WO2020215351A1 - Immunopotentiator, preparation method therefor, avian influenza vaccine and use thereof - Google Patents

Abstract

Disclosed is an immunopotentiator comprising a bursin pentapeptide and an astragalus polysaccharide, wherein the weight ratio of the bursin pentapeptide to the astragalus polysaccharide is 1:5 to 1:20. The immunopotentiator is simple to prepare and can improve the expression level of antibody titer for vaccine immunity. Also disclosed are a method for preparing the immunopotentiator, an avian influenza vaccine using the immunopotentiator and the application of the immunopotentiator in the avian influenza vaccine.

Description

Immune enhancer, preparation method, bird flu vaccine and application Technical field

The present invention relates to the technical field of veterinary biological products, in particular to an immune enhancer, a preparation method, avian influenza vaccine and application.

Background technique

Avian influenza is an important infectious disease caused by the avian influenza virus, which seriously harms the development of the chicken industry. Vaccine immunization is one of the most effective means to control the epidemic. However, with the promotion and use of vaccines, viruses continue to mutate under the pressure of vaccine selection, and their virulence has increased, which has brought challenges to the prevention and control of epidemics. To solve the above dilemma, clinically, the vaccine strains are constantly replaced. For the prevention and control of avian influenza, the replacement of vaccine strains can indeed have an immediate effect in a short period of time, but in the long run, this will also accelerate the rate of mutation of the avian influenza virus, and the prevention and treatment will be trapped in a cycle.

Immune enhancers can improve the function of the body's immune system and enhance the body's non-specific immunity to diseases. The use of appropriate immune enhancers in combination with vaccines is one of the effective ways to enhance the immunity of existing vaccines. Improving vaccine immunity means that fewer vaccines can have better effects. Clinical use can reduce the number of immunizations and reduce stress, which not only saves costs, but also effectively controls diseases.

Bursalin (BP) is a small molecule peptide that exists in the bursa of poultry and is closely related to immune function. Bursin pentapeptide (BP5) is a member of the bursin family. It can induce the differentiation and proliferation of poultry B lymphocyte precursors, increase antibody levels, and enhance humoral immunity; promote the transformation activity of T lymphocytes, thereby enhancing cellular immunity .

Two representative polypeptides in the bursin family are BP-14 and BP-5.

Regarding BP-14, the right holder Jiangsu Vocational College of Agriculture and Animal Husbandry Technology applied for Chinese patent CN201310413608.2 in 2013, which discloses a polypeptide whose amino acid sequence is:

COOH-G-H-K-T-R-N-D-P-L-K-G-A-V-D-NH2.

The effect is recorded as follows: the immune enhancer BP14 is added to the livestock and poultry vaccine in different doses according to different target animals for joint vaccination; the antibody level of the immunized animals is higher than that of the vaccine group; the test animals produce higher levels of cytokines . The dosage for poultry is 0.1μg/feather, and for pigs is 5μg/head; referring to Figure 1, BP-14+ vaccine (H5N1 subtype, Re-6 strain) is used, and the titer is 5-8log2.

Regarding BP-5, the right holder Li Deyuan applied for Chinese patent CN200810243575.0 in 2008 to disclose a bursal pentapeptide whose amino acid sequence is: CYS-LYS-ASP-VAL-TYR. The bursa pentapeptide (BP5) of the invention has simple structure, small molecular weight, no chemical toxicity, no immunogenicity, and simple preparation. It can be extracted from the bursa of chicken or other poultry, or can be chemically synthesized. The cost is very low and can be prepared in large quantities. Bursa pentapeptide (BP5) can promote the proliferation of T lymphocytes and B lymphocytes, improve the level of humoral and cellular immunity of the body, and can also improve the body's ability to resist peroxidative stress. It is a kind of broad application prospect Drugs or preparations for immunomodulation, immunotherapy and anti-peroxidation. It can be used in the fields of basic research, clinical treatment, nutrition and health care, and cosmetics.

The patent records the potential use of bursa pentapeptide in immunotherapy and immune regulation, but does not mention its application in avian influenza vaccine.

The right holder Henan University of Science and Technology applied for a Chinese patent CN201310069941.6 in 2013, which discloses recombinant Tα1-BP5 fusion peptides, genes, engineered bacteria and applications. The recombinant fusion peptide is a fusion of thymosin α1 and bursa pentapeptide BP5 through a flexible Linker. The present invention inserts the recombinant Tα1-BP5 fusion peptide gene into an expression vector, transforms Escherichia coli, and obtains a genetically engineered bacteria that efficiently expresses the recombinant Tα1-BP5 fusion peptide. The recombinant Tα1-BP5 fusion peptide is prepared through liquid culture and purification. The fusion peptide The thioredoxin was removed by enterokinase with a His tag at the N-terminal, and then purified by affinity chromatography to obtain a single recombinant Tα1-BP5 fusion peptide. The recombinant Tα1-BP5 fusion peptide of the invention can be used as a novel polypeptide immune adjuvant used with vaccines, can effectively enhance the body's cellular immunity and humoral immunity, and has broad application prospects.

The invention is based on the bursa pentapeptide BP5, and the gene recombination expression with thymosin α1 is carried out to achieve the purpose of immune enhancement.

It can be found from the above records that BP5 has a great potential for development of immune enhancement. Research and development in this field are conducting in-depth research in this area.

Therefore, the purpose of the present invention is to conduct a more in-depth study on BP5 and propose a new application formula for BP5 to improve the immune effect of the vaccine.

Summary of the invention

The purpose of the present invention is to provide an immune enhancer. At the same time, the present invention also discloses the preparation method of the immune enhancer, the avian influenza vaccine using the immune enhancer and the application of the avian influenza vaccine. The immune enhancer can effectively increase the antibody titer expression level of vaccine immunity. In addition, the immune enhancer is simple to prepare and has good industrial application prospects.

Before explaining the scheme of the present invention, the abbreviation is necessary to explain:

LB liquid medium: Luria-Bertani liquid medium;

IPTG: isopropyl thiogalactoside, Isopropylβ-D-Thiogalactoside;

SDS-PAGE electrophoresis: polyacrylamide gel electrophoresis;

Ni column: nickel column;

Binding buffer: nickel column purification protein buffer;

Elution buffer: elution buffer;

M: concentration unit, mol/L;

pET-32a: expression vector, fusion protein type prokaryotic high-efficiency expression vector, commercially available;

TE buffer (10mM Tris-HCl; 1mM EDTA): TE buffer, Tris-HCl concentration is 10mM, EDTA concentration is 1mM;

EcoR I: Restriction endonuclease, available on the market;

HindⅢ: Restriction endonuclease, commercially available;

T4 DNA Ligase: T4 DNA ligase, commercially available;

E.coli DH5α: Escherichia coli DH5α;

RosettaTM (DE3): Rosetta (DE3) E. coli expression strain;

APS: Astragalus polysaccharide;

BP5: bursin pentapeptide;

PBS: Phosphate buffered saline solution.

In order to achieve the above object, the technical solution provided by the present invention is: an immune enhancer, comprising bursin pentapeptide and astragalus polysaccharide; the weight ratio of the bursin pentapeptide and astragalus polysaccharide is 1:5 to 1:20.

It should be noted that the amino acid sequence of the bursin pentapeptide described herein is one or more tandem repeats of the amino acid sequence Cys-Lys-Arg-Val-Tyr, preferably 1-6 tandem repeats; In specific embodiments, five tandem repeats of the amino acid sequence Cys-Lys-Arg-Val-Tyr are used as the bursin pentapeptide used in the experiment.

In the existing theoretical inferences, the smaller the number of repetitive sequences, the more stable the structure of the protein can be maintained. Therefore, theoretically speaking, the protein immune enhancement effect of a single bursin pentapeptide sequence is the best.

In the aforementioned immune enhancer, the weight ratio of the bursin pentapeptide and astragalus polysaccharide is 1:15.

In the above-mentioned immune enhancer, it is composed of the following weight percentage components:

Bursin pentapeptide 1%;

Astragalus polysaccharide 15%;

The balance of water.

At the same time, the present invention also discloses an avian influenza vaccine, which contains the above-mentioned immune enhancer; the dosage of the immune enhancer in the avian influenza vaccine is 5-15 μl/feather.

It has been verified by experiments that within the dosage range of no less than 5μl/feather of the immune enhancer, it can achieve a substantial immune enhancement effect.

In the above-mentioned avian influenza vaccine, the antigen in the avian influenza vaccine is the H9N2 subtype avian influenza virus inactivated antigen or the H5N1 subtype avian influenza virus inactivated antigen.

In addition, the present invention also discloses a method for preparing the immune enhancer as described above, which is mixed and dissolved in water with cystin pentapeptide and astragalus polysaccharide.

In the preparation method of the above-mentioned immune enhancer, the following steps are included:

Step 1: Construct the gene of bursin pentapeptide;

Step 1 is specifically: taking 5'-TGCAAACGCGTGTAC-3' as the BP5 gene, adding EcoRI and HindⅢ restriction sites and protective bases before and after the fragment to obtain the fragment sequence F and the reverse complementary sequence R of the fragment sequence F. The reverse complement sequence R and the fragment sequence F constitute the nucleic acid fragment of the gene of bursin pentapeptide, the fragment length of the fragment sequence F is 93 bp, and the nucleic acid fragment of the gene of bursin pentapeptide is synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.

Fragment sequence F (refer to SEQ ID NO: 1 in the sequence list for details):

5'-AGC GAATTC (TGCAAACGCGTGTAC) ₅ AAGCTT CAC-3'

EcoR Ⅰ HindⅢ

The reverse complementary sequence R of the fragment sequence F (for details, please refer to the sequence list SEQ ID NO: 2):

5'-GTG AAGCTT (GTACACGCGTTTGCA) ₅ GAATTC GCT-3'

HindⅢ EcoRⅠ

The sequence of the EcoR Ⅰ restriction site is as follows: GAATTC;

The sequence of HindⅢ restriction site is shown as underlined: AAGCTT;

Step 2: Link the gene of bursin pentapeptide to the expression vector pET-32a to obtain the recombinant plasmid pET-32a-(BP5) ₅ ;

Step 2 specifically: Take 1μg of nucleic acid fragment and dissolve it in 50μL TE buffer solution, the buffer solution contains 10mM Tris-HCl and 1mM EDTA, and use EcoRI and HindⅢ endonuclease to double digestion of gene fragments; meanwhile, use EcoRⅠ and HindⅢ endonuclease for double digestion of pET-32a vector;

Mix the digested gene fragment and pET-32a vector, and add T4 DNA ligase to the mixed system to perform a ligation reaction; the ligation product is obtained after the ligation reaction is completed;

The ligation product was transformed into the standard E. coli strain E. coli DH5α, the positive colonies were verified with vector sequencing primers, and the positive clones were sequenced; the recombinant plasmid pET-32a-(BP5) _{5 with the} correct sequence was stored.

Step 3: Introduce the recombinant plasmid pET-32a-(BP5) ₅ into E. coli and induce expression to obtain an expression product;

Plasmid introduction: Take 5μL of recombinant plasmid pET-32a-(BP5) _{5 and} add it to 100μL of Escherichia coli engineered bacteria RosettaTM (DE3), mix well on ice for 30 minutes, and put the mixture in a water bath at 42°C for 90 s. After taking it out, quickly stand still on ice Leave for 3 minutes to complete the plasmid transformation to obtain the seed bacterial solution;

Induced expression: Take 0.1 mL of the seed bacteria liquid identified by PCR, the absorbance value of the seed bacteria liquid at 600nm wavelength OD ₆₀₀ ＝0.1, inoculate it into 5 mL LB liquid medium, shake culture at 37℃ to OD ₆₀₀ ＝0.4～0.6 At that time, IPTG was added to a final concentration of 1mM, and the expression was induced at 25°C for 6h. After centrifugation, the supernatant and the precipitate were collected and identified by SDS-PAGE electrophoresis. The product was present in the medium supernatant, and the expression was mainly soluble.

Step 4: Purify and dry the expression product to obtain bursin pentapeptide;

Product purification: Pack the Ni column, add slowly to the medium supernatant after the Binding buffer is equilibrated; after all samples flow through the Ni column, rinse with an appropriate amount of Binding buffer to remove impurities, and then slowly add the Elution buffer, and collect at the peak Eluent; the eluate is renatured by concentration gradient dialysis to obtain soluble (BP5) ₅ -thioredoxin with a size of about 22KDa; enterokinase is added to the purified product to remove thioredoxin to obtain (BP5) ₅ fusion Peptide; Ni column chromatography and dialysis refolding of the above-mentioned product (BP5) ₅ fusion peptide are performed again to obtain a high purity (BP5) ₅ fusion peptide; the product freeze-drying process is the bursin pentapeptide used in the present invention.

Step 5: Mix the bursin pentapeptide and astragalus polysaccharide and dissolve it in water for injection.

The amino acid sequence of the (BP5) ₅ fusion peptide prepared by the above method is (for details, please refer to the sequence list SEQ ID NO: 3):

COOH-E-F-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-K-L-NH2;

Namely: COOH-E-F-(C-K-R-V-Y)5-K-L-NH2

The repeating sequence is C-K-R-V-Y.

Finally, the present invention also discloses an application of the immune enhancer as described above, which is used as an immune enhancing adjuvant for avian influenza vaccine.

The beneficial effects of the present invention are:

The invention adopts the combined use of cystatin pentapeptide and astragalus polysaccharide, which has a synergistic effect on the effect, and the compatibility with the avian influenza vaccine can obtain an ideal immune effect.

Specifically, the avian influenza vaccine using the immune enhancer of the present invention has a 3 titer (log 2) higher than the avian influenza vaccine without the immune enhancer during the entire immune cycle. .

Description of the drawings

Figure 1 is a table of antibody levels of the H9N2 subtype avian influenza vaccine immunity test of the present invention;

Figure 2 is a table of antibody levels of the H5N1 subtype avian influenza vaccine immunity test of the present invention;

Figure 3 is a table of antibody levels of the H9N2 subtype avian influenza vaccine of the present invention with the addition of bursin pentapeptide immunoassay;

Figure 4 is a table of antibody levels of the H9N2 subtype avian influenza vaccine of the present invention added with Astragalus polysaccharides in the immune test;

Figure 5 is an SDS-PAGE electrophoresis diagram of the expression of recombinant (BP5) ₅ fusion peptide in E. coli.

Detailed ways

The present invention will be described below in conjunction with specific embodiments: but it can be understood that these specific embodiments are only used to illustrate the present invention, not to limit the present invention. Those skilled in the art can fully improve the specific embodiments or technical features of the present invention under the enlightenment of the present invention, but these improved or replaced technical solutions still belong to the protection scope of the present invention.

Example one

H9N2 subtype avian influenza vaccine

The components of 500 vaccines include:

3 parts oil phase (90ml), 2 parts water phase (60ml);

The oil phase contains 84.6ml white oil (EXonMobil), 5.4ml Spencer-80 (Zhaoqing Chaoneng Industrial Co., Ltd.);

The water phase contains 52.6ml H9N2 embryo fluid inactivated antigen (10 ⁷ EID ₅₀ /0.1mL, Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.), 2.4ml Tween-80 (Zhaoqing Super Energy Industrial Co., Ltd.), and 5ml immune enhancer.

The water phase contains 10μl/feather of the immune enhancer, and its components are: bursin pentapeptide 1wt%; astragalus polysaccharide 15wt%; and the balance water.

The preparation method of the immune enhancer is:

Step 1: Construct the gene of bursin pentapeptide;

Take 5'-TGCAAACGCGTGTAC-3' as the BP5 gene, and add EcoRI and HindⅢ restriction sites and protective bases before and after the fragment. The fragment length is 93 bp, which is synthesized by Bioengineering (Shanghai) Co., Ltd.

Fragment sequence F:

5'-AGC GAATTC (TGCAAACGCGTGTAC) ₅ AAGCTT CAC-3'

EcoR Ⅰ HindⅢ

Reverse complementary sequence R:

5'-GTG AAGCTT (GTACACGCGTTTGCA) ₅ GAATTC GCT-3'

HindⅢ EcoRⅠ

Step 2: Link the gene of bursin pentapeptide to the expression vector pET-32a to obtain the recombinant plasmid pET-32a-(BP5) ₅ ;

Take 1μg of the nucleic acid fragment and dissolve it in 50μL TE buffer (10mM Tris-HCl; 1mM EDTA), digest with EcoRI and HindⅢ enzymes, digest the pET-32a vector with the same enzymes; mix the digested fragments with the vector and use T4 DNA Ligase ligation; the ligation product is transformed into E.coli DH5α, the positive colonies are verified with vector sequencing primers, and the positive clones are sequenced; the recombinant plasmid pET-32a-(BP5) _{5 with the} correct sequence is stored.

Step 3: Introduce the recombinant plasmid pET-32a-(BP5) ₅ into E. coli and induce expression to obtain an expression product;

Plasmid introduction: Take 5μL of recombinant plasmid pET-32a-(BP5) _{5 and} add it to 100μL of Escherichia coli engineered bacteria RosettaTM (DE3), mix well on ice for 30 minutes, and put the mixture in a water bath at 42°C for 90 s. After taking it out, quickly stand still on ice Leave for 3 min to complete plasmid transformation.

Induction of expression: Take 0.1 mL of the seed bacteria liquid (OD ₆₀₀ ＝0.1) that is positive by PCR, inoculate it into 5 mL LB liquid medium, culture with shaking at 37°C to OD ₆₀₀ ＝0.4～0.6, add IPTG to the final concentration of 1mM, Induce expression at 25℃ for 6h. After centrifugation, the supernatant and the precipitate were collected and identified by SDS-PAGE electrophoresis. The product was present in the culture supernatant and was mainly expressed in soluble form. The results are shown in Figure 5. The meaning of each symbol in Figure 5 is: M: protein molecular weight standard; 1: culture supernatant; 2: bacterial pellet; 3: negative control;

Step 4: Purify and dry the expression product to obtain bursin pentapeptide;

Product purification: Pack the Ni column, add the culture supernatant after the Binding buffer is equilibrated; after all the samples flow through the Ni column, rinse with an appropriate amount of Binding buffer to remove impurities, and then slowly add the Elution buffer, collect and elute at the peak The eluate is renatured by concentration gradient dialysis to obtain soluble BP5-thioredoxin with a size of about 22KDa; enterokinase is added to the purified product to remove thioredoxin to obtain (BP5) ₅ fusion peptide; The product is subjected to Ni column chromatography and dialysis refolding to obtain a high-purity (BP5) ₅ fusion peptide; the product freeze-drying process is the bursin pentapeptide used in the present invention.

Step 5: Mix the bursin pentapeptide and astragalus polysaccharide and dissolve it in water for injection.

The BP5 used in the following examples, comparative examples, and immunoassays are all the bursin pentapeptide prepared in step 4 of this example.

Example 2

The H9N2 subtype avian influenza vaccine is substantially the same as in Example 1. The immune enhancer components are: bursin pentapeptide 1 wt%; astragalus polysaccharide 5 wt%; the balance water. The immune enhancer in the vaccine is 15μl/feather.

Example 3

The H9N2 subtype avian influenza vaccine is roughly the same as in Example 1. The immune enhancer components are: cystin pentapeptide 1wt%; astragalus polysaccharide 20wt%; the balance water. The immune booster in the vaccine is 5μl/feather.

Example 4

H5N1 subtype avian influenza vaccine

The components of 500 vaccines include:

3 parts oil phase (90ml), 2 parts water phase (60ml);

The oil phase contains 84.6ml white oil (EXonMobil), 5.4ml Spencer-80 (Zhaoqing Chaoneng Industrial Co., Ltd.);

The water phase contains 52.6ml H5N1 embryo fluid inactivated antigen (10 ⁸ EID ₅₀ /0.1mL, Guangdong Wenshi Dahuanong Biotechnology Co., Ltd.), 2.4ml Tween-80 (Zhaoqing Super Energy Industrial Co., Ltd.), 5ml immune enhancer .

The water phase contains 10μl/feather of the immune enhancer, and its components are: bursin pentapeptide 1wt%; astragalus polysaccharide 15wt%; and the balance water. The preparation method of the immune enhancer is the same as in Example 1.

Comparative example 1

The H9N2 subtype avian influenza vaccine is basically the same as in Example 1. The immune enhancer does not contain astragalus polysaccharides, and its components are: cystin pentapeptide 1 wt%; the balance water.

Comparative example 2

The H9N2 subtype avian influenza vaccine is roughly the same as in Example 1. The immune enhancer does not contain cystin pentapeptide, and its components are: its components are: astragalus polysaccharide 15wt%; the balance is water.

1. Immunization test of H9N2 subtype avian influenza vaccine

1.1 Animal experiment grouping

40 7-day-old SPF chickens were randomly divided into four groups, each with 10 chickens: A: conventional H9N2 subtype inactivated avian influenza vaccine, subcutaneous injection on the back of the neck, 0.3ml/feather; B: inactivated vaccine + APS-BP5 (Example 1); C: APS-BP5 control group (10μl/0.3ml/feather, APS-BP5 concentration content is the same as in Example 1); D: PBS control group (each immunized 0.3ml/feather).

1.2 Sample collection

Before immunization and 1-10 weeks after immunization, 1 mL of blood was collected from each chicken wing vein every week, centrifuged at 3000 rpm for 10 minutes to separate serum, and stored at -20°C.

1.3 Results

Determination of antibody levels. The specific antibody level was detected by the hemagglutination inhibition test (HI test). As shown in Figure 1, 3 weeks after immunization, the avian influenza virus (H9) antibody level of the APS-BP5 vaccine group began to be significantly higher than that of the conventional vaccine group (p <0.01), and continued until the end of the test. From 3 weeks after immunization to the end of the test, the antibody level of the APS-BP5 vaccine group was always higher than that of the conventional vaccine group by more than 3 titers. In the fourth week, its antibody level was higher than that of the conventional vaccine group by 7 titers. The degree is greater than 11 (log2).

2. Immunization test of H9N2 subtype avian influenza vaccine bursin pentapeptide adjuvant

2.1 Animal experiment grouping

40 7-day-old SPF chickens were randomly divided into four groups, each with 10 chickens: A: conventional H9N2 subtype inactivated avian influenza vaccine, subcutaneous injection at the back of the neck, 0.3ml/feather; B: inactivated vaccine + BP5 (right Proportion 1); C: BP5 control group (10μl/0.3ml/feather, the concentration of BP5 is the same as that of Comparative Example 1); D: PBS control group (each immunized 0.3ml/feather).

2.2 Sample collection

Before immunization and 1-10 weeks after immunization, 1 mL of blood was collected from each chicken wing vein every week, centrifuged at 3000 rpm for 10 minutes to separate serum, and stored at -20°C.

2.3 Results

Determination of antibody levels. The level of specific antibodies was detected by the hemagglutination inhibition test (HI test), as shown in Figure 2. 2 weeks after immunization, the level of avian influenza virus (H9) antibodies in the bursin pentapeptide adjuvant vaccine group began to gradually be higher than that of the conventional vaccine Group (p<0.01), and continued until the end of the experiment. From 3 weeks after immunization to the end of the experiment, the antibody level of the bursin pentapeptide vaccine group was always higher than the conventional vaccine group by about 2 titers.

3. Immunization test of astragalus polysaccharide adjuvant for H9N2 subtype avian influenza vaccine

3.1 Animal experiment grouping

40 7-day-old SPF chickens were randomly divided into four groups, each with 10 chickens: A: conventional H9N2 subtype inactivated avian influenza vaccine, subcutaneous injection at the back of the neck, 0.3ml/feather; B: inactivated vaccine + APS (right Proportion 2); C: APS control group (10μl/0.3ml/feather, the APS concentration content is the same as that of Comparative Example 2); D: PBS control group (each immunized 0.3ml/feather).

3.2 Sample collection

Before immunization and 1-10 weeks after immunization, 1 mL of blood was collected from the vein of each chicken wing every week, centrifuged at 3000 rpm for 10 minutes to separate the serum, and stored at -20°C.

3.3 Results

Determination of antibody levels. The specific antibody level was detected by the hemagglutination inhibition test (HI test), as shown in Figure 3. After immunization, the avian influenza virus (H9) antibody level of the astragalus polysaccharide adjuvant vaccine group was slightly higher than that of the conventional vaccine group, and it could continue To the end of the test.

4. Immunization test of H5N1 subtype avian influenza vaccine

4.1 Animal experiment grouping

40 7-day-old SPF chickens were randomly divided into four groups, each with 10 chickens: A: conventional H5N1 subtype inactivated avian influenza vaccine, subcutaneous injection at the back of the neck, 0.3ml/feather; B: H5N1 inactivated vaccine + APS- BP5 (Example 4); C: APS-BP5 control group (10μl/0.3ml/feather, the concentration of APS-BP5 is the same as in Example 4); D: PBS control group (each immunized 0.3ml/feather)

4.2 Sample collection

Before immunization and 1-10 weeks after immunization, 1 mL of blood was collected from the vein of each chicken wing every week, centrifuged at 3000 rpm for 10 minutes to separate the serum, and stored at -20°C.

4.3 Results

Determination of antibody levels. The specific antibody level was detected by the hemagglutination inhibition test (HI test). As shown in Figure 4, 2 weeks after immunization, the avian influenza virus (H5) antibody level of the APS-BP5 vaccine group began to be significantly higher than that of the conventional vaccine group (p <0.01), and continued until the end of the test. From 3 weeks after immunization to the end of the test, the antibody level of the APS-BP5 vaccine group was always higher than the conventional vaccine group by more than 3 titers.

Through the above immunization test, we can get the following conclusions:

Bursalin (BP) is a small molecule peptide that exists in the bursa of poultry and is closely related to immune function. Bursin pentapeptide (BP5) is a member of the bursin family. It can induce the differentiation and proliferation of poultry B lymphocyte precursors, increase antibody levels, and enhance humoral immunity; promote the transformation activity of T lymphocytes, thereby enhancing cellular immunity .

Polysaccharide compound is a kind of good immunomodulator, and its role in improving and improving the immune function of animals has attracted more and more attention. Astragalus polysaccharide is the main bioactive component in the root extract of Astragalus. Studies have shown that Astragalus polysaccharide can stimulate the activity of macrophages, promote the proliferation of T cells, promote the expression of surface antigens in lymphocytes, induce strong humoral and cellular immune responses, and play an important role in non-specific and specific immune responses. effect. In general, Astragalus polysaccharide has multiple functions such as immune regulation, improvement of immune response, and alleviation of immune dysfunction caused by environmental stress.

The combined use of the two immune enhancers has a synergistic effect on the effect, and the combination with the avian flu vaccine can obtain an ideal immune effect.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

1. An immune enhancer, characterized in that it comprises bursin pentapeptide and astragalus polysaccharide; the weight ratio of the bursin pentapeptide and astragalus polysaccharide is 1:5 to 1:20.
2. The immune enhancer according to claim 1, wherein the weight ratio of the bursin pentapeptide and astragalus polysaccharide is 1:15.
3. The immune enhancer according to claim 1 or 2, characterized in that it is composed of the following weight percentage components:

   Bursin pentapeptide 1%;

   Astragalus polysaccharide 15%;

   The balance of water.
4. An avian influenza vaccine, characterized in that: the vaccine contains the immune enhancer according to any one of claims 1-3; the dosage of the immune enhancer in the avian influenza vaccine is 5-15 μl/feather.
5. The avian influenza vaccine according to claim 4, wherein the antigen in the avian influenza vaccine is an H9N2 subtype avian influenza virus inactivated antigen or an H5N1 subtype avian influenza virus inactivated antigen.
6. A method for preparing the immune enhancer according to any one of claims 1 to 3, characterized in that: bursin pentapeptide and astragalus polysaccharide are mixed and dissolved in water.
7. The preparation method of the immune enhancer according to claim 6, characterized in that it comprises the following steps:

   Step 1: Construct the gene of bursin pentapeptide;

   Step 2: Link the gene of bursin pentapeptide to the expression vector pET-32a to obtain the recombinant plasmid pET-32a-(BP5) ₅ ;

   Step 3: Introduce the recombinant plasmid pET-32a-(BP5) ₅ into E. coli and induce expression to obtain an expression product;

   Step 4: Purify and dry the expression product to obtain bursin pentapeptide;

   Step 5: Mix the bursin pentapeptide and astragalus polysaccharide and dissolve it in water for injection.
8. The method for preparing an immune enhancer according to claim 7, wherein the step 1 and step 2 are specifically:

   Step 1: Using 5'-TGCAAACGCGTGTAC-3' as the BP5 gene, add EcoR Ⅰ and Hind Ⅲ restriction sites and protective bases before and after the fragment to obtain the fragment sequence F. The fragment length is 93 bp. It is produced by Shenggong Biotech (Shanghai) Limited company synthesis and provide;

   Step 2: Take 1μg of the nucleic acid fragment and dissolve it in 50μL TE buffer, and digest it with EcoR I and Hind III enzymes to obtain the digested fragments;

   Digest the pET-32a vector with EcoR Ⅰ and Hind Ⅲ enzymes;

   The digested fragment is mixed with the pET-32a vector after the enzyme digestion treatment, and the digested fragment and pET-32a vector are ligated with T4 DNA Ligase to obtain the ligation product;

   The ligation product was transformed into E.coli DH5α, the positive colonies were verified with vector sequencing primers, and the positive clones were sequenced; the recombinant plasmid pET-32a-(BP5) _{5 with the} correct sequence was stored.
9. The method for preparing an immune enhancer according to claim 8, wherein the step 3 is specifically:

   Plasmid introduction: Take the recombinant plasmid pET-32a-(BP5) _{5 and} add it to Escherichia coli RosettaTM (DE3), mix well on ice, put the mixture in a water bath at 42°C, take it out, and quickly stand on ice to complete the plasmid transformation. Obtain seed bacteria liquid;

   Induced expression: Take the seed bacteria liquid identified by PCR and inoculate it into the culture medium. When it is cultured to OD ₆₀₀ ＝0.4～0.6, add IPTG and induce expression at 25°C; after identification, the product exists in the supernatant of the culture medium. Soluble is expressed mainly.
10. An application of the immune enhancer according to any one of claims 1 to 3, which is used as an immune enhancer adjuvant for avian influenza vaccine.

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