A goat cytokine composition with immune-enhancing effects and its application

Abstract

This invention relates to the fields of veterinary immunology and molecular biology, and discloses a goat cytokine composition with immune-enhancing effects and its applications. The composition comprises recombinant goat cytokines IL-9 and IL-21, whose amino acid sequences are shown in SEQ ID NO. 8 and SEQ ID NO. 12, respectively. These two cytokines upregulate the expression of IL-2, IL-4, IFN-γ, IL-17, and TNF-α in goat PBMCs, stimulating Th1, Th2, and Th17 immune responses in goats. When used in combination with an inactivated goat infectious pleuropneumonia vaccine, this goat cytokine composition exhibits good immune-enhancing effects and shows potential application prospects. This goat cytokine composition has advantages such as ease of preparation, high yield, good stability, and low production cost.

Description

Technical Field

[0001] This invention relates to the fields of veterinary immunology and molecular biology, and more specifically, to a goat cytokine composition with immune-enhancing effects and its applications. Background Technology

[0002] Immunization is an effective measure for the prevention and control of livestock and poultry diseases, forming a strong defense against disease and preventing its spread. Adjuvants are molecules or compounds injected into animals before or simultaneously with the antigen that can enhance the body's immune response to the antigen or alter the type of immune response. In vaccine research, adjuvants are needed to assist vaccines in generating a more effective immune response, addressing issues such as single antigens and weak immunogenicity, and enhancing the vaccine's immunogenicity.

[0003] Cytokines are small protein molecules secreted by both immune and non-immune cells, possessing broad biological effects. They play a crucial role in immune signaling, regulating immune responses and enhancing protective immunity. Studies have demonstrated that IL-1, IL-2, IL-4, IL-12, and IFN-γ have the potential to serve as adjuvants for viral, bacterial, and parasitic vaccines, increasing antibody titers and cellular immunity levels, thus exhibiting significant immune-enhancing effects. IL-8 is a chemokine that regulates physiological and pathological processes and is an important inflammatory mediator, significantly increased in inflammatory sites, serum, and body fluids. IL-9 promotes inflammatory and allergic responses, supports T cell growth, and increases the release of IgE and IgG1 from B cells. IL-21 participates in regulating B cell proliferation and differentiation, inducing immunoglobulin production, and regulating T cell activation, differentiation, and function. IL-23, composed of the p19 subunit and the p40 subunit of IL-12, promotes Th17 cell proliferation and induces the expression of pro-inflammatory factors such as IL-17A, IL-17F, and IL-22. IL-27, composed of two subunits—p28 and EBV-induced gene (EBI3)—is a pleiotropic cytokine that can promote or inhibit various immune cells under different conditions. It promotes early Th1 cell differentiation, inhibiting influenza virus infection; inhibits CD4+ T cell differentiation into Th2, Th9, and Th17 cells, improving symptoms of allergic diseases; and promotes IL-10 expression in Th cells, thereby inhibiting the production of pro-inflammatory cytokines. Whether goat cytokines such as IL-8, 9, 21, 23, and 27 can act as immune enhancers remains to be studied.

[0004] Therefore, providing a goat cytokine composition with immune-enhancing effects and its application has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention proposes a goat cytokine composition with immune-enhancing effects and its application, aiming to solve the problems in the prior art.

[0006] This invention proposes a goat cytokine composition with immune-enhancing effects, comprising:

[0007] Recombinant goat cytokines IL-9 and IL-21, wherein the amino acid sequence of recombinant goat cytokines IL-9 is shown in SEQ ID NO.8 and the amino acid sequence of recombinant goat cytokines IL-21 is shown in SEQ ID NO.12.

[0008] This invention also proposes a method for preparing a goat cytokine composition with immune-enhancing effects:

[0009] Total RNA was extracted from goat peripheral blood PBMCs and reverse transcribed into cDNA. PCR amplification was performed using primers shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.10 to obtain the IL-21 protein gene fragment. The IL-21 protein gene fragment was inserted into the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-21 containing the goat IL-21 gene.

[0010] Peripheral blood PBMCs were isolated from goats, total RNA was extracted, and reverse transcribed into cDNA. Based on the goat IL-9 encoding gene (GeneID: 102179848), the goat IL-9 gene fragment was obtained by RT-PCR and cloned into the EcoRI and XhoI restriction sites of the pET30a(+) vector to obtain the recombinant expression plasmid pET30a-IL-9 containing the goat IL-9 gene. The primer sequences used for RT-PCR amplification are shown in SEQ ID NO. 5 and SEQ ID NO. 6, and the gene sequence encoding the goat IL-9 protein is shown in SEQ ID NO. 7.

[0011] The recombinant goat cytokines IL-9 and IL-21 were obtained by transforming Escherichia coli with pET30a-IL-9 and pET28a-IL-21 and then induced to express and isolate them.

[0012] Preferably, the induced expression and separation / purification are as follows:

[0013] The transformed E. coli were cultured until OD600 equaled 0.6. After induction with IPTG, the bacterial cells were collected by centrifugation. The bacterial cells were resuspended in supernatant Binding Buffer and then subjected to ultrasonic disruption. The ultrasonically disrupted suspension was centrifuged. The supernatant was the supernatant protein, and the precipitate was the inclusion body. The inclusion body was dissolved in inclusion body Binding Buffer and centrifuged. The supernatant was collected to obtain the inclusion body protein.

[0014] After filtering the inclusion body protein, the filtered inclusion body protein was loaded into a His Tag affinity chromatography column at a flow rate of 0.5 mL / min. The His Tag affinity chromatography column was washed with 5-10 column volumes of Binding Buffer, and then the target protein was eluted with Elution Buffer at a flow rate of 0.5 mL / min to obtain the recombinant goat cytokines IL-9 and IL-21.

[0015] The present invention also provides the application of the aforementioned goat cytokine composition with immune-enhancing effects in the preparation of nanoparticles with immune-enhancing effects.

[0016] Preferably, the method for preparing the nanoparticles with immune-enhancing effects includes the following preparation steps:

[0017] PLGA was dissolved in dichloromethane to prepare a 5% PLGA solution;

[0018] 5% PVA was added dropwise to the 5% PLGA solution, vortexed, and then subjected to a first ultrasonic disruption under ice bath conditions. Subsequently, the recombinant goat cell factor was added, vortexed, and then subjected to a second ultrasonic disruption under ice bath conditions to obtain the primary emulsion.

[0019] 5% PVA was added to the primary emulsion, followed by a third ultrasonic disruption under ice bath conditions to obtain the secondary emulsion.

[0020] The complex emulsion was stirred and evaporated, then frozen and ultracentrifuged. After the process was completed, the precipitate was collected, resuspended in water, and then freeze-dried to obtain the nanoparticles with immune-enhancing effects.

[0021] Preferably, the power of the first ultrasonic fragmentation is 40W, the ultrasonic time is 5s / 5s interval, and the total ultrasonic time is 5min; the power of the second ultrasonic fragmentation is 40W, the ultrasonic time is 5s / 5s interval, and the total ultrasonic time is 3min; the power of the third ultrasonic fragmentation is 40W, the ultrasonic time is 5s / 5s interval, and the total ultrasonic time is 5min.

[0022] Preferably, the centrifugal force of the cryogenic ultracentrifugation is 30,000 r / min, and the centrifugation time is 30 min.

[0023] Preferably, the freeze-drying process involves placing the resuspended precipitate at -80°C for 2 hours, then transferring it to a vacuum freeze dryer with a cold trap temperature of -45°C and an ultimate vacuum of 0.1 mBar for 24 hours.

[0024] The present invention also provides the application of the aforementioned immunomodulatory nanoparticles in the preparation of animal immunomodulators.

[0025] Preferably, the animal is a goat.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention contains two cytokines, IL-9 and IL-21, which can upregulate the expression of host cytokines such as IL-2, IL-4, IFN-γ, IL-17, and TNF-α. Moreover, IL-9 can promote inflammatory and allergic responses, support T cell growth, and increase the release of B cell IgE and IgG1. IL-21 can participate in regulating B cell proliferation and differentiation, inducing immunoglobulin production, and regulating T cell activation, differentiation, and function. The two work synergistically to promote T cell and B cell differentiation, resulting in a significant immune-enhancing effect.

[0028] 2. In this invention, inclusion bodies are obtained by first resuspending the supernatant in Binding Buffer, then sonicating and centrifuging to collect the supernatant. The inclusion bodies are then dissolved in Binding Buffer and centrifuged to collect the supernatant, thus obtaining a method for inducing the expression of inclusion body proteins. The filtered inclusion body proteins are then loaded onto a His Tag affinity chromatography column at a flow rate of 0.5 mL / min. The His Tag affinity chromatography column is washed with 5-10 column volumes of Binding Buffer, and then the target proteins are eluted with Elution Buffer at a flow rate of 0.5 mL / min. The resulting recombinant goat cytokines IL-9 and IL-21 have high expression levels, are readily available, low in cost, and have good safety, and can significantly improve the immunoprotective effect of vaccines. Attached Figure Description

[0029] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0030] Figure 1 This is an image showing the electrophoresis results on a 2% agarose gel provided in an embodiment of the present invention;

[0031] Figure 2 This is a graph showing the purity test results of the target protein provided in an embodiment of the present invention;

[0032] Figure 3 This is a diagram illustrating the effect of the cytokine IL-8 on the transcriptional levels of other cytokines in goats, provided in an embodiment of the present invention.

[0033] Figure 4 This is a diagram illustrating the effect of cytokine IL-9 on the transcriptional levels of other cytokines in goats, provided in an embodiment of the present invention.

[0034] Figure 5 This is a diagram illustrating the effect of cytokine IL-21 on the transcriptional levels of other cytokines in goats, provided in an embodiment of the present invention.

[0035] Figure 6 This is a diagram illustrating the effect of cytokine IL-23 on the transcriptional levels of other cytokines in goats, provided in an embodiment of the present invention.

[0036] Figure 7 This is a diagram illustrating the effect of cytokine IL-27 on the transcriptional levels of other cytokines in goats, provided in an embodiment of the present invention.

[0037] Figure 8 This diagram illustrates the influence of different cytokines on vaccine antibody levels, as provided in an embodiment of the present invention. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0039] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] This invention provides a goat cytokine composition with immune-enhancing effects, comprising:

[0044] Recombinant goat cytokines IL-9 and IL-21, wherein the amino acid sequence of recombinant goat cytokines IL-9 is shown in SEQ ID NO.8 and the amino acid sequence of recombinant goat cytokines IL-21 is shown in SEQ ID NO.12.

[0045] The nucleotide sequence of the recombinant goat cytokine IL-9 is shown in SEQ ID NO.7, and the nucleotide sequence of the recombinant goat cytokine IL-21 is shown in SEQ ID NO.11.

[0046] In this invention, the preparation method of the recombinant goat cytokines IL-9 and IL-21 is as follows:

[0047] Total RNA was extracted from goat peripheral blood PBMC cells and reverse transcribed into cDNA. PCR amplification was performed using primers shown in SEQ ID NO.9 and SEQ ID NO.10 to obtain the IL-21 protein gene fragment. The IL-21 protein gene fragment was inserted into the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-21 containing the goat IL-21 gene.

[0048] Peripheral blood PBMCs were isolated from goats, total RNA was extracted, and reverse transcribed into cDNA. Based on the coding gene for goat IL-9 (GeneID: 102179848), the open reading frame of goat IL-9 was obtained by RT-PCR and cloned into the EcoRI and XhoI restriction sites of the pET30a(+) vector to obtain the recombinant expression plasmid pET30a-IL-9 containing the goat IL-9 gene. The primer sequences used for RT-PCR amplification are shown in SEQ ID NO.5 and SEQ ID NO.6, and the nucleotide sequence of the goat IL-9 protein is shown in SEQ ID NO.7.

[0049] The recombinant goat cytokines IL-9 and IL-21 were obtained by transforming Escherichia coli with pET28a-IL-21 and pET30a-IL-9, inducing expression, and then isolating and purifying them.

[0050] Specifically, the method for synthesizing goat PBMC cDNA template is as follows:

[0051] (1) Use a vacuum anticoagulation blood collection tube to collect 50 mL of goat blood.

[0052] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0053] (3) Take 5 mL of the mixed solution from (2) and slowly add it along the tube wall into a centrifuge tube containing 5 mL of lymphocyte separation solution.

[0054] (4) Centrifuge at room temperature (2500 rpm) for 45 min and collect the circular lymphocyte layer (first layer: plasma layer, second layer: milky white circular lymphocyte layer, third layer: clear separation liquid layer, fourth layer: red blood cell layer).

[0055] (5) Wash the cells collected in (4) twice with about 5 times the volume of PBS (pH 7.4), and centrifuge at room temperature for 8 min (1500 rpm) each time.

[0056] (6) After the last wash, discard the supernatant to obtain isolated goat PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0057] (7) Use an RT-qPCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0058] Specifically, the PCR amplification reaction system used to prepare the recombinant expression plasmids pET28a-IL-21 and pET30a-IL-9 is as follows:

[0059] 1.0 μL of goat PBMC cell cDNA, 25 μL of PrimeSTAR Max Premix (2X), 2 μL of upstream primer F (10 pM), 2 μL of downstream primer R (10 pM), and 20 μL of sterile ultrapure water were mixed thoroughly. The mixture was then subjected to PCR at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min.

[0060] Take 50 μL of PCR product and electrophoresis it on a 2% agarose gel. Under UV light, cut the agarose gel at the target band and use the gel extraction kit from Takara Bio Inc. to recover and purify the target fragment. Follow the instructions in the manual. Digest the target gene and pET-28a plasmid with double enzymes, and recover the target gene and pET-28a large fragment again. Ligate overnight at 4℃.

[0061] In this invention, the induced expression and separation / purification are as follows:

[0062] The transformed E. coli were cultured until OD600 equaled 0.6. After induction with IPTG, the bacterial cells were collected by centrifugation. The bacterial cells were resuspended in supernatant Binding Buffer and then subjected to ultrasonic disruption. The ultrasonically disrupted suspension was centrifuged. The supernatant was the supernatant protein, and the precipitate was the inclusion body. The inclusion body was dissolved in inclusion body Binding Buffer and centrifuged. The supernatant was collected to obtain the inclusion body protein.

[0063] After filtering the inclusion body protein, the filtered inclusion body protein was loaded into a His Tag affinity chromatography column at a flow rate of 0.5 mL / min. The His Tag affinity chromatography column was washed with 5-10 column volumes of Binding Buffer, and then the target protein was eluted with Elution Buffer at a flow rate of 0.5 mL / min to obtain the recombinant goat cytokines IL-9 and IL-21.

[0064] Specifically, the supernatant binding buffer is prepared as follows: Weigh 29.2g NaCl and 1.36g imidazole, dissolve them in 0.02M K+-free PBS buffer, and bring the volume to 1L.

[0065] The preparation method for the supernatant elutioning buffer is as follows: Weigh 29.2g NaCl and 34g imidazole, dissolve them in 0.02M K+-free PBS buffer, and bring the volume to 1L.

[0066] The inclusion body binding buffer is prepared as follows: Weigh 29.2g NaCl, 1.36g imidazole and 480g urea, dissolve them in 0.02M K+-free PBS buffer and bring the volume to 1L.

[0067] The inclusion body elution buffer is prepared as follows: Weigh 29.2g NaCl, 34g imidazole and 480g urea, dissolve them in 0.02M K+-free PBS buffer and bring the volume to 1L.

[0068] Specifically, the transformed *E. coli* were cultured until OD600 reached 0.6, then induced with 1 mM IPTG for 5 h. The cells were collected by centrifugation at 4000 rpm for 15 min, and resuspended in approximately 40 ml of binding buffer. The cells were then sonicated at 600 W for 3 seconds followed by 5 seconds intervals for 25 min. The resulting suspension was centrifuged at 8000 g at 4°C for 20 min. The supernatant was the supernatant protein, and the precipitate was the inclusion bodies. The precipitate was dissolved overnight at 4°C with inclusion body binding buffer, centrifuged at 8000 g at 4°C for 20 min, and the supernatant was collected as the inclusion body protein.

[0069] After filtering the inclusion body protein through 0.45 μm and 0.22 μm filter membranes, the protein sample was slowly passed through a His Tag affinity chromatography column (5 mL) stored at 4 °C and in 20% ethanol at a flow rate of 0.5 mL / min. The column was washed with 5-10 column volumes of Binding Buffer at a flow rate of 2 mL / min. The target protein was then eluted with Elution Buffer at a flow rate of 0.5 mL / min.

[0070] The present invention also provides the application of the aforementioned goat cytokine composition with immune-enhancing effects in the preparation of nanoparticles with immune-enhancing effects.

[0071] In this invention, the preparation method of the nanoparticles with immune-enhancing effects includes the following preparation steps:

[0072] PLGA was dissolved in dichloromethane to prepare a 5% PLGA solution;

[0073] 5% PVA was added dropwise to the 5% PLGA solution, vortexed, and then subjected to a first ultrasonic disruption under ice bath conditions. Subsequently, the recombinant goat cell factor was added, vortexed, and then subjected to a second ultrasonic disruption under ice bath conditions to obtain the primary emulsion.

[0074] 5% PVA was added to the primary emulsion, followed by a third ultrasonic disruption under ice bath conditions to obtain the secondary emulsion.

[0075] The complex emulsion was stirred and evaporated, then frozen and ultracentrifuged. After the process was completed, the precipitate was collected, resuspended in water, and then freeze-dried to obtain the nanoparticles with immune-enhancing effects.

[0076] In this invention, the power of the first ultrasonic fragmentation is preferably 40W, the ultrasonic time is 5s / interval, and the total ultrasonic time is preferably 5min; the power of the second ultrasonic fragmentation is preferably 40W, the ultrasonic time is 5s / interval, and the total ultrasonic time is preferably 3min; the power of the third ultrasonic fragmentation is preferably 40W, the ultrasonic time is 5s / interval, and the total ultrasonic time is preferably 5min.

[0077] In this invention, the centrifugal force of the cryogenic ultracentrifugation is preferably 30,000 r / min, and the centrifugation time is preferably 30 min.

[0078] In this invention, the freeze-drying is preferably performed by placing the resuspended precipitate at -80°C for 2 hours, then transferring it to a vacuum freeze dryer with a cold trap temperature of -45°C, an ultimate vacuum of 0.1 mBar, and freeze-drying for 24 hours.

[0079] Specifically, the preferred method for preparing the nanoparticles with immune-enhancing effects is as follows:

[0080] (1) Weigh 50 mg of PLGA and place it in a 10 mL EP tube. Add 1 mL of dichloromethane (operate in a fume hood) to dissolve the PLGA and prepare a 5% PLGA solution.

[0081] (2) Add 2 mL of 5% PVA dropwise to the dissolved PLGA while vortexing and mixing for 1 min; under ice bath conditions, sonicate for 5 min (ultrasonic power 40 W, sonication for 5 s, interval 5 s).

[0082] (3) Add 5mg of recombinant protein dropwise while vortexing and mix for 1min; under ice bath conditions, sonicate for 3min (ultrasonic power 40W, sonication for 5s, interval of 5s) to form a milky white proemulsion.

[0083] (4) Add 2 mL of 5% PVA to the primary emulsion while vortexing, and sonicate in an ice bath for 5 min (ultrasonic power 40W, sonication for 5 s, interval of 5 s) to form a secondary emulsion.

[0084] (5) Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0085] (6) Centrifuge at 30,000 r / min for 30 min under refrigeration; after centrifugation, collect the supernatant and precipitate separately.

[0086] (7) Measure the volume of the supernatant, detect the protein content in the supernatant using the BCA protein quantification kit, and calculate the encapsulation efficiency of the PLGA-encapsulated recombinant protein.

[0087] (8) Resuspend the precipitate obtained after ultracentrifugation in 2 mL of deionized water, place it in a 5 mL vial, place it at -80℃ for 2 h, and then transfer it to a vacuum freeze dryer with a cold trap temperature of -45℃ and an ultimate vacuum of 0.1 mBar for 24 h.

[0088] The present invention also provides the application of the aforementioned immunomodulatory nanoparticles in the preparation of animal immunomodulators.

[0089] In this invention, the animal is preferably a goat.

[0090] Example 1: Preparation of recombinant goat cytokines IL-9 and IL-21

[0091] 1. Primer synthesis: Based on the coding genes of goat IL-9 (number 102179848) and IL-21 (100861248) proteins in GenBank, protein prediction analysis was performed using software. The signal peptide was removed, and specific primers were designed using Primer 5.0 software based on the mature peptide segments. The upstream and downstream primer (F, R) sequences for amplifying goat IL-9 are SEQ ID NO.5 and SEQ ID NO.6, respectively, and the upstream and downstream primer (F, R) sequences for amplifying goat IL-21 are SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0092] 2. Synthesize goat PBMC cDNA template:

[0093] (1) Use a vacuum anticoagulation blood collection tube to collect 50 mL of goat blood.

[0094] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0095] (3) Take 5 mL of the mixed solution from (2) and slowly add it along the tube wall into a centrifuge tube containing 5 mL of lymphocyte separation solution.

[0096] (4) Centrifuge at room temperature (2500 rpm) for 45 min and collect the circular lymphocyte layer (first layer: plasma layer, second layer: milky white circular lymphocyte layer, third layer: clear separation liquid layer, fourth layer: red blood cell layer).

[0097] (5) Wash the cells collected in (4) twice with about 5 times the volume of PBS (pH 7.4), and centrifuge at room temperature for 8 min (1500 rpm) each time.

[0098] (6) After the last wash, discard the supernatant to obtain isolated goat PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0099] (7) Use an RT-qPCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0100] 3. Cloning and expression of goat cytokine genes:

[0101] Take 1.0 μL of the cDNA template, 25 μL of PrimeSTAR Max Premix (2X), 2 μL of upstream primer F (10 pM), 2 μL of downstream primer R (10 pM), and 20 μL of sterile ultrapure water, mix thoroughly, and pre-denature at 95℃ for 3 min on a PCR instrument; denature at 95℃ for 15 s, anneal at 58℃ for 15 s, extend at 72℃ for 30 s, for 35 cycles; extend at 72℃ for 5 min, take 50 μL of the obtained PCR product, electrophoresis on a 2% agarose gel, cut the agarose gel at the target band under UV light, and recover and purify the target fragment using the gel extraction kit from Takara Bio Inc. (Dalian), following the instructions. Digest the target gene and pET-28a plasmid with double enzymes, recover the target gene and pET-28a large fragment again, ligate overnight at 4℃ to obtain the recombinant expression plasmid pET28a-IL-21 containing the goat IL-21 gene;

[0102] Based on the gene encoding goat IL-9 (GeneID: 102179848), the open reading frame of goat IL-9 was obtained by RT-PCR and cloned into the EcoRI and XhoI restriction sites of the pET30a(+) vector to obtain the recombinant expression plasmid pET30a-IL-9 containing the goat IL-9 gene. The primer sequences used for RT-PCR amplification are shown in SEQ ID NO.5 and SEQ ID NO.6, and the gene sequence encoding goat IL-9 protein is shown in SEQ ID NO.7.

[0103] The recombinant expression plasmids pET28a-IL-21 and pET30a-IL-9 were used to transfect competent Escherichia coli BL21, respectively. Positive clones were picked, plasmids were extracted, and the bacteria were identified by enzyme digestion and sequencing.

[0104] 4. Purification of the expression product

[0105] When *E. coli* containing the recombinant plasmid reached an OD600 of 0.6, the cells were induced with 1 mM IPTG for 5 h. After centrifugation at 4000 rpm for 15 min, the cells were collected. The cells were resuspended in approximately 40 ml of binding buffer and sonicated at 600 W for 25 min at 3 s intervals followed by 5 s intervals. The lysed suspension was centrifuged at 8000 g at 4°C for 20 min. The supernatant was the supernatant protein, and the precipitate was the inclusion bodies. The precipitate was dissolved overnight at 4°C in inclusion body binding buffer, centrifuged at 8000 g at 4°C for 20 min, and the supernatant was collected as the inclusion body protein.

[0106] The inclusion body proteins were filtered through 0.45 μm and 0.22 μm filter membranes, and then the protein sample was slowly passed through a His Tag affinity chromatography column (5 mL) stored in 20% ethanol at 4 °C at a flow rate of 0.5 mL / min. The column was washed with 5-10 column volumes of Binding Buffer at a flow rate of 2 mL / min. The target proteins were then eluted with Elution Buffer at a flow rate of 0.5 mL / min to obtain the recombinant goat cytokines IL-9 and IL-21.

[0107] Example 2 Preparation of nanoparticles with immune-enhancing effects (recombinant goat cytokine IL-9)

[0108] Solution preparation: 5% PVA: Dissolve 1g PVA in 20 mL of deionized water and heat and stir at 95℃ to dissolve.

[0109] Preparation steps:

[0110] S1. Weigh 50 mg of PLGA and place it in a 10 mL EP tube. Add 1 mL of dichloromethane (operate in a fume hood) to dissolve the PLGA and prepare a 5% PLGA solution.

[0111] S2. Add 2 mL of 5% PVA dropwise to the dissolved PLGA while vortexing and mixing for 1 min. Under ice bath conditions, sonicate for 5 min (ultrasonic power 40 W, sonication for 5 s, interval 5 s).

[0112] S3. While vortexing, add 5mg of recombinant goat cytokine IL-9 dropwise and vortex for 1min. Under ice bath conditions, sonicate for 3min (ultrasonic power 40W, sonication for 5s, interval 5s) to form a milky white proemulsion.

[0113] S4. Add 2 mL of 5% PVA to the primary emulsion while vortexing, and sonicate in an ice bath for 5 min (ultrasonic power 40W, sonication for 5 s, interval of 5 s) to form a secondary emulsion.

[0114] S5. Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0115] S6. Centrifuge at 30,000 r / min for 30 min using a freeze-thawed ultracentrifuge. After centrifugation, collect the supernatant and precipitate separately.

[0116] S7. The precipitate obtained after ultracentrifugation was resuspended in 2 mL of deionized water, placed in a 5 mL vial, and placed at -80℃ for 2 h. Then it was transferred to a vacuum freeze dryer and freeze-dried for 24 h to obtain recombinant goat cytokine IL-9 protein nanoparticles.

[0117] Example 3 Preparation of nanoparticles with immune-enhancing effects (recombinant goat cytokine IL-21)

[0118] The only difference from the example is that the 5 mg recombinant goat cytokine IL-9 added in step S3 is replaced with 5 mg recombinant goat cytokine IL-21.

[0119] Effect test

[0120] We selected goat cytokines IL-8, IL-9, IL-21, IL-23 p19, IL-23 p40, IL-27 p28, and IL-27 EBI3 for testing and comparison screening.

[0121] Test Example 1: Preparation of Recombinant Cytokine Proteins

[0122] 1.1 Synthetic Primers

[0123] Based on the encoding genes of goat IL-8 (102178438), IL-9 (102179848), IL-21 (100861248), IL-23 p19 (102190023), IL-23 p40 (100860845), IL-27 p28 (102183199), and IL-27 EBI3 (102174109) proteins in GenBank, protein prediction analysis was performed using software, the signal peptide was removed, and specific primers were designed using Primer 5.0 software based on the mature peptide.

[0124] The upstream and downstream primer (F, R) sequences for amplifying goat IL-8 are listed in SEQ ID NO.1 and SEQ ID NO.2, respectively; the upstream and downstream primer (F, R) sequences for amplifying goat IL-9 are listed in SEQ ID NO.5 and SEQ ID NO.6, respectively; the upstream and downstream primer (F, R) sequences for amplifying goat IL-21 are listed in SEQ ID NO.9 and SEQ ID NO.10, respectively; the upstream and downstream primer (F1, R1) sequences for amplifying goat IL-23p19 are listed in SEQ ID NO.13 and SEQ ID NO.14, respectively; the upstream and downstream primer (F2, R2) sequences for amplifying goat IL-23p40 are listed in SEQ ID NO.15 and SEQ ID NO.16, respectively; the upstream and downstream primer (F1, R1) sequences for amplifying goat IL-27p28 are listed in SEQ ID NO.20 and SEQ ID NO.21, respectively; and the upstream and downstream primer (F2, R2) sequences for amplifying goat IL-27EBI3 are listed in SEQ ID NO.1 and SEQ ID NO.2, respectively. Listed as NO.22 and SEQ ID NO.23.

[0125] The goat IL-8 gene sequence is shown in SEQ ID NO.3, which encodes the goat IL-8 protein. Its amino acid sequence is shown in SEQ ID NO.4, consisting of 79 amino acids. The goat IL-9 gene sequence is shown in SEQ ID NO.7, which encodes the goat IL-9 protein. Its amino acid sequence is shown in SEQ ID NO.8, consisting of 124 amino acids. The goat IL-21 gene sequence is shown in SEQ ID NO.11, which encodes the goat IL-21 protein. Its amino acid sequence is shown in SEQ ID NO.12, consisting of 129 amino acids. The goat IL-23 p19 gene sequence is shown in SEQ ID NO.17, and the goat IL-23 p40 gene sequence is shown in SEQ ID NO.18. These two gene sequences are linked by (G4S)3, and the recombinant goat IL-23 protein amino acid sequence is shown in SEQ ID NO.19, consisting of 491 amino acids. The goat IL-27 p28 gene sequence is shown in SEQ ID NO.24, and the goat IL-27 EBI3 gene sequence is shown in SEQ ID NO. As shown in NO.25, the two gene segments are linked by (G4S)3. The amino acid sequence of the recombinant goat IL-27 protein is shown in SEQ ID NO.26, consisting of 450 amino acids.

[0126] 1.2 Synthesis of goat PBMC cDNA template

[0127] (1) Use a vacuum anticoagulation blood collection tube to collect 50 mL of goat blood.

[0128] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0129] (3) Take 5 mL of the mixed solution from (2) and slowly add it along the tube wall into a centrifuge tube containing 5 mL of lymphocyte separation solution.

[0130] (4) Centrifuge at room temperature (2500 rpm) for 45 min and collect the circular lymphocyte layer (first layer: plasma layer, second layer: milky white circular lymphocyte layer, third layer: clear separation liquid layer, fourth layer: red blood cell layer).

[0131] (5) Wash the cells collected in (4) twice with about 5 times the volume of PBS (pH 7.4), and centrifuge at room temperature for 8 min (1500 rpm) each time.

[0132] (6) After the last wash, discard the supernatant to obtain isolated goat PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0133] (7) Use an RT-qPCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0134] 1.3 Cloning and Expression of Goat Cytokine Genes

[0135] The following reaction system was used for PCR amplification of the IL-8, IL-9, IL-21, IL-23 p19, IL-23 p40, IL-27 p28, and IL-27 EBI3 genes. The reaction system consisted of 1.0 μL of goat PBMC cell cDNA, 25 μL of PrimeSTAR Max Premix (2X), 2 μL of upstream primer F (10 pM), 2 μL of downstream primer R (10 pM), and 20 μL of sterile ultrapure water, all thoroughly mixed. The PCR was performed at 95°C for 3 min; followed by 95°C denaturation for 15 s, 58°C annealing for 15 s, and 72°C extension for 30 s, for 35 cycles; and a final extension at 72°C for 5 min.

[0136] Recombinant IL-23 and IL-27 were obtained by overlapping extension PCR reaction: using the two subunit fragments amplified by PCR and recovered by gel as splicing templates, and using F1 and R2 as upstream and downstream primers, PCR amplification and ligation were performed. The reaction conditions were: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 5 min.

[0137] 50 μL of the PCR products of the obtained IL-8, IL-21, IL-23, and IL-27 genes were respectively electrophoresed on a 2% agarose gel. The agarose gel containing the target band was excised under UV light, and the target fragment was recovered and purified using a gel extraction kit from Takara Bio Inc. (Dalian), following the manufacturer's instructions. The target gene and pET-28a plasmid were digested with double enzymes, and the target gene and pET-28a large fragment were recovered again. Ligation was performed overnight at 4°C. The ligation product was transformed into competent E. coli BL21.

[0138] Take 50 μL of the obtained IL-9 PCR product, electrophoresis it on a 2% agarose gel, and excise the agarose gel containing the target band under UV light. Purify the target fragment using a gel extraction kit from Takara Bio Inc. (Dalian), following the manufacturer's instructions. Digest the target gene and pET30a(+) plasmid separately with double enzymes, and recover the target gene and pET30a(+) large fragment again. Ligate overnight at 4°C. Transform competent E. coli BL21 cells into the ligation product.

[0139] The electrophoresis results on the 2% agarose gel are as follows: Figure 1 As shown, Figure 1 M: DNA molecular weight standard DL5000; 1: IL-8; 2: IL-9; 3: IL-21; 4: IL-23; 5: IL-27.

[0140] 1.4 Purification of the expression product

[0141] When the *E. coli* containing the recombinant plasmid was cultured to an OD600 of 0.6, it was induced with 1 mM IPTG for 5 h. The cells were then collected by centrifugation at 4000 rpm for 15 min. The cells were resuspended in 40 ml of supernatant binding buffer and sonicated at 600 W for 3 seconds followed by 5 seconds intervals for 25 min. The lysed suspension was centrifuged at 8000 g at 4 °C for 20 min. The supernatant was the supernatant protein, and the precipitate was the inclusion bodies. The precipitate was dissolved overnight at 4 °C in inclusion body binding buffer, centrifuged at 8000 g at 4 °C for 20 min, and the supernatant was collected as the inclusion body protein.

[0142] All inclusion body protein samples were filtered through 0.45 μm and 0.22 μm filter membranes, and then slowly passed through a His Tag affinity chromatography column (5 mL) stored at 4℃ in 20% ethanol at a flow rate of 0.5 mL / min. The column was washed with 5 column volumes of Binding Buffer at a flow rate of 2 mL / min, and the target protein was eluted with Elution Buffer at a flow rate of 0.5 mL / min. The obtained target protein was subjected to SDS-PAGE electrophoresis, and the results are shown below. Figure 2As shown, SDS-PAGE electrophoresis yielded a relatively single band of the target protein, indicating that the prepared target protein has high purity.

[0143] Test Example 2: Screening of cytokines with immune-enhancing effects

[0144] 2.1 Isolation of goat PBMC cells

[0145] Goat PBMC cells were isolated according to the steps described in 1.2, and cell viability was determined to be greater than 95% by trypan blue staining and cell counting was performed.

[0146] The cell concentration was adjusted to 5 × 10⁶ cells by resuspending the cells in RPMI 1640 medium. 6 The sample was prepared at a concentration of 1 / mL and 1% penicillin-streptomycin antibiotics and 10% fetal bovine serum were added.

[0147] 2.2 Co-incubation of recombinant goat cytokines with goat PBMCs

[0148] Add 1 mL of PBMC (5 × 10⁻⁶) 6 Cells were seeded into 24-well cell culture plates, and recombinant goat cytokine was added sequentially to achieve final concentrations of 5, 10, 20, and 40 μg / mL. A cell control group and a zeroing well were also included. Cells were incubated at 37°C for 24 h. Cells were collected and transferred to centrifuge tubes, washed three times with PBS, and centrifuged (12000 rpm) for 1 min each time. The cell pellet was used for subsequent RNA extraction.

[0149] 2.3 Quantitative PCR detection of different types of cytokines

[0150] Total RNA was extracted from PBMCs and amplified to obtain cDNA using a reverse transcription kit with gDNA removal. The transcriptional levels of IL-2, IL-4, IL-10, IL-17, IFN-γ, TNF-α, and TGF-β were detected using real-time quantitative PCR. The quantitative PCR reaction mixture consisted of: 1.0 μL cDNA, 5 μL SYBR Green ProTaq HS premix, 0.2 μL upstream primer (10 pM), 0.2 μL downstream primer (10 pM), and ddH2O to a final volume of 10 μL. The mixture was thoroughly mixed. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing for 30 s; 35 cycles. Two... -ΔΔCt The method was used to analyze changes in cytokine transcription levels.

[0151] The results are as follows Figure 3-7As shown, recombinant goat IL-8, IL-9, and IL-21 significantly increased the levels of cytokines such as IL-2, IL-4, IL-17, INF-γ, and TNF-α, indicating that recombinant IL-8, IL-9, and IL-21 have an immunomodulatory effect on the immune responses of goat Th17, Th1, and Th2 subtypes. Recombinant goat IL-23 significantly increased the levels of cytokines such as IL-4, IL-17, INF-γ, and TNF-α, and inhibited IL-2 transcription, indicating that recombinant IL-23 has an immunomodulatory effect on the immune responses of goat Th17 and Th2 subtypes, and has a certain inhibitory effect on the Th1 immune response. Recombinant goat IL-27 significantly increased the transcription levels of IL-4 and IL-17, and inhibited the transcription of IL-2, INF-γ, and TGF-β, indicating that recombinant IL-27 has a certain immunomodulatory effect on the immune responses of goat Th17 and Th2 subtypes, and inhibits the Th1 immune response.

[0152] Test Example 3: Preparation of Nanoparticles Using PLGA-Coated Recombinant Cytokine Protein

[0153] 3.1 Solution Preparation

[0154] 5% PVA: Dissolve 1g PVA in 20 mL of deionized water and heat at 95℃ with stirring until dissolved.

[0155] 3.2 Preparation of Nanoparticles

[0156] S1. Weigh 50 mg of PLGA into a 10 mL EP tube, add 1 mL of dichloromethane (operate in a fume hood), dissolve the PLGA, and prepare a 5% PLGA solution.

[0157] S2. Add 2 mL of 5% PVA dropwise to the dissolved PLGA while vortexing and mixing for 1 min; under ice bath conditions, sonicate for 5 min (ultrasonic power 40 W, sonication for 5 s, interval 5 s).

[0158] S3. While vortexing, add 5mg of recombinant protein dropwise and vortex for 1min; under ice bath conditions, sonicate for 3min (ultrasonic power 40W, sonication for 5s, interval 5s) to form a milky white proemulsion.

[0159] S4. Add 2 mL of 5% PVA to the primary emulsion while vortexing, and sonicate in an ice bath for 5 min (ultrasonic power 40W, sonication for 5 s, interval of 5 s) to form a secondary emulsion.

[0160] S5. Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0161] S6. Centrifuge at 30,000 r / min for 30 min using a freeze-thawed ultracentrifuge. After centrifugation, collect the supernatant and precipitate separately.

[0162] S7. Measure the volume of the supernatant, detect the protein content in the supernatant using the BCA protein quantification kit, and calculate the encapsulation efficiency of the PLGA-encapsulated recombinant protein.

[0163] S8. Resuspend the precipitate obtained after ultracentrifugation in 2 mL of deionized water, place it in a 5 mL vial, incubate at -80°C for 2 h, and then transfer it to a vacuum freeze dryer for freeze drying for 24 h.

[0164] S9. Remove the lyophilized sample and store the PLGA nanosubunit vaccine at 4°C for later use. Simultaneously, prepare protein-free PLGA nanoparticles using the same method.

[0165] 3.3 Electron Microscopy Observation of Nanoparticles

[0166] A small amount of lyophilized powder, each encapsulating a recombinant protein, was taken out and observed using a scanning electron microscope. The particle size was between 50 nm and 300 nm, with a smooth surface and a spherical shape.

[0167] 3.4 Determination of protein encapsulation rate in nanoparticles

[0168] The volume of the supernatant collected after ultracentrifugation was measured, and the protein content in the supernatant was detected using a BCA protein assay kit. The encapsulation efficiency was calculated using the following formula:

[0169] ;

[0170] The specific results are as follows: the coverage rate of IL-8 is 87.6%, the coverage rate of IL-9 is 90.8%, the coverage rate of IL-21 is 77.0%, the coverage rate of IL-23 is 94.8%, and the coverage rate of IL-27 is 87.2%.

[0171] Test Example 4: Immunostimulatory effect of recombinant goat cytokines on inactivated goat contagious pleuropneumonia vaccine:

[0172] Thirty goats aged 3-6 months were randomly divided into 5 groups of 6 each. Each goat was subcutaneously injected with 2 mL of inactivated goat infectious pleuropneumonia vaccine and simultaneously injected with PLGA lyophilized powder coated with recombinant cytokine proteins prepared in Test Example 3, dissolved in sterile PBS. The grouping and amount of recombinant cytokines are shown in Table 1. Unanticoagulated whole blood was collected via jugular vein before injection (day 0) and at 7, 14, 21, 28, 35, 49, and 63 days after injection. The blood was centrifuged at 3000 g at 4°C for 15 min, and the serum was collected, aliquoted, and stored at -80°C. Goat infectious pleuropneumonia antibodies in the serum were detected according to the ELISA kit instructions. The results are shown in Table 1. Figure 8 As shown, the results showed that the antibody levels in group 4 (IL-9 and IL-21 combined) were significantly higher than those in the inactivated vaccine control group on days 21 and 28.

[0173] Table 1. Goat grouping and immunization

[0174] ;

[0175] The experimental materials and tools used in the above embodiments were sourced from:

[0176] Laboratory animals: 30 goats, 3-6 months old, purchased from a family farm in Lai'an County, Chuzhou City, Anhui Province;

[0177] Tools, enzymes, and reagents: Preparative endonucleases BamH I, EcoR I, Xho I, Sal I, and DNA Marker were purchased from Nanjing Novizan Biotechnology Co., Ltd.; PrimeSTAR® Max DNA Polymerase was purchased from Takara Bio; plasmid extraction kits and agarose gel extraction kits were products of EZNA™ (USA), and the BCA protein quantification kit was a product of Thermo Fisher Scientific (USA); TRIzol® reagents, RT-qPCR reverse transcription kits, and high-specificity dye-based quantitative PCR detection kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; HisTrap... TM FF protein affinity chromatography column was purchased from GE Healthcare, USA; poly(lactic-co-glycolic acid) copolymer (PLGA) was purchased from Merck Sigma-Aldrich Biotechnology Co., Ltd.; inactivated caprine contagious pleuropneumonia vaccine was purchased from Inner Mongolia Jinyu Biotechnology Co., Ltd.; and caprine contagious pleuropneumonia antibody ELISA kit was purchased from Quanzhou Ruixin Biotechnology Co., Ltd.

[0178] Major instruments and equipment: PCR amplification instrument (TaKaRa), benchtop refrigerated centrifuge (Eppendorf), electric pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory), ultrasonic homogenizer (Ningbo Xinzhi Scientific Instrument Research Institute); gel imaging system, protein electrophoresis system, and microplate reader (Bio-Red).

[0179] sequence list

[0180] SEQ ID NO.1 (IL-8 F primer)

[0181] 5'-CGCGGATCCgcagttctgtcaagaatgagt-3'

[0182] SEQ ID NO.2 (IL-8 R primer)

[0183] 5'-CCGCTCGAGtggatcttgcttctcagctctct-3'

[0184] SEQ ID NO.3 (IL-8 nucleotide sequence)

[0185] ;

[0186] SEQ ID NO.4 (IL-8 amino acid sequence)

[0187] AVLSRMSTELRCQCIKTHSTPFHPKFIKELRVIESGPHCENSEIIVKLTNGKEEVCLDPKEKWVQKVVQAFLKRAEKQDP

[0188] SEQ ID NO.5 (IL-9 F primer)

[0189] 5'-CCGGAATTCagccaggggtgtttcac-3'

[0190] SEQ ID NO.6 (IL-9 R primer)

[0191] 5'-CCGCTCGAGcactttgcctctcatattttcattc-3'

[0192] SEQ ID NO.7 (IL-9 nucleotide sequence)

[0193] ;

[0194] SEQ ID NO.8 (IL-9 amino acid sequence)

[0195] SQGCFTYVGVKNIVYLIDNLQKDTSSNCSCSTDAADCLCLPISSGNCSTACFQEGLSQLSNSTVSTRFPLILNQVKRIVVALKSNKCGSFACEQPCNQTTTGNTLTFLKTLLESFQNENMRGKV

[0196] SEQ ID NO.9 (IL-21 R primer)

[0197] 5'-CCGGAATTCcacaagtcaagcttccaag-3'

[0198] SEQ ID NO.10 (IL-21 F primer)

[0199] 5'-CCGCTCGAGggacagatgctgatgaatcatct-3'

[0200] SEQ ID NO.11 (IL-21 nucleotide sequence)

[0201] ;

[0202] SEQ ID NO.12 (IL-21 amino acid sequence)

[0203] HKSSFQGQDRLFIRLRQLIDIVDQLKNYVNDLDPEFLPAPEDVKRHCERSAFSCFQKVQLKSANNGDNEKIINILTKQLKRKLPPTNAGRRQKHELTCPSCDSYEKKPPKEYLERLKSLIQKMIHQHLS

[0204] SEQ ID NO.13 (IL-23p19 F1 primer)

[0205] 5'-cgcGGATCCcgggctgtgtcagagga-3'

[0206] SEQ ID NO.14 (IL-23p19 1R primer)

[0207] 5'-GCTGCCACCACCGCCggggctcagagttgctgct-3'

[0208] SEQ ID NO.15 (IL-23p40 F2 primer)

[0209] 5'-GGCGTGGTGGCAGCatatgggaactggagaaaaatgt-3'

[0210] SEQ ID NO.16 (IL-23p40 R2 primer)

[0211] 5'-CCGCTCGAGactgcaggacacagatg-3'

[0212] SEQ ID NO.17 (IL-23p19 nucleotide sequence)

[0213] ;

[0214] SEQ ID NO.18 (IL-23 p40 nucleotide sequence)

[0215] ;

[0216] ;

[0217] SEQ ID NO.19 (IL-23 amino acid sequence)

[0218] RAVSEDSSPAWAQGQQLSQQLCMLAWSAHLPMGHVDLPREEGGDETTDDVPRIQCEDGCDPQGLRDNSQPCLQRIHRGLVFYEKLLGSDIFTGEPSLFPDGPVDQLHASILGLRELLQPKGHHWEAEQTPSPIPSQPWQRLLLRLKILRSLQAFVAVAARVFAHGAATLSPGGGGSGGGGSGGGGSIWELEKNVYVVELDWYPNAPGETVVLTCDTPEEDGITWTSDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSRSLLLLHKKEDGIWSTDILKDQKEPKAKSFLKCEAKDYSGHFTCSWLTAISTNLKFSVKSSRGSSDPRGVTCGAASLSAEKVSMDHREYNKYTVECQEGSACPAAEESLPIEVVMEAVHKLKYENYTSSFFIRDIIKPDPPKNLQLRPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKNKREKKLFTDQTSAKVTCHKDANIRVQARDRYYSSFWSEWASVSCS

[0219] SEQ ID NO.20 (IL-27p28 F1 primer)

[0220] 5'-CCGGAATTCttcccaaggcccccagg-3'

[0221] SEQ ID NO.21 (IL-27p28 R1 primer)

[0222] 5'-GCTGCCACCACCGCCagaggtcactccatcaggg-3'

[0223] SEQ ID NO.22 (IL-27EBI3 F2 primer)

[0224] 5'-GGCGGTGGTGGCAGCcgcgagagagaaggcca-3'

[0225] SEQ ID NO.23 (IL-27EBI3 R2 primer)

[0226] 5'-ACGCGTCGACcttgcccagggtcatgg-3'

[0227] SEQ ID NO.24 (IL-27 p28 nucleotide sequence)

[0228] ;

[0229] SEQ ID NO.25 (IL-27 EBI3 nucleotide sequence)

[0230] ;

[0231] SEQ ID NO.26 (IL-27 amino acid sequence)

[0232] EFFPRPPGRPPLSLQELQREFKVSLHLAKKLLSVVRVQAHHFAESHLPGVNLDLLPLGEQLPNVSTTFQAWRGLSDPERLCFLSLTLRPFHTLLGGLGRQEFWTSSERMQLQ ATRLDLRDLQQHLHFQVLAAGFDLPEEHENEEEKGLLPGALGTPLQISAQVSWSRFLYTYRLLHSLELVLSRTVRDLLLLSRAGNSVQALGFPTPSSSPDGVTSGGGGSGGGG SGGGGSREREGHPLASSQPRVRCQASRYPVAVDCSWTLPPTPPNSTRPTSFIATYRLGVAAHGESWPCLQPTPEATSCVIPDVQMFSMVPYVLNITAVHPSGVSSSFMPFVP EHIIKPDPPEGVRLSPLPGQRLWVQWEPPRTWPFPEIFSLKYRIRYKRHGAARFRQVGPIEATSFTVKAVRPQAKYCIQVAAQDLTDYGEWSAWSLPAAASMTLGKVDKLAAA

[0233] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The use of nanoparticles with immune-enhancing effect in the preparation of an immune enhancer for inactivated vaccine of contagious pleuropneumonia of goats, characterized in that, The preparation method of the nanoparticles with immune enhancement effect comprises the following preparation steps: PLGA is dissolved in dichloromethane to prepare a 5% PLGA solution; 5% PVA is added dropwise into the 5% PLGA solution, and after vortex, first ultrasonic crushing is carried out under ice bath condition, then recombinant goat cytokine is added, and after vortex, second ultrasonic crushing is carried out under ice bath condition to obtain a primary emulsion; 5% PVA is added into the primary emulsion, and then third ultrasonic crushing is carried out under ice bath to obtain a multiple emulsion; After the multiple emulsion is stirred and volatilized, freezing ultracentrifugation is carried out, and after the end, the precipitate is collected, the precipitate is resuspended with water, and then freeze-drying is carried out to obtain the nanoparticles with immune enhancement effect; The power of the first ultrasonic crushing is 40W, ultrasonic crushing is carried out for 5s / interval 5s, and the total ultrasonic time is 5min; the power of the second ultrasonic crushing is 40W, ultrasonic crushing is carried out for 5s / interval 5s, and the total ultrasonic time is 3min; the power of the third ultrasonic crushing is 40W, ultrasonic crushing is carried out for 5s / interval 5s, and the total ultrasonic time is 5min; The centrifugal force of the freezing ultracentrifugation is 30000r / min, and the centrifugal time is 30min; The freeze-drying is as follows: after the resuspended precipitate is placed at-80℃ for 2 hours, it is transferred into a vacuum freeze-drier, the cold trap temperature is-45℃, the limit vacuum degree is 0.1mBar, and freeze-drying is carried out for 24h; The recombinant goat cytokine is recombinant goat cytokine IL-9 and IL-21, the amino acid sequence of the recombinant goat cytokine IL-9 is as shown in SEQ ID NO.8, and the amino acid sequence of the recombinant goat cytokine IL-21 is as shown in SEQ ID NO.

12.

2. The use of nanoparticles having an immunopotentiation effect according to claim 1 in the preparation of an immunopotentiator for inactivated vaccine of contagious pleuropneumonia of goats, characterized in that, The preparation of the recombinant goat cytokine IL-9 and IL-21 comprises the following steps: Total RNA of goat peripheral blood PBMC cells is extracted, reverse transcribed into cDNA, and PCR amplification is carried out with the primers shown in SEQ ID NO.9 and SEQ ID NO.10 to obtain an IL-21 protein gene fragment, and the IL-21 protein gene fragment is inserted into a pET-28a(+) vector to obtain a recombinant expression plasmid pET28a-IL-21 containing a goat IL-21 gene; Goat peripheral blood PBMC cells are separated, total RNA is extracted, and reverse transcribed into cDNA, and a goat IL-9 gene fragment is obtained by using an RT-PCR method, and the goat IL-9 gene fragment is cloned into a pET30a(+) vector between EcoR I and Xho I enzyme cutting sites to obtain a recombinant expression plasmid pET30a-IL-9 containing a goat IL-9 gene, wherein the sequences of the primers used for RT-PCR amplification are as shown in SEQ ID NO.5 and SEQ ID NO.6, and the coding gene sequence of the goat IL-9 protein is as shown in SEQ ID NO.7; The pET28a-IL-21 and pET30a-IL-9 are used to transform E. coli for induced expression and separation and purification to obtain the recombinant goat cytokine IL-9 and IL-21; The induced expression and separation and purification are as follows: The transformed E. coli was cultured to OD600 equal to 0.6, and then centrifuged after induction using IPTG. The bacteria were resuspended using supernatant Binding Buffer, and then subjected to ultrasonic disruption. The suspension after ultrasonic disruption was centrifuged to obtain supernatant and precipitated inclusion bodies. The inclusion bodies were dissolved using inclusion body Binding Buffer, and then centrifuged. The supernatant was obtained to obtain inclusion body protein; The inclusion body protein was filtered, and then loaded into a HisTag affinity chromatography column at a flow rate of 0.5 mL / min. The His Tag affinity chromatography column was washed using 5-10 times the column volume of Binding Buffer, and then the target protein was eluted using Elution Buffer at a flow rate of 0.5 mL / min. The recombinant goat cytokines IL-9 and IL-21 were obtained.

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