阅读说明：本技术 猪瘟病毒重组抗原及其制备方法和应用 (Classical swine fever virus recombinant antigen and preparation method and application thereof ) 是由 曹文龙 孔迪 滕小锘 易小萍 张大鹤 于 2020-06-15 设计创作，主要内容包括：本发明涉及一种猪瘟病毒重组抗原及其制备方法和应用,所述猪瘟病毒重组抗原为经过蛋白质改造的E2蛋白,所述蛋白质改造包括点突变,所述点突变为将E2蛋白的第166位氨基酸突变成天冬酰胺,且将第229位氨基酸突变成丙氨酸。本发明将E2蛋白关键的糖基化位点第229位的Asn氨基酸突变成Ala,能够去掉糖基化,并且将第166位的Asp氨基酸突变成Asn,引入一个新的糖基化位点,从而能够在增加蛋白的免疫原性的同时降低其过敏反应。(The invention relates to a classical swine fever virus recombinant antigen and a preparation method and application thereof, wherein the classical swine fever virus recombinant antigen is E2 protein modified by protein, the protein modification comprises point mutation, and the point mutation is to change the 166 th amino acid of the E2 protein into asparagine and change the 229 th amino acid into alanine. The invention changes the Asn amino acid at 229 th position of the key glycosylation site of the E2 protein into Ala to remove glycosylation, and changes the Asp amino acid at 166 th position into Asn to introduce a new glycosylation site, thereby increasing the immunogenicity of the protein and reducing the anaphylactic reaction.)

1. A classical swine fever virus recombinant antigen is E2 protein which is subjected to protein modification, wherein the protein modification comprises point mutation, and the point mutation is that the 166 th amino acid of the E2 protein is mutated into asparagine, and the 229 th amino acid is mutated into alanine.

2. The classical swine fever virus recombinant antigen of claim 1, wherein the protein engineering further comprises adding a T4 bacteriophage fibrin trimerization domain at the C-terminus of the E2 protein.

3. The classical swine fever virus recombinant antigen according to claim 2, wherein the amino acid sequence of the classical swine fever virus recombinant antigen is as set forth in SEQ ID NO:5, respectively.

4. The classical swine fever virus recombinant antigen according to claim 2, wherein the amino acid sequence of the classical swine fever virus recombinant antigen is as set forth in SEQ ID NO:3, respectively.

5. The classical swine fever virus recombinant antigen according to claim 2, wherein the amino acid sequence of the classical swine fever virus recombinant antigen is as set forth in SEQ ID NO:1 is shown.

6. An expression gene capable of expressing the classical swine fever virus recombinant antigen according to any one of claims 1 to 5.

7. The expressible gene of claim 6 having the nucleic acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO:6 or SEQ ID NO: shown in fig. 8.

8. An expression vector comprising the expressible gene of claim 6 or 7.

9. A host cell having incorporated in its genome the gene of claim 6 or 7.

10. The host cell of claim 9, wherein the host cell is a CHO cell.

11. A preparation method of a classical swine fever virus recombinant antigen is characterized by comprising the following steps: culturing the host cell of claim 9 or 10 under suitable conditions, collecting the culture medium and/or the lysate of said host cell, and isolating and purifying to obtain said recombinant antigen of classical swine fever virus.

12. Use of the classical swine fever virus recombinant antigen according to any one of claims 1 to 5, the expression gene according to claim 6 or 7, the expression vector according to claim 8, or the host cell according to claim 9 or 10 for the preparation of a product for the control of classical swine fever.

13. A classical swine fever vaccine, comprising the classical swine fever virus recombinant antigen of any one of claims 1 to 5 and a pharmaceutically acceptable adjuvant.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a classical swine fever virus recombinant antigen and a preparation method and application thereof.

Background

Classical Swine Fever (CSF) is an acute, febrile and highly contagious disease caused by Classical Swine Fever Virus (CSFV), and has major symptoms of high fever and hemorrhage, and the pathological cause is mainly degeneration of small vessel wall, which leads to hemorrhage, infarction and even necrosis of internal organs. The transmission routes are mainly contact transmission, digestive tract transmission or respiratory tract transmission. The disease can cause acute, subacute, chronic or even delayed infection, and can also cause non-pathogenic subclinical symptoms. Because pigs of different ages, sexes and breeds can be infected and attacked, the swine fever causes great economic loss to the pig industry. The disease is distributed in many countries and regions, swine fever is determined to be a type A legal epidemic disease in the International animal health code by the world animal health Organization (OIE), and is listed as a type of infectious disease in China, which is one of the most important and most harmful infectious diseases in swine diseases.

CSFV is an enveloped, positive-sense, single-stranded RNA virus with rounded virions classified as a pestivirus of the flaviviridae family. Research shows that the diameter of the virus particle is 30-50 nm, the virus particle is coated with a protein envelope, and glycoprotein protrusions with the length of 6-8 nm are arranged on the envelope. The CSFV genome is approximately 12.3kb in length and comprises a large Open Reading Frame (ORF) flanked by a 5 'noncoding region (5' -UTR) and a 3 'noncoding region (3' -UTR). The Open Reading Frame (ORF) encodes a polyprotein of 3898 amino acid residues (aa) which is processed to mature viral proteins by host cells and viral proteases, splitting into four structural proteins (C, E2, Erns, E1) and eight non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B, Npro, P7). The E2 glycoprotein is one of the most important envelope proteins of classical swine fever virus, and consists of 373 amino acids, has a size of about 55kDa, and contains 6N-linked and 1O-linked specific glycosylation sites. 4 antigen regions, namely an A region, a B region, a C region and a D region, exist on the antigen of the E2 protein, and the positions of the 4 antigen regions are between amino acid residues 690-866 at the N end of the E2 protein, which indicates that the part may be an important region for the E2 protein to show antigenicity. The E2 protein is the most important structural protein involved in CSFV replication and can induce virus to infect host cells. During the CSFV infection of host cells, the E2 protein is connected into homodimers or heterodimers by means of disulfide bonds between cysteines and distributed on the surfaces of virions, the structural domain formed by disulfide bonds maintains the stability of the antigenic structure of the E2 protein, and the dimers can effectively stimulate the generation of neutralizing antibodies aiming at CSFV in animals.

The adoption of immunization is the main measure for controlling the swine fever in China at present, and the quality of the vaccine directly influences the immune control effect of the epidemic disease. Since the 50 s, the hog cholera lapinized virus vaccine (the 'C' strain) which is self-developed in China is widely applied, however, the generation and the spread of the hog cholera cannot be effectively controlled after the long-term use of the hog cholera lapinized virus vaccine, the immunogenicity of the hog cholera lapinized virus vaccine is low, and the requirements of modern prevention and control and eradication of the hog cholera cannot be met.

Disclosure of Invention

Therefore, the swine fever virus recombinant antigen which has better immunogenicity and higher safety and is easy to express is needed to be provided.

A classical swine fever virus recombinant antigen is a protein-engineered E2 protein, wherein the protein engineering comprises point mutation, the point mutation is that the 166 th amino acid of the E2 protein is mutated into asparagine, and the 229 th amino acid is mutated into alanine.

In one embodiment, the protein engineering further comprises adding a T4 bacteriophage fibrin trimerization domain at the C-terminus of the E2 protein.

In one embodiment, the amino acid sequence of the recombinant antigen of classical swine fever virus is as set forth in SEQ ID NO:5, respectively.

In one embodiment, the amino acid sequence of the recombinant antigen of classical swine fever virus is as set forth in SEQ ID NO:3, respectively.

In one embodiment, the amino acid sequence of the recombinant antigen of classical swine fever virus is as set forth in SEQ ID NO:1 is shown.

The invention also provides an expression gene which can express the swine fever virus recombinant antigen.

In one embodiment, the nucleic acid sequence is as set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO:6 or SEQ ID NO: shown in fig. 8.

The invention also provides an expression vector containing the expression gene.

The invention also provides a host cell, wherein the genome of the host cell is doped with the expression gene.

In one embodiment, the host cell is a CHO cell.

The invention also provides a preparation method of the classical swine fever virus recombinant antigen, which comprises the following steps: culturing the host cell under a proper condition, collecting a culture solution and/or a lysate of the host cell, and then carrying out separation and purification to obtain the classical swine fever virus recombinant antigen.

The invention also provides application of the swine fever virus recombinant antigen, the expression gene, the expression vector or the host cell in preparation of products for preventing and treating swine fever.

The invention also provides a classical swine fever vaccine which comprises the classical swine fever virus recombinant antigen and a pharmaceutically acceptable adjuvant.

The E2 protein is a single subunit protein, which has poor immunogenicity when singly expressed, while the immunogenicity of the E2 protein which is expressed by recombination is related to the glycosylation level thereof, and the glycosylation modification level and the structure of a sugar chain are not completely consistent in different expression systems, so that the antigenicity of the proteins is different. The glycosylation structure is changed through rational mutation, in particular, Asn amino acid at 229 th position of key glycosylation site of E2 protein is mutated into Ala, glycosylation can be removed, Asp amino acid at 166 th position is mutated into Asn, a new glycosylation site is introduced, and therefore, the immunogenicity of the protein can be increased, and the anaphylactic reaction of the protein can be reduced. Immune experiments and tolerance experiments prove that the immunogenicity of the modified E2 protein is obviously improved, the allergic stress reaction of the modified E2 protein to pigs is reduced, and the safety is higher. The subunit vaccine prepared by the swine fever virus recombinant antigen has good immunogenicity, is safe and efficient, has very high cell expression level, can be used for large-scale batch production, is easy to control quality, is stable among batches, and has low production cost.

Drawings

FIG. 1 shows the results of gel electrophoresis of the PCR product obtained after PCR amplification of NE2 gene; wherein 1 is NE2 gene; 2 is negative control; m is a molecular weight marker;

FIG. 2 shows the results of gel electrophoresis of PCR products after PCR amplification of a plurality of colony samples transformed with NE2 protein gene; wherein 1-6 are products obtained after PCR amplification of colony samples transformed by NE2 gene, 7 is negative control, and M is molecular weight marker;

FIG. 3 is a map of the constructed eukaryotic expression vector PCI-NE 2-GS;

FIG. 4 shows the results of SDS-PAGE gel electrophoresis of cell culture supernatants of example 2, wherein 1 to 5 are cell culture supernatants of NE2 group, E2-1 group, E2-2 group, E2-3 group and E2-4 group, respectively; 6 to 8 are endoglycosidase Endo H_fCell culture supernatants of the treated NE2 group, E2-1 group, E2-2 group; 9 is a negative control; m is a molecular weight marker;

FIG. 5 shows Western Blot analysis results of recombinant CHO supernatant samples expressed in example 3, wherein 1-5 are recombinant CHO supernatant samples of NE2 group, E2-1 group, E2-2 group, E2-3 group and E2-4 group, respectively; 6 to 8 are endoglycosidase Endo H_fThe treated NE2 group, E2-1 group and E2-2 histone recombinant CHO supernatant samples; 9 is a negative control; m is a molecular weight marker;

FIG. 6 is a graph showing the results of Ni purification in example 4; wherein, 1 is sample loading, 2 is flow-through, 3 is 50mM imidazole elution impurity, 4 is 300mM imidazole elution target protein, and M is molecular weight marker;

FIG. 7 is a graph showing the results of molecular sieve chromatography of NE2 group in example 4;

FIG. 8 is a graph showing the results of the molecular sieve chromatography of group E2-1 in example 4;

FIG. 9 is an electron microscope result chart of NE2 histone in example 4;

FIG. 10 is a graph showing the results of neutralizing antibody titers in example 7.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The classical swine fever virus recombinant antigen is an E2 protein modified by a protein, wherein the protein modification comprises point mutation, the point mutation is that the 166 th amino acid of the E2 protein is mutated into asparagine, and the 229 th amino acid is mutated into alanine.

The term "antigen" refers to all substances that induce an immune response in the body, i.e., substances that are specifically recognized and bound by antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, proliferate and differentiate to produce immune response products (sensitized lymphocytes or antibodies), and specifically bind to the corresponding products in vitro and in vivo. Thus, antigenic substances possess two important properties: immunogenicity (immunogenicity) and immunoreactivity (immunoreactivity). Immunogenicity refers to the ability of an antigen to induce an organism to generate a specific immune response, produce antibodies and/or sensitize lymphocytes; immunoreactivity refers to the ability to undergo specific binding reactions with the corresponding immune effector substance (antibody or sensitized lymphocytes) in vitro and in vivo.

The E2 protein is a single subunit protein, which has poor immunogenicity when singly expressed, while the immunogenicity of the E2 protein which is expressed by recombination is related to the glycosylation level thereof, and the glycosylation modification level and the structure of a sugar chain are not completely consistent in different expression systems, so that the antigenicity of the proteins is different. The glycosylation structure is changed through rational mutation, in particular, Asn amino acid at 229 th position of key glycosylation site of E2 protein is mutated into Ala, glycosylation can be removed, Asp amino acid at 166 th position is mutated into Asn, a new glycosylation site is introduced, and therefore, the immunogenicity of the protein can be increased, and the anaphylactic reaction of the protein can be reduced. Immune experiments and tolerance experiments prove that the immunogenicity of the modified E2 protein is obviously improved, the allergic stress reaction of the modified E2 protein to pigs is reduced, and the safety is higher. The subunit vaccine prepared by the swine fever virus recombinant antigen has good immunogenicity, is safe and efficient, has very high cell expression level, can be used for large-scale batch production, is easy to control quality, is stable among batches, and has low production cost.

In a specific example, the protein engineering further comprises adding a T4 bacteriophage fibrin trimerization domain at the C-terminus of the E2 protein. By adding the T4 phage fibrin trimerization domain (T4 fibrosis domain) at the C-terminus of the E2 protein, a complex structure of trimers can be formed, further increasing immunogenicity. In addition, because the formation of trimer is prevented by the existence of Asn amino acid at 229 th site of glycosylation site of E2 protein, a T4 bacteriophage fibrin trimerization domain is added at the C terminal of the E2 protein on the basis of mutation of Asn amino acid at 229 th site of the protein to Ala, so that a complex trimer structure can be introduced, thereby obviously improving the immunogenicity of antigen.

In a specific example, the protein modification further comprises inserting a tandem repeat epitope of the E2 protein modified by the point mutation into the E2 protein, wherein the amino acid sequence of the modified protein is shown as SEQ ID NO:5, respectively. Immunogenicity can be further increased by inserting a tandem repeat epitope in the E2 protein, wherein the tandem repeat epitope contains Asn 166 introduced by the point mutation as described above.

The term "epitope" refers to an antigenic determinant, which is a specific chemical group on the surface or other parts of an antigenic substance molecule, having a certain composition and structure, capable of specifically binding to its corresponding antibody or sensitized lymphocyte, and the structurally defined antigenic determinant is called an epitope.

In a specific example, the protein modification further comprises inserting an epitope of Erns protein into the E2 protein, and the amino acid sequence of the modified protein is as shown in SEQ ID NO:3, respectively. The Erns protein exists in the form of homodimer in the virus particle, and after entering into the body, the Erns can stimulate the body to generate a neutralizing antibody, so that the immune pig can obtain the protection force on CSFV. Erns have RNase activity, which may play an important role in the replication of CSFV, and is associated with the persistence of infection in the host. By adding the antigenic epitope of the Erns protein into the E2 protein, on one hand, the antigenicity can be increased, and on the other hand, because the full-length Erns protein is not used, the immune pig and the virus-infected pig can be distinguished by detecting antibodies of other Erns epitopes, so that the E2 protein can be used for purification of pig farms.

In a specific example, the amino acid sequence of the classical swine fever virus recombinant antigen is as set forth in SEQ ID NO:1, the best effect is obtained.

The expression gene of one embodiment of the invention can express the classical swine fever virus recombinant antigen.

In one specific example, the nucleic acid sequence of the expressed gene is as set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO:6 or SEQ ID NO: shown in fig. 8. It will be appreciated that due to the degeneracy of the codons, the nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon optimized nucleic acid sequences, but are not limited thereto.

The expression vector of an embodiment of the present invention contains the expression gene.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the invention. In a specific embodiment, the expression vector may be pSV2-GS, pCI-GS, pcDNA4-GS, etc., preferably pCI-GS.

The host cell according to an embodiment of the present invention has the above-described expression gene incorporated into its genome.

The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

In one particular example, the host cell is a CHO cell. Specifically, the CHO cell line may be DG44, DXB11, CHO-K1, CHO-S cell line, etc., preferably CHO-S. The recombinant classical swine fever virus antigen protein obtained after the CHO cell expression modification is expressed by using a eukaryotic system, the glycosylation of the protein is sufficient, the immunogenicity of the antigen protein is good, the expression level is very high and reaches 3 g/L-4 g/L, the recombinant cell can be cultured in a suspension manner on a large scale, the complexity of vaccine preparation is greatly reduced, and the production cost is reduced.

The preparation method of the classical swine fever virus recombinant antigen provided by the embodiment of the invention comprises the following steps: culturing the host cell under proper conditions, collecting the culture solution and/or the lysate of the host cell, and then separating and purifying to obtain the classical swine fever virus recombinant antigen.

In a specific example, the method of separation and purification includes nickel column affinity chromatography, molecular sieve chromatography, and the like, but is not limited thereto and may be selected as needed.

The swine fever vaccine provided by the embodiment of the invention comprises the swine fever virus recombinant antigen and a pharmaceutically acceptable adjuvant.

In one specific example, the adjuvant can be one or a combination of two or more of white oil, aluminum stearate, span and tween, and the white oil adjuvant is preferably used.

Embodiments of the present invention will be described in detail below with reference to specific examples.