Improved gB protein of porcine pseudorabies virus and application thereof

Abstract

The invention provides an improved gB protein of porcine pseudorabies virus and application thereof, and belongs to the technical field of biological medicine. The improved gB protein of the porcine pseudorabies virus comprises one or more amino acid mutations or / and shearing substitutions on the basis of a wild type and can be maintained in a fusion precursor state, the yield is remarkably increased when mammalian cells are used for expressing target protein, and the generated protein is more stable and uniform and has a good application prospect. The level of a serum specific IgG antibody generated by induction in an immunized animal body is remarkably improved, and the antibody can be used for preparing a pseudorabies virus vaccine or a vaccine component or a vaccine detection reagent and detecting or preventing or treating diseases and / or symptoms caused or mediated by the pseudorabies virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a modified gB protein of pseudorabies virus of porcine, a nucleic acid molecule, a vector, a cell and an application thereof. Background Art

[0002] Pseudorabies in pigs is caused by Pseudorabies Virus (PRV), which can cause abortion in pregnant sows, stillbirth, infertility in breeding pigs, and death of piglets. The mortality rate of piglets under 15 days old is as high as 100%, the incidence rate of weaned piglets is 20%-40%, and the mortality rate is 10%-20%. There is currently no effective treatment for PRV, and vaccination is the fundamental measure to prevent and control pseudorabies in pigs.

[0003] Porcine pseudorabies virus belongs to the family Herpesviridae, the subfamily Alphaherpesvirinae, the genus Varicellavirus, and the porcine herpesvirus type I. Porcine pseudorabies virus is a linear double-stranded DNA virus with a total genome size of about 150Kb. The genome is divided into four parts, consisting of the main regions of the long unique region (UL), the short unique region (UL), the internal repeat sequence (IRS), and the terminal repeat sequence (TRS) (Ben et al., 1985). The gene of porcine pseudorabies virus can encode more than 70-100 proteins, which can be mainly divided into three categories, including capsid protein, envelope glycoprotein and various enzymes. Among them, 11 envelope proteins play an important role in the replication and transmission of PRV, which can be divided into essential glycoproteins (gB, gD, gH, gK, gL) and non-essential glycoproteins (gC, gE, gG, gI, gM, gN) (Nauwynck et al., 1997).

[0004] The gB protein of pseudorabies virus of porcine is encoded by the UL27 gene, which is about 2.8Kb in length. It is a type I glycoprotein located on the envelope of the virus particle and is the most abundant protein in the PRV envelope. It is highly homologous and has similar functions in different herpes viruses. It is a key protein for the virus to enter the host and spread. It has the function of inducing the body to produce neutralizing antibodies and immune responses (Mettenleiter et al., 1994; Yoon et al., 2006). Therefore, it has become the preferred glycoprotein for the development of PRV subunit vaccines and the evaluation of vaccine immune effects.

[0005] However, there are still great challenges in developing and producing recombinant vaccines of pseudorabies virus using the membrane fusion protein gB of pseudorabies virus as an antigen. In the process of promoting the fusion of virus and cell membrane, gB protein has two conformations, pre-fusion and post-fusion. The conventional method of preparing recombinant gB protein as vaccine antigen will only produce antigen protein in the post-fusion conformation, which greatly reduces or even inactivates the immunogenicity of the antigen, causing the vaccine to lose its protective efficacy. Precisely because of the important functions of gB protein and the unstable characteristics of pre-fusion conformation, obtaining a stable and uniform pre-fusion conformation gB protein trimer is a key factor in the successful development of vaccines, neutralizing antibodies and serological detection reagents, so as to maximize the stimulation of the immune system to produce a correct immune response and play a role in protecting the body. Therefore, designing and improving the stable conformation of the fusion precursor gB protein is an important direction for the development of recombinant protein vaccines of pseudorabies virus.

[0006] Over the years, many PRV recombinant protein vaccines have been developed and tried, and currently no PRV subunit vaccine is on the market. The PRV vaccines on the Chinese market are mainly inactivated vaccines and gene-deficient live vaccines. Although inactivated vaccines are safe, their immune efficacy is limited. There are certain risks in the safety of live vaccines, and there are risks of insufficient attenuation and genetic instability. There is an urgent need to develop more effective vaccines to ultimately achieve the goal of purifying the PRV virus in pig farms. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a pseudorabies virus gB protein mutant, which contains one or more amino acid mutations compared with the wild-type virus gB protein. The PRV gB protein mutant can form a trimer structure and maintain a fusion precursor state; at the same time, the yield is greatly improved when the target protein is expressed in mammalian cells, and the produced protein is more stable and uniform. When used as a vaccine or vaccine component, the immunogenicity is increased, and the immunized animal can be induced to produce a higher level of neutralizing antibodies. The PRV gB protein mutant of the present invention can be used to prepare a pseudorabies virus vaccine or a vaccine component for the prevention and / or treatment of diseases and / or conditions caused or mediated by PRV.

[0008] Definition of terms:

[0009] In the present invention, the term "gB protein of pseudorabies virus of porcine" or "PRV" generally refers to a porcine herpesvirus type I of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellavirus, and the virus is generally associated with porcine pseudorabies. The porcine pseudorabies virus may include a variety of forms with an envelope. The porcine pseudorabies virus genome generally encodes 11 structural proteins: gB, gD, gH, gK, gL, gC, gE, gG, gI, gM, and gN.

[0010] In the present invention, the term "protein mutant" may generally refer to a protein whose amino acid sequence or protein structure has one or more changes compared to the wild-type protein. The changes may include: deletion, insertion, substitution, truncation and / or deletion of one or more amino acids, processing or cutting of the protein structure. In the present invention, the protein mutant may refer to a mutant of the gB protein of pseudorabies virus.

[0011] In the present invention, the term "amino acid mutation" generally refers to the modification of an amino acid in a parent amino acid sequence. For example, the modification may include replacement, insertion and / or deletion of one or more amino acids. In the present invention, the amino acid mutation may include the deletion of at least one amino acid residue at a specified position of an amino acid sequence. For example, the amino acid residue may be a glycosylation site. In the present invention, the amino acid mutation may include the replacement of an amino acid at at least one amino acid position. In certain embodiments, the amino acid mutation may optimize the conformation of a protein composed of the amino acid sequence. Amino acid mutations may be produced using genetic or chemical methods well known in the art. For example, genetic methods may include site-directed mutagenesis, PCR and / or gene synthesis, etc.

[0012] In the present invention, the amino acid mutation comprises amino acid mutation by codon optimization. The term "codon optimization" generally means replacing one, at least one, or more than one codon in a parent polypeptide encoding nucleic acid by codons encoding the same amino acid residue with different relative usage frequencies to improve the expression of the nucleic acid encoding the polypeptide. In the present invention, as long as the amino acid mutation is the same as the present invention, all possible codons encoding the mutant amino acid are within the scope of the present invention.

[0013] In the present invention, the term "penetration of an Alpha helix structure" means that an Alpha helix structure with two bends changes under certain circumstances into a linear Alpha helix structure that is no longer bent.

[0014] In the present invention, the term "virus-like particle", "virus-like particle" or "VLP" generally refers to a hollow particle containing one or more structural proteins of a virus. The virus-like particle does not have viral nucleic acid and cannot replicate autonomously. The virus-like particle may be identical or similar in morphology to a true virus particle.

[0015] In the present invention, the term "proteolytic cleavage" generally refers to the process of cleaving an amino acid sequence using a protease.

[0016] In the present invention, the amino acid mutation comprises amino acid mutation by codon optimization. The term "codon optimization" generally means replacing one, at least one, or more than one codon in a parent polypeptide encoding nucleic acid by codons encoding the same amino acid residue with different relative usage frequencies to improve the expression of the nucleic acid encoding the polypeptide. In the present invention, as long as the amino acid mutation is the same as the present invention, all possible codons encoding the mutant amino acid are within the scope of the present invention.

[0017] In the present invention, the term "trimer" generally refers to a protein structure composed of three protein subunits of the same type or different types. For example, the protein trimer can be a herpes virus trimer gB protein. For example, the protein trimer can be linked together by a special chemical structure. In the present invention, the protein trimer can be a gB protein of pseudorabies virus.

[0018] In the present invention, the term "homotrimer" generally refers to a protein structure composed of three protein subunits of the same type. In the present invention, the homotrimer can be connected together by a special chemical structure. For example, the chemical structure can be a disulfide bond between monomers. For example, the homotrimer includes a base. In the present invention, the protein trimer can be the gB protein of pseudorabies virus.

[0019] In the present invention, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule. For example, the conformational change may include a change in the chemical bonds in the protein molecule or a change in the folding mode of a polypeptide.

[0020] In the present invention, the term "structural protein" generally refers to a protein that constitutes a component of a viral particle. The structural protein may include a structural protein of a herpes virus. The structural protein of the present invention may include gB, gD, gH, gK, gL, gC, gE, gG, gI, gM and gN.

[0021] In the present invention, the term "fusion precursor" generally refers to a structural protein of the virus before the infection of the host cell when membrane fusion has not yet occurred. Generally speaking, the fusion precursor of the wild-type PRV gB protein is a metastable protein, and its conformation is discontinuous after the virus infects the host cell and membrane fusion occurs, and gradually and irreversibly changes into a conformation of a low-energy stable state (conformation after fusion). In the present invention, a series of transformations are carried out to the PRV gB protein so that the gB protein trimer can stably be in the fusion precursor state.

[0022] In the present invention, the expression of the protein mutation site is usually expressed by "amino acid + amino acid position + (amino acid after mutation)". In the present invention, the mutation may include but is not limited to the addition, substitution, deletion and / or deletion of amino acids. For example, the term "H544P" usually refers to the mutation of the 544th amino acid H to the amino acid P.

[0023] In the present invention, the amino acid sequence and position of the gB protein of the PRV wild-type virus strain can be found in Accession number: sequence in QGL40152.1. The PRV gB protein mutant is obtained by mutation based on the amino acid type of the gB protein at a specific position in GenBank number QGL40152.1.

[0024] In the present invention, described homology generally refers to the similarity, similarity or association between two or more sequences.Comparison carried out in order to determine the sequence homology percentage can be realized in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for maximum alignment in the full-length sequence range being compared or in the target sequence region for realizing.Described homology can also be measured by the following method: FASTA and BLAST.

[0025] In the present invention, the protein mutant may comprise a single chain of gB protein, which is expressed as a complete modified gB protein subunit, which can form a trimer under the action of molecular forces. In some embodiments, the complete protein with a trimer higher-order structure is an effective component of the vaccine.

[0026] In the present invention, the term "mutation" generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule. Amino acid changes may include substitution, deletion, and insertion of one or more amino acids.

[0027] In the present invention, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

[0028] In the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication start site. The vector may also include components that assist it to enter cells or assist the target element to be integrated into host cells, such as viral particles, liposomes, protein coats or integrase, but not limited to these substances.

[0029] In the present invention, the term "detection reagent" generally refers to a reagent used for diagnosing diseases or conditions and evaluating antibody titers. The detection reagent may include the PRV gB protein mutant of the present invention as a detection antigen to detect antibodies against pseudorabies virus gB.

[0030] In the present invention, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the PRV gB protein mutant of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention and / or the cell of the present invention, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are generally non-toxic to the recipient at the dosage and concentration used.

[0031] In the present invention, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium, and / or nonionic surfactants.

[0032] In the present invention, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide. For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example 1-30, 1-20 or 1-10, for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0033] In the present invention, the homolog can be a protein or polypeptide having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide.

[0034] In the present invention, the term "comprising" generally means including the features specifically specified, but not excluding other elements.

[0035] In the present invention, the "flin cleavage site" may also be referred to as a "furin cleavage site".

[0036] In the present invention, the term "adjuvant" generally refers to any substance that assists or regulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen. In the present invention, the adjuvant can be used to enhance the antigenicity of the PRV gB protein mutant. In some embodiments, the adjuvant can include a mineral (e.g., alum, aluminum hydroxide, or phosphate) suspension. In some embodiments, the adjuvant can include an oil-in-water emulsion.

[0037] In one aspect, the present invention provides a mutant of the gB protein of pseudorabies virus (PRV).

[0038] The present invention is a novel protective vaccine designed on the basis of structural biology and in combination with immunological principles, which has high safety, wide protection and reliable preparation process. The yield of the recombinant protein vaccine provided by the present invention is significantly improved compared with the wild type. The mutant can be stably maintained in the fusion precursor state and can induce a higher level of neutralizing antibodies in animal immune tests.

[0039] The present invention uses the public sequence of the gB protein of the PRV epidemic strain (QGL40152.1), designs the antigen protein based on the observation of the three-dimensional structure data of the gB proteins of different homologous strains, and mutates or cuts the amino acids at some specific sites.

[0040] Compared with the amino acid sequence of the gB protein of the wild-type pseudorabies virus, the mutant comprises one or more amino acid site mutations.

[0041] For example, in the PRV gB protein mutant of the present invention, the mutation may include: a single amino acid mutation (a point mutation of a single amino acid) and / or multiple amino acid mutations (a collection of multiple continuous or discontinuous point mutations). For example, the single amino acid mutation may include: insertion, deletion and / or substitution. For example, the multiple amino acid mutations may include: insertion, deletion and / or substitution. For example, the insertion may include: insertion of a single amino acid and / or insertion of multiple amino acids. For example, the replacement may include: replacing a single amino acid with another amino acid other than itself, replacing a single amino acid with a combination of multiple continuous amino acids, replacing multiple discontinuous amino acids with another amino acid other than itself at the same time and / or replacing multiple discontinuous amino acids with a combination of multiple continuous amino acids at the same time. For example, the deletion may include: deletion of a single amino acid and / or deletion of multiple amino acids.

[0042] In the present invention, the mutation site and the type of amino acid after mutation can be determined by the three-dimensional structure of PRV gB protein. Due to the strong genetic variability of viruses, the amino acid mutation of the present invention is not limited to the change of the original amino acid type at a specific site.

[0043] The mutant comprises one or more of the following properties:

[0044] 1) The mutant of the gB protein of the porcine pseudorabies virus of the present invention is applicable to different PRV strains (i.e., the classical strain type 1 and the variant strain type 2);

[0045] 2) maintaining the protein trimer formed by the PRV gB protein mutant in a pre-fusion conformation;

[0046] 3) Increase the yield, stability and homogeneity of trimeric proteins during recombinant expression;

[0047] 4) To produce higher levels of neutralizing antibodies in the immunized animals to inhibit the infection of pseudorabies virus (enhanced immunogenicity).

[0048] The gB protein includes the sequence shown in SEQ ID NO.1; the mutant mutates at the ferin protease cleavage site of SEQ ID NO.1 and becomes ineffective, and the mutation is selected from any one or more of the following:

[0049] 1) A deletion mutation of any 1-31 amino acids between amino acid L486 and amino acid V516;

[0050] 2) any 1-31 amino acids between L486 and amino acid V516 are mutated to become a linker;

[0051] 3) Any one or more amino acids in the furin cleavage site between L486 and amino acid V516 are mutated to amino acids other than K or R.

[0052] The above mutations can reduce the conformational change from the fusion precursor to the fusion post-form caused by enzyme cleavage.

[0053] In the above 1), amino acid L486 to amino acid V516 may be missing, or amino acid P492 to amino acid P514 may be missing, etc.

[0054] In the above 2), the linker may include 1-15 amino acids; preferably, the linker is (GGSGG)n, where n is an integer of 1-3; more preferably, the linker is GGSGG.

[0055] In the above 3), two point mutations may be included; preferably, they are any two point mutations in positions 499-503; preferably, the point mutation is G; further preferably, they include R500G and R502G mutations, or R499G and R503G mutations.

[0056] Preferably, the mutant may also selectively include any one or more of the following: deletion mutation of one or more amino acids in the transmembrane region of the gB protein, mutation of any point to proline, mutation of one or more amino acids in repulsive release.

[0057] The deletion mutation of one or more amino acids in the transmembrane region of the gB protein increases the solubility of the expressed gB protein, makes it less likely to precipitate, makes it more uniform and improves the yield.

[0058] The deletion mutation of the transmembrane region of the gB protein is selected from any one or more of the following:

[0059] (1) Any 1-98 amino acid deletion mutation between amino acid M1 and amino acid S98;

[0060] (2) Any 1-167 amino acid deletion mutation between amino acid K748 and amino acid P914.

[0061] Preferably, the deletion mutation in the transmembrane region of the gB protein is selected from any one or more of the following:

[0062] A. Deletion mutation from amino acid M1 to amino acid G57;

[0063] B. Deletion mutation from amino acid V776 to amino acid P914;

[0064] C. Deletion mutation from amino acid M1 to amino acid S98;

[0065] D. a deletion mutation from amino acid V776 to amino acid P914;

[0066] E. Deletion mutation from amino acid A760 to amino acid P914;

[0067] F. a deletion mutation from amino acid L755 to amino acid P914;

[0068] G. a deletion mutation from amino acid V753 to amino acid P914;

[0069] H. Deletion mutation of amino acid K748 to amino acid P914.

[0070] The arbitrary point mutation is proline, which is an amino acid mutation that prevents the alpha helix from penetrating, so that the gB protein remains in the state of a fusion precursor. Preferably, any one or more amino acids in 1-45 amino acids between S528 and amino acid R572 are mutated to proline; more preferably, it is a H544P point mutation.

[0071] The mutation of one or more amino acids that release repulsion is preferably the mutation of R745 to an amino acid other than K or R.

[0072] The mutant may be a trimer structure.

[0073] The C-terminus of the trimer structure may or may not contain a heterologous trimerization domain.

[0074] The trimer structure can be achieved by self-folding, or by including a heterotrimeric domain, wherein the heterotrimeric domain is selected from any one or more of foldon, GCN4, and RSTN.

[0075] In another aspect, the present invention provides a genetic engineering product related to the aforementioned mutant.

[0076] According to the knowledge of those skilled in the art, the genetic engineering products can be understood as products obtained by using genetic engineering technology. The genetic engineering products include but are not limited to: nucleic acid molecules, vectors, and cells.

[0077] The nucleic acid molecule is used to encode the aforementioned mutant, and can make the mutant a monomer or a trimer.

[0078] The vector includes the aforementioned nucleic acid molecule and is used to express the aforementioned mutant.

[0079] The present invention is a recombinant pseudorabies virus protein produced by plasmid expression of a recombinant DNA construct. Any embodiment may also include at least one additional antigen or a combination of PRV infectious or non-infectious DNA fragments. On the other hand, the present invention is a trimeric recombinant protein comprising the separation or purification of porcine pseudorabies virus gB protein. On the other hand, the present invention is a virus-like particle comprising the separation or purification of porcine pseudorabies virus gB protein. In certain embodiments, the trimeric recombinant protein of the isolated or purified porcine pseudorabies virus gB protein can be used to prepare an immunoprotective vaccine with a pharmaceutically acceptable carrier. In one embodiment, the virus-like particles of the isolated or purified porcine pseudorabies virus gB protein can be used to prepare an immunoprotective vaccine with a pharmaceutically acceptable carrier. In another embodiment, the vaccine may include one or more other antigens. In certain embodiments, the vaccine immunized animals can effectively produce neutralizing antibodies against viral infection.

[0080] The cell includes the aforementioned vector, or it is understood that the cell includes the aforementioned nucleic acid molecule for expressing the aforementioned mutant. For example, the nucleic acid molecule or vector of the present invention can be introduced into the cell. For example, the cell can include: prokaryotic cells (e.g., Escherichia coli cells) and / or eukaryotic cells (e.g., HEK293T cells, Expi293F cells, CHO cells). In some embodiments, the vector of the present invention can be introduced into the host cell by methods known in the art, and the method can include: heat shock transformation, transient transfection and / or stable transfection. For example, the transfection method can include: electroporation, liposome transfection and / or calcium phosphate transfection. In some embodiments, the cell can include introducing the vector of the present invention into the host cell by methods known in the art (e.g., transient transfection) to express (e.g., transiently express) the PRV gB protein mutant of the present invention. For example, in some embodiments, the cell (e.g., Expi293F cell) can transiently express the PRV gB protein mutant of the present invention.

[0081] In another aspect, the present invention provides the use of the aforementioned mutant or genetic engineering product

[0082] The applications include, but are not limited to: detection of pseudorabies virus gB antibodies, and application in the preparation of vaccines or drugs for preventing, diagnosing or treating pseudorabies in pigs.

[0083] The pseudorabies virus gB antibody detection is to use the mutant as the antigen protein of the detection reagent to detect the antibody of the pseudorabies virus gB.

[0084] The vaccine includes the aforementioned mutants and may be a subunit vaccine or a virus-like particle (VLP) vaccine, but is not limited to the two forms listed. Those skilled in the art may obtain other types of related vaccines or conjugates and combined antigens, etc., according to conventional technical means.

[0085] In some embodiments, the vaccine comprises an mRNA vaccine encoded by a reverse-designed synthetic RNA molecule based on the designed mutant.

[0086] In some embodiments, the vaccine comprises a DNA vaccine encoded by a reverse-designed synthetic deoxyribonucleic acid molecule based on the designed mutant.

[0087] In some embodiments, the vaccine may also include gD, gB, gD, gH, gK, gL, gC, gE, gG, gI, gM and gN proteins of PRV.

[0088] Based on the above, the present invention also provides a drug or vaccine or a pseudorabies virus gB antibody detection kit comprising the aforementioned mutant or genetic engineering product.

[0089] The medicine or vaccine may also include other pharmaceutically acceptable carriers or adjuvants or excipients, including but not limited to: protective agents, stabilizers, preservatives, bactericides, inactivators, adjuvants and / or buffers. Unless incompatible with the PRV gB protein mutant, the vaccine, the isolated nucleic acid molecule, the vector, and the cell of the present invention, any conventional medium or reagent may be considered for use in the pharmaceutical composition of the present invention.

[0090] The vaccine may include the aforementioned mutants and compositions, and / or be formulated with various types of vaccine adjuvants (including water adjuvants, oil adjuvants, nano adjuvants, etc.) to form an immunogenic composition.

[0091] The present invention also provides the use of the aforementioned mutant or genetic engineering product or drug or vaccine or pseudorabies virus gB antibody detection kit in the prevention, diagnosis and treatment of porcine pseudorabies.

[0092] In yet another aspect, the present invention provides a method for preparing the aforementioned variant.

[0093] The preparation method includes expression through genetic engineering.

[0094] For example, the method may include the following steps:

[0095] 1) constructing the PRV B protein mutant plasmid; 2) transfecting the plasmid into cells; 3) collecting cell supernatant and purifying the PRV gB protein mutant protein.

[0096] Beneficial effects of the present invention:

[0097] The present invention provides a modified gB protein mutant of a fusion precursor pseudorabies virus, wherein the gB protein mutant comprises one or more amino acid mutations or shearing compared to the wild-type PRV virus gB protein. The PRV virus gB protein mutant can form a trimer structure and maintain a fusion precursor state; at the same time, the yield when the target protein is expressed using mammalian cells is greatly improved, and the produced protein is more stable and uniform. When used as a vaccine or vaccine component, it has high immunogenicity and can induce immunized animals to produce higher antibodies, and has the characteristics of high efficiency, broad spectrum, stability and high yield. The PRV virus gB protein mutant described in the present invention can be used to prepare a PRV vaccine for the prevention and / or treatment of diseases and / or conditions caused or mediated by PRV.

[0098] Those skilled in the art can easily discern other aspects and advantages of the present invention from the detailed description below. In the detailed description below, only exemplary embodiments of the present invention are shown and described. As will be appreciated by those skilled in the art, the content of the present invention enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figures 1A-1C What is shown are the stability results of the target protein in the supernatant of the PRV gB protein mutants described in the present invention, wherein 1A is the expression level result of the supernatants of 12 mutants at 0 day, 1B is the expression level attenuation percentage result of the supernatants of 12 mutants after high temperature stress at 50°C, and 1C is the expression level attenuation percentage result of the supernatants of 12 mutants after long-term placement at 37°C on the 1st, 3rd and 7th days.

[0100] Figure 2A-2B The results of gel filtration chromatography purification of the PRV gB protein of the present invention are shown, 2A is the YK028Ag009 wild-type gB protein, and 2B is the YK028Ag027 mutant gB protein.

[0101] Figure 3Shown are the results of gel electrophoresis (SDS-PAGE) of the PRV gB protein of the present invention, A is the YK028Ag009 wild-type gB protein, B is the YK028Ag012 mutant gB protein, C is the YK028Ag017 mutant gB protein, and D is the YK028Ag027 mutant gB protein.

[0102] Figure 4A-4B The results of the analysis of the PRV gB protein by high performance liquid chromatography (SEC-HPLC) of the present invention are shown. 4A is the wild-type gB protein of YK028Ag009, and 4B is the mutant gB protein of YK028Ag027.

[0103] Figure 5 The results of the antibody detection experiment of the serum of mice immunized with the PRV gB protein mutant of the present invention against pathogens are shown. 2W is the serum dilution binding antibody level 2 weeks after the initial immunization, and 5W is the serum dilution binding antibody level 2 weeks after the second booster immunization.

[0104] Figure 6A-6B The results of the particle size distribution of the PRV gB protein mutants of the present invention detected by a protein stability analyzer (PANTA) are shown, 6A is the YK028Ag012 mutant gB protein, and 6B is the YK028Ag027 mutant gB protein. DETAILED DESCRIPTION

[0105] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0106] In the present invention, the term "1H1 antibody" generally refers to a monoclonal antibody that specifically binds to the gB protein of pseudorabies virus, has a broad-spectrum neutralizing activity, and can be used as a specific detection antibody for the gB protein of pseudorabies virus. The amino acid sequences of the heavy and light chains of the 1H1 antibody can be found in Accession number: 5YSL_A and 5YSL_H. (Xiangdong Li, Yi Shi, Kegong Tian et al., 2017) In the present invention, the protein, polypeptide and / or amino acid sequence involved should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein or polypeptide.

[0107] Example 1 Design of improved gB protein mutants based on the three-dimensional structure of gB protein

[0108] Based on the amino acid sequence shown in SEQ ID NO: 1, the gB protein mutants in Table 1 were designed.

[0109] Table 1 Molecular number, mutation site, and titer of 20ml expression test of gB protein mutants

[0110]

[0111]

[0112] The mutant amino acid residue sequences designed above were codon optimized and synthesized using human as the host (commissioned by General Biotechnology (Anhui) Co., Ltd.), and then cloned into the pCDNA3.4 vector (from Thermo Fisher Scientific), and the plasmid was extracted for further transfection.

[0113] Example 2 Small volume expression of designed PRV gB protein mutant

[0114] HEK293 cells cultured in OPM-293CD05 serum-free medium were used for suspension transfection in 20 mL of the system in a 50 mL TPP permeable suspension culture shake tube. The live cell density was adjusted to about 6 million per mL on the day of transfection. Each transfection used 20 μg of plasmid and 100 μg of PEI-MAX cell transfection reagent. The plasmid and transfection reagent were diluted with 600 μL of OPM-293CD05 serum-free medium, respectively. The diluted transfection reagent was added to the diluted plasmid, gently mixed, and incubated at room temperature for 8 minutes to prepare the plasmid complex. After the incubation, the prepared plasmid complex was slowly dripped into the cell solution to be transfected, and the cell solution was placed at 37°C, 8% CO 2 The cells were cultured in a shaker at 200 rpm. 18-24 hours after transfection, 10% of the culture volume of BalanCD CHO Feed 4 supplement medium (0.8×) (V / V) was added to each bottle of transfected cells. On the fourth day after transfection, the culture supernatant was collected to detect the expression of Titer.

[0115] The amount of antigen in the sample that can bind to the 1H1 antibody was detected using the Gator, a label-free molecular interaction detector based on the BLI (biomembrane interference) principle. The specific method is to use the ProtienA probe to capture 4nM of 1H1, and then test the initial binding rate of the sample to the probe. The purified PRV-gB of known concentration was diluted to different concentrations, and the initial binding rate was detected and a standard curve was established. The concentration of the unknown sample is calculated based on the standard curve established with known concentrations, which is the Titer assay. The test results at a transfection volume of 20mL are shown in Table 1. Compared with the expression level of 86.06mg / L of the wild-type gB protein YK028Ag009 molecule, the expression level of the YK028Ag027 molecule improved by structural biology design is as high as 608.91mg / L, and the yield is increased by 7.08 times.

[0116] Example 3 Evaluation of the stability of target protein in the supernatant of PRV gB protein mutant expression

[0117] Expi 293F cells cultured in OPM-293CD05 serum-free medium (Thermo fisher, catalog number: 2045637) were used to perform suspension transfection in 20 mL of the system in a 50 mL TPP permeable suspension culture shake tube. The live cell density was adjusted to about 6 million per mL on the day of transfection. Each transfection used 20 μg of plasmid and 100 μg of PEI-MAX (purchased from Polysciences, catalog number: 24765-1). The plasmid and transfection reagent were diluted using 600 μL of OPM-293CD05 serum-free medium (purchased from Shanghai Aopuma Biotechnology Co., Ltd., catalog number: 81075-001). The diluted transfection reagent was added to the diluted plasmid, gently mixed, and incubated at room temperature for 8 minutes to prepare the plasmid complex. After the incubation, the prepared plasmid complex was slowly dripped into the cell solution to be transfected, and the cell solution was placed at 37°C, 8% CO 2The cells were cultured in a shaker at 200 rpm. 18-24 hours after transfection, 10% of the culture volume of BalanCD CHO Feed4 (purchased from FUJIFILM Irvine Scientific, Inc, catalog number: 94134-10L) (0.8×) (V / V) was added to each bottle of transfected cells. The cells were harvested on the fourth day after transfection. The supernatant harvested after centrifugation was sterilized and filtered through a 0.22 μm filter. Each was divided into 5 tubes, 600 μL / tube. The Titer (D0) of the harvested supernatant was determined by binding with the antibody mAb-1H1. The stability test conditions were: 50°C for 1h, 37°C for 1 day, 37°C for 3 days, and 37°C for 7 days. The changes in the expression level of each sample were determined by binding with the antibody mAb-1H1. A total of 12 PRV antigen mutants, YK028Ag009, YK028Ag012, YK028Ag017, YK028Ag018, YK028Ag019, YK028Ag020, YK028Ag021, YK028Ag025, YK028Ag026, YK028A g027, YK028Ag028, and YK028Ag029, were evaluated, and three replicates were made for each mutant.

[0118] Stability results such as Figure 1A-1B As shown, 1A is the expression level of the supernatants of 12 mutants of PRV at 0 days, 1B is the expression attenuation percentage of the supernatants of 12 mutants after 1h of high temperature stress at 50℃, and 1C is the expression attenuation percentage of the supernatants of 12 mutants after long-term storage at 37℃ for 1 day, 3 days, and 7 days. The results showed that the average expression level of each mutant was 93-427 mg / L, among which the YK 028Ag027 mutant was the highest; the average expression attenuation percentage after 50℃ high temperature stress was 88%-105%, and the expression attenuation percentage after long-term storage at 37℃ for D1, D3, and D7 was 105%-129%, 114%-151%, and 111%-146%, respectively, indicating that the stability of the designed mutants at 50℃ high temperature stress for 1h and 37℃ long-term storage for D7 was good.

[0119] Example 4 Large-volume expression of PRV gB protein mutants in mammalian cells

[0120] For large-volume transfection, fast-growing Expi 293F cells (Thermo fisher catalog number: 2045637) with good cell morphology were used as transfection hosts. The viable cell density of the seed cells was adjusted to 3 million per milliliter before transfection, and 400 mL of cell solution was used for transfection for each project. Each project used 500 μg of plasmid and 2 mL of EZ Trans cell transfection reagent (purchased from Shanghai Li Ji Biotechnology Co., Ltd., catalog number: AC04L092), which were diluted with 15 mL of OPM-293CD05 culture medium, gently mixed, and then the diluted transfection reagent was gently added to the diluted plasmid, and incubated at room temperature for 10-15 minutes to prepare the plasmid complex. After the incubation, the prepared plasmid complex was slowly dripped into the cell solution to be transfected, and the cell solution was placed at 37°C, 8% CO 2 The cells were cultured in a shaker at 100 rpm. 18-22 hours after transfection, 10% of the culture volume of 293Feed medium (purchased from China Biotechnology Co., Ltd., catalog number: HC106001) (V / V) was added to each bottle of transfected cells, and the cell status was monitored every day. When the cell viability dropped to 70%, the cell culture fluid was harvested and transferred to the purification section for purification.

[0121] Example 5 Purification of recombinant pseudorabies virus gB protein

[0122] The culture harvest (cell harvest fluid) of the recombinant pseudorabies virus gB protein expressed by 293F cells was used for separation and purification of the recombinant protein. The specific steps are as follows:

[0123] (1) Cell fluid harvesting

[0124] Use a high-speed centrifuge to centrifuge at 4000 rpm for 50 min, collect the centrifugal supernatant and filter it using a 0.22 μm bottle top filter to obtain the cell clarified harvest solution.

[0125] (2) Ultrafiltration / diafiltration

[0126] Used by Peristaltic pump, PALL Filtron The tangential flow ultrafiltration system composed of a clamp and a T-SERIES OMEGA 30kD membrane package was used to ultrafilter and concentrate the cell clarified harvest solution and wash the solution into a 1×PBS (pH 7.4) buffer to avoid interference of components in the cell clarified harvest solution with subsequent purification.

[0127] (3) Nickel affinity chromatography

[0128] According to the protein tag design, Ni-NTA was used to capture and purify the target protein through the His tag. The washed filtrate was collected and slowly loaded into 10 mL of HisPur pre-equilibrated with 1× PBS, pH 7.4 at 4°C. TM In the Ni-NTAResin gravity column, nickel affinity chromatography was performed and the flow-through was collected. After loading, impurities were washed with 10 column volumes of elution buffer (10mmol / L Imidazole, 1×PBS pH 7.4, 350mM NaCl), and finally the target protein was eluted with elution buffer (500mmol / L Imidazole, 1×PBS pH 7.4, 350mM NaCl).

[0129] The eluted target protein was purified by 30 kD Millipore Ultrafiltration was performed using an Ultra filter tube to concentrate the product to about 4 mL. The concentrated product was tested for UV absorption at UV280 nm using NP80Touch.

[0130] (4) Gel filtration chromatography

[0131] Gel filtration chromatography separates proteins according to their molecular weight. Based on nickel affinity chromatography capture, the target trimer recombinant pseudorabies virus gB protein that can be used for immunization is obtained.

[0132] The Unique AutoPure protein purification system and the Cytica HiLoad were balanced using 1× PBS, pH 7.4. TM 16 / 600Superdex TM 200 pg gel filtration chromatography column, the concentrated nickel affinity chromatography elution product is loaded onto the chromatography column. The effluent product is collected over time in a 4 mL / tube manner, and the collected samples are analyzed by SDS-PAGE protein gel electrophoresis.

[0133] The gel filtration chromatography results are as follows Figure 2A-2B As shown, 2A is the wild-type gB protein of YK028Ag009, and 2B is the mutant gB protein of YK028Ag027. The retention time at the absorption peak of the target trimer recombinant pseudorabies virus gB protein is about 70 minutes. The absorption peak of the wild-type gB protein of YK028Ag009 is lower at 70 minutes, indicating that its content in the target trimer recombinant pseudorabies virus gB protein is lower; the absorption peak of the target protein of the mutant YK028Ag027 is relatively high at about 70 minutes, indicating that its content in the target trimer recombinant pseudorabies virus gB protein is higher.

[0134] The yield was calculated based on the concentration (mg / mL) of the collected product and the product volume, and the yield was normalized to the amount per liter based on the expression volume in order to compare the yields of different mutant gB proteins.

[0135] Table 2 Expression levels after affinity purification results normalization

[0136] Molecular number Yield after protein purification (mg / L) YK028Ag009 5.79 YK028Ag010 4.23 YK028Ag012 28.67 YK028Ag017 34.19 YK028Ag027 72.04

[0137] The results of protein purification are shown in Table 2. Compared with the wild-type gB protein YK028Ag009 molecule with an expression level of 5.79 mg per liter, the expression level of the YK028Ag027 molecule improved by structural biology design was 72.04 mg per liter, and the yield was increased by 12.44 times. Similar to the titer quantitative results in the small test, the yield was also greatly improved.

[0138] The results showed that the improved design of the YK028Ag027 mutant successfully increased the yield of the target trimeric recombinant pseudorabies virus gB protein, laying the foundation for the development of the production process.

[0139] Example 6 Analysis of the status of pseudorabies virus gB protein mutants using protein gel electrophoresis

[0140] In order to determine the status of the pseudorabies virus gB protein mutant, the corresponding gB protein after purification was subjected to protein gel electrophoresis (SDS-PAGE) analysis. The purified gB protein was pretreated. Two 30 μL samples were taken in parallel for each sample to be analyzed. 7.5 μL of reducing (containing DDT reducing agent) and non-reducing (without DDT reducing agent) protein electrophoresis loading buffer were added respectively. After thorough mixing, the samples were placed in a metal bath at 100°C and heated for 15 minutes. After heating, the samples were centrifuged instantly to allow all the samples to settle to the bottom of the tube. The pretreated samples were subjected to precast gel (SurePAGE TM , Bis-Tris, 10×8, 4%-20%, 12wells), load 3μL of standard control marker, and load 10μL of each of the remaining samples (if the component concentration is too high, reduce the loading volume appropriately, otherwise increase it) and perform electrophoresis at a constant voltage of 180 volts for about 45 minutes (based on the migration of the electrophoresis indicator dye to the bottom of the gel). After completion, separate the SDS-PAGE electrophoresis gel, wash with clean water, and stain with Fastblue protein staining solution after washing. After electrophoresis, the gel is completely immersed in the staining solution. After heating to boiling in a microwave oven, take out and stain at a low speed for about 5 minutes. After sufficient staining, remove the staining solution, wash the gel with deionized water, add deionized water after washing, place the gel in a microwave oven and heat it to boiling, decolorize for 15-20 minutes, repeat 2 to 3 times, until the blue staining solution background is completely removed. Place the decolorized gel on a white board for band observation and photography.

[0141] The results of protein gel electrophoresis analysis were as follows Figure 3 As shown, A is the wild-type g B protein of YK028Ag009 protein, B is the mutant g B protein of YK028Ag012 protein, C is the mutant g B protein of YK028Ag017 protein, and D is the mutant g B protein of YK028Ag027 protein. The molecular weight of g B protein is about 80 kDa. The non-reduction electrophoresis target band of the wild-type g B protein of YK028Ag009 protein is consistent with the theoretical molecular size, but there are three bands in the DTT reduction electrophoresis. The possible reason for the analysis is that most of the wild-type proteins undergo conformational changes from fusion precursor to fusion post-body, so the band breaks after DTT reduction electrophoresis, and becomes three bands after DTT reduction electrophoresis. The electrophoresis molecular weight of the designed mutant is not significantly different from that of the wild-type, and the band of the designed g B protein mutant is almost not broken after DDT reduction, indicating that the protein of the designed g B protein mutant still maintains the conformation of the fusion precursor for the most part. The results showed that the purity and concentration of the designed gB protein mutants were significantly improved and maintained in the pre-fusion conformation.

[0142] Example 7 Analysis of the status of pseudorabies virus gB protein mutants using SEC-HPLC chromatography

[0143] In order to determine the status of pseudorabies virus gB protein mutants, the purified corresponding gB protein was subjected to SEC-HPLC chromatography. A Shimadzu high-performance liquid chromatograph and a Sepax SRT-C SE C-500 (5 μm, 7.8*300mm) for detection and analysis. The detection wavelength was set to 280nm, the mobile phase was 20mM phosphate, 300mM sodium chloride, pH7.4, the flow rate was 0.8mL / min, the injection volume was 20μg, and the isocratic elution was 25min. The test samples were processed as follows: the samples were filtered using a 0.22μm needle filter, mixed, and sampled in a liquid phase vial containing an inner insert for later use.

[0144] SEC-HPLC chromatographic results are as follows Figure 4A-4B As shown, 4A is the wild-type g B protein of YK028Ag009, and 4B is the mutant g B protein of YK028Ag027. The absorption peak of the wild-type gB protein is more complex, and other impurity peaks show that the purity of the affinity purified product is low and the uniformity is poor; while the purity of the target protein peak of the YK028Ag027 mutant is significantly improved, and the uniformity of the YK028Ag027 mutant is also improved compared with the wild-type gB protein.

[0145] Example 8 Immunization of mice using purified pseudorabies virus gB protein antigen

[0146] In order to further verify the protective effect of the designed mutant molecules on animals, the preferred mutant (YK028Ag012) was used to immunize mice with purified samples, and then the sera of the immunized mice were collected to test the resistance to virus-infected target cells in vitro to determine the potential protective effect on animals.

[0147] The mouse immunization method is as follows:

[0148] Twenty healthy BALB / c female mice aged 4-8 weeks, weighing about 15g, were randomly divided into 4 groups. The mice were immunized by intramuscular injection of 100 μL of the test sample on days 0 and 21, respectively, and the control group was injected with 100 μL of PBS. Blood was collected on days 14 (before the second immunization) and 35, and serum was separated and collected. The animal immunization experimental method is shown in Table 3 below:

[0149] Table 3 Animal immunization experimental methods

[0150]

[0151] The adjuvant was prepared according to the instructions of ISA201 adjuvant (purchased from SEPPIC, catalog number: ISA201VG): the ratio of protein to adjuvant was 1:1 (50 μL protein + 50 μL adjuvant).

[0152] During the experiment, there were no abnormalities in the appearance, behavior, feeding, secretions, excretions, etc. of the mice, and they had good tolerance, indicating that this recombinant protein has good safety in mouse experiments.

[0153] Example 9 ELISA detection of anti-PRV recombinant protein IgG antibody levels in mouse serum after immunization

[0154] Pre-coating: dilute the recombinant protein to 2 μg / mL using 1×PBS; add the diluted recombinant protein solution to a flat-bottom 96-well ELISA plate at a volume of 100 μL per well; seal the ELISA plate with a sealing film and incubate overnight at 4°C.

[0155] Blocking: Add 350 μL of PBST to each well of the ELISA plate, wash on a 96-well plate shaker for 5 minutes, discard the washing solution, and wash off the remaining liquid with absorbent paper. Repeat the washing three times; add 300 μL of blocking solution to each well, seal with a sealing film, and incubate at room temperature for 1 hour.

[0156] Sample incubation: Use blocking solution to dilute the serum to be tested in a gradient of 1:10. Add 350μL of PBST to each well of the ELISA plate, wash it on a 96-well plate shaker for 5 minutes, pour out the washing solution, and wash the remaining liquid with absorbent paper, repeat the washing three times. Take 100μL of the diluted sample and add it to the 96-well plate in turn, seal it with a sealing film, and incubate it at 37℃ for 1h.

[0157] Secondary antibody incubation: Add 350 μL of PBST to each well of the ELISA plate, wash on a 96-well plate shaker for 5 minutes, discard the washing solution, and wash the remaining liquid with absorbent paper, repeat the washing three times. Dilute the HRP-conjugated secondary antibody (purchased from BETHYL, item number: 44) with blocking solution at a ratio of 1:5000, add 100 μL of secondary antibody dilution solution to each well, seal with sealing film, and incubate at 37°C for 1 hour.

[0158] Color development: Add 350 μL PBST to each well of the ELISA plate, wash on a 96-well plate shaker for 5 min, discard the washing solution, and wash off the remaining liquid with absorbent paper, repeat the washing three times; add 50 μL TMB color development solution to each well, incubate at room temperature in the dark for 15 min; add 50 μL TMB stop solution to each well.

[0159] Data reading and concentration calculation: Use an ELISA reader to read the absorbance at 450 nm and make a bar graph.

[0160] like Figure 5 As shown, 2W is the serum dilution binding antibody level 2 weeks after the initial immunization, and 5W is the serum dilution binding antibody level 2 weeks after the second booster immunization. The anti-PRV recombinant protein IgG antibody level in the serum of the blank control group mice in the first 2 weeks and the second 2 weeks was low, and the test results of the first 2 weeks and the second 2 weeks can be seen that the PRV recombinant protein vaccine optimized in the present invention can produce higher antibody levels in the body of the mice after immunization, among which the group with the highest IgG antibody level is the YK028Ag012 (recombinant protein) + adjuvant group, which is 4.1 times that of the inactivated vaccine group, indicating that the serum IgG antibody level produced in the body of the mouse after immunization with the PRV recombinant protein vaccine optimized in the present invention is significantly improved.

[0161] Example 10 Analysis of the stability status of pseudorabies virus gB protein mutants using the Panta instrument

[0162] The thermal stability results of mutant gB proteins were detected using a Panta instrument. Sample No. 1 was the purified YK028Ag012 mutant gB protein, and sample No. 2 was the purified YK028Ag027 mutant gB protein. The Panta instrument was used for thermal denaturation and particle size detection, with a heating range of 25°C-95°C and a heating rate of 1°C / min.

[0163] The particle size and uniformity test results are shown in Table 4 and Figure 6A-6B As shown, Fig. 6A It is the mutant gB protein of YK028Ag012 protein. Figure 6BThe results in Table 4 show that the PDI values ​​of YK028Ag017 and YK028Ag027 mutants are less than 0.1, indicating that the YK028Ag017 and YK028Ag027 mutant samples are highly homogeneous, and the homogeneity of the YK028Ag017 and YK028Ag027 mutants is better.

[0164] Table 4 Particle size distribution results of pseudorabies virus gB protein mutants detected by Panta instrument

[0165]

[0166]

[0167] Note: Φ represents mean, σ represents variance. Radius: hydrodynamic radius; PDI: polydispersity index, evaluation standard, <0.1 represents high uniformity, 0.1-0.25 represents relatively uniformity, >0.25 represents polydispersity and aggregates, >1 represents unsuitable for DLS detection.

[0168] The thermal stability results are shown in Table 5. The thermal denaturation temperatures (T m The values ​​of Turbidity signal after heating were all high, greater than 60°C, indicating that the protein had good thermal stability. The change in Turbidity signal value after heating was less than 10mAU, so it was considered that there was no significant change and T was not counted. turbidity The value of , indicating that no obvious aggregation occurs; the results of thermal stability show that the YK028Ag012 and YK028Ag027 mutant gB proteins have good thermal stability and do not aggregate and precipitate.

[0169] Table 5 Thermal stability results of pseudorabies virus gB protein mutants detected by Panta instrument

[0170] Sample name <![CDATA[T m ]]> <![CDATA[T onset ]]> <![CDATA[T size ]]> YK028Ag012 73.41 66.00 69.66 YK028Ag017 75.03 64.85 55.78 YK028Ag018 72.52 59.68 63.26 YK028Ag020 75.41 60.04 71.03 YK028Ag021 73.90 68.51 71.21 YK028Ag025 76.48 65.50 71.70 YK028Ag026 76.79 62.37 72.71 YK028Ag027 73.24 67.87 69.12 YK028Ag028 75.07 69.37 71.83 YK028Ag029 76.90 61.08 72.48

[0171] Note: Φ represents mean, σ represents variance. Tm: melting temperature, the higher the evaluation standard, the better; Tonset: the temperature at which unfolding begins, the higher the evaluation standard, the better; Tsize: the temperature at which the average particle size begins to change, the higher the evaluation standard, the better.

[0172] In summary, the gB protein fusion precursor trimer recombinant protein vaccine of porcine pseudorabies virus improved by structural biology design has the advantages of safety, stability, and high efficiency, and greatly improves the production of this recombinant protein, and has very good application prospects.

[0173] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention are obvious to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. A mutant of the gB protein of pseudorabies virus, characterized in that: The gB protein includes the sequence shown in SEQ ID NO.1; the mutant mutates at the ferin protease cleavage site of SEQ ID NO.1 and becomes ineffective, and the mutation is selected from any one or more of the following: 1) A deletion mutation of any 1-31 amino acids between amino acid L486 and amino acid V516; 2) any 1-31 amino acids between L486 and amino acid V516 are mutated to become a linker; 3) Any one or more amino acids in the furin cleavage site between L486 and amino acid V516 are mutated to amino acids other than K or R.

2. The mutant according to claim 1, characterized in that In the above 1), amino acid L486 to amino acid V516 are deleted or amino acid P492 to amino acid P514 are deleted; in the above 2), the linker includes 1-15 amino acids; preferably, the linker is (GGSGG)n, n is an integer of 1-3; more preferably, the linker is GGSGG; in the above 3), two point mutations are included; preferably, they are any two point mutations in positions 499-503; preferably, the point mutation is G; further preferably, R500G and R502G mutations, or R499G and R503G mutations are included.

3. The mutant according to claim 1, characterized in that It also includes deletion mutations of one or more amino acids in the transmembrane region of the gB protein.

4. The mutant according to claim 3, characterized in that The deletion mutation of the transmembrane region of the gB protein is selected from any one or more of the following: (1) Any 1-98 amino acid deletion mutation between amino acid M1 and amino acid S98; (2) Any 1-167 amino acid deletion mutation between amino acid K748 and amino acid P914.

5. The mutant according to claim 4, characterized in that The deletion mutation of the transmembrane region of the gB protein is selected from any one or more of the following: A. Deletion mutation from amino acid M1 to amino acid G57; B. Deletion mutation from amino acid V776 to amino acid P914; C. Deletion mutation from amino acid M1 to amino acid S98; D. a deletion mutation from amino acid V776 to amino acid P914; E. Deletion mutation from amino acid A760 to amino acid P914; F. a deletion mutation from amino acid L755 to amino acid P914; G. a deletion mutation from amino acid V753 to amino acid P914; H. Deletion mutation of amino acid K748 to amino acid P914.

6. The mutant according to claim 1, characterized in that It also includes any point mutation to proline; preferably any one or more amino acids in 1-45 amino acids between S528 and amino acid R572 are mutated to proline; more preferably, it is the H544P point mutation.

7. The mutant according to claim 1, characterized in that It also includes mutations of one or more amino acids that release repulsion, preferably mutation of R745 to an amino acid other than K or R.

8. The mutant according to any one of claims 1 to 7, characterized in that It has a trimer structure.

9. The mutant according to claim 8, characterized in that The trimer structure is achieved by including or not including a heterotrimeric domain, wherein the heterotrimeric domain is selected from any one or more of foldon, GCN4, and RSTN.

10. A nucleic acid molecule encoding the mutant according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A cell comprising the vector of claim 11.

13. Use of the mutant according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the cell according to claim 12 in the detection of pseudorabies virus gB antibody or in the preparation of a vaccine or drug for the prevention, diagnosis or treatment of porcine pseudorabies.

14. The use according to claim 13, characterized in that The vaccine comprises the mutant and composition described in any one of claims 1-9, and / or an immunogenic composition prepared with various types of vaccine adjuvants. The vaccine is a subunit vaccine or a virus-like particle vaccine or a conjugate and a combined antigen.

15. A drug or vaccine or a pseudorabies virus gB antibody detection kit comprising the mutant according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, or the cell according to claim 12.

16. The method for preparing a mutant according to any one of claims 1 to 9, characterized in that: Including expression through genetic engineering.

17. Use of the mutant according to any one of claims 1 to 9, or the nucleic acid molecule according to claim 10, or the vector according to claim 11, or the cell according to claim 12, or the drug or vaccine according to claim 15, or the pseudorabies virus gB antibody detection kit in the prevention, diagnosis and treatment of pseudorabies in pigs.

Images