A chicken 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 chicken cytokine composition with immune-enhancing effects and its applications. The immune-enhancing chicken cytokine composition includes recombinant chicken cytokines IL-9 and IL-21, whose amino acid sequences are shown in SEQ ID NO.4 and SEQ ID NO.8, respectively. These two cytokines can upregulate the expression of IL-1β, IL-2, IL-4, IL-6, IFN-γ, IL-17A, and TNF-α in chicken PBMCs, stimulating Th1, Th2, and Th17 immune responses in chickens. When used in combination with a Newcastle disease-avian influenza bivalent inactivated vaccine, this recombinant chicken cytokine composition exhibits a significant immune-enhancing effect and has potential application prospects. This chicken 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 chicken cytokine composition with immune-enhancing effects and its applications. Background Technology

[0002] Immunization is a crucial means of controlling infectious diseases, serving as a robust defense against disease and preventing its spread. Adjuvants are molecules or compounds injected into animals before or simultaneously with the antigen that enhance the body's immune response or alter the type of immune response. In vaccine research, adjuvants are needed to assist vaccines in evoking a more effective immune response, addressing issues such as single antigens and weak immunogenicity, and enhancing the vaccine's immunogenicity.

[0003] Cytokines are a class of proteins with broad biological activity produced by immune cells or immune-related cells in the body. They play important roles in the generation and regulation of humoral and cellular immunity, and are indispensable components for the body's immunological functions. In the past 20 years, studies have demonstrated that cytokines such as IL-2, IL-7, IL-18, IFN, and GM-CSF have shown good adjuvant effects in inactivated or genetically engineered vaccines for related avian diseases, increasing antibody titers and cellular immunity levels, and exhibiting good immune-enhancing effects. IL-9, a multifunctional cytokine mainly produced by Th9 cells, was initially identified as a T-cell growth factor, and subsequently found to play a key role in regulating humoral and cellular immunity. It can simultaneously enhance both adaptive cellular and humoral immunity: promoting CD8⁺ T-cell activation and killing, and promoting B-cell proliferation and differentiation to produce high-affinity antibodies; it can also directionally regulate Th1 / Th2 responses, adapting to different antigen requirements. Furthermore, it can maintain the survival of memory T / B cells, regulate the function of macrophages and natural killer cells, participate in the development and maturation of immune organs, and prolong the protection period of vaccines; it also has good biocompatibility and a well-defined target. IL-21 can enhance the killing activity of NK cells, participate in the regulation of B cell proliferation and differentiation, induce immunoglobulin production, regulate T cell activation, differentiation and function, and can also increase and maintain CD8. + T cells contribute to the sustained immune response against tumors, thus playing a crucial role in host defense, anti-tumor activity, and antiviral activity. They also play a vital role in both innate and adaptive immunity. Whether chicken IL-9 and IL-21 cytokines can serve as immune enhancers remains to be studied.

[0004] Therefore, providing a chicken 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 chicken cytokine composition with immune-enhancing effects and its application, aiming to solve at least one of the problems in the background art.

[0006] This invention proposes a chicken cytokine composition with immune-enhancing effects, comprising the following preparation steps: including recombinant chicken cytokines IL-9 and IL-21, wherein the amino acid sequence of the recombinant chicken cytokine IL-9 is as shown in SEQ ID NO.4, and the amino acid sequence of the recombinant chicken cytokine IL-21 is as shown in SEQ ID NO.8.

[0007] Preferably, the recombinant chicken cytokines IL-9 and IL-21 are prepared as follows: Based on the encoding genes of chicken IL-9 (Gene ID: 416307) and chicken IL-21 (Gene ID: 554218), two pairs of specific primers are designed using Primer 5.0 software, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.6. Total RNA is extracted from chicken peripheral blood PBMCs and reverse transcribed into cDNA. RT-PCR amplification is performed using the primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.6 to obtain IL-9 and IL-21 protein gene fragments. The IL-9 protein gene fragment is inserted between the BamH I and Not I restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-9 containing the chicken IL-9 gene. The IL-21 protein gene fragment was inserted between the BamH Ⅰ and Xho Ⅰ restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-21 containing the chicken IL-21 gene.

[0008] The recombinant chicken cytokines IL-9 and IL-21 were obtained by transforming Escherichia coli with pET28a-IL-21 and pET28a-IL-9 for induced expression and then isolated and purified.

[0009] Preferably, the induction of expression and isolation purification are performed by culturing the transformed Escherichia coli to OD200. 600 When the concentration is 0.6, the bacterial cells are collected by centrifugation after induction with IPTG. The bacterial cells are resuspended in Binding Buffer and then sonicated. The sonicated suspension is centrifuged, and the supernatant is the supernatant protein. The precipitate is the inclusion body. The inclusion body is dissolved in Binding Buffer and centrifuged. The supernatant is collected to obtain the inclusion body protein.

[0010] Binding Buffer: Weigh 29.2 g NaCl and 1.36 g imidazole, dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0011] Elutioning Buffer: Weigh 29.2 g NaCl and 34 g imidazole, dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0012] Inclusion body binding buffer: Weigh 29.2 g NaCl, 1.36 g imidazole and 480 g urea, dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0013] Inclusion body elution buffer: Weigh 29.2 g NaCl, 34 g imidazole, and 480 g urea, and dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[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 chicken cytokines IL-9 and IL-21.

[0015] The present invention also provides the application of the chicken cytokine composition with immune-enhancing effect described above in the preparation of nanoparticles with immune-enhancing effect.

[0016] Preferably, the preparation method of the nanoparticles with immune-enhancing effects includes the following steps: dissolving PLGA in dichloromethane to prepare a 5% PLGA solution; adding 5% PVA dropwise to the 5% PLGA solution, vortexing, and then performing a first ultrasonic disruption under ice bath conditions; subsequently adding the recombinant chicken cytokine, vortexing, and then performing a second ultrasonic disruption under ice bath conditions to obtain a primary emulsion; adding 5% PVA to the primary emulsion, and then performing a third ultrasonic disruption under ice bath conditions to obtain a secondary emulsion; stirring and evaporating the secondary emulsion, then freezing and ultracentrifuging it, collecting the precipitate after the process, resuspending the precipitate in water, and then freeze-drying it to obtain the nanoparticles with immune-enhancing effects.

[0017] 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.

[0018] Preferably, the centrifugal force of the cryogenic ultracentrifugation is 30,000 r / min, and the centrifugation time is 30 min.

[0019] 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.

[0020] The present invention also provides the application of the aforementioned immunomodulatory nanoparticles in the preparation of animal immunomodulators.

[0021] Preferably, the animal is a chicken.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present 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-17A, and TNF-α, and synergistically promote the differentiation of T cells and B cells, thus having a significant immune-enhancing effect.

[0023] 2. The recombinant chicken cytokines IL-9 and IL-21 prepared in this invention have high expression levels, are easy to obtain, have low cost, good safety, and can significantly improve the immune protection effect of vaccines. Attached Figure Description 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: Figure 1 This is an image showing the electrophoresis results on a 2% agarose gel provided in an embodiment of the present invention; Figure 2 This is a graph showing the purity test results of the target protein provided in an embodiment of the present invention; Figure 3 A graph showing the changes in the transcriptional level of the cytokine IL-9 provided in an embodiment of the present invention; Figure 4 This is a graph showing the changes in the transcriptional level of the cytokine IL-21 provided in an embodiment of the present invention; Figure 5This is a graph showing the changes in the transcriptional level of the cytokine IL-19 provided in an embodiment of the present invention; Figure 6 This is a graph showing the changes in the transcriptional level of the cytokine IL-22 provided in an embodiment of the present invention; Figure 7 A graph showing the levels of Newcastle disease (ND) antibodies against different cytokines provided in this embodiment of the invention; Figure 8 A graph showing the antibody levels of different cytokines for avian influenza (AI) provided in an embodiment of the present invention. Detailed Implementation 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The present invention provides a chicken cytokine composition with immune-enhancing effects, comprising: recombinant chicken cytokines IL-9 and IL-21, wherein the amino acid sequence of the recombinant chicken cytokine IL-9 is as shown in SEQ ID NO.4, and the amino acid sequence of the recombinant chicken cytokine IL-21 is as shown in SEQ ID NO.8.

[0029] The nucleotide sequence of the recombinant chicken cytokine IL-9 is shown in SEQ ID NO.3, and the nucleotide sequence of the recombinant chicken cytokine IL-21 is shown in SEQ ID NO.7.

[0030] In this invention, the recombinant chicken cytokines IL-9 and IL-21 are prepared as follows: Based on the encoding genes of chicken IL-9 (Gene ID: 416307) and chicken IL-21 (Gene ID: 554218), two pairs of specific primers are designed using Primer 5.0 software, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.6. Total RNA is extracted from chicken peripheral blood PBMCs and reverse transcribed into cDNA. RT-PCR amplification is performed using the primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.6 to obtain IL-9 and IL-21 protein gene fragments. The IL-9 protein gene fragment is inserted between the BamH I and Not I restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-9 containing the chicken IL-9 gene. The IL-21 protein gene fragment was inserted between the BamH Ⅰ and Xho Ⅰ restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-21 containing the chicken IL-21 gene.

[0031] The recombinant chicken cytokines IL-9 and IL-21 were obtained by transforming Escherichia coli with pET28a-IL-21 and pET28a-IL-9 for induced expression and then isolated and purified.

[0032] Specifically, the method for synthesizing chicken PBMC cDNA template is as follows: (1) Collect 20 mL of chicken blood using a vacuum anticoagulation blood collection tube.

[0033] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0034] (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.

[0035] (4) Centrifuge at room temperature (2500 rpm) for 20 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).

[0036] (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.

[0037] (6) After the last wash, discard the supernatant to obtain isolated chicken PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0038] (7) Use an RT-PCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0039] Specifically, the PCR amplification reaction system used to prepare the recombinant expression plasmids pET28a-IL-21 and pET28a-IL-9 was as follows: 2.5 μL of chicken PBMC cell cDNA, 25 μL of PrimeSTAR Max Premix (2X), 2.5 μL of upstream primer F (10 pM), 2.5 μL of downstream primer R (10 pM), and 17.5 μL of sterile ultrapure water, mixed thoroughly. The mixture was pre-denatured at 95℃ for 5 min on a PCR instrument; followed by denaturation at 95℃ for 10 s, annealing at 58℃ for 15 s, extension at 72℃ for 45 s, for 35 cycles; and a final extension at 72℃ for 5 min.

[0040] 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℃.

[0041] In this invention, the induced expression and isolation purification are performed by culturing the transformed Escherichia coli to OD200. 600 When the concentration was 0.6, bacterial cells were collected by centrifugation after induction with IPTG. The cells were resuspended in Binding Buffer and then sonicated. The sonicated suspension was centrifuged; the supernatant was the supernatant protein, and the precipitate was the inclusion bodies. The inclusion bodies were dissolved in Binding Buffer and centrifuged. The supernatant was collected to obtain the inclusion body protein. Binding Buffer: 29.2 g NaCl and 1.36 g imidazole were weighed and dissolved in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0042] Elutioning Buffer: Weigh 29.2 g NaCl and 34 g imidazole, dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0043] Inclusion body binding buffer: Weigh 29.2 g NaCl, 1.36 g imidazole and 480 g urea, dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0044] Inclusion body elution buffer: Weigh 29.2 g NaCl, 34 g imidazole, and 480 g urea, and dissolve in 0.02 M potassium-free solution. + Bring the PBS buffer to a final volume of 1L.

[0045] 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 chicken cytokines IL-9 and IL-21.

[0046] Specifically, the transformed E. coli were cultured to OD200. 600 When the concentration is 0.6, after induction with 1 mM IPTG for 5 h, the bacterial cells are collected by centrifugation at 4000 rpm for 15 min. The cells are resuspended in approximately 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 is centrifuged at 8000 g at 4 °C for 20 min. The supernatant is the supernatant protein, and the precipitate is the inclusion body. The precipitate is dissolved in inclusion body binding buffer overnight at 4 °C, centrifuged at 8000 g at 4 °C for 20 min, and the supernatant is collected as the inclusion body protein.

[0047] The inclusion body proteins were filtered sequentially through 0.45 μm and 0.22 μm filter membranes. The protein sample was then slowly passed through a 5 mL His Tag affinity chromatography column (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 protein was then eluted with Elution Buffer at a flow rate of 0.5 mL / min.

[0048] The present invention also provides the application of the chicken cytokine composition with immune-enhancing effect described above in the preparation of nanoparticles with immune-enhancing effect.

[0049] In this invention, the preparation method of the nanoparticles with immune-enhancing effects includes the following preparation steps: PLGA was dissolved in dichloromethane to prepare a 5% PLGA solution; 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 chicken cell factor was added, vortexed, and then subjected to a second ultrasonic disruption under ice bath conditions to obtain the colostrum. 5% PVA was added to the primary emulsion, followed by a third ultrasonic disruption under ice bath conditions to obtain the secondary emulsion. 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.

[0050] 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.

[0051] In this invention, the centrifugal force of the cryogenic ultracentrifugation is preferably 30,000 r / min, and the centrifugation time is preferably 30 min.

[0052] 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.

[0053] Specifically, the preferred method for preparing the nanoparticles with immune-enhancing effects is as follows: (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.

[0054] (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).

[0055] (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.

[0056] (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.

[0057] (5) Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0058] (6) Centrifuge at 30,000 r / min for 30 min under refrigeration; after centrifugation, collect the supernatant and precipitate separately.

[0059] (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.

[0060] (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.

[0061] The present invention also provides the application of the aforementioned immunomodulatory nanoparticles in the preparation of animal immunomodulators.

[0062] In this invention, the animal is preferably a chicken.

[0063] Example 1: Preparation of recombinant chicken cytokines IL-9 and IL-21 1. Primer synthesis: Based on the coding genes for chicken IL-9 (Gene ID: 416307) and chicken IL-21 (Gene ID: 554218) 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 chicken IL-9 are SEQ ID NO.1 and SEQ ID NO.2, respectively, and the upstream and downstream primer (F, R) sequences for amplifying chicken IL-21 are SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0064] 2. Synthesize chicken PBMC cDNA template: (1) Collect 20 mL of chicken blood using a vacuum anticoagulation blood collection tube.

[0065] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0066] (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.

[0067] (4) Centrifuge at room temperature (2500 rpm) for 20 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).

[0068] (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.

[0069] (6) After the last wash, discard the supernatant to obtain isolated chicken PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0070] (7) Use an RT-PCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0071] 3. Cloning and Expression of Chicken Cytokine Genes: Take 2.5 μL of cDNA from chicken PBMC cells, 25 μL of PrimeSTAR MaxPremix (2X), 2.5 μL of upstream primer F (10 pM), 2.5 μL of downstream primer R (10 pM), and 17.5 μL of sterile ultrapure water, and mix thoroughly. Perform pre-denaturation at 95℃ for 5 min on a PCR instrument; followed by denaturation at 95℃ for 10 s, annealing at 58℃ for 15 s, and extension at 72℃ for 45 s, for 35 cycles; then perform a final extension at 72℃ for 5 min. Take 50 μL of the obtained PCR product and perform electrophoresis on a 2% agarose gel. Under UV light, cut the agarose gel at the target band location and purify the target fragment using a gel extraction kit from Takara Bio Inc. (Dalian), following the manufacturer's instructions. Digest the target gene and pET-28a plasmid separately with double enzymes, and then recover the target gene and pET-28a large fragment again. Ligate overnight at 4℃. Recombinant expression plasmids pET28a-IL-9 containing the chicken IL-9 gene and pET28a-IL-21 containing the chicken IL-21 gene were obtained. The recombinant expression plasmids pET28a-IL-9 and pET28a-IL-21 were used to transfect competent Escherichia coli BL21, respectively. Positive clones were selected, plasmids were extracted, and the bacteria were identified by enzyme digestion and sequencing.

[0072] 4. Purification of the expression product: E. coli containing the recombinant plasmid were cultured to OD200. 600When the concentration is 0.6, after induction with 1 mM IPTG for 5 h, the bacterial cells are collected by centrifugation at 4000 rpm for 15 min. The cells are resuspended in approximately 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 is centrifuged at 8000 g at 4 °C for 20 min. The supernatant is the supernatant protein, and the precipitate is the inclusion body. The precipitate is dissolved in inclusion body binding buffer overnight at 4 °C, centrifuged at 8000 g at 4 °C for 20 min, and the supernatant is collected as the inclusion body protein.

[0073] The inclusion body proteins 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 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 chicken cytokines IL-9 and IL-21. Example 2 Preparation of nanoparticles with immune-enhancing effects (recombinant chicken cytokine IL-9) Solution preparation: 5% PVA: Dissolve 1g PVA in 20 mL of deionized water and heat and stir at 95℃ to dissolve.

[0074] Preparation steps: 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.

[0075] 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).

[0076] S3. While vortexing, add 5mg of recombinant chicken 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 promulgation.

[0077] 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.

[0078] S5. Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0079] S6. Centrifuge at 30,000 r / min for 30 min using a freeze-thawed ultracentrifuge. After centrifugation, collect the supernatant and precipitate separately.

[0080] 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 with a cold trap temperature of -45℃ and an ultimate vacuum of 0.1 mBar for 24 h to obtain recombinant chicken cytokine IL-9 protein nanoparticles.

[0081] Example 3 Preparation of nanoparticles with immune-enhancing effects (recombinant chicken cytokine IL-21) The only difference from the example is that the 5mg recombinant chicken cytokine IL-9 added in step S3 is replaced with 5mg recombinant chicken cytokine IL-21.

[0082] Effect test Chicken cytokines IL-9, IL-21, IL-19, and IL-22 were selected for testing and comparison screening.

[0083] Test Example 1: Preparation of Recombinant Cytokine Proteins 1.1 Synthetic Primers Based on the coding genes of chicken IL-9 (416307), IL-21 (554218), IL-19 (107055197), and IL-22 (417838) 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.

[0084] The upstream and downstream primer (F, R) sequences for amplifying chicken IL-9 are listed in SEQ ID NO.1 and SEQ ID NO.2, respectively; the upstream and downstream primer (F, R) sequences for amplifying chicken IL-21 are listed in SEQ ID NO.5 and SEQ ID NO.6, respectively; the upstream and downstream primer (F, R) sequences for amplifying chicken IL-19 are listed in SEQ ID NO.9 and SEQ ID NO.10, respectively; and the upstream and downstream primer (F1, R1) sequences for amplifying chicken IL-22 are listed in SEQ ID NO.13 and SEQ ID NO.14, respectively.

[0085] The chicken IL-9 gene sequence is shown in SEQ ID NO.3, which encodes the chicken IL-9 protein. Its amino acid sequence is shown in SEQ ID NO.4, consisting of 118 amino acids. The chicken IL-21 gene sequence is shown in SEQ ID NO.7, which encodes the chicken IL-21 protein. Its amino acid sequence is shown in SEQ ID NO.8, consisting of 126 amino acids. The chicken IL-19 gene sequence is shown in SEQ ID NO.11, which encodes the chicken IL-19 protein. Its amino acid sequence is shown in SEQ ID NO.12, consisting of 152 amino acids. The chicken IL-22 gene sequence is shown in SEQ ID NO.15, which encodes the chicken IL-22 protein. Its amino acid sequence is shown in SEQ ID NO.16, consisting of 169 amino acids.

[0086] 1.2 Synthesis of chicken PBMC cDNA template (1) Use a vacuum anticoagulation blood collection tube to collect 20 mL of chicken blood.

[0087] (2) Mix the anticoagulated blood with PBS in equal proportions under sterile conditions.

[0088] (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.

[0089] (4) Centrifuge at room temperature (2500 rpm) for 20 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).

[0090] (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.

[0091] (6) After the last wash, discard the supernatant to obtain isolated chicken PBMC cells. Extract total RNA using the TRIzol method according to the instructions and determine the RNA concentration.

[0092] (7) Use an RT-PCR reverse transcription kit to reverse transcribe RNA into cDNA.

[0093] 1.3 Cloning and Expression of Chicken Cytokine Genes The following reaction system was used for PCR amplification of the IL-9, IL-21, IL-19, and IL-22 genes. The reaction system consisted of 2.5 μL of chicken PBMC cell cDNA, 25 μL of PrimeSTAR Max Premix (2X), 2.5 μL of upstream primer F (10 pM), 2.5 μL of downstream primer R (10 pM), and 17.5 μL of sterile ultrapure water, all thoroughly mixed. The PCR reaction was performed at 95°C for 5 min, followed by 95°C denaturation for 10 s, 58°C annealing for 15 s, and 72°C extension for 45 s, for 35 cycles; followed by a final extension at 72°C for 5 min.

[0094] Take 50 μL of the PCR product obtained above, and perform electrophoresis on a 2% agarose gel. Under UV light, cut the agarose gel containing the target band, and recover and purify the target fragment using a gel extraction kit from Takara Bio Inc. (Dalian), following the manufacturer's instructions. Digest the target gene and pET-28a plasmid separately with double enzymes, and recover the target gene and pET-28a large fragment again. Ligate overnight at 4°C. Transform the ligation product into competent E. coli BL21. The electrophoresis results on the 2% agarose gel are as follows: Figure 1 As shown. M: DNA molecular weight standard DL5000; 1: IL-9; 2: IL-19; 3: IL-21; 4: IL-22.

[0095] 1.4 Purification of the expression product The above-mentioned E. coli containing the recombinant plasmid were cultured to OD200. 600 When the concentration is 0.6, the cells were induced with 1 mM IPTG for 5 h, centrifuged at 4000 rpm for 15 min to collect the cells, and resuspended in 40 ml of supernatant binding buffer. The cells were then 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 in inclusion body binding buffer overnight at 4°C, centrifuged at 8000 g at 4°C for 20 min, and the supernatant was collected as the inclusion body protein.

[0096] 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 purity of the target protein was determined, and the results are as follows. Figure 2As shown. SDS-PAGE electrophoresis yielded relatively single bands of the target protein. M: relative molecular mass of the protein; 1: recombinant IL-9 protein before purification; 2: recombinant IL-9 protein after purification; 3: recombinant IL-19 protein before purification; 4: recombinant IL-19 protein after purification; 5: recombinant IL-21 protein before purification; 6: recombinant IL-21 protein after purification; 7: recombinant IL-22 protein before purification; 8: recombinant IL-22 protein after purification.

[0097] Test Example 2: Screening of cytokines with immune-enhancing effects 2.1 Isolation of chicken PBMC cells Chicken 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.

[0098] The cell concentration was adjusted to 5 × 10⁶ cells / mL 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.

[0099] 2.2 Co-incubation of recombinant chicken cytokines with chicken PBMCs Add 1 mL of PBMC (5 × 10⁻⁶) 6 Cells were seeded into 24-well cell culture plates, and recombinant chicken cytokines were added sequentially to achieve final concentrations of 5, 10, 20, 40, and 80 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.

[0100] 2.3 Quantitative PCR detection of different types of cytokines Total RNA was extracted from PBMCs and amplified to obtain cDNA using a reverse transcription kit with gDNA removal. The transcriptional levels of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, 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 as follows: 94 °C pre-denaturation for 30 s; cycling reaction: 94 °C for 5 s, 60 °C for 30 s, 40 cycles; melting curve: 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s. Two... -ΔΔCt The method was used to analyze changes in cytokine transcription levels.

[0101] The results are as follows Figure 3-6 As shown, IL-9 significantly downregulated IL-10 mRNA levels at concentrations of 5, 20, 40, and 80 μg / mL, and significantly upregulated IL-1β, IL-2, IL-4, TNF-α, IFN-λ, and TGF-β mRNA levels at concentrations of 5, 10, 20, and 40 μg / mL, and significantly upregulated IL-17A mRNA levels at concentrations of 5 and 10 μg / mL. IL-19 significantly upregulated IL-1β, IL-2, IL-4, IL-6, IL-17A, TNF-α, IFN-λ, and TGF-β mRNA levels, and upregulated IL-10 mRNA levels at concentrations of 40 and 80 μg / mL. IL-21 significantly upregulated the mRNA levels of IL-1β, IL-17A, IL-6, TNF-α, IFN-λ, and TGF-β at all concentrations, upregulated the mRNA levels of IL-10 and IL-4 at 40 and 80 μg / mL, and significantly upregulated the mRNA level of IL-2 at 10, 20, 40, and 80 μg / mL. IL-22 significantly downregulated the mRNA level of IL-10 at all concentrations and significantly upregulated the mRNA levels of IL-1β, IL-2, IL-4, IL-6, IL-17A, TNF-α, IFN-λ, and TGF-β.

[0102] Test Example 3: Preparation of Nanoparticles Using PLGA-Coated Recombinant Cytokine Protein 3.1 Solution Preparation 5% PVA: Dissolve 1g PVA in 20 mL of deionized water and heat at 95℃ with stirring until dissolved.

[0103] 3.2 Preparation of Nanoparticles 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.

[0104] 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).

[0105] 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.

[0106] 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.

[0107] S5. Place the ultrasonically treated emulsion in a fume hood and stir while evaporating until the organic solvent has completely evaporated.

[0108] S6. Centrifuge at 30,000 r / min for 30 min using a freeze-thawed ultracentrifuge. After centrifugation, collect the supernatant and precipitate separately.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 3.3 Electron Microscopy Observation of Nanoparticles: 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.

[0113] 3.4 Determination of protein encapsulation rate in nanoparticles 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:

[0114] The specific results are as follows: the coverage rate of IL-9 is 70.9%, the coverage rate of IL-21 is 96.8%, the coverage rate of IL-19 is 93.8%, and the coverage rate of IL-22 is 84.4%.

[0115] Test Example 4: Immunostimulatory effect of recombinant chicken cytokines on Newcastle disease-avian influenza bivalent inactivated vaccine Ninety chicks weighing 87.39±3.78g were randomly divided into 6 groups: a control group (PBS group) and 5 experimental groups, with 15 chicks in each group. Each chick in the experimental groups received a subcutaneous injection of 0.5mL of Newcastle disease-avian influenza bivalent inactivated vaccine in the neck and back, along with PLGA lyophilized powder coated with recombinant cytokine proteins prepared in Test Example 3, dissolved in sterile PBS. The group assignments and amounts of recombinant cytokines are shown in Table 1. Fasting weights were measured before injection (day 0) and at 7, 14, 21, 28, 35, 42, and 49 days after injection. Unanticoagulated whole blood was collected via cardiac sampling, centrifuged at 3000g, 4℃ for 15 minutes, and serum was collected, aliquoted, and stored at -80℃. Weight gain rate (the percentage increase in body weight this week compared to last week) and total weight gain rate (the percentage increase in body weight at the end of the experiment compared to the body weight at day 0 at the start of the experiment) were calculated for each stage. Weight gain rate = (final weight - initial weight) / initial weight * 100%. The levels of ND and AI antibodies were detected using a standard hemagglutination inhibition assay. Figure 7 , Figure 8 As shown in Table 2, the results showed that the fourth combination (IL-9 and IL-21 combined) significantly improved the levels of ND and AI antibodies and had the best effect among all cytokine combinations, without adverse effects on chicken growth performance.

[0116]

[0117]

[0118] The experimental materials and tools used in the above embodiments were sourced from: Laboratory animals: 90 healthy Hy-Line Brown chickens that were 1 day old, purchased from Nanjing Tegeli Company and raised at the Animal Experiment Center of Nanjing Agricultural University; Tools, enzymes, and reagents: Preparative endonucleases and DNA markers 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 from EZNA™, USA; BCA protein quantification kits were from Thermo Fisher Scientific, USA; TRIzol® reagents, RT-PCR reverse transcription kits, and high-specificity dye-based quantitative PCR detection kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; HisTrap TMFF protein affinity chromatography column was purchased from GE Healthcare, USA; poly(lactic-co-glycolic acid) (PLGA) was purchased from Merck Sigma-Aldrich Biotechnology Co., Ltd.; Newcastle disease-avian influenza bivalent inactivated vaccine (batch number: 101082165) was purchased from Yangzhou Youbang Biotechnology Co., Ltd.; ND antigen was purchased from Qingdao Lijian Biotechnology Co., Ltd.; and AI (H9 subtype) antigen was purchased from Harbin Weike Biotechnology Co., Ltd.

[0119] 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).

Claims

1. A chicken cytokine composition with immune-enhancing effects, characterized in that, The invention includes recombinant chicken cytokines IL-9 and IL-21, the amino acid sequence of which is shown in SEQ ID NO.4 and the amino acid sequence of which is shown in SEQ ID NO.

8.

2. The chicken cytokine composition with immune-enhancing effects according to claim 1, characterized in that, The recombinant chicken cytokines IL-9 and IL-21 were prepared as follows: Based on the encoding genes of chicken IL-9 (Gene ID: 416307) and chicken IL-21 (Gene ID: 554218), two pairs of specific primers were designed using Primer 5.0 software, as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.

6. Total RNA was extracted from chicken peripheral blood PBMCs and reverse transcribed into cDNA. RT-PCR amplification was performed using the primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO.6 to obtain IL-9 and IL-21 protein gene fragments. The IL-9 protein gene fragment was inserted between the BamH I and Not I restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-9 containing the chicken IL-9 gene. The IL-21 protein gene fragment was inserted between the BamH I and Xho I restriction sites of the pET-28a(+) vector to obtain the recombinant expression plasmid pET28a-IL-21 containing the chicken IL-21 gene. pET28a-IL-21 and pET28a-IL-9 were then transformed into *E. coli* for induced expression, and the recombinant chicken cytokines IL-9 and IL-21 were isolated and purified.

3. The chicken cytokine composition with immune-enhancing effects according to claim 2, characterized in that, The induction of expression and isolation / purification consisted of culturing the transformed *E. coli* to OD200. 600 When the concentration is equal to 0.6, bacterial cells are collected by centrifugation after induction with IPTG. The bacterial cells are resuspended in Binding Buffer and then sonicated. The sonicated suspension is centrifuged, and the supernatant is the supernatant protein, while the precipitate is the inclusion body. The inclusion body is dissolved in Binding Buffer and centrifuged, and the supernatant is collected to obtain the inclusion body protein. The inclusion body protein is filtered and loaded into 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 protein is eluted with Elution Buffer at a flow rate of 0.5 mL / min to obtain the recombinant chicken cytokines IL-9 and IL-21 proteins.

4. The use of a chicken cytokine composition with immune-enhancing effects as described in any one of claims 1-3 in the preparation of nanoparticles with immune-enhancing effects.

5. The application according to claim 4, characterized in that, The preparation method of the immunomodulatory nanoparticles includes the following steps: dissolving PLGA in dichloromethane to prepare a 5% PLGA solution; adding 5% PVA dropwise to the 5% PLGA solution, vortexing, and then performing a first ultrasonic disruption under ice bath conditions; subsequently adding the recombinant chicken cytokine protein, vortexing, and then performing a second ultrasonic disruption under ice bath conditions to obtain a primary emulsion; adding 5% PVA to the primary emulsion, and then performing a third ultrasonic disruption under ice bath conditions to obtain a secondary emulsion; stirring and evaporating the secondary emulsion, then freezing and ultracentrifuging, collecting the precipitate after the process, resuspending the precipitate in water, and then freeze-drying to obtain the immunomodulatory nanoparticles.

6. The application according to claim 5, characterized in that, The first ultrasonic fragmentation has a power of 40W, with 5s of ultrasound per 5s interval, and a total ultrasound time of 5min; the second ultrasonic fragmentation has a power of 40W, with 5s of ultrasound per 5s interval, and a total ultrasound time of 3min; the third ultrasonic fragmentation has a power of 40W, with 5s of ultrasound per 5s interval, and a total ultrasound time of 5min.

7. The application according to claim 6, characterized in that, The centrifugal force of the cryogenic ultracentrifugation is 30,000 r / min, and the centrifugation time is 30 min.

8. The application according to claim 7, characterized in that, 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.

9. The application of the nanoparticle with immune-enhancing effect as described in claim 4 in the preparation of animal immune enhancers.

10. The application according to claim 9, characterized in that, The animal in question is a chicken.

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