American-type PRRSV N protein antigenic epitope and applications thereof

Abstract

The invention discloses an American-type PRRSV N protein antigenic epitope and applications thereof, and aims to solve the technical problem of insufficient research on PRRSV N protein cell epitopes.The invention designs a short peptide, and the amino acid sequence of the short peptide is as shown in SEQ NO.1 or is an amino acid sequence which is derived from the amino acid sequence as shown in the SEQ ID NO.1 and has the same function. The American-type PRRSV N protein antigenic epitope is screened, and comprises the amino acid sequence of the short peptide. The short peptide or the American-type PRRSV N protein antigenic epitope are applied in preparing an antibody for preventing and / or treating American-type PRRSV. The invention provides a novel N-protein antigenic epitope, completes the identification of the epitope, improves the epitope map of the N protein, lays a foundation for the research of American-type PRRSV cross-protective epitope vaccines, and has great significance fordifferentiated detection on American-type PRRSV and European-type PRRSV.

Description

technical field

[0001] The invention relates to the technical field of cellular immunity, in particular to an epitope of N protein antigen of American type PRRSV and application thereof. Background technique

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is the most serious viral disease in the swine industry in the world. Sows infected with PRRSV are characterized by reproductive disorders, mainly premature birth, late abortion, stillbirth, mummified fetus, fever, anorexia, etc. Infected piglets show respiratory symptoms and neurological symptoms. In the course of the disease, the skin of both ears will appear cyanotic and purple, so it is also called "blue ear disease". Contact infection, semen transmission, and air transmission are the main transmission routes of PRRSV, and transporters such as birds, rodents, and humans may all become vectors of the disease. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the disease...

Examples

Embodiment 1

[0043] Example 1: Selection and preparation of immunogen

[0044] 1. Selection of Immunogen

[0045] The inventor finds through long-term, a large amount of research, practice, experiment:

[0046] First, among the proteins encoded by the PRRSV genome, the nucleocapsid protein encoded by ORF7, namely the N protein, is highly conserved;

[0047] Secondly, the immune reactivity induced by N protein in the body is much higher than that of other proteins, so it is easy to obtain high-affinity monoclonal antibodies;

[0048] Thirdly, the content of N protein is the highest after PRRSV infection, accounting for 40% to 60% of the total structural protein, which is the dominant target for PRRSV antigen detection.

[0049] Based on the above three points, N protein becomes the preferred target protein.

[0050] 2. Preparation of Immunogen

[0051] The accession number of the nucleotide sequence of N protein on Genebank is: EF112445.

[0052] (1) Construct the pET-28a-N recombinant...

Embodiment 2

[0059] Example 2: Preparation and Identification of Monoclonal Antibody

[0060] 1. Animal Immunization

[0061] (1) Add the immunogen N protein to Freund's complete adjuvant for the first immunization, and emulsify it to make the immune antigen;

[0062] (2) Three 4- to 8-week-old female BALB / c mice were immunized by multi-point subcutaneous injection on the back, with an immune dose of 50 μg / mouse;

[0063] (3) BALB / c mice were boosted with the same method and dose after emulsification with Freund's incomplete adjuvant and immune antigen every 3 weeks;

[0064] (4) After the fourth immunization, 3 to 4 days before cell fusion, BALB / c mice were super-immunized with immunogen without adjuvant by tail vein injection, and the immunization dose was 100 μg / mouse;

[0065] (5) Determination of titer and sensitivity of multiple antiserum:

[0066] One week after the last booster immunization, tail-cut blood was collected from 3 mice respectively, and then the potency and sensitiv...

Embodiment 3

[0087] Example 3: Purification of Antibodies

[0088] Caprylic acid-ammonium sulfate method for antibody purification, the operation method is as follows:

[0089] (1) Take out the frozen monoclonal antibody ascites or antiserum, and centrifuge at 6000 r / min for 30 minutes to obtain the supernatant, which is diluted 5 times with sodium acetate buffer (0.06mol / L pH4.8).

[0090] (2) Adjust the pH to 4.5 with NaOH (5 mol / L), stir slowly at room temperature for 0.5 h, and add n-octanoic acid to a final concentration of 25 μL / mL.

[0091] (3) Centrifuge at 6000 r / min for 30 minutes at 4°C, and keep the supernatant.

[0092] (4) Filter the obtained supernatant with medium-speed filter paper, add 10×PBS buffer solution (pH7.4), the volume is 1 / 10, mix well and adjust the pH to 7.4.

[0093] (5) Add solid ammonium sulfate to the mixture at 4°C, the addition ratio is 0.2778 g / mL, and can be added several times, and the addition is completed within 30 minutes.

[0094] (6) Centrifug...

technical field

[0001] The invention relates to the technical field of cellular immunity, in particular to an epitope of N protein antigen of American type PRRSV and application thereof. Background technique

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is the most serious viral disease in the swine industry in the world. Sows infected with PRRSV are characterized by reproductive disorders, mainly premature birth, late abortion, stillbirth, mummified fetus, fever, anorexia, etc. Infected piglets show respiratory symptoms and neurological symptoms. In the course of the disease, the skin of both ears will appear cyanotic and purple, so it is also called "blue ear disease". Contact infection, semen transmission, and air transmission are the main transmission routes of PRRSV, and transporters such as birds, rodents, and humans may all become vectors of the disease. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the disease...

Examples

Embodiment 1

[0043] Example 1: Selection and preparation of immunogen

[0044] 1. Selection of Immunogen

[0045] The inventor finds through long-term, a large amount of research, practice, experiment:

[0046] First, among the proteins encoded by the PRRSV genome, the nucleocapsid protein encoded by ORF7, namely the N protein, is highly conserved;

[0047] Secondly, the immune reactivity induced by N protein in the body is much higher than that of other proteins, so it is easy to obtain high-affinity monoclonal antibodies;

[0048] Thirdly, the content of N protein is the highest after PRRSV infection, accounting for 40% to 60% of the total structural protein, which is the dominant target for PRRSV antigen detection.

[0049] Based on the above three points, N protein becomes the preferred target protein.

[0050] 2. Preparation of Immunogen

[0051] The accession number of the nucleotide sequence of N protein on Genebank is: EF112445.

[0052] (1) Construct the pET-28a-N recombinant...

Embodiment 2

[0059] Example 2: Preparation and Identification of Monoclonal Antibody

[0060] 1. Animal Immunization

[0061] (1) Add the immunogen N protein to Freund's complete adjuvant for the first immunization, and emulsify it to make the immune antigen;

[0062] (2) Three 4- to 8-week-old female BALB / c mice were immunized by multi-point subcutaneous injection on the back, with an immune dose of 50 μg / mouse;

[0063] (3) BALB / c mice were boosted with the same method and dose after emulsification with Freund's incomplete adjuvant and immune antigen every 3 weeks;

[0064] (4) After the fourth immunization, 3 to 4 days before cell fusion, BALB / c mice were super-immunized with immunogen without adjuvant by tail vein injection, and the immunization dose was 100 μg / mouse;

[0065] (5) Determination of titer and sensitivity of multiple antiserum:

[0066] One week after the last booster immunization, tail-cut blood was collected from 3 mice respectively, and then the potency and sensitiv...

Embodiment 3

[0087] Example 3: Purification of Antibodies

[0088] Caprylic acid-ammonium sulfate method for antibody purification, the operation method is as follows:

[0089] (1) Take out the frozen monoclonal antibody ascites or antiserum, and centrifuge at 6000 r / min for 30 minutes to obtain the supernatant, which is diluted 5 times with sodium acetate buffer (0.06mol / L pH4.8).

[0090] (2) Adjust the pH to 4.5 with NaOH (5 mol / L), stir slowly at room temperature for 0.5 h, and add n-octanoic acid to a final concentration of 25 μL / mL.

[0091] (3) Centrifuge at 6000 r / min for 30 minutes at 4°C, and keep the supernatant.

[0092] (4) Filter the obtained supernatant with medium-speed filter paper, add 10×PBS buffer solution (pH7.4), the volume is 1 / 10, mix well and adjust the pH to 7.4.

[0093] (5) Add solid ammonium sulfate to the mixture at 4°C, the addition ratio is 0.2778 g / mL, and can be added several times, and the addition is completed within 30 minutes.

[0094] (6) Centrifug...

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