacc tggccagaag ctccacaatg tggaggccac atcctacgcc 1860

ctcttggctc tgctggtagt caaagacttt gactctgtcc ctcctattgt gcgctggctc 1920

aatgagcaga gatactacgg aggtggctat ggatctaccc aggccacttt catggtgttc 1980

caagccttgg cccaatacca gaaggatgtc cctgatcaca aggatctgaa cctggatgtg 2040

tccatccacc tgcccagccg cagcgctcca gtcaggcatc gtatcctctg ggaatctgct 2100

agccttctgc gggtagacat gatgagattc aatattaatg actttcttcc cccaggacct 2160

cccagccccc agggtgacag gggagtgggc tccagtgctg ttattctaga tgataacttt 2220

gtaacacctc ccagccccat ccactattga ttacttccaa ccaaacaaca aaagaaatca 2280

gctgcctccc agccccgtag accacgtagg cctcggcact gcgttcgaaa atagtatata 2340

ccctcccagc cccaaatctt aaagaccccc cacttaaccc tagaagaaaa ctattcatta 2400

ggaggcctcc cagccccatg aacgtcattt ccgttggaac ccctcccagc cccagctttg 2460

aatattataa gatactaaag atgaaagtta cactcagccc tgtaatttct ccggctcagc 2520

ctcccagccc caagccgtta cttcaccccc aaaccaattc tggcgggacc tcccagcccc 2580

cacccaggac aaagcctctt ctttttctcc agacctccca gccccgaccc caccgttcaa 2640

tggggagcac tgctttggag catttatgtc ccagaaaagg acgaactaaa gggaagacct 2700

aaagaaggac gaactaa 2717

<210> 17

<211> 2715

<212> DNA

<213> Artificial sequence

<400> 17

atgcacctga taccccattg gatccccctg gtcgccagcc tcggcctgct cgccggcggc 60

tcgtccgcgt ccgccgccga ggaagccttc gacctctgga acgatgcgcc aaggcctgcg 120

tgctcgacct caaggacggc gtgcgttcca gccgcatgag cgtcgacccg gccatcgccg 180

acaccaacgg ccagggcgtg ctgcactact ccatggtcct ggagggcggc aacgacgcgc 240

tcaagctggc catcgacaac gccctcagca tcaccagcga cggcctgacc atccgcctcg 300

aaggcggcgt cgagccgaac aagccggtgc gctacagcta cacgcgccag gcgcgcggca 360

gttggtcgct gaactggctg gtaccgatcg gccacgagaa gccctcgaac atcaaggtgt 420

tcatccacga actgaacgcc ggcaaccagc tcagccacat gtcgccgatc tacaccatcg 480

agatgggcga cgagttgctg gcgaagctgg cgcgcgatgc caccttcttc gtcagggcgc 540

acgagagcaa cgagatgcag ccgacgctcg ccatcagcca tgccggggtc agcgtggtca 600

tggcccaggc ccagccgcgc cgggaaaagc gctggagcga atgggccagc ggcaaggtgt 660

tgtgcctgct cgacccgctg gacggggtct acaactacct cgcccagcag cgctgcaacc 720

tcgacgatac ctgggaaggc aagatctacc gggtgctcgc cggcaacccg gcgaagcatg 780

acctggacat caagcccacg gtcatcagtc atcgcctgca cttccccgag ggcggcagcc 840

tggccgcgct gaccgcgcac caggcctgcc acctgccgct ggagactttc acccgtcatc 900

gccagccgcg cggctgggaa caactggagc agtgcggcta tccggtgcag cggctggtcg 960

ccctctacct ggcggcgcga ctgtcatgga accaggtcga ccaggtgatc cgcaacgccc 1020

tggccagccc cggcagcggc ggcgacctgg gcgaagcgat ccgcgagcag ccggagcagg 1080

cccgtctggc cctgaccctg gccgccgccg agagcgagcg cttcgtccgg cagggcaccg 1140

gcaacgacga ggccggcgcc gccaactgta gacatgatga gattcaatat taatgacttt 1200

cttcccccag gacctagcag cccccagggt gacaggggag tgggctccag tgctgttatt 1260

ctagatgata actttgtaac acctagcagc ccctccacta ttgattactt ccaaccaaac 1320

aacaaaagaa atcagctgcc tcccagcccc gtagaccacg taggcctcgg cactgcgttc 1380

gaaaatagta tataccctag cagccccaat cttaaagacc ccccacttaa ccctagaaga 1440

aaactattca ttaggaggcc tagcagcccc atgaacgtca tttccgttgg aacccctagc 1500

agccccagct ttgaatatta taagatacta aagatgaaag ttacactcag ccctgtaatt 1560

tctccggctc agcctagcag ccccagccgt tacttcaccc ccaaaccaat tctggcggga 1620

cctagcagcc cccacccagg acaaagcctc ttctttttct ccagacctag cagcccccga 1680

ccccaccgtt caatggggag cactgctttg gagcatttat gtcccagaaa ccccctccgg 1740

ctgtggggag cagaacatga tcggcatgac gcccacagtc atcgctgtgc actacctgga 1800

cagcaccgaa caatgggaga agttcggcct ggagaagagg caggaagcct tggagctcat 1860

caagaagggg tacacccagc aactggcctt cagacaaaag aactcagcct ttgccgcctt 1920

ccaggaccgg ctgtccagca ccctgctgac agcctatgtg gtcaaggtct tcgctatggc 1980

agccaacctc atcgccatcg actcccaggt cctctgtggg gccgtcaaat ggctgatcct 2040

ggagaagcag aagcctgatg gagtcttcga ggagaatggg cccgtgatac accaagaaat 2100

gattggtggc ttcaagaaca ctgaggagaa agacgtgtcc ctgacagcct ttgttctcat 2160

cgcgctgcag gaggctaaag acatctgtga accacaggtc aatagcctgt tgcgcagcat 2220

caataaggca agagacttcc tcgcagacta ctacctagaa ttaaaaagac catatactgt 2280

ggccattgct ggttatgccc tggctctatc tgacaagctg gatgagccct tcctcaacaa 2340

acttctgagc acagccaaag aaaggaaccg ctgggaggaa cctggccaga agctccacaa 2400

tgtggaggcc acatcctacg ccctcttggc tctgctggta gtcaaagact ttgactctgt 2460

ccctcctatt gtgcgctggc tcaatgagca gagatactac ggaggtggct atggatctac 2520

ccaggccact ttcatggtgt tccaagcctt ggcccaatac cagaaggatg tccctgatca 2580

caaggatctg aacctggatg tgtccatcca cctgcccagc cgcagcgctc cagtcaggca 2640

tcgtatcctc tgggaatctg ctagccttct gcggagggaa gacctaaaga aggacgaact 2700

aaaaggacga actaa 2715

<210> 18

<211> 2715

<212> DNA

<213> Artificial sequence

<400> 18

atgcacctga taccccattg gatccccctg gtcgccagcc tcggcctgct cgccggcggc 60

tcgtccgcgt ccgccgccga ggaagccttc gacctctgga acgatgcgcc aaggcctgcg 120

tgctcgacct caaggacggc gtgcgttcca gccgcatgag cgtcgacccg gccatcgccg 180

acaccaacgg ccagggcgtg ctgcactact ccatggtcct ggagggcggc aacgacgcgc 240

tcaagctggc catcgacaac gccctcagca tcaccagcga cggcctgacc atccgcctcg 300

aaggcggcgt cgagccgaac aagccggtgc gctacagcta cacgcgccag gcgcgcggca 360

gttggtcgct gaactggctg gtaccgatcg gccacgagaa gccctcgaac atcaaggtgt 420

tcatccacga actgaacgcc ggcaaccagc tcagccacat gtcgccgatc tacaccatcg 480

agatgggcga cgagttgctg gcgaagctgg cgcgcgatgc caccttcttc gtcagggcgc 540

acgagagcaa cgagatgcag ccgacgctcg ccatcagcca tgccggggtc agcgtggtca 600

tggcccaggc ccagccgcgc cgggaaaagc gctggagcga atgggccagc ggcaaggtgt 660

tgtgcctgct cgacccgctg gacggggtct acaactacct cgcccagcag cgctgcaacc 720

tcgacgatac ctgggaaggc aagatctacc gggtgctcgc cggcaacccg gcgaagcatg 780

acctggacat caagcccacg gtcatcagtc atcgcctgca cttccccgag ggcggcagcc 840

tggccgcgct gaccgcgcac caggcctgcc acctgccgct ggagactttc acccgtcatc 900

gccagccgcg cggctgggaa caactggagc agtgcggcta tccggtgcag cggctggtcg 960

ccctctacct ggcggcgcga ctgtcatgga accaggtcga ccaggtgatc cgcaacgccc 1020

tggccagccc cggcagcggc ggcgacctgg gcgaagcgat ccgcgagcag ccggagcagg 1080

cccgtctggc cctgaccctg gccgccgccg agagcgagcg cttcgtccgg cagggcaccg 1140

gcaacgacga ggccggcgcc gccaactgta gacatgatga gattcaatat taatgacttt 1200

cttcccccag gaagccctcc cagccagggt gacaggggag tgggctccag tgctgttatt 1260

ctagatgata actttgtaac aagccctccc agctccacta ttgattactt ccaaccaaac 1320

aacaaaagaa atcagctgag ccctcccagc gtagaccacg taggcctcgg cactgcgttc 1380

gaaaatagta tatacagccc tcccagcaat cttaaagacc ccccacttaa ccctagaaga 1440

aaactattca ttaggaggag ccctcccagc atgaacgtca tttccgttgg aaccagccct 1500

cccagcagct ttgaatatta taagatacta aagatgaaag ttacactcag ccctgtaatt 1560

tctccggctc agagccctcc cagcagccgt tacttcaccc ccaaaccaat tctggcggga 1620

agccctccca gccacccagg acaaagcctc ttctttttct ccagaagccc tcccagccga 1680

ccccaccgtt caatggggag cactgctttg gagcatttat gtcccagaaa ccccctccgg 1740

ctgtggggag cagaacatga tcggcatgac gcccacagtc atcgctgtgc actacctgga 1800

cagcaccgaa caatgggaga agttcggcct ggagaagagg caggaagcct tggagctcat 1860

caagaagggg tacacccagc aactggcctt cagacaaaag aactcagcct ttgccgcctt 1920

ccaggaccgg ctgtccagca ccctgctgac agcctatgtg gtcaaggtct tcgctatggc 1980

agccaacctc atcgccatcg actcccaggt cctctgtggg gccgtcaaat ggctgatcct 2040

ggagaagcag aagcctgatg gagtcttcga ggagaatggg cccgtgatac accaagaaat 2100

gattggtggc ttcaagaaca ctgaggagaa agacgtgtcc ctgacagcct ttgttctcat 2160

cgcgctgcag gaggctaaag acatctgtga accacaggtc aatagcctgt tgcgcagcat 2220

caataaggca agagacttcc tcgcagacta ctacctagaa ttaaaaagac catatactgt 2280

ggccattgct ggttatgccc tggctctatc tgacaagctg gatgagccct tcctcaacaa 2340

acttctgagc acagccaaag aaaggaaccg ctgggaggaa cctggccaga agctccacaa 2400

tgtggaggcc acatcctacg ccctcttggc tctgctggta gtcaaagact ttgactctgt 2460

ccctcctatt gtgcgctggc tcaatgagca gagatactac ggaggtggct atggatctac 2520

ccaggccact ttcatggtgt tccaagcctt ggcccaatac cagaaggatg tccctgatca 2580

caaggatctg aacctggatg tgtccatcca cctgcccagc cgcagcgctc cagtcaggca 2640

tcgtatcctc tgggaatctg ctagccttct gcggaaggac gaactaaaag gacgaactaa 2700

agggaagacc taaag 2715

Claims (10)

1. A fusion protein, comprising a pseudomonas exotoxin a domain segment, a porcine circovirus type 2 Cap protein dominant epitope segment, a porcine circovirus type 3Cap protein dominant epitope segment, and a carboxy-terminal portion;

wherein the dominant antigen epitope segment of the porcine circovirus type 2 Cap protein is expressed by a nucleotide sequence shown by SEQ ID NO.2, and the dominant antigen epitope segment of the porcine circovirus type 3Cap protein is expressed by a nucleotide sequence shown by SEQ ID NO. 3.

2. The fusion protein of claim 1, wherein the fusion protein comprises a pseudomonas exotoxin a domain comprising pseudomonas exotoxin a domain i and pseudomonas exotoxin a domain ii;

preferably, the Pseudomonas exotoxin A domain segment is expressed by the nucleotide sequence shown in SEQ ID No. 1.

3. The fusion protein of claim 1, wherein the carboxy-terminal portion is expressed by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID No. 4;

preferably, the carboxy-terminal portion is expressed by the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

4. The fusion protein of any one of claims 1-3, wherein the Pseudomonas exotoxin A domain segment, the porcine circovirus type 2 Cap protein dominant epitope segment, the porcine circovirus type 3Cap protein dominant epitope segment, and the carboxy-terminal portion are arranged in the order PEA-PCV2 Cap-PCV3 Cap-carboxy-terminal, linked by a flexible amino acid;

preferably, the flexible amino acid has an amino acid sequence shown as SEQ ID NO. 7.

More preferably, a complement protein C3d is further included between the Pseudomonas exotoxin A domain segment and the porcine circovirus type 2 Cap protein dominant epitope segment;

further preferably, the complement protein C3d is expressed by the nucleotide sequence shown in SEQ ID NO. 8.

5. A method for producing the fusion protein according to any one of claims 1 to 4, wherein the gene encoding the fusion protein is expressed in a host.

6. The method of claim 5, wherein the gene encoding the fusion protein is expressed using a mammalian expression system;

preferably, the gene encoding the fusion protein is expressed using a CHO cell expression system;

preferably, an expression vector containing a gene encoding the fusion protein is provided, the expression vector is introduced into CHO cells, and then the CHO cells are screened to obtain a CHO cell strain which stably expresses the fusion protein, and the CHO cell strain is expressed to obtain the fusion protein;

preferably, the production method further comprises the step of acclimatizing the CHO cell stably expressing the fusion protein to suspension culture.

7. A protein expression system capable of expressing the fusion protein of any one of claims 1-4.

8. Use of the fusion protein according to any one of claims 1 to 4 or the fusion protein produced by the production method according to claim 5 or 6 in any one of the following (A) to (D):

A) preparing a porcine circovirus vaccine;

B) preparing antibodies of porcine circovirus;

C) preparing a kit for detecting porcine circovirus;

D) preparing diagnostic antigen of porcine circovirus.

9. A vaccine comprising the fusion protein of any one of claims 1-4.

10. The vaccine according to claim 9, wherein the concentration of the fusion protein in the vaccine is 10-100 μ g/ml, preferably 20-80 μ g/ml, more preferably 50 μ g/ml;

preferably, the vaccine further comprises adjuvants including at least one of vaccine adjuvants, stabilizers and antibiotics;

preferably, the vaccine adjuvant comprises aluminium hydroxide gel, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, MF59 adjuvant or ISA201R VG, preferably ISA201R VG.

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