CN108383913A - The preparation and application of Recombinant Swine IL-23 enhancing PCV2 vaccine immunity adjuvants - Google Patents

Abstract

The invention discloses the preparation and application of a recombinant porcine IL-23 enhancing PCV2 vaccine immune adjuvant. The present invention obtains the gene fragments of P40 and P19 of the Tibetan pig IL-23 gene for the first time by using the gene cloning technology, and uses the 2A self-cut expression technology to connect the P40 and P19 genes to obtain the complete IL-23 gene sequence, and then uses In- The fusion cloning method constructs the complete IL-23 gene into the eukaryotic secretory expression vector VR1020, wraps the IL-23 plasmid with chitosan to prepare chitosan nanoparticles, and inoculates experimental animals with PCV2 vaccine at the same time, using pigs as experimental animals , the results showed that IL-23 eukaryotic transfection plasmid can effectively enhance the immune response of pigs to PCV2 vaccine and promote the growth of vaccinated animals; it provides a new technology for the development of new and efficient PCV2 immune adjuvants.

Description

Preparation and Application of Recombinant Porcine IL-23 Enhanced Immune Adjuvant of PCV2 Vaccine

technical field

The invention relates to the preparation and application of a recombinant porcine IL-23 enhancing PCV2 vaccine immune adjuvant.

Background technique

Pork products are the main source of meat consumption for urban and rural residents and are closely related to people's lives. Since the 21st century, the annual number of live pigs and slaughtered pigs in my country has always accounted for about 50% of the world's total, ranking first in the world. While my country's pig industry has achieved world-renowned achievements, there are still many factors that restrict the healthy development of my country's pig industry, among which disease is the most important factor. Porcine circovirus type 2 (PCV2) infection is a relatively common type of infectious disease. PCV2 is the main pathogen that causes Postweaning multisystemic wasting syndrome (PMWS) in weaned piglets. Infection has caused significant economic losses to the swine industry worldwide. PCV2 is not only the pathogen that causes PMWS, but also closely related to diseases such as porcine dermatitis and nephrotic syndrome, granulomatous enteritis, porcine respiratory disease syndrome, reproductive disorders in sows, necrotizing lymphadenitis and congenital tremor in piglets. Diseases related to PCV2 are collectively referred to as porcine circovirus disease (PCVD).

At present, the main means of prevention and control of PCVD is vaccination. Scholars at home and abroad have made remarkable achievements in the research of PCV2 vaccines. PCV2 marker vaccine, etc. However, there are certain limitations in the use of vaccines. In my country, PCV2 whole-virus inactivated vaccines are widely used, while other vaccines are used less. However, after immunizing pigs with PCV2 inactivated vaccine, the feed intake of pigs will be significantly reduced, and some pigs will occasionally die. Therefore, choosing an immune adjuvant with low toxicity and good immune effect is the only way to solve this problem. way. Commonly used immune adjuvants include liposomes, chitosan, etc., but less research has been done on cytokine adjuvants.

Interleukin-23 (interleukin-23, IL-23) is a newly discovered cytokine, which is a heterodimer composed of two subunits p40 and p19, and exerts biological effects by binding to corresponding receptors , IL-23 receptor (IL-23R) is composed of two chains, IL-23R and β1, and the co-expression of the two chains can produce a high-affinity binding site. IL-23 is mainly secreted by antigen-presenting cells, such as DC cells derived from peripheral blood mononuclear cells. The study found that the expression of IL-23R chain in CD4 ⁺ CD4RB ^lo memory T cells was significantly higher than that in CD4 ⁺ CD4RB ^hi naive T cells, indicating that IL-23 mainly acts on memory T cells. IL-23 is a pro-inflammatory cytokine that induces and promotes pro-inflammatory Th1 and Th17 cells to secrete cytokines to participate in the regulation of anti-infection and other immune defenses; it can enhance the activity of NK cells and induce the differentiation of Th1 cells; it can inhibit TGF-β inhibits the activity of nTreg cells; IL-23 can also regulate innate immune lymphocytes (NK cells, NKT cells and γδT cells) and ILC cells, and play an important role in infection immunity and tumor immunity.

Contents of the invention

The purpose of the present invention is to provide preparation and application of a recombinant porcine IL-23 enhancing PCV2 vaccine immune adjuvant.

The present invention provides a protein, comprising the following four segments: IL-23 protein P40 subunit, self-cleaving linker peptide 2A, TPA signal peptide and IL-23 protein P19 subunit.

The protein includes the following four sections from upstream to downstream: IL-23 protein P40 subunit, self-cleavage connecting peptide 2A, TPA signal peptide and IL-23 protein P19 subunit.

The protein is composed of the following four segments: IL-23 protein P40 subunit, self-cleavage connecting peptide 2A, TPA signal peptide and IL-23 protein P19 subunit.

Any of the above IL-23 proteins is derived from Tibetan pigs.

The amino acid sequence of the P40 subunit of the IL-23 protein is 1-323 of the sequence 2.

The amino acid sequence of the self-cleaving connecting peptide 2A is the 324th-344th position of sequence 2.

The amino acid sequence of the TPA signal peptide is 345-369 in sequence 2.

The amino acid sequence of the P19 subunit of the IL-23 protein is 370-562 in sequence 2.

The protein is as follows (1) or (2):

(1) A protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

(2) A protein derived from (1) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing and has the same function.

DNA molecules encoding any of the above proteins also belong to the protection scope of the present invention.

The DNA molecule is the DNA molecule described in any one of the following 1) to 3):

1) The DNA molecule shown in sequence 1 in the sequence listing;

2) a DNA molecule that hybridizes to the DNA molecule defined in 1) under stringent conditions and encodes a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing;

3) The DNA molecules derived from Tibetan pigs and 1) have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, A DNA molecule having at least 97%, at least 98% or at least 99% homology and encoding a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing.

The above-mentioned stringent conditions can be in a solution of 6×SSC, 0.5% SDS, hybridization at 65 μ, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing DNA molecules of the above-mentioned proteins also belong to the protection scope of the present invention.

The recombinant vector is a recombinant plasmid obtained by inserting the double-stranded DNA shown in Sequence 1 of the sequence listing into the multiple cloning site of the eukaryotic expression vector.

The recombinant vector can specifically be a recombinant plasmid obtained by inserting the double-stranded DNA shown in Sequence 1 of the sequence listing into the BamHI restriction site of the VR1020 plasmid.

The present invention also protects the application of the protein or the DNA molecule or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in the production of products; the function of the product is any of the following 1)-9) :

1) As a PCV2 vaccine adjuvant;

2) Improve the immune effect of PCV2 vaccine products;

3) Promote animal growth;

4) Increase animal weight;

5) Increase the number of red blood cells;

6) Increase the number of white blood cells;

7) Improve the expression of Toll-like receptor gene;

8) Improve immune memory-related cytokine gene expression;

9) Increase the gene expression of immune signal transduction molecules.

The present invention also protects a product, including the protein or the DNA molecule or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria; the use of the product is any one of the following 1)-9);

1) As a PCV2 vaccine adjuvant;

2) Improve the immune effect of PCV2 vaccine;

3) Promote animal growth;

4) Increase animal weight;

5) Increase the number of red blood cells;

6) Increase the number of white blood cells;

7) Improve the expression of Toll-like receptor gene;

8) Improve immune memory-related cytokine gene expression;

9) Increase the gene expression of immune signal transduction molecules.

Any one of the Toll-like receptor genes described above can specifically be TLR2 or TLR7.

Any one of the immune memory-related cytokine genes mentioned above can specifically be STAT1, STAT2, STAT3, STAT4 or Bcl-2.

The present invention also protects a preparation method of a PCV2 vaccine adjuvant, comprising the steps of: adopting chitosan to coat the recombinant carrier.

In the method, the mass ratio of the chitosan to the recombinant carrier is 30:1.

The preparation method of described PCV2 vaccine adjuvant is specifically as follows:

1. Mix the aqueous solution of the recombinant carrier with an appropriate amount of TPP solution, and incubate at a constant temperature of 55°C for 20 minutes to obtain a premixed solution of the plasmid and TPP; the mass ratio of the recombinant carrier to TPP is 1:3; the TPP solution has a concentration of It is 10mg/mlTPP aqueous solution.

2. Slowly add the premix solution obtained in step 2 into the chitosan solution dropwise under magnetic stirring in a water bath at 55°C, and mix evenly so that the mass ratio of chitosan to recombinant plasmid is 30:1, and then incubate at constant temperature for 10 minutes Standby, obtain the chitosan nanoparticle of wrapping recombinant carrier; Described chitosan solution is the chitosan solution that concentration is 2.4mg/ml; The configuration method of described chitosan solution is: dissolving chitosan In 1% (pH5.5) glacial acetic acid aqueous solution, the chitosan concentration was set to 2.4 mg/ml to obtain a 2.4 mg/ml chitosan solution.

The invention also protects the PCV2 vaccine adjuvant prepared by the method.

The invention also protects a product comprising said PCV2 vaccine adjuvant and PCV2 vaccine.

The present invention first utilizes the gene cloning technology to obtain the gene fragments of P40 and P19 of the Tibetan pig IL-23 gene, and uses 2A self-cutting expression technology to connect the P40 and P19 genes to obtain the complete IL-23 gene sequence, and then uses In- The fusion cloning method constructs the complete IL-23 gene into the eukaryotic secretory expression vector VR1020, wraps the IL-23 plasmid with chitosan to prepare chitosan nanoparticles, and inoculates experimental animals with PCV2 vaccine at the same time, using pigs as experimental animals , the results show that IL-23 eukaryotic transfection plasmid can effectively enhance the immune response of pigs to PCV2 vaccine and promote the growth of inoculated animals; it provides a new technology for the development of new and efficient PCV2 immune adjuvants.

Description of drawings

Figure 1 Electrophoresis pattern of p40 and p19 genes (1.0% agarose gel), M: Trans 2K, 1: p19, 2: p40.

Fig. 2 Electrophoresis pattern of p40, p19, 2A-TPA fragments (1.0% agarose gel), M: Trans5K, 1-2: P40, 3-5: 2A-TPA, 6-7: P19.

Figure 3 GFP expression in HKE293 cells, A: GFP expression in HEK293 cells 24 hours after transfection, B: GFP expression in HEK293 cells 48 hours after transfection, C: GFP expression in HEK293 cells 72 hours after transfection expression in cells.

Fig. 4 RT-PCR product electrophoresis detection pattern (1.0% agarose gel) of cells transfected with recombinant IL-23 plasmid, M: Trans 5K, 1: cell control, 2: VR1020 empty vector control, 3: VRIL-23.

Figure 5 Lymphoblast proliferation.

Fig. 6 Particle size distribution of chitosan-wrapped IL-23 plasmid.

Figure 7 Changes in the number of experimental pig blood cells.

Fig. 8 Changes in the expression level of the experimental pig Toll-like receptor gene.

Figure 9. Changes in expression levels of genes related to immune memory in experimental pigs.

Fig. 10 Changes in the expression levels of the experimental porcine immune signal transduction molecule genes.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set to repeat the experiment three times, and the results were averaged.

Embodiment 1, the construction of eukaryotic recombinant plasmid VRIL-23

The recombinant plasmid VRIL-23 is a recombinant plasmid obtained by inserting the fusion gene IL-23 into the BamHI restriction site of the VR1020 plasmid. The fusion gene IL-23 is shown in sequence 1 of the sequence listing, encoding the fusion protein IL-23 shown in sequence 2 of the sequence listing. Recombinant plasmid VRIL-23 is used to express fusion protein IL-23 in eukaryotic cells.

In sequence 2 of the sequence listing, amino acid residues 1-323 constitute the P40 fragment, amino acid residues 324-344 constitute the 2A fragment (self-cleaving connecting peptide), and amino acid residues 345-369 constitute the TPA fragment ( signal peptide), the 370-562 amino acid residues constitute the P19 fragment.

In sequence 1 of the sequence listing, nucleotides 1-969 encode the P40 segment, nucleotides 970-1032 encode the 2A segment, nucleotides 1033-1107 encode the TPA segment, and nucleotides 1108-1689 Acid encodes the P19 fragment.

Those skilled in the art can use any method to prepare the recombinant plasmid VRIL-23, such as directly artificially synthesizing the fusion gene IL-23 shown in Sequence 1 of the Sequence Listing, and then insert it forward into the BamHI restriction site of the VR1020 plasmid.

The method for preparing recombinant plasmid VRIL-23 adopted by the inventors of the present invention is provided below as an example.

1. Extraction of RNA from peripheral blood lymphocytes

1. Isolation and culture of peripheral blood lymphocytes of Tibetan pigs (Sichuan Academy of Animal Husbandry Research Institute of Pig Breeding, this pig breed was introduced from Ganzi Prefecture, Sichuan Province)

(1) Take 40mL of blood from the anterior vena cava of Tibetan pigs and add 40mL of 1640 medium to dilute in equal amounts;

(2) Add 5 mL of lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd., product number: WBC1110) into a 15 mL centrifuge tube, then slowly add 5 mL of blood diluted in step (1), and centrifuge at room temperature at 1500 r on a horizontal centrifuge 30min;

(3) After centrifugation, four clear layers can be seen (from top to bottom: plasma layer, lymphocyte layer, separation liquid layer, red blood cell layer), carefully absorb the lymphocyte layer, a total of 2mL;

(4) Add 10 mL of 1640 medium for washing, and centrifuge at room temperature for 10 min at 1500 r;

(5) Discard the supernatant, add 10mL 1640 medium, mix by pipetting, and centrifuge at 1500r for 10min at room temperature;

(6) Discard the supernatant, add 20 mL of 1640 complete medium containing 10% (volume percentage) fetal bovine serum and penicillin/streptomycin (penicillin concentration is 100U/ml, streptomycin concentration is 0.1mg/ml) by blowing and mixing Mix well, adjust the cell concentration to 2×10 ⁷ mL ^-1 , then add 20 μL of LPS (from sigma, product number: L2880) with a concentration of 1 mg/mL, so that the concentration of LPS in the culture system is 1 μg/mL, at 37°C, Cultivate for 4 hours in a 5% CO ₂ incubator.

2. Extraction of total RNA from peripheral blood lymphocytes of Tibetan pigs

(1) After collecting the supernatant of adherent cells, add 1mL Trizol to the remaining cells, blow the cells off the plate with a pipette tip, and then transfer them to a 1.5mLEP tube;

(3) Add 200 μL of chloroform, shake vigorously for 15 sec, let stand at room temperature for 5 min, and centrifuge at 12000 g at 4°C for 15 min;

(4) Carefully absorb 400 μL of supernatant, then add 400 μL of isopropanol, mix well, let stand at room temperature for 10 minutes, and centrifuge at 12000 g at 4°C for 10 minutes;

(5) Discard the supernatant, add 1 mL of 75% ethanol, and centrifuge at 12000 g at 4 μ for 5 min;

(6) Discard the supernatant, and add 20 μL of RNase-free Water to dissolve the RNA pellet after it has dried naturally;

(7) The purity and concentration of RNA were detected by ultraviolet spectrophotometer, and the integrity of RNA was detected by 1.0% agarose gel electrophoresis.

2. PCR amplification of P40 gene PCR product 1 and P19 gene PCR product 1

Use the TaKaRa PrimeScript RT reagent Kit to synthesize cDNA from the RNA obtained in step 1.

According to the gene sequence of p40 and p19 of porcine IL-23 published on GenBank, design specific primers (see Table 1) with Primer5.0 software.

Table 1 Tibetan pig PCR primers

Using cDNA as a template, use the specific primers corresponding to p40 and p19 to amplify the target gene by PCR respectively. The p19 gene PCR product 1 (position 1108-1689 of sequence 1) containing the last 3 stop codon bases of product 1) and 582 bp (Fig. 1).

Table 2 Target gene PCR amplification system

PCR cycling conditions

The PCR product 1 of the p40 gene and the PCR product 1 of the p19 gene amplified above were respectively connected to a T vector (pBLUE-T vector, Beijing Boaolong Immunology Technology Co., Ltd., BD-M10015) to obtain the P40-T vector and the p19 gene -T carrier.

3. Construction of eukaryotic recombinant plasmid VRIL-23 of IL-23 gene

1. Synthesis of 2A-TPA

Design and synthesize three primers, respectively named 2A-TPA-1, 2A-TPA-2, 2A-TPA-3, and use these three primers to amplify the fusion gene of 2A-TPA by PCR. Primer sequences (see Table 3):

Table 3 2A-TPA PCR primers

The PCR reaction system is 50 μL (see Table 4):

Table 4 2A-TPA reaction system

PCR reaction conditions:

After 2% agarose gel electrophoresis, the gel was cut and recovered to obtain 2A-TPA (its nucleotide sequence is 970-1107 of Sequence 1).

2. Amplification of P40 gene PCR product 2 and P19 gene PCR product 2

1) Design and synthesis of primers

Using the In-fusion method, design overlapping primers, and insert the 2A short peptide gene between the P40 and P19 genes into the vector VR1020 together. The 5' end of the P40 gene overlaps with the vector by 16 bp, named 40-F, 3' There is 10 bp overlapping with 2A-TPA at the end, named 40-R; there is 10 bp overlapping with P40 at the 5' end of 2A-TPA, and it is named 2A-F; there is 10 bp overlapping with P19 at the 3' end of 2A-TPA, and it is named 2A-R ; There is 10 bp overlapping with 2A-TPA at the 5' end of the P19 gene, named 19-F, and 16 bp overlapping with VR1020 at the 3' end, named 19-R. Primer sequences (see Table 5):

Table 5 Primer Sequence

2) Amplification of the target fragment

Using the P40-T vector obtained in step 2 as a template and using 40-F and 40-R as primers to amplify, a 995bp P40 amplification product 2 was obtained ( FIG. 2 ).

Using the P19T vector obtained in the above two as a template, and using 19-F and 19-R as primers to amplify, a 608bp P19 amplification product 2 was obtained (Fig. 2).

Using the 2A-TPA obtained in the above 1 as a template, and using 2A-F and 2A-R as primers to amplify, a 158bp 2A-TPA amplification product 2 was obtained (Fig. 2).

3) Carrier skeleton acquisition

The VR1020 plasmid (VR1020 is provided by Vical Company, USA) was single digested with BamHI to obtain the VR1020 linear vector backbone.

4) Ligation of the fragment and the carrier

The P40 amplification product 2, P19 amplification product 2, and 2A-TPA amplification product 2 obtained in the above 2) were connected with the VR1020 linear vector backbone to obtain the eukaryotic recombinant plasmid VRIL-23.

The above connection system is 10 μL (see Table 6):

Table 6 Fragment and carrier connection system

Connection conditions: 50°C, 15min.

4. Activity detection of eukaryotic recombinant plasmid VRIL-23

1. Expression activity of eukaryotic recombinant plasmid VRIL-23 in HEK293 cells

Transfect HEK293 cells (human embryonic kidney cells, Shanghai Bayley Bio Fetal Bovine Serum Company, HEK-293).

1) Fluorescence microscope detection

Before transfection, it was observed that the cells grew well, and there were few dead cells. The cells after transfection for 24h, 48h and 72h were observed with a fluorescence microscope.

The results are shown in Figure 3, showing that GFP began to be expressed 24 hours after transfection, indicating that the plasmid could be successfully expressed in the cells.

2) Detection of the target gene in HEK293

The cells were collected at 48 hours after transfection, the total RNA of the cells was extracted, the cDNA was obtained by reverse transcription as a template, and the IL-23-specific primers (F: 5'-ATGCACCTTCAGCAGCTGGTTGTC-3', R: 5'-TTACTGGCTCAGAGTTGCTGCTC-3') were used for PCR amplification. Empty vector VR1020 plasmid and HEK-293 cells were used as controls.

The results are shown in Figure 4. It can be seen that the VRIL-23 recombinant plasmid can be successfully expressed in HEK293 cells.

2. Lymphoblast stimulation experiment

(1) Prepare and collect Tibetan pig peripheral blood lymphocytes according to the method recorded in step 1;

(2) The plasmid to be tested and the pEGFP-N3-GFP expression plasmid were transfected into HEK293 cells (human embryonic kidney cells, Shanghai Baili Biological Fetal Bovine Serum Company, HEK-293) according to the instructions of the liposome transfection kit, respectively. The culture supernatant was collected 24h, 48h, and 72h after transfection.

The plasmids to be tested are: the eukaryotic recombinant plasmid VRIL-23 (VRIL-23) prepared in Step 3, and the empty vector VR1020 plasmid (VR1020).

At the same time, a control group (Cell) with no plasmid transfection, a control group (control) with only the empty vector VR1020 plasmid and the pEGFP-N3-GFP expression plasmid were set.

(3) Add 50 μl of Tibetan pig peripheral blood lymphocytes and 50 μl of the supernatant collected in step (2) to the experimental wells of the 96-well plate according to the layout. Set up 3 replicate wells for each sample, and set up control wells, and place them in the cell culture box for 48 hours;

(4) After 48 hours, take out the 96-well plate, add 10 μl CCK8 to each well and gently blow evenly to continue culturing for 2 hours;

(5) Take out the 96-well plate, and use Bio-Reader 3550 to detect the OD _450nm of each well.

The results are shown in Figure 5. The results showed that the proliferation of the VRIL-23 group was significantly higher than that of the control group VR1020, which indicated that the supernatant of HEK293 cells transfected with the experimental plasmid could significantly stimulate the proliferation of porcine lymphocytes (P<0.05).

5. Detection of endotoxin

Use the "gel method" to detect the endotoxin content of the VRIL-23 plasmid. For the method, refer to the "Instructions for Limulus Reagent". The specific method is as follows:

(1) Positive control sample preparation: take endotoxin standard substance (Shanghai Haring Biotechnology Co., Ltd., article number: 150600), and prepare a solution with a concentration of 2 times sensitivity λ=0.25Eu/ml in water for inspection;

(2) Negative control: check water;

(3) Test sample: According to the test sample endotoxin limit value, the effective dilution factor of the calculator (MVD=cL/λ) is required, and the sample is diluted for later use;

(4) Take the LAL reagent, break off the neck of the ampoule and mark it, 1 of which is used as a positive control tube, 1 is used as a negative control tube, 2 are used as a sample tube, and 1 is used as a positive control tube for a sample, 5 in total;

(5) Add 0.2ml of water for inspection, 0.1ml of water for inspection + 0.1ml of endotoxin solution with a concentration of 2 times λ, and 0.1ml of water for inspection + 0.1ml to the negative control tube, positive control tube, and sample tube as required. Test sample (eukaryotic recombinant plasmid VRIL-23 prepared in step 3), shake gently to be tested;

(6) Put the sample to be tested in the previous step into the thermostat vertically, incubate at 37°C for 60 minutes, and read the result.

As a result, there was no gel formation in the sample tube, indicating that the eukaryotic recombinant plasmid VRIL-23 prepared in Step 3 did not contain endotoxin.

Example 2, the application of eukaryotic recombinant expression plasmid VRIL-23 as a vaccine adjuvant

1. Preparation of vaccine adjuvants

Use the ion cross-linking method to wrap the VRIL-23 plasmid, and prepare nanoparticles as a vaccine adjuvant. The specific method is:

(1) Preparation of the solution:

10mg/ml TPP solution: Dissolve TPP in ddH ₂ O to make the TPP concentration 10mg/ml to obtain a 10mg/ml TPP solution;

Chitosan solution of 2.4mg/ml: Chitosan is dissolved in 1% (pH5.5) glacial acetic acid aqueous solution, makes chitosan concentration be 2.4mg/ml, obtains the chitosan solution of 2.4mg/ml ;

Filter the prepared solution with a 0.22m microporous membrane to sterilize;

(2) Premixing of plasmid and TPP solution: mix the eukaryotic recombinant plasmid VRIL-23 solution (solvent is water) with an appropriate amount of TPP solution, and incubate at 55°C for 20 minutes to obtain a premixed solution of plasmid and TPP; The mass ratio of nuclear recombinant plasmid VRIL-23 and TPP is 1:3;

Chitosan-coated plasmid: Slowly add the premix of the above-mentioned plasmid and TPP into the chitosan solution under the condition of magnetic stirring in a water bath at 55°C, and mix evenly so that the mass ratio of chitosan to plasmid is 30:1, and then Incubate at constant temperature for 10 minutes and set aside to obtain chitosan nanoparticles wrapped with VRIL-23;

(3) Sampling detection: Take an appropriate amount of chitosan nanoparticles wrapped with VRIL-23 and use a Malvern particle size analyzer to detect the Zeta potential and particle size of the nanoparticles.

Result as shown in Figure 6, the particle size of the chitosan nanoparticle that wraps VRIL-23 is 216.7nm, and Zeta potential is 24.5mv, shows that it meets the requirement of transfection cell, and this particle is named after CS-IL-23 ( Image 6).

2. Biological effects of vaccine adjuvants expressed in pigs

1. Animal grouping

Select 10 Dudachang three-way hybrid pigs (provided by Sichuan Pig Research Institute) that were not immunized with PCV2 vaccine at the age of 3 weeks, and randomly divided them into 2 groups with 5 pigs in each group, in which group A was the experimental group and group D was the control group group, processed as follows (Table 7):

Table 7 Experimental Animal Grouping and Inoculation Preparations

Before inoculation, it was recorded as week 0, and 5 ml of blood from the anterior vena cava was collected at weeks 0, 1, 2, 4, 8, and 12 and stored in anticoagulant tubes for subsequent analysis. Weigh the body weight of each animal before and after immunization, and make a record.

The statistical results of body weight changes are shown in Table 8. As can be seen from Table 8, there was no significant difference in body weight between the experimental group (Group A) and the control group (Group D) before inoculation (P>0.05), and in 84 days after inoculation, the weight gain of Group A was significantly higher than that of D (P<0.05), the results showed that the inoculation of CS-IL-23 had a significant effect on promoting the growth of piglets.

Table 8 Body weight change table of experimental animals

2. Routine blood analysis

On days 0, 7, 14, 28, 35, 56, and 84 of immunization, 50 μl of fresh blood was taken, and the number of white blood cells and red blood cells in the blood of the experimental group was detected by automatic blood cell classification detection technology.

The results are shown in Figure 7. It can be seen from Figure 7 that the number of leukocytes in group A was significantly higher than that in group D on day 35 and day 84 after inoculation (P<0.05). On the 35th day, 56th day and 84th day, the red blood cells in group A increased significantly (P<0.05). There was no significant difference in hemoglobin content between group A and group D (P>0.05).

3. Detection of immune-related gene expression

Blood from anterior vena cava was collected at 0, 1, 2, 4, 8, and 12 weeks after immunization, RNA was extracted, and cDNA was obtained by reverse transcription.

According to the pig gene sequence reported in GenBank, design and synthesize specific primers for quantitative PCR, as shown in Table 9.

Table 9 Porcine immune-related gene primers

Using cDNA as a template, the primers in Table 9 above were used to perform quantitative PCR to detect the expression changes of related genes, and PPIA was used as an internal reference gene, and the relative expression of genes was calculated by the 2 ^-ΔΔCT method.

Figure 8 shows the expression changes of Toll-like receptor genes TLR2 and TLR7. Compared with the control group, the expression of TLR2 gene in experimental group A increased significantly from day 7 to day 14 after inoculation with CS-IL-23 (P<0.05). From day 28 to day 84, the expression of TLR7 in the experimental group was significantly higher than that in the control group under the stimulation of CS-IL-23 (P<0.05).

Figure 9 shows the changes in gene expression of immune memory-related cytokines. The results showed that after inoculation with CS-IL-23, the expression levels of cytokines in the experimental group were significantly higher than those in the control group.

The expression changes of immune signal transduction molecule genes STAT1, STAT2, STAT3, STAT4 and Bcl-2 are shown in Figure 10. The results showed that the expression of STAT gene in group A was significantly higher than that in group D after inoculation with CS-IL-23 (P<0.05), and the expression of Bcl-2 in group A was significantly higher than that in group A on day 14 and day 84. Group D (P<0.05).

<110> Sichuan University

<120> Preparation and Application of Recombinant Porcine IL-23 Enhanced PCV2 Vaccine Adjuvant

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ctgctgctgc tactgccctg gacctctcag ggccgggctg tgcctgaggg cagcagccct 1200

gcttgggctc aaggccagca gctctcacag cagctctgca cgctggcctg gactgcacat 1260

ctaccaatgg gacatgtgga tctaccaaga gaagaggggag atgatgagac tacaagtgaa 1320

gtcccccata tccagtgcgg ggatggctgt gatcctcagg gactcaggga caacagtcag 1380

tcctgcttgc aaaggatcca ccaaggcctg gttttttatg agaagctgct gggctcagac 1440

attttcacag gggagccttc tctacaccct gatggctctg tgggccagct tcacgcctcc 1500

ctactgggcc tcaggcaact cttgcagccc gagggtcacc actgggagac tgagcagacg 1560

ccaagcccca gtcccagcca gccctggcaa cgcctccttc tccgcctcaa gatccttcgc 1620

agcctccagg cctttgtggc tgtagctgcc cgggtcttcg cccatggagc agcaactctg 1680

agccagtaa 1689

<210> 2

<211> 562

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 2

Met His Leu Gln Gln Leu Val Val Ser Trp Phe Ser Leu Val Trp Leu

1 5 10 15

Ala Ser Pro Ile Val Ala Ile Trp Glu Leu Glu Lys Asn Val Tyr Val

20 25 30

Val Glu Leu Asp Trp Tyr Pro Asn Ala Pro Gly Glu Met Val Val Leu

35 40 45

Thr Cys Asn Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Ser Asp Gln

50 55 60

Ser Ser Glu Val Leu Gly Thr Gly Lys Thr Leu Thr Ile His Val Lys

65 70 75 80

Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys Arg Lys Gly Gly Ala Val

85 90 95

Leu Ser Gln Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp

100 105 110

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

115 120 125

Leu Lys Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp

130 135 140

Leu Thr Ala Ile Ser Thr Asp Leu Lys Phe Ser Val Lys Ser Ser Arg

145 150 155 160

Gly Ser Ala Asp Pro Arg Gly Val Thr Cys Gly Thr Ala Met Leu Ser

165 170 175

Glu Asp Leu Gly Glu Tyr Lys Tyr Arg Val Glu Cys Gln Glu Gly Ser

180 185 190

Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Val Leu Glu

195 200 205

Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile

210 215 220

Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Asn Pro

225 230 235 240

Leu Lys Asn Ser Arg His Val Glu Ile Ser Trp Glu Tyr Pro Asp Thr

245 250 255

Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Met Phe Gly Val Gln Val

260 265 270

Gln Gly Lys Asn Lys Arg Glu Lys Lys Asp Lys Leu Phe Thr Asp Gln

275 280 285

Thr Ser Ala Lys Val Thr Cys His Lys Asp Ala Asn Ile Arg Val Gln

290 295 300

Ala Arg Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val

305 310 315 320

Ser Cys Asn Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

325 330 335

Asp Val Glu Glu Asn Pro Gly Pro Met Asp Ala Met Lys Arg Gly Leu

340 345 350

Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe Val Ser Pro Ser Gly

355 360 365

Thr Met Leu Gly Ser Arg Ala Val Met Leu Met Leu Leu Leu Leu Leu Leu

370 375 380

Leu Pro Trp Thr Ser Gln Gly Arg Ala Val Pro Glu Gly Ser Ser Pro

385 390 395 400

Ala Trp Ala Gln Gly Gln Gln Leu Ser Gln Gln Leu Cys Thr Leu Ala

405 410 415

Trp Thr Ala His Leu Pro Met Gly His Val Asp Leu Pro Arg Glu Glu

420 425 430

Gly Asp Asp Glu Thr Thr Ser Glu Val Pro His Ile Gln Cys Gly Asp

435 440 445

Gly Cys Asp Pro Gln Gly Leu Arg Asp Asn Ser Gln Ser Cys Leu Gln

450 455 460

Arg Ile His Gln Gly Leu Val Phe Tyr Glu Lys Leu Leu Gly Ser Asp

465 470 475 480

Ile Phe Thr Gly Glu Pro Ser Leu His Pro Asp Gly Ser Val Gly Gln

485 490 495

Leu His Ala Ser Leu Leu Gly Leu Arg Gln Leu Leu Gln Pro Glu Gly

500 505 510

His His Trp Glu Thr Glu Gln Thr Pro Ser Pro Ser Pro Ser Gln Pro

515 520 525

Trp Gln Arg Leu Leu Leu Arg Leu Lys Ile Leu Arg Ser Leu Gln Ala

530 535 540

Phe Val Ala Val Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu

545 550 555 560

Ser Gln

Claims (10)

1. A protein, comprising the following four segments: IL-23 protein P40 subunit, self-cleaving connecting peptide 2A, TPA signal peptide and IL-23 protein P19 subunit. 2. The protein according to claim 1, characterized in that: the protein is as follows (1) or (2): (1) A protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing; (2) A protein derived from (1) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing and has the same function. 3. A DNA molecule encoding the protein of claim 1 or 2. 4. The DNA molecule according to claim 3, characterized in that: The DNA molecule is the DNA molecule described in any one of the following 1) to 3): 1) The DNA molecule shown in sequence 1 in the sequence listing; 2) A DNA molecule that hybridizes to the DNA molecule defined in 1) under stringent conditions and encodes a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing; 3) The DNA molecules derived from Tibetan pigs and 1) have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, A DNA molecule having at least 97%, at least 98% or at least 99% homology and encoding a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing. 5. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein of claim 1 or 2. 6. the application of the protein according to claim 1 or 2 or the DNA molecule described in claim 3 or 4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria according to claim 5 in the preparation of products; The function of the product is any one of the following 1)-9): 1) As a PCV2 vaccine adjuvant; 2) Improve the immune effect of PCV2 vaccine products; 3) Promote animal growth; 4) Increase animal weight; 5) Increase the number of red blood cells; 6) Increase the number of white blood cells; 7) Improve the expression of Toll-like receptor gene; 8) Improve immune memory-related cytokine gene expression; 9) Increase the gene expression of immune signal transduction molecules. 7. A product comprising the protein according to claim 1 or 2 or the DNA molecule according to claim 3 or 4 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria according to claim 5; said product The purpose is any of the following 1)-9); 1) As a PCV2 vaccine adjuvant; 2) Improve the immune effect of PCV2 vaccine; 3) Promote animal growth; 4) Increase animal weight; 5) Increase the number of red blood cells; 6) Increase the number of white blood cells; 7) Improve the expression of Toll-like receptor gene; 8) Improve immune memory-related cytokine gene expression; 9) Increase the gene expression of immune signal transduction molecules. 8. A preparation method of a PCV2 vaccine adjuvant, comprising the steps of: adopting chitosan to wrap the recombinant carrier according to claim 5. 9. The PCV2 vaccine adjuvant prepared by the method according to claim 8. 10. A product comprising the PCV2 vaccine adjuvant of claim 9 and the PCV2 vaccine.