CN107266581B - Preparation method and application of Tibetan pig IL-12 recombinant plasmid enhanced PCV2 vaccine immunologic adjuvant - Google Patents

Abstract

The invention discloses application of Tibetan pig IL-12 and recombinant plasmids thereof in preparation of PCV2 vaccine immunologic adjuvants. The invention provides a protein provided by the invention, which consists of IL-12 protein P40 subunit, 2A, TPA subunit and IL-12 protein P35 subunit; the amino acid sequence of the 2A is the 324-344 position of the sequence 2; the amino acid sequence of the TPA is 345-369 th site of the sequence 2. According to the experiment, chitosan nanoparticles are prepared by wrapping IL-12 plasmids with chitosan, and are inoculated to experimental animals together with PCV vaccines, mice and pigs are respectively adopted as the experimental animals, and the results show that IL-12 eukaryotic transfection plasmids can effectively enhance the immune response of the PCV vaccines, so that a thought is provided for developing novel and efficient PCV immune adjuvants.

Description

Preparation method and application of Tibetan pig IL-12 recombinant plasmid enhanced PCV2 vaccine immunologic adjuvant

Technical Field

The invention belongs to the technical field of biology, particularly belongs to molecular immunology, and relates to application of Tibetan pig IL-12 and recombinant plasmids thereof in preparation of PCV2 vaccine immunologic adjuvants.

Background

Pork products are the main source of meat consumption of urban and rural residents and are closely related to the life of people. Since the 21 st century, annual number of stockings and number of slaughterings of domestic pigs always account for about 50% of the world total amount and are the first in the world. While the pig industry in China has attracted attention, a plurality of factors restricting the healthy development of the pig industry in China exist, wherein diseases are the most main factors. At present, the level of diagnosis and prevention of swine diseases in China has a larger gap compared with the level of swine diseases in developed countries in the world, swine infectious diseases are still quite serious, and the infection of the circovirus type 2 (PCV 2) is a common infectious disease. PCV2 is a main pathogen causing Postweaning Multisystemic Wasting Syndrome (PMWS), is often mixed with porcine highly pathogenic reproductive and respiratory syndrome, porcine foot and mouth disease, porcine parvovirus disease and the like clinically to cause serious economic loss to the pig industry all over the world. PCV2 is not only a causative agent of PMWS, but also closely associated with diseases such as Porcine dermatitis and nephrotic syndrome, granulomatous enteritis, Porcine respiratory disease syndrome, sow reproductive disorders, necrotizing lymphadenitis, and congenital tremor of piglets, and these diseases associated with PCV2 are collectively referred to as Porcine circovirus disease (PCVD).

At present, the main means for preventing and controlling PCVD is vaccination, scholars at home and abroad conveniently obtain remarkable achievements in PCV2 vaccine research, and successfully develop PCV2 inactivated vaccine, PCV2 attenuated vaccine, PCV2 genetic engineering vaccine, PCV1-2 chimeric vaccine, PCV2 marker vaccine and the like. However, the use of the vaccine has certain limitations, and the PCV2 whole virus inactivated vaccine is widely used in our country, while other vaccines are used less frequently. After the PCV2 inactivated vaccine is used for immunizing a swinery, the situations that the feed intake of pigs is obviously reduced and the death of individual pigs happens can occur, therefore, the selection of an immunologic adjuvant with low toxicity and good immunologic effect is a poor way for solving the problem. The commonly used immune adjuvants include liposome, chitosan and the like, but the research on cytokine adjuvants is less.

Cytokines are active multifunctional proteins produced by the hematopoietic system and immune system of the organism itself. There are over ten cytokines currently found, of which Interleukins (IL), tumor necrosis factor (TNF- α) and interferon (IFN- γ) are of paramount importance to animals. IL-12 is an important member of the IL-12 cytokine family, is a heterodimer composed of two subunits, p40 and p35, can enhance the activity of NK cells, induce the differentiation of Th1 cells, and play an important role in many inflammatory responses and immune regulation. The cytokine is naturally present in an organism and has a regulating effect on immunity, and the cytokine is more and more concerned in recent years, but the half-life of the cytokine in vivo is short, the time efficiency is short, and the cost is high, so that a thought is provided for cloning a cytokine gene into a plasmid and transfecting the cytokine gene into the organism for expression so as to solve the problem.

Disclosure of Invention

It is an object of the present invention to provide Tibetan pig IL-12.

The protein (named as IL-12') provided by the invention consists of IL-12 protein P40 subunit, 2A (self-cutting connecting peptide), TPA (signal peptide acting secretion signal) and IL-12 protein P35 subunit;

the amino acid sequence of the 2A is the 324-344 position of the sequence 2;

the amino acid sequence of the TPA is 345-369 th site of the sequence 2.

In the protein, the IL-12 protein is from Tibetan pigs;

or, the protein is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(2) and (b) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

DNA molecules encoding the above-described proteins are also within the scope of the present invention.

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

1) DNA molecule shown in sequence 1 in the sequence table;

2) a DNA molecule which is hybridized with the DNA molecule defined in 1) under strict conditions and codes the protein consisting of the amino acid sequence shown in the sequence 2 in the sequence table;

3) a DNA molecule which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA molecule defined in 1) and encodes a protein consisting of the amino acid sequence shown as the sequence 2 in the sequence table.

The stringent conditions may be hybridization at 65 ℃ in a solution of 6 XSSC, 0.5% SDS, followed by washing the membrane once with each of 2 XSSC, 0.1% SDS, and 1 XSSC, 0.1% SDS.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing DNA molecules of the above-mentioned proteins or transfection reagents containing said recombinant vectors are also within the scope of the present invention.

The recombinant vector is obtained by inserting a DNA molecule encoding the protein into a eukaryotic expression vector;

or, the recombinant vector is a vector obtained by inserting a DNA molecule encoding the protein into a eukaryotic expression vector; the eukaryotic expression vector is VR1020 plasmid.

The transfection reagent containing the recombinant vector is a transfection reagent obtained by wrapping the recombinant vector by chitosan;

the preparation method comprises the following steps:

1. uniformly mixing a VRIL-12 solution (water is used as a solvent) and a proper amount of TPP solution, and incubating at the constant temperature of 55 ℃ for 20min to obtain a premixed solution of plasmid and TPP; the mass ratio of VRIL-12 to TPP is 1: 3;

10mg/ml TPP solution: TPP is dissolved in ddH2O to make the concentration of TPP 10mg/ml, thus obtaining TPP solution of 10 mg/ml;

2. slowly dripping the premixed solution of the plasmid and TPP into a chitosan solution under the magnetic stirring condition of 55 ℃ water bath, uniformly mixing to ensure that the mass ratio of chitosan to the plasmid is 30:1, and then incubating for 10min at constant temperature for later use to obtain chitosan nanoparticles wrapping VRIL-12;

2.4mg/ml chitosan solution: chitosan was dissolved in 1% (pH5.5) glacial acetic acid to a chitosan concentration of 2.4mg/ml, yielding a chitosan solution of 2.4 mg/ml.

Or the transfection reagent containing the recombinant vector is a transfection reagent obtained by encapsulating the recombinant vector by liposome.

In the transfection reagent, the mass ratio of the chitosan to the recombinant vector is 10: 1;

or the liposome is DMRIE, and the mass ratio of the DMRIE to the plasmid is 2.5: 1.

the protein or the DNA molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium or the transfection reagent containing the recombinant vector is applied to the preparation of any one of the following 1) to 7);

1) PCV2 vaccine adjuvant;

2) products for improving PCV2 vaccine immune effect;

3) products for promoting animal growth;

4) products for increasing the weight of an animal;

5) increasing lymphocyte, basophil granulocyte, CD3⁺T cell, CD4⁺T or CD8⁺The product of the number of T cells;

6) products for increasing the content of hemoglobin, IgG1, IgG2a or PCV antibodies;

7) products that increase the expression of IL-12, IL7, IL23, IL-4, IL15, IL6, IL10, TLR3, TLR9, TLR1, TLR6, TLR2, TLR7, CD62L, IFN- γ, TNF- α, TGF β -1, STAT1, STAT1, STAT2, STAT3, STAT4, CD45, Bcl-2 or MyD 88.

It is another object of the present invention to provide a PCV2 vaccine adjuvant.

The PCV2 vaccine adjuvant provided by the invention comprises the protein or the DNA molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium or a transfection reagent containing the recombinant vector.

The experiment firstly utilizes a gene cloning technology to obtain gene fragments of P40 and P35 of the Tibetan pig IL-12 gene, connects the P40 and the P35 gene by using a 2A self-shearing expression technology to obtain a complete IL-12 gene sequence, then constructs the complete IL-12 into a eukaryotic secretory expression vector VR1020 by using an In-fusion cloning method, uses chitosan to wrap IL-12 plasmid to prepare chitosan nanoparticles and inoculates the chitosan nanoparticles and PCV vaccine to experimental animals at the same time, and respectively adopts mice and pigs as the experimental eukaryotic animals, and the result shows that the IL-12 transfection plasmid can effectively enhance the immune response of the PCV vaccine, thereby providing a thought for developing a novel high-efficiency PCV immune adjuvant.

Drawings

FIG. 1 is an electrophoretogram (1.0% agarose gel) of p40 and p35 genes, M: trans 2K, 2: p35, 3: p 40.

FIG. 2 shows electrophoretograms of p40, p35, and 2A-TPA fragments (1.0% agarose gel), M: Trans5K, 1-2: p40, 3-5: 2A-TPA, 6-7: p35.

FIG. 3 shows the expression of GFP in HKE293 cells, A: HEK293 growth status before transfection, B: GFP expression in HEK293 cells 24 hours after transfection, C: GFP expression in HEK293 cells 48 hours after transfection, D: GFP expression in HEK293 cells 72 hours after transfection.

FIG. 4 shows the pattern of the PCR products (1.0% agarose gel), M: trans5K, 1: cell control, 2: VR1020 empty vector control, 3: VRIL-12.

FIG. 5 shows lymphoblastoid cell proliferation.

FIG. 6 shows the particle size distribution of chitosan-coated IL-12 plasmid.

FIG. 7 shows the expression level changes of IL-12 gene in Tibetan pig PBMCS cells.

FIG. 8 shows the expression level changes of the Toll-like receptor gene in Tibetan pig PBMCS cells.

FIG. 9 shows the expression level changes of IL-7, IL-15 and IL-23 in Tibetan pig PBMCS cells.

FIG. 10 shows the expression level changes of Th1 type cytokines in Tibetan pig PBMCS cells.

FIG. 11 shows the expression level changes of Th2 type cytokines in Tibetan pig PBMCS cells.

FIG. 12 shows the expression level of TGF-. beta.1 in Tibetan pig PBMCS cells.

FIG. 13 shows the expression level change of the Tibetan pig PBMCS cell MyD 88.

FIG. 14 shows the body weight change of mice.

FIG. 15 shows the change in the number of blood cells in peripheral blood of mice.

FIG. 16 shows the content changes of IgG, IgG1 and IgG2a in mouse serum.

FIG. 17 shows the change in the amount of PCV-2 antibody in mouse serum.

FIG. 18 shows the change in the number of Th and Tc cell subsets in peripheral blood of mice.

FIG. 19 shows the change in the expression level of IL-12 gene in peripheral blood of mice.

FIG. 20 shows the change in expression level of Toll-like receptor gene in peripheral blood of mouse.

FIG. 21 shows the change in the expression level of an immunological memory-associated gene in peripheral blood of a mouse.

FIG. 22 shows the change in the expression level of Th1 type cytokine in mouse peripheral blood.

FIG. 23 shows the change in expression level of Th2 type cytokine in peripheral blood of mice.

FIG. 24 shows the change in the expression level of TGF-1 in mouse peripheral blood.

FIG. 25 shows the change in the expression level of STAT1 in mouse peripheral blood.

FIG. 26 shows the change in the number of blood cells in experimental pigs.

FIG. 27 shows the T cell changes in peripheral blood of experimental pigs.

FIG. 28 shows IgG changes in experimental porcine serum.

FIG. 29 is a graph of anti-PCV-2 antibody changes in experimental porcine serum.

FIG. 30 shows the change of gene expression levels of experimental porcine TLR2 and TLR 7.

FIG. 31 shows the change in the expression level of experimental porcine cytokine genes.

FIG. 32 shows the expression level changes of the genes of the pig immune signaling molecules.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of eukaryotic recombinant plasmid VRIL-12

First, extracting RNA of peripheral blood lymphocytes

1. Isolation and culture of Tibetan pig peripheral blood lymphocytes

(1) Collecting 40mL of anterior cavity venous blood of the Tibetan pigs, and adding 1640 culture medium for equivalent dilution;

(2) adding 5mL of lymphocyte separation solution into a 15mL centrifuge tube, slowly adding the blood after equivalent dilution, and centrifuging for 30min at room temperature of 1500r on a horizontal centrifuge;

(3) after centrifugation, clear 4 layers of layering (from top to bottom: plasma layer, lymphocyte layer, separation liquid layer, erythrocyte layer) can be seen, and the lymphocyte layer is carefully sucked up, wherein the total volume is 2 mL;

(4) adding 10mL 1640 culture medium for washing, and centrifuging at room temperature of 1500r for 10 min;

(5) discarding the supernatant, adding 10mL 1640 culture medium, uniformly mixing by blowing, and centrifuging at 1500r at room temperature for 10 min;

(6) discarding the supernatant, adding 20mL 1640 complete medium containing 10% fetal calf serum and double antibody, mixing, adjusting cell concentration to 2 × 10⁷mL^-1Then, 20uL of LPS at a concentration of 1mg/mL was added to give a final concentration of 1ug/mL of LPS at 37 ℃ in 5% CO₂Culturing in an incubator for 4 h.

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

(1) After collecting supernatant from adherent cells, adding 1mL of Trizol to the remaining cells, blowing the cells off the plate by using a gun head, and transferring the cells to a 1.5mL EP tube;

(3) adding 200uL chloroform, shaking vigorously for 15sec, standing at room temperature for 5min, and centrifuging at 12000g at4 deg.C for 15 min;

(4) carefully sucking 400uL of supernatant, adding 400uL of isopropanol, uniformly mixing, standing at room temperature for 10min, and centrifuging at 12000g at4 ℃ for 10 min;

(5) discarding the supernatant, adding 1mL of 75% ethanol, and centrifuging at 12000g and 4 ℃ for 5 min;

(6) removing the supernatant, and adding 20uL of RNase-free Water for dissolving after the RNA precipitate is naturally air-dried;

(7) the purity and concentration of RNA were determined by UV spectrophotometer and the integrity of RNA was determined by 1.0% agarose gel electrophoresis.

Secondly, PCR amplification of P35 gene PCR product 1 and P40 gene PCR product 1

cDNA was synthesized from the RNA obtained above using TaKaRa PrimeScript RT reagent Kit.

Specific primers were designed using Primer5.0 software based on the published gene sequences of p40 and p35 of porcine IL-12 in GenBank (see Table 1).

TABLE 1 Tibetan pig PCR primers

Using cDNA as template, PCR amplification of the target gene was carried out using specific primers corresponding to p40 and p35, respectively, at 20uL (see Table 2), to obtain 972bp p40 gene PCR product 1 (position 1-969 of SEQ ID NO: 1) and 771bp p35 gene PCR product 1 (position 1108-1878 of SEQ ID NO: 1) (FIG. 1).

TABLE 2 PCR amplification System for target genes

PCR cycling conditions

The P40 gene PCR product 1 and the P35 gene PCR product 1 obtained by the amplification are respectively connected to a T vector (pBLUE-T vector, Beijing BOOolong immune technology Co., Ltd., BD-M10015) to obtain a P40-T vector and a P35-T vector.

Construction of eukaryotic recombinant plasmid VRIL-12 of IL-12 gene

1. Synthesis of 2A-TPA VRIL-12

Three primers, named 2A-TPA-1, 2A-TPA-2, 2A-TPA-3, were designed and synthesized according to literature procedures and used for PCR amplification of the fusion gene of 2A-TPA. Primer sequences (see table 3):

TABLE 32A-TPA PCR primers

The PCR reaction system was 50uL (see Table 4):

TABLE 42A TPA reaction System

And (3) PCR reaction conditions:

after 2% agarose gel electrophoresis, cutting and recovering the gel to obtain 2A-TPA (the nucleotide sequence is the 970 nd-1107 site of the sequence 1).

2. Amplification of PCR product 2 from the P40 Gene and PCR product 2 from the P35 Gene

1) Design and Synthesis of primers

An In-fusion method is used for designing an overlapping primer, a 2A short peptide gene is connected between two genes P40 and 35 and then the two genes are inserted into a vector VR1020 together, the 5 'end of the P40 gene is overlapped with the vector by 16bp and is named as 40-F, and the 3' end is overlapped with 2A-TPA by 10bp and is named as 40-R; the 5 'end of the 2A-TPA is overlapped with the P40 by 10bp and is named as 2A-F, the 3' end of the 2A-TPA is designed with a primer which is overlapped with the P35 gene by 10bp and is named as 2A-R; the P35 gene has 10bp overlap with 2A-TPA at the 5 'end and is named as 35-F, and 16bp overlap with VR1020 at the 3' end and is named as 35-R. Primer sequences (see table 5):

TABLE 5 primer sequences

2) Amplification of fragments of interest

The two obtained P40-T vectors were used as templates, and 40-F and 40-R were used as primers for amplification to obtain 995bp P40 amplification product 2 (FIG. 2).

The two obtained P35T vectors were used as templates, and 35-F and 35-R were used as primers for amplification to obtain 797bp P35 amplification product 2 (FIG. 2).

The 2A-TPA obtained in the above 1 was used as a template, and 2A-F and 2A-R were used as primers for amplification to obtain 158bp 2A-TPA amplification product 2 (FIG. 2).

3) Obtaining the vector backbone

The VR1020 plasmid (VR1020 from Vical, USA) was digested with BamHI to obtain VR1020 linear vector backbone.

4) Ligation of fragments to vectors

Connecting the P40 amplification product 2, the P35 amplification product 2, the 2A-TPA amplification product 2 obtained in the step 2) with a VR1020 linear vector skeleton to obtain the eukaryotic recombinant plasmid VRIL-12.

The above linker was 10uL (see Table 6):

TABLE 6 fragment and vector ligation System

Connection conditions are as follows: 50 ℃ for 15 min.

Sequencing eukaryotic recombinant plasmid VRIL-12, wherein the plasmid is obtained by inserting a DNA fragment shown in a sequence 1 into BamHI enzyme cutting sites of VR1020 plasmid.

The DNA fragment shown in the sequence 1 is named as a fusion fragment IL-12 ', and the protein coded by the fusion fragment is named as IL-12' protein.

Wherein P40 is the 1 st-969 th site of the sequence 1, 2A is the 970 nd-1032 th site of the sequence 1, TPA is the 1033 rd-1107 site of the sequence 1, P35 is the 1108 th-1878 th site of the sequence 1;

the protein encoded by the fusion fragment is named as IL-12' protein, and the amino acid sequence of the protein is shown as sequence 2 in the sequence table (P40 is shown as 1-323 th position of the sequence 2, 2A is shown as 324-344 th position of the sequence 2, TPA is shown as 345-369 th position of the sequence 2, and P35 is shown as 370-625 th position of the sequence 2).

Four, activity detection of eukaryotic recombinant plasmid VRIL-12

1. Expression activity of eukaryotic recombinant plasmid VRIL-12 in HEK293 cells

The eukaryotic recombinant plasmid VRIL-12 and pEGFP-N3-GFP expression plasmid (Clontech, 6080-1, as a positive transfection control) prepared in the three procedures described above were added simultaneously to transfect HEK293 cells (human embryonic kidney cells, Shanghai Bayer organism fetal bovine serum, HEK-293) using a lipofection kit (cationic liposome DMRIE: purchased from Invitrogen, USA, SGMB01-GMB-GCR 4118).

1) Fluorescence microscopy assay

Cells were observed to grow well and to be less dead before transfection, and cells were observed 24h, 48h and 72h after transfection with a fluorescence microscope.

The results are shown in FIG. 3, which shows that GFP expression begins 24h after transfection, indicating that the plasmid can be successfully expressed in the cells.

2) Detection of target gene in HEK293

Supernatants were collected at 24h, 48h, 72h after transfection in 1 above, and cells were collected 48h after transfection.

Total RNA of cells transfected for 48h was extracted, reverse-transcribed to give cDNA as a template, and PCR-amplified using IL-12 specific primers (35-F and 35-R). Control with empty vector VR 1020.

The results are shown in FIG. 4, and it can be seen that the constructed VRIL-12 recombinant plasmid can be successfully expressed in HEK293 cells.

2. Lymphoblast stimulation assay

VRIL-12 group:

(1) collecting peripheral blood lymphocytes of the Tibetan pigs according to the previous method;

(2) according to the typesetting, 50. mu.l of Tibetan pig peripheral blood lymphocytes and 50. mu.l of the supernatant collected 24h, 48h and 72h after the transfection collected in the above 1 were added to the 96-well plate experimental wells, respectively. Each sample is provided with 3 repeated holes and a control hole, and is placed in a cell culture box for culturing for 48 hours;

(3) after 48h, taking out a 96-well plate, adding 10 mul of CCK8 into each well, and slightly and uniformly blowing to continuously culture for 2 h;

(4) the 96-well plate was removed and each well was examined for OD450 by Bio-Reader 3550.

VR1020 group: unlike the VRIL-12 group, the transfection supernatant was replaced with the control transfection supernatant.

The control transfection supernatant was collected after transfection of plasmid VR1020 into HEK 29324, 48, and 72 hours.

Cell is HEK293 Cell, control is VR1020 blank plasmid.

Cell survival was measured by CCK8, and the results are shown in fig. 5, wherein proliferation of VRIL-12 group is obvious compared with VR1020 group, which indicates that the supernatant of experiment plasmid transfected HEK293 cells can significantly stimulate porcine lymphocyte proliferation (P < 0.05). Prove that VRIL-12 can correctly express IL-12 and has immunostimulation activity; is the basic evidence that the later recombinant plasmid can be used as immunological adjuvant when being expressed in vivo cells.

Fifth, detection of endotoxin

The endotoxin content of the VRIL-12 plasmid is detected by a gel method, which is referred to limulus reagent instruction, and the specific method is as follows:

(1) preparation of positive control sample: preparing endotoxin standard product (150600, Shanghai Haalin Biotechnology Co., Ltd.) with water to obtain a solution with a concentration 2 times of sensitivity λ of 0.25 Eu/ml;

(2) negative control: water for examination;

(3) detecting a sample: according to the requirement calculator effective dilution multiple (MVD ═ cL/lambda) of the endotoxin limit value of the detected sample, diluting the sample for standby;

(4) taking a limulus reagent, breaking off ampoule necks and marking, wherein 1 branch is taken as a positive control tube, 1 branch is taken as a negative control tube, 2 branches are taken as a test tube, and 1 branch is taken as a test article positive control tube, and the total number is 5;

(5) respectively adding 0.2ml of detection water, 0.1ml of endotoxin solution with the concentration of 2 times lambda and 0.1ml of detection water and 0.1ml of detection sample (VRIL-12 plasmid) into a negative control tube, a positive control tube and a sample tube to be detected according to requirements, and gently shaking uniformly to detect;

(6) vertically placing the sample to be detected in the last step into a thermostat, incubating for 60 minutes at 37 ℃, and reading the result.

No gel formation was observed in the sample tube, indicating that the eukaryotic recombinant plasmid VRIL-12 contains no endotoxin.

Example 2 application of eukaryotic recombinant plasmid VRIL-12 as vaccine adjuvant

Preparation of vaccine adjuvant

1. Preparation of VRIL-12-coated chitosan nanoparticle (CS-IL-12)

The VRIL-12 plasmid is wrapped by an ion crosslinking method to prepare nanoparticles serving as a vaccine adjuvant, and the specific method comprises the following steps:

(1) preparing a solution:

10mg/ml TPP solution: TPP is dissolved in ddH2O to make the concentration of TPP 10mg/ml, thus obtaining TPP solution of 10 mg/ml;

2.4mg/ml chitosan solution: dissolving chitosan in 1% (pH5.5) glacial acetic acid water solution to make chitosan concentration be 2.4mg/ml, to obtain 2.4mg/ml chitosan solution;

filtering the prepared solution with 0.22m microporous membrane for sterilization;

(2) plasmid and TPP solution pre-mix: uniformly mixing a VRIL-12 solution (water is used as a solvent) and a proper amount of TPP solution, and incubating at the constant temperature of 55 ℃ for 20min to obtain a premixed solution of plasmid and TPP; the mass ratio of VRIL-12 to TPP is 1: 3;

chitosan-coated plasmid: slowly dripping the premixed solution of the plasmid and TPP into a chitosan solution under the magnetic stirring condition of 55 ℃ water bath, uniformly mixing to ensure that the mass ratio of chitosan to the plasmid is 30:1, and then incubating for 10min at constant temperature for later use to obtain chitosan nanoparticles wrapping VRIL-12;

(4) sampling and detecting: and taking a proper amount of chitosan nanoparticles wrapping VRIL-12, and detecting the Zeta potential and the particle size of the nanoparticles by using a Malvern particle sizer.

As shown in FIG. 6, the particle size of the chitosan nanoparticle encapsulating VRIL-12 is 107.78 + -14.92 nm, the Zeta potential is +26.4 + -2.41 mv, and the dispersity is 0.217 + -0.011, which indicates that the particle meets the requirements of transfected cells, and the particle is named as CS-IL-12.

2. Preparation of DMRIE plasmid Complex (DMRIE-IL-12)

The VRIL-12 recombinant plasmid 4. mu.g was diluted in 500. mu.L RPMI 1640 reduced serum medium. Correspondingly, 10. mu.L of DMRIE (cationic liposome DMRIE: purchased from Invitrogen, USA, SGMB01-GMB-GCR4118) was diluted separately into 500. mu.L of RPMI 1640 reduced serum medium. Standing at room temperature for 5min after respectively mixing uniformly, adding the diluted plasmid into corresponding diluted DMRIE, standing at room temperature for 30min after lightly mixing uniformly to obtain DMRIE plasmid complex (mass ratio of DMRIE to plasmid is 2.5: 1) as transfection cell reagent.

Second, the biological effect of VRIL-12-coated chitosan nanoparticle and DMRIE plasmid complex expressed in Tibetan pig PBMCS cells

1. Transfected Tibetan pig PBMCS cells

The PBMCS of the Tibetan pig peripheral blood was collected according to the previous method and divided into the following groups:

experimental group (CS-IL-12+ PCV 2): transfecting the prepared VRIL-12-coated chitosan nanoparticle with the tibetan pig PBMCS cells after 12h culture, culturing for 4.5h, adding 10 mul to dilute 10⁴ Double PCV 2.

Experimental group (DMRIE-IL-12+ PCV): the cells of Tibetan pig PBMCS cultured for 12h were transfected by the DMRIE plasmid complex (DMRIE-IL-12) prepared in the above 1, cultured for 4.5h, and 10. mu.L of the diluted 10⁴ Double PCV 2.

PCV2 control (PCV2, control): culturing 10 μ L of RPMI 1640 complete medium and cells cultured with Tibetan pig PBMCS for 12h for 4.5h, adding 10 μ L of diluted 10⁴ Double PCV 2.

Blank control group: blank PBMCS.

The experimental group and the control group are respectively centrifuged (10000g,30sec) for 24h, 48h, 72h, 96h, 120h, 144h and 168h after the virus adding time, cells are collected, and are lysed by 1mL of Trizol and are frozen at-80 ℃ for later use. The layout is shown in Table 7.

TABLE 7 Tibetan pig PBMCS transfection typesetting schematic

CS-IL-12+PCV2	DMRIE-IL-12+PCV2	PCV2	Blank control
				CS-IL-12+PCV2	DMRIE-IL-12+PCV2	PCV2	Blank control
CS-IL-12+PCV2	DMRIE-IL-12+PCV2	PCV2	Blank control

The transfection methods of the above groups are as follows:

chitosan nanoparticle transfection PBMCS: regulating pH of the prepared chitosan nanoparticle solution to about 6.0 with NaOH solution, changing cell culture medium to corresponding serum-free culture medium before transfection, adding chitosan nanoparticle solution containing 2ug plasmid for transfection, and changing to complete culture medium after 3h for continuous culture;

liposome transfection reagent DMRIE transfection PBMCS:

(1) preparation of PBMCS cells: centrifuging (250g,15min) PBMCS, washing the cells with RPMI 1640 serum-free medium once, then resuspending the cells with RPMI 1640 serum-free medium, counting, and diluting to the cell density of 1.5x10⁷Per mL;

(2) transfection: 0.2mL of cell suspension was added to a 6-well cell culture plate, DMRIE plasmid complex (containing 2ug of plasmid) was added, cells were cultured at 37 ℃ under 5% CO2 for 4.5h, 2mL of preheated RPMI 1640 medium containing 15% heat-inactivated FBS and 1.5% penicillin streptomycin was added, and the mixture was mixed and cultured.

2. Detecting expression of immune-related genes

And respectively extracting RNA of the 2 different groups of transfected cells cultured in different time periods, and carrying out reverse transcription to obtain first-strand cDNA.

According to the pig gene sequence reported in GenBank, the fluorescent quantitative PCR specific primers were designed and synthesized, see Table 8

TABLE 8 fluorescent quantitation primers

The diluted cDNA was used as a template, and the target gene was amplified with specific primers shown in Table 8, and the fluorescent quantitative PCR reaction system was 15. mu.L (see Table 9):

TABLE 9 Gene amplification System of interest

Reaction conditions are as follows:

after the reaction is finished, beta-action is taken as an internal reference gene and is expressed by 2^-ΔΔCTThe relative expression level of the gene was calculated.

The fluorescence quantitative standard curve result shows that the correlation coefficient of each gene PCR is above 0.99, the amplification efficiency is between 95% and 105%, and the fluorescence quantitative requirement is met.

The difference of the expression level of the genes between different groups is analyzed and compared by a Tukey multiple comparison-based one-factor analysis of variance and two-factor analysis of variance method. When P <0.05, the relative expression levels of the genes between the different groups were considered to be significantly different.

As can be seen from FIG. 7(control is blank cell culture supernatant), the expression level of IL-12 gene in experimental group is significantly higher than that in control group (P <0.05) after Tibetan pig PBMCS is transfected with IL-12 plasmid; and the significant difference exists among experimental groups, and the DMRIE-IL-12 group is significantly higher than the CS-IL-12 group (the former is high in efficiency but high in price, so that the former is not practical and expensive, and the latter CS-IL-12 is economic, safe and effective) (P < 0.05).

As can be seen from fig. 8(control is blank cell culture supernatant), after tibetan pig PBMCS is transfected with IL-12 plasmid, the expression levels of the Toll-like receptor genes TLR3 and TLR9 in the experimental group are significant compared with the expression levels of TLR3 and TLR9 in the control group (P < 0.05); and the significant difference exists among the experimental groups, and the DMRIE-IL-12 group is significantly higher than the CS-IL-12 group (P < 0.05).

As can be seen from fig. 9(control is blank cell culture supernatant), the expression levels of the experimental group immunological memory related genes IL7, IL15 and IL23 were significant for the control group (P <0.05) after IL-12 plasmid was transfected by tibetan pig PBMCS; and the expression levels of IL7, IL15 and IL23 are significantly different among experimental groups, and the CS-IL-12 group is significantly higher than the DMRIE-IL-12 group (P < 0.05); IL7 at 96h DMRIE-IL-12 group was significantly higher than CS-IL-12 group (P < 0.05); IL15 at 72h DMRIE-IL-12 group was significantly higher than CS-IL-12 group (P < 0.05); IL23 was significantly higher in the DMRIE-IL-12 group than in the CS-IL-12 group at 48h and 96h (P < 0.05).

As can be seen from FIG. 10(control is blank cell culture supernatant), the expression levels of Th1 type cytokines IL2 and TNF-. alpha.in the experimental group were significant compared with the expression levels of IL2 and TNF-. alpha.in the control group (P <0.05) after IL-12 plasmid was transfected by Tibetan pig PBMCS; and the expression level of IL2 and TNF-alpha is significantly different among the experimental groups, and the DMRIE-IL-12 group is significantly higher than the CS-IL-12 group (P < 0.05); IL2 at 168h the CS-IL-12 group was significantly higher than the DMRIE-IL-12 group (P < 0.05); TNF-alpha at 72h and 144h CS-IL-12 group was significantly higher than DMRIE-IL-12 group (P < 0.05).

As can be seen from fig. 11(control is blank cell culture supernatant), after IL-12 plasmid was transfected by tibetan pig PBMCS, the expression levels of the Th2 cytokines IL4, IL6 and IL10 in the experimental group were significant (P < 0.05); and the expression level of IL4 and IL6 is significantly different between experimental groups, and the DMRIE-IL-12 group is significantly higher than the CS-IL-12 group (P < 0.05); IL6 at 48h the CS-IL-12 group was significantly higher than the DMRIE-IL-12 group (P < 0.05); IL10 was significantly higher in the CS-IL-12 group than in the DMRIE-IL-12 group at 72h, 120h and 144h (P <0.05), and significantly higher in the DMRIE-IL-12 group than in the CS-IL-12 group at 24h, 48h and 168h (P < 0.05).

As can be seen from FIG. 12(control is blank cell culture supernatant), the significant control group (P <0.05) of the expression level of the feedback regulatory gene TGF beta-1 in the experimental group after IL-12 plasmid transfection with Tibetan pig PBMCS; and significant difference exists among experimental groups, the DMRIE-IL-12 group is significantly higher than the CS-IL-12 group (P < 0.05); TGF beta-1 at 72h and 144h CS-IL-12 group was significantly higher than DMRIE-IL-12 group (P < 0.05).

As can be seen from fig. 13(control is blank cell culture supernatant), the expression level of the experimental group immune signaling molecule MyD88 is significant after IL-12 plasmid transfection by tibetan pig PBMCS, compared with the expression level of the control group MyD88 (P < 0.05); and the expression level of MyD88 is significantly different among experimental groups, and the CS-IL-12 group is significantly higher than the DMRIE-IL-12 group (P < 0.05); MyD88 was significantly higher in the DMRIE-IL-12 group than in the CS-IL-12 group at 48h and 168h (P < 0.05).

Thirdly, the biological effect of the vaccine adjuvant expressed in the mouse body

1. Grouping immunization of experimental mice

50 female age Kunming mice of 28 days, weighing about 25-30g, were randomly grouped into 5 groups of 10 mice each. Experimental groups were numbered a1 and a2, control groups were numbered C1 and C2, respectively, and the mouse immunization schedule is shown in table 10. Recording as 0 week before injection, periodically collecting blood every 1-5 weeks after injection, collecting blood of about 350 μ L each group, weighing each mouse body weight every week for fixed time before and after immunizing animals, and recording.

TABLE 10 mouse grouping and immunization schedule

Before and after immunization, the body weight of each mouse was weighed at a fixed time per week, and the body weight of each group of mice was calculated as the mean and variance, and the body weight change of the mice was shown in fig. 14. The results show that the body weight of the mice increases with the increase of the feeding time, and the body weight of the mice in the experimental group is not obviously different from that of the control group (P > 0.05).

2. Routine analysis of blood of laboratory mice

50ul of fresh blood is taken, the conditions of blood change levels such as lymphocyte number, erythrocyte number, hemoglobin content and the like in the blood of the experimental mice are detected through a blood cell automatic classification detection technology, and the peripheral blood immunological change conditions of each experimental mouse in the experimental period are determined.

Blood the change in the number of blood cells in peripheral blood of mice was routinely analyzed (see FIG. 15). The results show that after immunization, the lymphocyte number of the experimental group is obviously higher than that of the control group (P <0.05), and no obvious difference exists between the experimental groups (P > 0.05); the number of basophils in the experimental group is obviously higher than that in the control group (P <0.05), and the number of CS/P-IL-12 groups among the experimental groups is obviously higher than that in the CS-IL-12 group (P < 0.05); the hemoglobin content and the number of red blood cells of the experimental group are not obviously different from those of the control group (P > 0.05).

3. Flow cytometry analysis

50 μ L of fresh anticoagulated blood was taken and administered against mouse fluorescently labeled CD4⁺、CD3⁺And CD8⁺The monoclonal antibody is subjected to cell staining, the Th and Tc subgroup cells in the immune cells of the experimental mice are distinguished by flow cytometry, and the change condition of cellular immune response is observed through the dynamic change of the Th and Tc subgroup cells. The flow cytometry comprises the following specific steps:

(1) taking 50 mu L of fresh anticoagulation blood, adding a premixed antibody (containing 0.5 mu L of anti-Mouse CD3e-FITC, 0.625 mu L of anti-Mouse CD8-PE, 0.50.625 mu L of anti-Mouse CD 4-PerCP-Cy5.50.625 mu L), shaking, uniformly mixing and keeping out of the sun for 30 min;

(2) adding 500 μ L erythrocyte lysate, standing in dark for 10min, centrifuging at 3500r for 5min, and removing supernatant;

(3) adding 1ml PBS buffer solution, suspending the cells gently, removing residual erythrocyte fragments, centrifuging at 3500r for 5min, and removing supernatant;

(4) adding 300 mu L of PBS into the cell sediment, mixing uniformly, storing at4 ℃ in a dark place, and detecting on a machine within 24 h.

Flow cytometry detection of CD4 in mouse peripheral blood⁺T and CD8⁺The number of T cell subsets varied (see figure 18). The results show CD4 in peripheral blood of mice after immunization⁺T and CD8⁺The number of T cell subsets is increased; experimental group CD4⁺T and CD8⁺The number of T cells is obviously higher than that of the control group CD4⁺T and CD8⁺T cell number (P)<0.05), and the CS/P-IL-12 group is significantly higher than the CS-IL-12 group (P) among experimental groups<0.05)。

4. Determination of IgG, IgG1, IgG2a and PCV-Ab in serum

Taking 100 mu L of fresh anticoagulation blood, centrifuging at 3500r for 10min to separate serum, and storing the serum at-80 ℃ for later use. The serum levels of IgG, IgG1, IgG2a and PCV-Ab were determined with reference to the ELISA kit instructions.

ELISA detected changes in IgG, IgG1, and IgG2a levels in mouse serum (see FIG. 16). The result shows that after immunization, the contents of IgG, IgG1 and IgG2a of mice in a control group are not obviously increased and 6 are reduced, while the contents of an experimental group are obviously increased; the IgG content in the serum of the experimental group mouse is obviously higher than that of the control group (P <0.05), and the CS-IL-12 group between the experimental groups is obviously higher than that of the CS/P-IL-12 group (P < 0.05); the content of IgG1 and IgG2a in the serum of the mice in the experimental group is obviously higher than that in the control group (P <0.05), and no obvious difference exists between the experimental groups (P > 0.05).

The ELISA measured the PCV antibody content in the serum of the mice after immunization (see FIG. 17). The result shows that after the PCV vaccine is immunized, effective PCV antibody is detected in the serum of the mouse; the PCV antibody content in the serum of the experimental group mouse is obviously higher than that in the control group PCV antibody (P <0.05), and the CS/P-IL-12 group among the experimental groups is obviously higher than that in the CS-IL-12 group PCV antibody (P < 0.05).

5. Detection of expression level of immune-related gene

RNA of the blood of the mice 1-5 weeks after the immunization of the groups 1 is extracted and reverse transcription is carried out to obtain cDNA.

R according to the mouse gene sequence reported in GenBank, the specific primers for the synthetic quantitative PCR were designed and synthesized, see Table 11.

TABLE 11 mouse immune-related Gene primers

Quantitative PCR detects the change of expression of related genes.

As can be seen from FIG. 19, the expression level of IL-12 cytokine in the mice in the experimental group was significantly higher than that of IL-12 in the control group (P <0.05) after the immunization; there was no significant difference in the expression level of IL-12 between the control groups (P > 0.05); between experimental groups, the expression level of IL-12 in the CS/P-IL-12 group was significantly higher than that in the CS-IL-12 group (P <0.05) on days 7, 21 and 28 of immunization, respectively.

As can be seen from fig. 20, after immunization, the expression levels of the Toll-like receptor genes TLR1 and TLR6 of the experimental group mice were significant compared with the expression levels of TLR1 and TLR6 of the control group (P < 0.05); no significant difference in the expression level of TLR1 and TLR6 between controls (P > 0.05); on day 21, TLR1 and TLR6 expression reached a maximum; there was no significant difference in the expression levels of TLR1 and TLR6 between experimental groups (P > 0.05).

As can be seen from fig. 21, after immunization, the expression levels of the immunological memory-associated genes CD62L and IL15 of the experimental mice were significant for the control group (P < 0.05); no significant difference in the expression level of CD62L and IL15 between the control groups (P > 0.05); the expression level of CD62L was significantly higher between the experimental groups than that of the CS/P-IL-12 group CD62L (P <0.05) on days 7 and 14; the expression level of CD62L is not significantly different from that of CD62L in an experimental group and a control group at the 35 th day (P > 0.05); the expression level of IL15 was significantly higher between the experimental groups than that of the CS/P-IL-12 group IL15 (P <0.05) on days 14, 28 and 35.

As can be seen from FIG. 22, after immunization, the expression levels of Th1 type cytokines IFN-gamma and TNF-alpha of the mice in the experimental group are significant compared with the expression levels of IFN-gamma and TNF-alpha in the control group (P < 0.05); the expression levels of IFN-gamma and TNF-alpha are not significantly different between the control groups (P > 0.05); between experimental groups, the expression level of IFN-gamma of the CS-IL-12 group is obviously higher than that of IFN-gamma of the CS/P-IL-12 group (P < 0.05); the expression level of TNF-alpha is not obviously different among experimental groups (P > 0.05).

As can be seen from fig. 23, after immunization, the expression levels of the Th2 type cytokines IL6 and IL10 in the experimental group were significant compared with the expression levels of IL6 and IL10 in the control group (P < 0.05); there was no significant difference in the expression levels of IL6 and IL10 between the controls (P > 0.05); the expression level of IL6 is not obviously different from the experimental group (P >0.05) in the expression level of IL 6; the expression level of IL10 is obviously higher than that of the CS/P-IL-12 group IL10 between experimental groups at 7 days, 28 days and 35 days (P < 0.05); the expression level of IL10 was significantly higher than that of the CS/P-IL-12 group IL10 (P <0.05) between the experimental groups at day 14.

As can be seen from FIG. 24, after immunization, the mice in the experimental group fed back the expression level of TGF beta-1, which is significant in comparison with the expression level of TGF beta-1 in the control group (P < 0.05); the expression level of TGF beta-1 between control groups is not obviously different (P > 0.05); there was no significant difference in the expression level of TGF-1 between experimental groups (P > 0.05).

As can be seen from fig. 25, the expression level of STAT1 in the experimental group mice was significant compared to the expression level of STAT1 in the control group (P <0.05) after immunization; there was no significant difference in the expression level of STAT1 between the controls (P > 0.05); at day 7, the expression level of STAT1 was not significantly different between the experimental group and the control group (P > 0.05); the expression level of STAT1 in the CS/P-IL-12 group was significantly higher than that in the CS-IL-12 group STAT1 between the experimental groups at day 14 and day 28 (P < 0.05); on day 35, the expression level of STAT1 in the CS-IL-12 group was significantly higher than that in the CS/P-IL-12 group STAT1 between the experimental groups (P < 0.05).

Fourth, the biological effect of the vaccine adjuvant expressed in the pig body

1. Grouping animals

10 Duda Chang three-way hybrid pigs (provided by pig research institute in Sichuan province) of 3-week-old non-immune PCV2 vaccines were selected and randomly divided into 2 groups of 5, wherein group A was an experimental group and group B was a control group, and the treatment was as follows (Table 12):

TABLE 12 grouping of experimental animals and inoculation preparations

Day 0 before inoculation, 5ml of anterior vena cava blood was taken at weeks 0, 1, 2, 4, 8, and 12 and stored in anticoagulation tubes for subsequent analysis. The weight of each animal was weighed before and after immunization and recorded.

As can be seen from Table 13, the weight difference between the experimental group (group A) and the control group (group D) before inoculation is not significant (P >0.05), and the weight increase of the group A is significantly higher than that of the group D (P <0.05) within 84 days after inoculation, and the result shows that the inoculation of VRIL-12-CNP has the effect of promoting the growth of piglets.

TABLE 13 weight change table for experimental animals

Note: the different lower case letters in the same column indicate significant difference (P < 0.05).

2. Routine blood analysis

The specific method is the same as the previous method.

As can be seen in fig. 26, the red blood cell numbers in group a were significantly higher than those in group D at days 7, 14, 56 and 84 after inoculation (P < 0.05). Group a hemoglobin content increased significantly on days 7, 14 and 35 (P < 0.05). The difference in leukocyte numbers between group A and group D was not significant (P > 0.05).

3. Flow cytometry analysis

The specific method is the same as the previous method.

FIG. 27 shows CD3⁺T cell, CD4⁺T cells and CD8⁺Changes in the number of T cells and CD8⁺/CD4⁺A change in the ratio. Three cell numbers all increased significantly after inoculation, group A CD3⁺T cell, CD4⁺T cells and CD8⁺The T cell number was significantly higher than that of the control group D (P) at 28 days, 56 days and 84 days after inoculation<0.05), CD8 after group A vaccination⁺/CD4⁺The ratio of (A) was also significantly higher than that of the control group D, and the difference was significant at day 56 (P)<0.05)。

4. Determination of IgG and PCV-Ab in serum

The specific method is the same as the previous method.

Changes in immunoglobulin IgG in serum as shown in fig. 28, IgG levels were significantly higher in group a than in group D (P <0.05) during the test period (days 0 to 84). IgG levels increased from day 14 post inoculation and reached the highest level at day 56.

FIG. 29 is a graph of the change in the level of specific anti-PCV-2 antibody. At day 0, both the experimental and control groups were PCV2 antibody negative (S/P values less than 0.16), PCV2 antibody levels began to increase after inoculation, and specific PCV2 antibody levels were significantly higher in group a than in group D (P <0.05) at days 7, 28, 56, and 84.

5. Detection of expression level of immune-related gene

Taking the blood of the anterior vena cava at 0, 1, 2, 4, 8 and 12 weeks of immunization, extracting RNA, and carrying out reverse transcription to obtain cDNA.

Based on the gene sequence of pig reported in GenBank, the specific primers for quantitative PCR were designed and synthesized, see Table 14.

TABLE 14 porcine immune-related gene primers

Quantitative PCR was performed using cDNA as a template and the primers shown in Table 14, and the change in expression level of the relevant gene was detected using PPIA as an internal reference gene and 2^-ΔΔCTThe method calculates the relative expression quantity of the gene.

FIG. 30 shows the expression level changes of the Toll-like receptor genes TLR2 and TLR 7. Compared with the control group, the expression level of TLR2 and TLR7 genes of the experimental group A is remarkably increased from 7 days to 84 days after VRIL-12-CNP inoculation (P < 0.05). The TLR2 gene increases significantly higher than the control group (P <0.05) at day 28 and day 84, and the TLR7 expression level of the experimental group is significantly higher than the control group (P <0.05) under the stimulation of VRIL-12-CNP at day 7, day 28 and day 84.

As shown in FIG. 31, the cytokine gene expression level varied, and after inoculation of VRIL-12-CNP, the cytokine expression levels in the experimental group were significantly higher than those in the control group. The IL-2 gene expression level of group A was significantly higher than that of group D at day 56 (P < 0.05); the IL-4 gene was significantly higher on days 14, 28 and 56 than group D (P < 0.05); the expression level of the IL-6 gene is obviously increased from 7 days (P < 0.05); the expression level of IL-12 gene was significantly higher on days 7, 28, 56 and 84 than in control group D (P < 0.05).

The changes in the expression levels of the genes STAT1, STAT2, STAT3, STAT4, CD45 and Bcl-2 are shown in FIG. 32. The STAT1 gene expression level of the experimental group A is significantly higher than that of the group D (P <0.05) on days 7, 14, 56 and 84 after VRIL-12-CNP inoculation, and the STAT2, STAT3 and STAT4 genes are also significantly higher than that of the group D (P <0.05) on days 7 to 56; at 14 days after inoculation, the Bcl-2 gene expression level of the group A is obviously higher than that of the group D (P < 0.05); the expression level of CD45 in group A was significantly higher than that in group D from day 7 to day 84 (P < 0.05).

Sequence listing

<110> Sichuan university

<120> preparation method and application of Tibetan pig IL-12 recombinant plasmid enhanced PCV2 vaccine immunologic adjuvant

<160> 2

<170> PatentIn version 3.5

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<211> 1878

<212> DNA

<213> Artificial sequence

<220>

<223>

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atgcaccttc agcagctggt tgtctcctgg ttttccctgg tttggctggc atctcccatt 60

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gcccctggag aaatggtggt cctcacctgc aacacccctg aagaagacgg catcacgtgg 180

acctcagacc agagcagtga ggtcttgggc actggcaaaa ccctgaccat ccacgtcaaa 240

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ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaagaccag 360

aaagagccca aaaacaagag ctttctaaaa tgtgaggcaa agaattactc cggacgtttc 420

acctgctggt ggctgacggc aatcagtact gatttgaaat tcagtgtcaa aagcagcaga 480

ggctccgctg acccccgtgg cgtgacatgt ggcacggcaa tgctctcaga ggacctcggg 540

gagtataagt acagagtgga gtgtcaggag ggcagtgcct gcccagccgc tgaggagagc 600

ctgcccattg aggtcgtgct ggaagctgtt cacaagctta agtatgaaaa ctacaccagc 660

agcttcttca tcagggacat catcaaacca gaccctccca agaatctgca gctgaaccca 720

ttaaagaatt ctcgacacgt ggagatcagc tgggagtacc ctgacacctg gagcacccca 780

cattcctact tttccctgat gtttggtgtt caagttcagg gcaagaacaa aagagaaaag 840

aaagataaac tcttcacgga ccaaacctca gccaaggtta catgccacaa ggatgccaac 900

atccgcgtgc aagcccggga ccgctactac agctcctcct ggagtgaatg ggcatctgtg 960

tcctgcaatg gaagcggaga gggcagggga agtcttctaa catgcgggga cgtggaggaa 1020

aatcccgggc caatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 1080

gcagtcttcg tttcgcccag cggtaccatg tgttcccctg ggttcggcct ccaaactagc 1140

gccctcacct gcgacgtcca tccatctgcg tccagccgcc gactcgctgt caccgcagcg 1200

ccgctcagca tgtgcccgct gcgcaacctc ctccttgtgg ccaccctggt cctcctcaac 1260

cacctggacc atctcagttt gggcaggagc ctccctgcaa ccacagcagg cccaggaatg 1320

ttcaaatgcc tcaaccactc ccaaaatctg ctgaaggccg tcagcaacac acttcagaag 1380

gccaaacaaa ccctagaatt ttactcctgc acttcagaag agatcgatca tgaagatatc 1440

accaaagata aaaccagcac agtggaggcc tgcttaccac ttgaactagc cacgaatgag 1500

agttgcctgg ctgccagaga gacctcttta ataactaatg gaaattgcct gacttctgga 1560

aagacctctt ttatgacaac cctgtgcctt agcagtatct acgaggactt gaagatgtac 1620

catgtggagt tccaggccat gaatgcaaag cttctgatgg atcctaagag gcagatcttt 1680

ctggatcaga acatgctgac agctattact gagctgatgc aggctctgaa tttcaacagt 1740

gagactgtgc cacagaagcc ctccctggaa gaactggatt tttataagac taaaatcaag 1800

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tatctgaatt cttcctaa 1878

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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 Cys Ser Pro Gly Phe Gly Leu Gln Thr Ser Ala Leu Thr Cys

370 375 380

Asp Val His Pro Ser Ala Ser Ser Arg Arg Leu Ala Val Thr Ala Ala

385 390 395 400

Pro Leu Ser Met Cys Pro Leu Arg Asn Leu Leu Leu Val Ala Thr Leu

405 410 415

Val Leu Leu Asn His Leu Asp His Leu Ser Leu Gly Arg Ser Leu Pro

420 425 430

Ala Thr Thr Ala Gly Pro Gly Met Phe Lys Cys Leu Asn His Ser Gln

435 440 445

Asn Leu Leu Lys Ala Val Ser Asn Thr Leu Gln Lys Ala Lys Gln Thr

450 455 460

Leu Glu Phe Tyr Ser Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile

465 470 475 480

Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu

485 490 495

Ala Thr Asn Glu Ser Cys Leu Ala Ala Arg Glu Thr Ser Leu Ile Thr

500 505 510

Asn Gly Asn Cys Leu Thr Ser Gly Lys Thr Ser Phe Met Thr Thr Leu

515 520 525

Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr His Val Glu Phe

530 535 540

Gln Ala Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe

545 550 555 560

Leu Asp Gln Asn Met Leu Thr Ala Ile Thr Glu Leu Met Gln Ala Leu

565 570 575

Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Pro Ser Leu Glu Glu Leu

580 585 590

Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe

595 600 605

Arg Ile Arg Ala Val Thr Ile Asp Arg Met Met Ser Tyr Leu Asn Ser

610 615 620

Ser

625

Claims (10)

1. A protein which is composed of an amino acid sequence shown as a sequence 2 in a sequence table.

2. A DNA molecule encoding the protein of claim 1.

3. The DNA molecule of claim 2, wherein: the DNA molecule is shown as a sequence 1 in a sequence table.

4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a DNA molecule encoding the protein of claim 1 or a transfection reagent comprising said recombinant vector.

5. The recombinant vector according to claim 4, wherein: the recombinant vector is obtained by inserting a DNA molecule encoding the protein of claim 1 into a eukaryotic expression vector.

6. The recombinant vector according to claim 5, wherein: the eukaryotic expression vector is VR1020 plasmid.

7. A transfection reagent according to claim 4, characterized in that:

the transfection reagent containing the recombinant vector is a transfection reagent obtained by wrapping the recombinant vector of claim 5 by chitosan;

or the transfection reagent containing the recombinant vector is a transfection reagent obtained by encapsulating the recombinant vector by liposome.

8. A transfection reagent according to claim 7, characterized in that:

the mass ratio of the chitosan to the recombinant vector is 10: 1;

or the liposome is DMRIE, and the mass ratio of the DMRIE to the plasmid is 2.5: 1.

9. use of the protein of claim 1 or the DNA molecule of claim 2 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 or a transfection reagent containing said recombinant vector for the preparation of any one of the following 1) -2);

1) PCV2 vaccine adjuvant;

2) a product for improving the immune effect of PCV2 vaccine.

10. A PCV2 vaccine adjuvant comprising the protein of claim 1 or the DNA molecule of claim 2 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 or a transfection reagent containing said recombinant vector.

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