阅读说明：本技术 大肠杆菌偏嗜性可溶性猪pd-1重组蛋白的制备及应用 (Preparation and application of escherichia coli preference soluble pig PD-1 recombinant protein ) 是由 王选年 朱艳平 刘佳 何勇 岳锋 孙国鹏 郭东光 李鹏 李文明 刘金宵 于 2021-01-15 设计创作，主要内容包括：本发明涉及一种大肠杆菌偏嗜性可溶性猪PD-1重组蛋白的制备及应用,其中可溶性猪PD-1重组蛋白的氨基酸序列如SEQ ID NO：1所示,利用基因克隆技术得到经过密码子偏嗜性修饰的猪PD-1基因的可溶性重组蛋白,该可溶性猪PD-1重组蛋白能有效提高PCV2疫苗免疫后PCV2的抗体水平以及机体免疫力,增强猪对PCV2疫苗的免疫应答,提高接种动物的免疫力,可解决现有技术PCV2疫苗免疫后效果不理想的问题。(The invention relates to preparation and application of escherichia coli preference soluble pig PD-1 recombinant protein, wherein the amino acid sequence of the soluble pig PD-1 recombinant protein is shown as SEQ ID NO: 1, the soluble recombinant protein of the pig PD-1 gene modified by codon preference is obtained by utilizing a gene cloning technology, and the soluble pig PD-1 recombinant protein can effectively improve the antibody level and the organism immunity of PCV2 after the immunization of PCV2 vaccine, enhance the immune response of pig to PCV2 vaccine, improve the immunity of inoculated animals and solve the problem of non-ideal effect after the immunization of PCV2 vaccine in the prior art.)

1. The soluble porcine PD-1 recombinant protein is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. A DNA molecule encoding the soluble porcine PD-1 recombinant protein of claim 1, which has the nucleotide sequence set forth in SEQ ID NO: 2, respectively.

3. A method of preparing the soluble porcine PD-1 recombinant protein of claim 1, comprising the steps of:

(1) cloning the DNA molecule of claim 2 into expression plasmid pET-32a (+) to obtain recombinant expression vector pET-32a-PD 1;

(2) transforming the recombinant expression vector pET-32a-PD1 obtained in the step (1) into an escherichia coli Rosetta engineering strain, and selecting a single colony to be cultured at 37 ℃ to serve as seed liquid;

(3) inoculating the seed solution obtained in the step (2) into YT culture solution containing Amp according to the volume ratio of 1:100, and culturing at 37 ℃ to OD₆₀₀Adding IPTG (isopropyl thiogalactoside) with the value of 0.5-1.0 for induction culture;

(4) purifying and concentrating the induction expression product by chromatography to obtain the soluble porcine PD-1 recombinant protein with escherichia coli preference.

4. The method according to claim 3, wherein the conditions for the induction culture in the step (3) are: the induction temperature is 28 ℃, the induction time is 4h, and the IPTG concentration is 0.5 mmol/L.

5. The use of the soluble porcine PD-1 recombinant protein of claim 1 in the preparation of a PCV2 vaccine radiopharmaceutical.

6. Use of the soluble porcine PD-1 recombinant protein of claim 1 in the preparation of a PCV2 vaccine enhancer or adjuvant.

7. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a DNA molecule of the soluble porcine PD-1 recombinant protein of claim 2.

8. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a DNA molecule of the soluble porcine PD-1 recombinant protein of claim 2 for the preparation of a corresponding product.

Technical Field

The invention relates to preparation and application of escherichia coli partial tropism soluble pig PD-1 recombinant protein, and belongs to the field of biological medicine.

Background

Porcine circovirus type 2 (PCV 2) disease is one of the important epidemic diseases which endanger the world pig industry at present, and the pathogenicity of PCV2 is directly related to the strain type. PCV2 was typed on the basis of sequence differences of ORF 2. PCV2 mainly includes five subtypes PCV2a, PCV2b, PCV2c, PCV2d and PCV2e, of which PCV2b is the currently predominant genotype. At present, the new situation that different gene subtypes are simultaneously epidemic in China is presented, mixed infection of PCV2a and PCV2b is increased, and PCV2d is also increased in infection and becomes a main subtype of infection. In China, the PCV2b strain in the PCV2 epidemic strain has significantly higher toxicity than PCV2a, which is also probably an important reason for serious harm caused by porcine circovirus disease. Therefore, there is a need to develop new vaccines or vaccine adjuvants for the prevention of PCV 2.

PCV2 causes immunosuppression of the body after infection, and forms serious economic loss. At present, the research on T cell immunosuppressive receptors and their regulatory mechanisms after porcine PCV2 infection is not clear. PCV2 infection caused CD4⁺And CD8⁺The number of T lymphocytes decreases, causing immunosuppression and immune damage. Immune regulation of PCV2 and/or a host co-infected with a pathogen is considered critical for the development of clinical disease. Mixed infection of classical swine fever virus and pig breedingThe infection of the porcine influenza virus alone or the respiratory syndrome virus can cause the expression level of porcine T cell surface inhibitory receptor Cytotoxic T lymphocyte associated antigen 4 (cytotoxin T lymphocyte-associated antigen-4, CTLA-4, also known as CD152) and Programmed cell death factor 1(Programmed death 1, PD-1, also known as CD279) to be increased. When PCV2 is naturally infected, the expression levels of PD-L1 and the ligand PD-L2 are increased, and the PD-1/PD-L1 pathway is activated to play a role in immune negative regulation and play an important role in causing the immune suppression of the organism. Blocking the interaction between inhibitory receptors and their related ligands with anti-PD-1/PD-L1 antibodies or soluble PD-1 proteins can reverse T cell failure and enhance cell-mediated immune responses.

Therefore, the invention aims to prepare the soluble pig PD-1 recombinant protein to block the influence of the immunosuppressive receptor PD-1 and the ligand on the transcription change of the related cell factors when PCV2 is infected, evaluate the regulation effect of the soluble pig PD-1 recombinant protein on the immunosuppression generated by PCV2, enhance the immune response of organisms, lay a foundation for the development of a novel PCV2 vaccine enhancer or adjuvant, and provide a new strategy for the prevention and treatment of PCV 2.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide preparation of escherichia coli partial tropism soluble porcine PD-1 recombinant protein and application thereof in PCV2 vaccine, aims to improve PCV2 antibody level and organism immunity after PCV2 vaccine immunization, and solves the problem that the effect of the PCV2 vaccine in the prior art is not ideal after immunization.

In order to achieve the purpose, the invention adopts the technical scheme that:

the soluble porcine PD-1 recombinant protein has an amino acid sequence shown as SEQ ID NO: 1 is shown.

The DNA molecule of the recombinant protein of the coded soluble pig PD-1, the nucleotide sequence of the DNA molecule is shown as SEQ ID NO: 2, respectively.

The preparation method of the soluble porcine PD-1 recombinant protein comprises the following steps:

(1) cloning the DNA molecule of claim 2 into expression plasmid pET-32a (+) to obtain recombinant expression vector pET-32a-PD 1;

(2) transforming the recombinant expression vector pET-32a-PD1 obtained in the step (1) into an escherichia coli Rosetta engineering strain, and selecting a single colony to be cultured at 37 ℃ to serve as seed liquid;

(3) inoculating the seed solution obtained in the step (2) into YT culture solution containing Amp according to the volume ratio of 1:100, and culturing at 37 ℃ to OD₆₀₀Adding IPTG (isopropyl thiogalactoside) with the value of 0.5-1.0 for induction culture;

(4) purifying and concentrating the induction expression product by chromatography to obtain the soluble porcine PD-1 recombinant protein with escherichia coli preference.

The conditions in the induction culture in the step (3) are as follows: the induction temperature is 28 ℃, the induction time is 4h, and the IPTG concentration is 0.5 mmol/L.

The soluble porcine PD-1 recombinant protein is applied to the preparation of PCV2 vaccine immune drugs.

The soluble porcine PD-1 recombinant protein is applied to preparation of PCV2 vaccine reinforcing agents or adjuvants.

The recombinant vector, the expression cassette, the transgenic cell line or the recombinant strain of the DNA molecule of the soluble porcine PD-1 recombinant protein.

The recombinant vector, the expression cassette, the transgenic cell line or the recombinant strain of the DNA molecule of the soluble pig PD-1 recombinant protein are applied to the preparation of corresponding products.

The invention has the beneficial effects that:

the soluble recombinant protein of the pig PD-1 gene modified by codon preference is obtained by utilizing a gene cloning technology, and the soluble pig PD-1 recombinant protein can effectively enhance the immune response of pigs to PCV2 vaccines and improve the immunity of inoculated animals. Wherein the soluble porcine PD-1 recombinant protein can promote the proliferation of PBMCs in vivo, improve the gene transcription level and the protein expression level of positive immunoregulation cell factors IL-2, IL-12 and IFN-gamma, and promote CD4 in peripheral blood⁺And CD8⁺The proportion of the T lymphocyte subpopulation is increased, and a new technology is provided for developing a novel and efficient PCV2 immunopotentiator or adjuvant.

Drawings

FIG. 1 shows the results of soluble pig PD-1 recombinant protein purification and Western blotting detection;

wherein, 1: the result of purification of sample 1; 2: the purification results of sample 2; 3: pET-32a/Rosetta induces expression products; 4: rosetta inducible expression products;

FIG. 2 shows the binding of soluble porcine PD-1 recombinant protein to PBMCs in vitro;

wherein, A: PCV2 infects PBMCs alone; b: soluble porcine PD-1 recombinant protein coacts with PCV2 on PBMCs;

FIG. 3 shows the proliferation of PBMCs promoted by the soluble porcine PD-1 recombinant protein in vitro;

wherein, A: soluble porcine PD-1 recombinant protein coacts with PCV2 on PBMCs; b: PCV2 infects PBMCs alone;

FIG. 4 shows the in vitro PCV2 clearance by soluble porcine PD-1 recombinant protein;

wherein, indicates that the difference is significant (p <0.05), indicates that the difference is very significant (p < 0.01); FIGS. 5-11, below, are the same;

FIG. 5 is a graph of the effect of soluble porcine PD-1 recombinant protein on cytokine transcription levels in vitro;

FIG. 6 shows the effect of soluble porcine PD-1 recombinant protein on cytokine secretion levels in vitro;

FIG. 7 is a graph of the effect of soluble porcine PD-1 recombinant protein on PCV2 neutralizing antibody levels following immunization of healthy piglets with PCV2 vaccine;

wherein, A: PCV2 vaccine group; b: PCV2 vaccine and 0.2mg/kg soluble pig PD-1 recombinant proteome; c: PCV2 vaccine was mixed with 0.4mg/kg soluble porcine PD-1 recombinant proteome D: PCV2 vaccine and porcine serum albumin group; FIGS. 8-12 below are the same;

FIG. 8 shows the effect of soluble porcine PD-1 recombinant protein on the proliferation of PBMCs after immunization of healthy piglets with PCV2 vaccine;

FIG. 9 shows that CCK-8 detects the proliferation of soluble porcine PD-1 recombinant protein on cells;

FIG. 10 is a graph showing the effect of soluble porcine PD-1 recombinant protein on cytokine transcription levels following immunization of healthy piglets with PCV2 vaccine;

FIG. 11 shows the effect of soluble porcine PD-1 recombinant protein on cytokine secretion levels following immunization of healthy piglets with PCV2 vaccine;

FIG. 12 shows that the soluble pig PD-1 recombinant protein and PCV2 vaccine are used for immunizing healthy piglets together and then carrying out CD4 treatment on the healthy piglets⁺And CD8⁺Influence of the proportion of T cells.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Example 1 preparation of soluble porcine PD-1 recombinant protein

(1) Escherichia coli codon modification of pig PD-1 protein extracellular region gene

The pig PD-1 protein extracellular region gene is modified under the principle that the codon preference table of escherichia coli, the content of a gene sequence G + C cannot be higher than 60 percent and the original codon of the gene is respected, the codon preference of the escherichia coli is also modified during the design of a gene sequence primer, and the specific primer sequence of the enzyme cutting site is shown in table 1. The major amino acid sites were engineered as follows: ACA → ACC, GAG → GAA, CCC → CCG, CTC → CTG, CGC → CGT, AGG → CGT. The nucleotide sequence of the DNA molecule of the escherichia coli preference soluble porcine PD-1 protein is shown as SEQ ID NO: 2, respectively. The designed and modified gene sequence is sent to the company of biological engineering (Shanghai) GmbH for synthesis.

TABLE 1 porcine PD-1 extracellular region gene fragment amplification primers

(2) Inducible expression of soluble porcine PD-1 protein

The DNA of the escherichia coli tropism soluble porcine PD-1 protein modified in example 1 is amplified by PCR and cloned into pMD-18T to construct a cloning plasmid. By PCR, digestion and sequencing, the correct PD-1 gene was identified and cloned by digestion into pET-32a (+). And screening by PCR and sequence determination to obtain a group expression vector pET-32a-PD 1. pET-32a-PD1 is transformed into an engineering strain of Escherichia coli Rosetta after sequencing verification. Single colonies were picked and cultured overnight at 37 ℃ as seed solutions. Inoculating the seed solution into fresh 2 XYT containing Amp (100 μ g/mL) at a volume ratio of 1:100The culture medium (16g tryptone, 10g yeast extract, 5g NaCl, 100mg ampicillin, constant volume to 1000mL) was cultured at 37 ℃ to OD₆₀₀And adding IPTG (isopropyl thiogalactoside) with different concentrations for induction culture, wherein the value is 0.5-1.0. The optimal induction conditions are determined as follows: the induction was carried out at 28 ℃ for 4h with an IPTG concentration of 0.5 mmol/L.

(3) Purification and concentration determination of soluble porcine PD-1 recombinant protein

Inducing a large amount of recombinant bacteria pET-32a-PD1/Rosetta to express and obtain PD-1 recombinant protein, and passing the expression product throughAnd (3) purifying and concentrating the Ni-NTA Resin by column chromatography to finally obtain the recombinant target protein with the purity of 95%. The optimum imidazole concentration in the elution buffer was determined to be 200 mmol/L.

The purified protein content was determined using a Beckman Coulter company DU800 UV spectrophotometer. After zeroing with PBS buffer, the values of A260 (absorbance at 260 nm) and A280 (absorbance at 280 nm) of the samples were measured. According to the formula: the protein concentration (mg/mL) was (1.45 × a280 to 0.74 × a260) × dilution, and the concentration of the purified PD-1 protein was measured to be 0.9 mg/mL.

The amino acid sequence of the obtained soluble porcine PD-1 recombinant protein is shown as SEQ ID NO: 1 is shown.

Example 2 in vitro Activity identification method of soluble porcine PD-1 recombinant protein as vaccine enhancer

(1) Western blotting (Western blotting) for identifying PD-1 recombinant protein activity

Western blotting identifies soluble pig PD-1 recombinant protein of induced expression, and the specific steps are as follows:

1) preparing glue: firstly, sequentially adding the components to prepare 12% separation gel in a beaker according to a system shown in a table 2, absorbing excessive water in a glass plate by using filter paper after the gel is solidified, and then starting to prepare concentrated gel according to a table 3.

TABLE 212% preparation of the separation gel

Components	Volume of
		Sterile water	4.9mL
30% acrylamide	6.0mL
		1.5mM Tris-HCl buffer	3.8mL
10% sodium dodecyl sulfate	150.0μL
		10% ammonium persulfate	150.0μL
Tetramethyl ethylene diamine	6.0μL

Formulation of Table 35% concentrated gum

Components	Volume of
		Sterile water	4.2mL
30% acryloyl groupAmines as pesticides	1.0mL
		1.0mM Tris-HCl buffer	0.75mL
10% sodium dodecyl sulfate	60.0μL
		10% ammonium persulfate	60.0μL
Tetramethyl ethylene diamine	6.0μL

2) After sample loading and SDS polyacrylamide gel electrophoresis are finished, the glass plate is pried open to slightly take off the protein gel, the PVDF membrane (polyvinylidene fluoride membrane) is activated for 60s by methanol, and the protein gel, the sponge pad, the filter paper and the PVDF membrane are respectively placed in a membrane transfer buffer solution to be soaked for at least 30 min. Then clamping the spongy cushion, the filter paper, the protein gel and the PVDF membrane in sequence, putting the clamped spongy cushion, the filter paper, the protein gel and the PVDF membrane into a membrane rotating instrument, and rotating the membranes for 1h under the condition of 350 mA.

3) And (3) sealing: after the membrane transfer was completed, the PVDF membrane was removed and washed with TBST (TBS + Tween-20). 5% skimmed milk powder-TBST, sealing with shaking table at 37 deg.C for 2 hr; the TBST solution is washed for 3 times and 5 min/time.

4) And (3) hybridization: diluting an anti-PD-1 monoclonal antibody prepared in a laboratory by 1:1000 times in volume ratio, adding the diluted anti-PD-1 monoclonal antibody into an incubation box with a PVDF membrane, covering the incubation box with the PVDF membrane, and incubating overnight at 4 ℃; the primary antibody was collected and the membrane washed 3 times 5 min/time with TBST. Adding a horseradish peroxidase-labeled secondary antibody diluted by the volume ratio of 1:5000 into an incubation box, covering a PVDF membrane, incubating for 2h at 37 ℃ in a shaking table, collecting the secondary antibody, and washing the membrane for 3 times and 5 min/time by TBST.

5) Color development: the film was removed with tweezers and was completely immersed and in full contact with the DAB chromogenic solution. And (5) after the color development is completed, immediately flushing the reaction product by using double distilled water, and photographing to analyze the result. The results are shown in FIG. 1.

(2) Combination of soluble porcine PD-1 recombinant protein and PBMCs

The isolated porcine PBMCs were counted and adjusted to 1X 10 with incomplete medium RPMI1640⁶Per mL, blocking the PD-1/PD-L1 pathway with soluble porcine PD-1 recombinant protein after addition of PCV2 cytotoxic (group B). After culturing for 36h, cells were collected, washed 3 times with PBS, and diluted to 1:1000 volume ratio for anti-His polyclonal antibody by adding PBS. Binding for 1h at 37 ℃ and washing with PBS was repeated 3 times; adding IgG-FITC diluted by 1:50 times according to the volume ratio, combining for 1h at 37 ℃, and repeatedly washing for 3 times by PBS; adding 1mL PBS suspension, filtering with 200 mesh filter screen, and counting by 2X 10⁴And (3) detecting the fluorescence signal intensity of each cell by flow cytometry, analyzing the binding condition of the soluble porcine PD-1 recombinant protein and the PBMCs, and simultaneously setting a PCV2 independent infection group (group A). The results are shown in FIG. 2.

(3) Proliferation effect of soluble pig PD-1 recombinant protein on PBMCs

The isolated porcine PBMCs were counted and adjusted to 1X 10 with incomplete medium RPMI1640⁶Per mL, 100 μ L of cell suspension was added per well in a 96-well plate. After PCV2 was inoculated, PCV 2-infected PBMCs (group B) were treated with soluble porcine PD-1 recombinant protein to a final mass concentration of 10. mu.g/mL, labeled with CFSE dye, and cultured for 4d before flow cytometry to detect PBMCs proliferation. A PCV2 virus control group (group a) was also set. The results are shown in FIG. 3.

(4) Fluorescent quantitative PCR (polymerase chain reaction) detection of virus load of PCV2 in PBMCs after blocking of soluble pig PD-1 recombinant protein

After counting PBMCs from pigs, the incomplete medium was adjusted to 1X 10 in RPMI1640⁶one/mL of the recombinant protein was placed in a 12-well plate, and 10. mu.g/mL of soluble porcine PD-1 recombinant protein was added to the PBMCs to which PCV2 was added, followed by culturing for 36 hours. And (3) repeatedly freezing and thawing the pore plate at-80 ℃ for 3 times, carrying out 12000r/m, centrifuging at 4 ℃ for 20min, collecting supernatant, extracting virus genome DNA, carrying out fluorescent quantitative PCR (polymerase chain reaction), carrying out 3 times of repetition on each sample, recording CT (computed tomography) values, and calculating the PCV2 virus load in PBMCs according to a standard curve. The results are shown in FIG. 4.

(5) Regulation effect of soluble pig PD-1 recombinant protein on PBMCs cytokine transcription level

After counting PBMCs from pigs, the incomplete medium was adjusted to 1X 10 in RPMI1640⁶Each well of the cell suspension is added with 100 mu L of cell suspension in a 96-well plate, and PCV2 cytotoxicity is added to set as PCV 2; adding soluble pig PD-1 recombinant protein to make its final mass concentration be 10 μ g/mL, setting PCV2+ soluble pig PD-1 recombinant protein group, each gradient has 3 repeats, setting normal cell control group and soluble pig PD-1 recombinant protein separately stimulated PBMCs group, 37 deg.C, 5% CO₂Culturing in incubator for 36h, collecting cell, extracting RNA, reverse transcribing to cDNA, performing fluorescent quantitative PCR with beta-actin (beta-actin) as endogenous control, and adopting 2^-ΔΔCtThe analysis method calculates the relative expression value of the fluorescent quantitative PCR detection and compares the relative expression value with the beta-action for analysis, and defines the mRNA content in the normal control group as 1 time, and can obtain the transcription levels of IL-2, IL-12 and IFN-gamma at different time points. The results are shown in FIG. 5.

1) Extraction of cDNA from PBMCs

Cracking of cells: blowing and beating the cells in the heavy suspension culture plate by using a pipette gun, transferring the cell suspension into a centrifuge tube, centrifuging at 12000r/m and 4 ℃ for 2min, and discarding the supernatant. Washing with PBS once, centrifuging at 12000r/m for 2min at 4 deg.C, and discarding the supernatant. To the collected cells, the appropriate amount of lysis Buffer RL (to which 50 × DTT solution has been added) was added and thoroughly pipetted well until no solid precipitate was evident in the lysate. Extraction was performed according to the instructions of the RNA extraction kit.

Reverse transcription of cellular RNA: an appropriate amount of the extracted RNA was taken to prepare a reverse transcription reaction solution according to Table 4.

TABLE 4 reverse transcription System

Components	Volume of
		5 x quantitative RT-PCR reaction reagent	2μL
Total RNA amount	7μL
		Single distilled water without RNA enzyme	Volume to 10. mu.L

After gentle mixing, reverse transcription reaction is carried out immediately, incubation is carried out for 15min at 37 ℃, water bath is carried out for 5s at 85 ℃, cooling is carried out at 4 ℃ to obtain cDNA, and the cDNA is stored at-80 ℃ for later use.

2) Fluorescent quantitative PCR detection of cytokines

The cDNA was diluted to 100ng or less, and a real-time fluorescent quantitative PCR reaction solution (prepared on ice) was prepared as shown in Table 5. The primer sequences of cytokines are shown in Table 6.

TABLE 5 real-time fluorescent quantitative PCR reverse transcription reaction system

Components	Volume of
		TB Green dye Ex Taq II enzyme mixture	10.0μL
PCR upstream primer	0.8μL
		PCR downstream primer	0.8μL
ROX reference dye II (50X)	0.4μL
		DNA	2.0μL
Sterile water	6.0μL
		Total volume	20.0μL

TABLE 6 real-time fluorescent quantitative PCR primer sequence Listing of beta-actin and cytokine

After the above systems are mixed gently and uniformly, carrying out fluorescent quantitative PCR amplification, wherein the amplification reaction program comprises the following steps: pre-denaturation at 95 ℃ for 30s, 40 cycles were performed: denaturation at 95 ℃ for 5s, and reaction at 60 ℃ for 34 s; dissolution curve: 95 ℃ for 30s, 60 ℃ for 1min and 95 ℃ for 15 s.

As can be seen from FIG. 5, the PCV2 infected group of IL-12mRNA was lower than the normal group at 24-72 h, especially the 72h transcription level was significantly lower than the normal group (p < 0.01); IFN-gamma mRNA at 36h and 72h, PCV2 infected group was significantly reduced (p <0.01) compared with normal group, at 24h (p <0.05), and 12, 48 and 60h were not significantly changed. IL-2 is obviously reduced 12-72 h after PCV2 infection, and is extremely obviously down-regulated at 36h and 72h (p is less than 0.01). The results show that PCV2 can increase the PD-1 and PD-L1, activate the PD-1/PD-L1 pathway and inhibit the transcription levels of cytokines IL-2, IL-12 and IFN-gamma in vitro.

(6) Regulation effect of soluble porcine PD-1 recombinant protein on PBMCs cytokine secretion level

Collecting cell culture supernatant: collecting with sterile tube, and centrifuging at 3000r/m for 20 min. And calculating the concentrations of the cytokines IL-2, IL-12 and IFN-gamma according to the detection of an ELISA cytokine detection kit. The results are shown in FIG. 6.

As can be seen from FIG. 6, after PCV2 infection, PD-1/PD-L1 pathway was blocked by porcine soluble porcine PD-1 recombinant protein, and culture supernatant was collected and recorded by ELISA kit detection. The results show that: IL-2 and IL-12 in the sPD-1 protein group were significantly elevated (P <0.01) relative to the normal PCV2 group after blockade; IFN-gamma was significantly upregulated (P < 0.05). Results consistent with fluorescent quantitative PCR were obtained by ELISA detection.

Example 3 in vivo Activity identification method of soluble porcine PD-1 recombinant protein as vaccine enhancer

(1) Grouping animals

The immune amount of the soluble porcine PD-1 recombinant protein is determined to be 0.2mg/kg by consulting literature and equivalent dose conversion among animals. 40 weaned piglets of 45-day-old non-immune PCV2 vaccine are selected and randomly divided into 4 groups of 10 piglets, wherein the group A is a PCV2 vaccine immune group, the groups B and C are test groups, the group D is an unrelated protein control group, and the specific grouping conditions are shown in Table 7.

TABLE 7 grouping of test animals and inoculation formulations

Group of	Number of animal heads	Molecular formulation	Inoculation mode	Protein immunizing dose
					A	10 heads	PCV2 vaccine	Intramuscular injection	0mg/kg
B	10 heads	PCV2 vaccine + soluble porcine PD-1 recombinant protein	Intramuscular injection	0.2mg/kg
					C	10 heads	PCV2 vaccine + soluble porcine PD-1 recombinant protein	Intramuscular injection	0.4mg/kg
D	10 heads	PCV2 vaccine + porcine serum albumin	Intramuscular injection	0.2mg/kg

(2) PCV2 antibody level detection

Blood was collected after 14d after inoculation and plasma was separated to measure PCV2 antibody levels in piglets of different immunization groups. The ELISA antibody detection method specifically comprises the following steps: PCV2cap protein coated ELISA plate (1 mug/mL), 100 mug/hole, 4 ℃ overnight, PBST plate washing 3 times, 5 min/time; adding 200 μ L of 5% skimmed milk-PBST into each well, sealing, incubating at 37 deg.C for 2 hr, and washing the plate with PBST for 3 times (5 min/time); adding 100 μ L of diluted serum with different fold ratios into each well, incubating at 37 deg.C for 30min, washing the plate with PBST for 3 times, 5 min/time (avoiding cross-well during washing the plate); adding HRP-labeled anti-mouse IgG with the volume ratio of 1:5000 into the mixture, incubating the mixture for 30min at 37 ℃, discarding the mixture, and washing the plate for 3 times and 5 min/time by PBST; adding TMB developing solution, incubating at 37 deg.C in dark for 8min, adding 2mol/L H₂SO₄The reaction was stopped at 50. mu.L/well and the OD read at 450 nm. And (4) judging a result: simultaneous dilutionSerum OD to be tested for release₄₅₀nm value/negative serum OD₄₅₀The nm value (P/N) is more than or equal to 2.1. The ELISA antibody titer of the serum is determined as the maximum dilution of the serum with the P/N value being more than or equal to 2.1.

From fig. 7, it can be seen that the soluble porcine PD-1 recombinant protein can increase the antibody level after PCV2 vaccine immunization by about 1.2 times (P <0.01), and the antibody level of the soluble porcine PD-1 recombinant protein group is significantly increased (P <0.05) relative to the unrelated protein control group.

(3) Isolation of porcine PBMCs

After inoculation, 5mL of anterior vena cava whole blood was withdrawn at 14d, and the specific steps were as follows:

1) anticoagulated whole blood (10% sodium citrate anticoagulant as 10%) was drawn in the porcine anterior vena cava and diluted with an equal volume of 2% serum in PBS.

2) The diluted blood is slowly added above the equal volume of the lymphocyte separating medium, so that the diluted blood is flatly laid above the separating medium, and the interface between the diluted blood and the separating medium is kept clear. Horizontal centrifugation at room temperature, 2000r/m, 25 min.

3) After centrifugation, significant stratification will occur: the diluted plasma layer, the lymphocyte layer and the transparent separation liquid layer are arranged from top to bottom in sequence, and the red blood cells and the granulocytes are arranged at the bottom.

4) Gently pipette the plasma layer and the middle leukocyte layer of the separation medium, i.e., lymphocytes, into a 15mL sterile centrifuge tube, and wash the leukocyte layer with 10mL of 2% serum-containing PBS. 1500r/m, and centrifuging for 10 min.

5) Discarding the supernatant, adding 3 times volume of erythrocyte lysate according to the amount of erythrocytes in leukocytes, performing lysis on ice, slightly shaking for several times every few minutes, performing lysis for about 15min until the leukocyte is white, centrifuging at 4 ℃ and 2000r/m for 10min, and carefully and thoroughly washing the supernatant.

6)5mL PBS containing 2% serum was resuspended in cells at 1500 rpm and centrifuged for 10 min.

7) Repeat step 6).

8) The supernatant was discarded and the cells were resuspended for use.

(4) Flow cytometry is used for analyzing proliferation effect of soluble pig PD-1 recombinant protein on PBMCs

To determine whether soluble porcine PD-1 recombinant protein could promote cell proliferation response following immunization with PCV2 vaccine, PBMCs were isolated at 5mL of pre-luminal venous whole blood drawn at 14d and adjusted to 1X 10 with incomplete medium RPMI1640⁶Each 1mL of the cells was added with 2. mu.L of a CFSE stock solution (final concentration: 10. mu.M), and the proliferation activity was measured by the CFSE method and CCK-8. After culturing the CFSE-treated PBMCs for 4d, T cell proliferation was examined by flow cytometry. The detection results are shown in fig. 8: a: 31.26 ± 4.10%, B: 73.12 ± 13.80%, C: 51.94 ± 27.92%, D: 39.87 ± 10.86% (mean proliferation percentage of multiple replicates), the results showed that proliferation of T cells showed: the 0.2mg/mL soluble pig PD-1 recombinant protein group is obviously higher than that of a vaccine control group (p)<0.01) and proliferation is evident compared with the irrelevant control group (p)<0.05), while the other immunization groups did not differ significantly (p) compared to the vaccine control group>0.05)。

(5) CCK-8 detection of influence of soluble pig PD-1 recombinant protein on cell proliferation

The isolated PBMCs were adjusted to 1X 10 with RPMI1640 medium of 10% fetal bovine serum and 10% negative porcine serum⁶cells/mL, then added to 96-well plates in 3 replicates per group, while setting media controls, 37 ℃, 5% CO₂After culturing in an incubator for 4 days, adding 10 microliter of CCK-8 solution into each well, continuing culturing for 2 hours, and measuring OD of each well by using a microplate reader₄₅₀nm, proliferation index (SI) ═ test OD₄₅₀nm value/OD of vaccine control group₄₅₀And (5) nm value. From the above data analysis, the effect of endotoxin toxicity on cell proliferation was obtained. The results are shown in FIG. 9, which shows that the soluble porcine PD-1 recombinant protein can promote the proliferation of PBMCs and enhance the immune effect.

(6) Fluorescent quantitative PCR detection of transcription change of cell factor

In order to research the influence of the pig soluble PD-1 recombinant protein on the immune effect of the mononuclear cells in vivo, PBMCs are separated and extracted at 14d, the transcription change conditions of IL-2, IL-12 and IFN-gamma mRNA are detected by a fluorescent quantitative PCR method, and the immune state of an organism is judged.

The results are shown in fig. 10, and compared with the vaccine control group, the PD-1 transcription change of the group added with soluble porcine PD-1 recombinant protein was not obvious after 14d inoculation, but the transcription of PD-L1 was significantly reduced (p <0.01) compared with the vaccine and unrelated protein control group. The expression level of IL-2 in the group added with the low-dose soluble porcine PD-1 recombinant protein is remarkably up-regulated (p <0.01) relative to the vaccine control group and the irrelevant control group, while the group added with 0.2mg/kg soluble porcine PD-1 recombinant protein can remarkably up-regulate the expression of IL-12 and IFN-gamma (p <0.01) relative to the vaccine control group, but the increase of IFN-gamma is more obvious (p <0.05) compared with the irrelevant control group, and the up-regulation of IL-12 is not obvious although the increase is realized (p > 0.05). However, the modulation on the high-dose soluble porcine PD-1 recombinant protein group is not obvious (p is greater than 0.05), which indicates that the soluble porcine PD-1 recombinant protein can improve the transcription level of the cell factor.

(7) Influence of soluble pig PD-1 recombinant protein on change of plasma cytokine content of piglets

After PCV2 infection, soluble porcine PD-1 recombinant protein is used for blocking a PD-1/PD-L1 pathway, plasma is separated at 14d, ELISA is used for detecting the secretion of IL-2, IL-12 and IFN-gamma, the influence of the soluble porcine PD-1 recombinant protein on immune response in vivo is studied, and meanwhile, PSA irrelevant protein and an immune vaccine control group are arranged. The plasma obtained by separation is used for detecting the changes of IL-2, IL-12 and IFN-gamma by an ELISA method, and the specific operation steps are carried out according to the kit instruction of Wuhanyun clone science and technology company Limited.

The results are shown in fig. 11, compared with the vaccine control group, the expression level of the IL-2 in the low-dose soluble pig PD-1 recombinant protein group is obviously increased (p <0.01), and the expression level of the IL-2 in the rest immune groups is increased and has no obvious difference (p > 0.05). The secretion of IL-12 in the 0.2mg/kg soluble porcine PD-1 recombinant protein group is obviously increased (p is less than 0.05), but the 0.4mg/kg soluble porcine PD-1 recombinant protein group is close to the normal level, and the difference is not significant (p is more than 0.05). The soluble porcine PD-1 recombinant protein can improve the protein levels of cytokines IL-2 and IL-12.

(8) Detection of CD4 in peripheral blood using flow cytometry⁺And CD8⁺Change in T lymphocyte subpopulation

Extracting PBMCs at 14d, separating the porcine PBMCs, and purifyingSterile PBS washing 3 times, the cell concentration is adjusted to 1X 10⁶one/mL, 0.2. mu.g anti-CD4-PE and 0.2. mu.g anti-CD8-APC were added according to the antibody instructions, incubated at 4 ℃ for 30min, and a blank control and a single-stain control were set simultaneously.

As shown in FIG. 12, the results showed that porcine CD4⁺、CD8⁺T cell subset proportion profile, vaccine control group (a): 0.8 ± 0.30, 0.2mg/kg soluble porcine PD-1 recombinant proteome (B): 1.63 ± 0.11, 0.2mg/kg soluble porcine PD-1 recombinant proteome (C): 1.55 ± 0.20, irrelevant protein control group (D): 1.2 ± 0.63. The results showed that the other immunization group, CD4, was compared with the vaccine control group (A)⁺/CD8⁺The proportion of T cells, especially the low dose proteome, was increased more significantly (P)<0.01) but not significantly changed compared to the unrelated proteome (P)>0.05), which shows that the soluble porcine PD-1 recombinant protein can obviously improve CD4⁺/CD8⁺Proportion of T cells.

The analysis proves that the soluble porcine PD-1 recombinant protein can promote the proliferation of PBMCs, improve the gene transcription level and the protein expression level of positive immunoregulation cell factors IL-2, IL-12 and IFN-gamma and promote CD4 in peripheral blood⁺And CD8⁺The proportion of T lymphocyte subpopulations is increased.

Sequence listing

<110> college of New county

<120> preparation and application of escherichia coli partial tropism soluble porcine PD-1 recombinant protein

<130> production of recombinant protein

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His Phe Ile Leu Asn Trp Tyr Arg Leu Ser Pro Ser Asn Gln Thr Asp

20 25 30

Lys Leu Ala Ala Phe Ser Glu Asp Gly Ser Gln Pro Gly Arg Asp Pro

35 40 45

Arg Phe His Val Thr Pro Leu Pro Asn Gly Arg Asp Phe His Met Ser

50 55 60

Val Val Ala Thr Arg Arg Asn Asp Ser Gly Thr Tyr Phe Cys Gly Ala

65 70 75 80

Ile Tyr Leu Pro Pro Lys Thr Gln Ile Asn Glu Ser His Gln Ala Lys

85 90 95

Leu Thr Val Thr Glu Arg Val Leu Glu Leu Pro Thr Glu His Pro Ser

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Cys Pro Pro Arg Pro Glu Gly His Leu Glu Gly Gln Val Leu Val Ile

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Thr Ser

130

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