Application of antigen gene and polypeptide thereof in prevention of canine parvovirus disease

Abstract

The invention discloses an application of an antigen gene and a polypeptide thereof in preventing canine parvovirus disease. The antigen polypeptide provided by the invention can be used as a neutralizing epitope vaccine; the neutralizing epitope vaccine is a protein consisting of 207 amino acid residues; a gene for coding the antigen polypeptide contains 621 nucleotides. The antigen polypeptide provided by the invention, namely the neutralizing epitope vaccine, has the advantages of strong antigenicity, good prevention effect, safety, high yield and the like, and provides a new choice for prevention of canine parvovirus diseases.

Description

Technical Field

[0001] The invention relates to application of an antigen gene and a polypeptide thereof in preventing canine parvovirus disease. Background Art

[0002] Canine parvovirus disease is a highly contagious disease caused by canine parvovirus (CPV) and is one of the most dangerous infectious diseases for puppies. Canine parvovirus (CPV) appeared in the United States in 1978 and spread rapidly among dogs around the world. It is mainly characterized by canine non-suppurative myocarditis and acute hemorrhagic enteritis. It is highly contagious and lethal, causing serious economic losses to the pet industry and dog breeding industry.

[0003] At present, the disease is mainly prevented by vaccination. Traditional vaccines include inactivated vaccines and attenuated vaccines. Although they are highly effective and immunogenic, both use intact viruses as vaccines. There may be situations where the virus is not completely inactivated or the virus is rejuvenated, which has the risk of infecting animals and spreading the virus, and there are safety hazards to a certain extent. VP2 is the main host protective antigen of CPV, which is closely related to the tissue tropism and host range of the virus. The main antigenic epitopes in VP2 can induce the production of neutralizing antibodies. Therefore, it is very important to use genetic engineering technology to obtain protective neutralizing epitope proteins, which can fundamentally eliminate the spread of pathogens, and the protein has a small molecular weight and high yield. The present invention is to connect the protective antigenic epitopes of the VP2 protein identified in this experiment in series to prepare a subunit vaccine, thereby obtaining a safe and effective neutralizing epitope vaccine, which lays the foundation for the effective prevention of CPV. Summary of the invention

[0004] The purpose of the present invention is to provide an application of an antigen gene and its polypeptide in preventing canine parvovirus disease. After immunizing BABL / c female mice with the vaccine, a high antibody level is produced 14 days later, and the antibody level reaches a peak on the 21st day. Compared with the full-length vaccine group and the attenuated vaccine group, the neutralizing epitope vaccine group can stimulate the body to produce a stronger humoral immune response and cellular immune response.

[0005] The canine parvovirus disease neutralizing epitope vaccine and its genomic nucleotide sequence and protein sequence are within the protection scope of the present invention.

[0006] The polypeptide constructed by the present invention, i.e., the canine parvovirus disease neutralizing epitope vaccine, is obtained by using the neutralizing antibodies scFv-16, scFv-hb5, scFv-132, and scFv-149 stored in this laboratory to perform antigen epitope mapping on the VP2 gene (GenBank sequence number is NC_001539.1) to obtain 5 neutralizing epitopes, and then connecting them together to express the obtained protein;

[0007] The neutralizing epitope vaccine is as follows (a) or (b): (a) a protein consisting of amino acid residues 1 to 207 from the N-terminus of sequence 2 in the sequence list; (b) a protein derived from (a) with the same activity after one or more amino acid residues are replaced and / or deleted and / or added;

[0008] The gene encoding the neutralizing epitope vaccine also falls within the protection scope of the present invention.

[0009] In the gene, the DNA molecule encoding the neutralizing epitope vaccine is as follows: (1) or (2) or (3): (1) a DNA molecule shown in nucleotides 1 to 621 from the 5' end of sequence 1 in the sequence list; (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein with the same activity; (3) a DNA molecule that has at least 90% homology with the DNA sequence defined in (1) and encodes a protein with the same activity.

[0010] The above stringent conditions may be hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0011] The expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the above-mentioned genes all fall within the protection scope of the present invention.

[0012] The related preparations of the canine parvovirus disease neutralizing epitope vaccine are within the scope of protection of the present invention.

[0013] The application of the polypeptide and polypeptide gene in preventing canine parvovirus disease is within the scope of protection of the present invention.

[0014] The canine parvovirus disease neutralizing epitope vaccine constructed by the present invention can be regarded as a subunit vaccine modified by genetic engineering, and its corresponding preparations, such as freeze-dried powder injection preparations, liquid vaccine preparations, etc., all fall within the scope of protection of this patent.

[0015] The present invention provides an antigen polypeptide, namely a neutralizing epitope vaccine, which can stimulate mice to produce higher antibody levels and achieve better protective effects. It also has the advantages of strong antigenicity, controllable industrial production quality, high yield, and avoidance of pathogen transmission. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a neutralization epitope mapping map.

[0017] Figure 2 This is a DNA gel map of the construction of the neutralizing epitope vaccine. M is a molecular weight marker, and lanes 1, 2, and 3 are the gene fragments of the neutralizing epitope vaccine.

[0018] Figure 3 The SDS-PAGE map of the neutralizing epitope vaccine. M is the molecular weight marker, and lane 1 is the protein molecule of the neutralizing epitope vaccine.

[0019] Figure 4 This is a Western Blot map of the neutralizing epitope vaccine. Lane 1 is the protein of the neutralizing epitope vaccine, and lane 2 is the negative control.

[0020] Figure 5 This is the antibody titer of neutralizing epitope vaccine (VP2D) in experimental BABL / c female mice.

[0021] Figure 6 This is the content of immune cells in the experimental BABL / c female mice at different time points after they were immunized with neutralizing epitope vaccine (VP2D). Specific implementation plan

[0022] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0023] Example 1: Localization of Antigen Neutralizing Epitopes

[0024] The expression level of truncated proteins in the periplasm of cells was detected by FCM. We transformed Escherichia coli DH5α cells with the pAPEx-V1-V11 plasmid and added the positive colonies to the liquid culture medium for culture. When the OD value 600 was 0.4-0.6, IPTG was added for induction. After culturing at 37°C for 4 hours, spheroplasts were prepared according to standard procedures and incubated with FITC-anti-Flag IgG (1:2000) and 1% BSA for 1 hour, detected by FCM, and a negative control was set up at the same time. According to the expression amount of V1-V11 protein in the periplasmic cavity of the bacterial inner membrane, different volumes of bacterial liquid were collected, spheroplasts were prepared, and they were incubated with 2μg ScFv, anti-his antibody (1:500) and FITC-labeled goat anti-mouse antibody (1:2000) respectively, and then detected by FCM.

[0025] The results are as follows Figure 1 As shown, the neutralizing epitopes of scFv-16 are V2 and V3, the neutralizing epitope of scFv-hb5 is V8, the neutralizing epitope of scFv-132 is V10, and the neutralizing epitope of scFv-149 is V11.

[0026] Example 2: Construction and identification of neutralizing epitope vaccines

[0027] Primers were designed based on the selected 5 segments of neutralization epitope vaccine genes, and the VP2 gene was used as a template. The 5 segments of neutralization epitope vaccine genes were connected together using the overlap PCR method. The neutralization epitope vaccine gene fragments were recovered using a gel recovery kit, and the neutralization epitope vaccine gene fragments were connected to the pET28a vector to construct an expression vector recombinant plasmid.

[0028] The results are as follows Figure 2 As shown, the neutralizing epitope vaccine gene size was approximately 621 bp, and the expression vector recombinant plasmid was successfully constructed.

[0029] Example 3: Expression, purification and identification of neutralizing epitope vaccines

[0030] The recombinant plasmid was introduced into Escherichia coli Rosetta to obtain recombinant bacteria. The obtained recombinant bacteria were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 120 r / min with shaking until OD 600nm =0.35; add IPTG to a final concentration of 0.25mmol / L, and culture at 37℃, 120r / min for 4h. Centrifuge the bacterial solution at 4℃, 4000r / min for 30min, and collect the bacterial precipitate. Resuspend the bacterial precipitate with PBS solution, add lysozyme solution (purchased from Amresco) and make the lysozyme concentration 1mg / mL, place at 4℃ for 1h, then perform ultrasonic disruption (25 watts of power, 3min), centrifuge at 4℃, 10000g for 30min, and collect the precipitate. Wash the precipitate with washing solution 3 times, dissolve the precipitate with denaturing solution, and place at 4℃ overnight. Add the denatured protein dropwise to the 10-fold system refolding solution and place at 4℃ overnight. Dialyze the refolded protein into PBS solution, filter, and store in a -70 refrigerator. See the results. Figure 3 .

[0031] After the purified protein was subjected to 15% SDS-PAGE electrophoresis, the protein was transferred to a nitrocellulose membrane (NC membrane) using a membrane transfer apparatus, with a current of 200 mA and a transfer time of 40 minutes. The NC membrane was blocked with 5% skimmed milk powder and incubated at 37°C for 3 hours. Mouse anti-VP2 polyclonal antibody (1:1000) was added and incubated at 4°C overnight. The NC membrane was washed as above, HRP-labeled anti-mouse secondary antibody (1:7500) was added and incubated at 37°C for 1 hour, and the NC membrane was washed 4 times with PBST solution, each time for 10 minutes. The membrane was exposed and developed using ECL luminescence detection reagent, and GPV VP2 was set as a negative control. The results are shown in the figure. Figure 4 .

[0032] Example 4: Determination of antibody titer and immune cell content after immunization of mice with neutralizing epitope vaccine

[0033] 72 6-week-old BABL / c female SPF mice were randomly divided into 6 groups (n=12), including the first group immunized with 100 μL (43.2 pmol / μL) of purified VP2D protein vaccine, the second group immunized with 100 μL (43.2 pmol / μL) of purified VP2 protein vaccine, the third group immunized with 100 μL of commercial canine parvovirus vaccine, the fourth group immunized with 100 μL (43.2 pmol / μL) of VP2D protein + complete Freund's adjuvant vaccine, the fifth group immunized with 100 μL (43.2 pmol / μL) of VP2D protein + incomplete Freund's adjuvant vaccine, and all mice in the sixth group were immunized with 100 μL of PBS. Venous blood was collected at 7, 14 and 21 days after immunization, and serum was separated.

[0034] Anti-CPV antibody levels were detected by ELISA. Figure 5 . ELISA results showed that the antibody level of the neutralizing epitope vaccine group rose to a higher level 14 days after immunization. Compared with the VP2 vaccine group and the attenuated vaccine group, the VP2D vaccine group could stimulate the body to produce a stronger humoral immune response. FCM was used to detect the content of immune cells in mouse leukocytes, including CD4+T cells, CD8+T cells, CD11b+ cells and F4 / 80+ cells. Results are shown in Figure 6 , 7 to 21 days after immunization, the levels of CD4+T cells, CD8+T cells, CD11b+ cells and F4 / 80+ cells in the experimental group showed an upward trend. Compared with the attenuated vaccine group, the VP2D+CFA vaccine group and the VP2D+IFA vaccine group stimulated the body to produce a stronger cellular immune response.

Claims

1. An antigenic polypeptide, which is a protein obtained by selecting 5 neutralizing epitopes from the VP2 gene and connecting them in series for expression; The antigen polypeptide is as follows (a) or (b): (a) a protein consisting of amino acid residues 1-207 from the N-terminus of sequence 2 in the sequence list; (b) a protein derived from (a) with the same activity after one or more amino acid residues are replaced and / or deleted and / or added.

2. A gene encoding the antigenic polypeptide according to claim 1.

3. The gene according to claim 2, characterized in that: In the gene, the DNA molecule encoding the antigen polypeptide is the following (1) or (2) or (3): (1) the DNA molecule represented by nucleotides 1 to 621 from the 5' end of sequence 1 in the sequence list; (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein having the same activity; (3) A DNA molecule that has at least 90% homology with the DNA sequence defined in (1) and encodes a protein with the same activity.

4. An expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the gene according to claim 3.

5. Use of the antigen polypeptide according to claim 1 and the gene of the antigen polypeptide according to claim 3 in the preparation of related products.

6. Use of the antigenic polypeptide according to claim 1 and the gene of the antigenic polypeptide according to claim 3 in preventing canine parvovirus disease.

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