CN109966484B - Immunopotentiator, preparation method, avian influenza vaccine and application - Google Patents
Immunopotentiator, preparation method, avian influenza vaccine and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biological products for animals, and discloses an immunopotentiator which comprises a cyst element pentapeptide and astragalus polysaccharide; the weight ratio of the capsaicine pentapeptide to the astragalus polysaccharide is 1:5-1:20; the immunopotentiator can effectively improve the antibody titer expression level of vaccine immunity. The immunopotentiator is simple to prepare and has good industrial application prospect, and meanwhile, the invention also discloses a preparation method of the immunopotentiator, an avian influenza vaccine adopting the immunopotentiator and application of the immunopotentiator in the aspect of avian influenza vaccine.
Description
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to an immunopotentiator, a preparation method, an avian influenza vaccine and application.
Background
Avian influenza is an important infectious disease caused by avian influenza virus, seriously jeopardizing the development of chicken industry. Vaccine immunization is one of the most effective means of controlling epidemic diseases. However, with the popularization and use of vaccines, viruses are continuously mutated under the selection pressure of the vaccines, so that the virulence is enhanced, and challenges are brought to the prevention and control of epidemic diseases. Solves the above dilemma, and is clinically treated by continuously replacing vaccine strains. For the prevention and treatment work of the avian influenza, the replacement of vaccine strains can have an immediate effect in a short time, but in the long term, the variation speed of the avian influenza virus is accelerated, and the prevention and treatment are in a circulatory dilemma.
The immunopotentiator can improve the immune system function of the organism and strengthen the nonspecific immunity of the organism to diseases, and the proper immunopotentiator is selected to be used together with the vaccine, so that the immunopotentiator is one of effective ways for enhancing the immune efficacy of the existing vaccine. The vaccine immunity efficacy is improved, which means that fewer vaccines can have better effects, the clinical application can reduce the number of immunization times and the stress, thereby saving the cost and effectively controlling diseases.
Bursin (BP) is a small molecule skin substance found in the bursa of Fabricius of birds and has a close relationship with immune function. The bursin pentapeptide (BP 5) is one of bursin families, and has the functions of inducing the differentiation and proliferation of B lymphocyte precursors of poultry, improving the antibody level and enhancing humoral immunity; promoting the transformation activity of T lymphocytes, thereby enhancing the effect of cellular immunity.
Two polypeptides of the bursin family that are representative are BP-14 and BP-5.
Regarding BP-14, the national academy of science and technology of agriculture and livestock of the right Jiangsu in 2013 applied for Chinese patent CN201310413608.2 discloses a polypeptide, which has the amino acid sequence:
COOH-G-H-K-T-R-N-D-P-L-K-G-A-V-D-NH2。
the effect is according to the description: adding an immunopotentiator BP14 into the livestock and poultry vaccine according to different dosages of different target animals for joint inoculation; the antibody level of the immunized animals is higher than that of the vaccine group; the subject animals produced higher levels of cytokines. The dosage of the poultry is 0.1 mug/serving and the pig is 5 mug/serving; referring to FIG. 1 thereof, BP-14+ vaccine (H5N 1 subtype, re-6 strain) was used with a titer of 5-8log2.
Regarding BP-5, the applicant Li Deyuan applied for Chinese patent CN200810243575.0 in 2008 discloses a bursal pentapeptide, which has the amino acid structure sequence as follows: CYS-LYS-ASP-VAL-TYR. The bursa of Fabricius pentapeptide (BP 5) has the advantages of simple structure, small molecular weight, no chemical toxicity, no immunogenicity and simple preparation, can be extracted from bursa of Fabricius of chickens or other birds, can be chemically synthesized, has low cost during chemical synthesis, and can be prepared in a large scale. The bursa of Fabricius pentapeptide (BP 5) can promote proliferation of T lymphocytes and B lymphocytes, improve the humoral immunity and cellular immunity level of an organism, and also can improve the peroxidation stress resistance of the organism, and is a medicament or preparation with wide application prospect and immunoregulation, immunotherapy and peroxidation resistance. Can be used in the fields of basic research, clinical treatment, nutrition and health care, beauty and cosmetics, etc.
This patent describes the potential use of bursa pentapeptides in immunotherapy and immunomodulation, but does not mention use in avian influenza vaccines.
The patent CN201310069941.6 of the university of Henan science and technology of the right people applied for China in 2013 discloses recombinant T alpha 1-BP5 fusion peptide, gene, engineering bacteria and application. The recombinant fusion peptide is formed by fusing thymosin alpha 1 and Fabricius bursa pentapeptide BP5 through a flexible Linker. The recombinant T alpha 1-BP5 fusion peptide gene is inserted into an expression vector, escherichia coli is transformed, the genetically engineered bacterium for efficiently expressing the recombinant T alpha 1-BP5 fusion peptide is obtained, the recombinant T alpha 1-BP5 fusion peptide is prepared through liquid culture and purification, thioredoxin is removed from the fusion peptide through enterokinase with an N-terminal His tag, and the fusion peptide is purified through affinity chromatography, so that the single recombinant T alpha 1-BP5 fusion peptide can be obtained. The recombinant T alpha 1-BP5 fusion peptide can be used as a novel polypeptide immunoadjuvant matched with vaccine, can effectively enhance cellular immunity and humoral immunity level of organisms, and has wide application prospect.
The invention is based on bursa of Fabricius pentapeptide BP5, and performs gene recombination expression with thymosin alpha 1, thereby achieving the purpose of enhancing immunity.
From the above, BP5 was found to have a very large development potential for immune enhancement. Research and development in this area is under intense study.
The present invention was therefore based on the object of carrying out a more intensive study on BP5 and proposing a new formulation for the application of BP5 in order to improve the vaccine immune effect.
Disclosure of Invention
The invention aims to provide an immunopotentiator, and simultaneously discloses a preparation method of the immunopotentiator, an avian influenza vaccine adopting the immunopotentiator and application of the immunopotentiator in the aspect of avian influenza vaccine. The immunopotentiator can effectively improve the antibody titer expression level of vaccine immunity. The immunopotentiator is simple to prepare and has good industrial application prospect.
Before describing the scheme of the invention, the following description is necessary:
LB liquid medium: luria-Bertani liquid Medium;
IPTG: isopropyl Thiogalactoside, isopropyless-D-Thiogalactoside;
SDS-PAGE electrophoresis: polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis;
ni column: a nickel column;
binding buffer: purifying protein buffer solution by a nickel column;
an execution buffer: eluting the buffer;
m: concentration unit, mol/L;
pET-32a: expression vectors, fusion protein type prokaryotic high-efficiency expression vectors, are commercially available;
TE buffer (10 mM Tris-HCl;1mM EDTA): TE buffer, tris-HCl concentration 10mM, EDTA concentration 1mM;
EcoRI: restriction endonucleases, commercially available;
hind III: restriction endonucleases, commercially available;
t4 DNA Ligase: t4 DNA ligase, commercially available;
coli DH 5. Alpha: coli DH 5. Alpha;
rosetta (DE 3): rosetta (DE 3) E.coli expression strain;
APS: astragalus polysaccharide;
BP5: a capsulorhein pentapeptide;
PBS: phosphate buffered saline.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows: an immunopotentiator comprises a capsulorhein and astragalus polysaccharide; the weight ratio of the capsaicine pentapeptide to the astragalus polysaccharide is 1:5-1:20.
It should be noted that: the amino acid sequence of the capsaicine pentapeptide is one or more tandem repeats of amino acid sequence Cys-Lys-Arg-Val-Tyr, preferably 1-6 tandem repeats; in the specific examples herein, 5 tandem repeats of the amino acid sequence Cys-Lys-Arg-Val-Tyr were used as the experimental capsulopent.
In the existing theory, the smaller the number of repeated sequences, the more stable the structure of the protein can be maintained, so that the effect of enhancing the protein immunity of the single bursin pentapeptide sequence is the best in theory.
In the immunopotentiator, the weight ratio of the capsulopent to the astragalus polysaccharide is 1:15.
The immunopotentiator comprises the following components in percentage by weight:
a capsulorhein pentapeptide 1%;
15% of astragalus polysaccharide;
the balance of water.
Meanwhile, the invention also discloses an avian influenza vaccine, which contains the immunopotentiator; the dosage of the immunopotentiator in the avian influenza vaccine is 5-15 μl/serving.
Experiments prove that the immunopotentiator can play a substantial immunopotentiator effect within the dosage range of not less than 5 mu l/plume.
In the avian influenza vaccine, the antigen in the avian influenza vaccine is an inactivated antigen of H9N2 subtype avian influenza virus or an inactivated antigen of H5N1 subtype avian influenza virus.
In addition, the invention also discloses a preparation method of the immunopotentiator, which is characterized in that the capsaicine pentapeptide and the astragalus polysaccharide are mixed and dissolved in water.
The preparation method of the immunopotentiator comprises the following steps:
step 1: constructing a gene of a bursin pentapeptide;
the step 1 specifically comprises the following steps: 5'-TGCAAACGCGTGTAC-3' is taken as BP5 gene, ecoRI and HindIII restriction enzyme sites and protective bases are respectively added before and after the fragment to obtain a fragment sequence F and a reverse complementary sequence R of the fragment sequence F, the reverse complementary sequence R and the fragment sequence F form a nucleic acid fragment of the gene of the bursin pentapeptide, the fragment length of the fragment sequence F is 93BP, and the nucleic acid fragment of the gene of the bursin pentapeptide is synthesized by a division of biological engineering (Shanghai) Co.
Fragment sequence F (specific reference can be made to sequence table SEQ ID NO: 1):
reverse complement sequence R of fragment sequence F (specific reference can be made to sequence table SEQ ID NO: 2):
the sequence of the EcoRI cleavage site is shown underlined: GAATTC;
the sequence of the HindIII cleavage site is shown underlined: AAGCTT;
step 2: connecting the gene of the bursin pentapeptide to an expression vector pET-32a to obtain a recombinant plasmid pET-32a- (BP 5) 5 ；
The step 2 is specifically as follows: 1. Mu.g of the nucleic acid fragment was dissolved in 50. Mu.L of TE buffer containing 10mM Tris-HCl and 1mM EDTA, and the gene fragment was subjected to double digestion with EcoRI and HindIII endonucleases; simultaneously, double enzyme digestion treatment is carried out on the pET-32a vector by using EcoRI and HindIII endonucleases;
mixing the digested gene fragment and the pET-32a vector, and adding T4 DNA ligase into the mixed system for connection reaction; obtaining a connection product after the connection reaction is finished;
the ligation product is transformed into E.coli DH5 alpha, positive colonies are verified by using a vector sequencing primer, and the positive clones are sequenced; recombinant plasmid pET-32a- (BP 5) with correct preservation sequence 5 。
Step 3: recombinant plasmid pET-32a- (BP 5) 5 Introducing into escherichia coli for induction expression to obtain an expression product;
plasmid introduction: taking 5 mu L of recombinant plasmid pET-32a-(BP5) 5 Adding into 100 μl engineering bacteria Rosetta (DE 3) of Escherichia coli, mixing on ice for 30min, standing for 3min after taking out in water bath at 42deg.C for 90s, and completing plasmid transformation to obtain seed bacterial liquid;
induction of expression: taking 0.1mL of seed bacterial liquid with positive PCR identification, and the light absorption value OD of the seed bacterial liquid at 600nm wavelength 600 =0.1, inoculated into 5mL of LB liquid medium, shake-cultured at 37 ℃ to OD 600 When=0.4 to 0.6, IPTG was added to a final concentration of 1mM and induced to express at 25 ℃ for 6h. After centrifugation, the supernatant and pellet were collected and identified using SDS-PAGE electrophoresis, the product was present in the medium supernatant and was predominantly expressed as soluble.
Step 4: purifying and drying the expression product to obtain the capsaicine pentapeptide;
and (3) purifying a product: filling a Ni column, and slowly adding a culture medium supernatant after Binding buffer balancing; after all samples flow through the Ni column, washing with a proper amount of Binding buffer to remove the impurity protein, slowly adding the washing buffer, and collecting the eluent at the peak value; the eluent is subjected to concentration gradient dialysis renaturation to obtain solubility (BP 5) 5 Thioredoxin, about 22KDa in size; adding enterokinase to the purified product, and removing thioredoxin to obtain (BP 5) 5 Fusion peptides; the above product (BP 5) was again treated 5 The fusion peptide is subjected to Ni column chromatography and dialysis renaturation to obtain high purity (BP 5) 5 Fusion peptides; the product is freeze-dried to obtain the capsaicine pentapeptide used in the invention.
Step 5: mixing and dissolving the capsaicine pentapeptide and astragalus polysaccharide in water for injection.
Obtained by the method (BP 5) 5 The amino acid sequence of the fusion peptide is (specific reference can be made to the sequence table 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 repeated sequence is C-K-R-V-Y.
Finally, the invention also discloses an application of the immunopotentiator as any one of the above, which is used as an immunopotentiator adjuvant of the avian influenza vaccine.
The beneficial effects of the invention are as follows:
the invention adopts the matching use of the bursin pentapeptide and the astragalus polysaccharide, plays a synergistic effect in effect, and can obtain ideal immune effect when being matched with the avian influenza vaccine.
Specifically, the avian influenza vaccine using the immunopotentiator of the present invention will express 3 titers (log 2) higher in immune expression compared to an avian influenza vaccine without immunopotentiator added throughout the immune cycle.
Drawings
FIG. 1 is a diagram showing the variation of antibodies in an immunoassay for an H9N2 subtype avian influenza vaccine of the present invention with the addition of a bursin pentapeptide fusion peptide and astragalus polysaccharide;
FIG. 2 is a diagram showing the variation of antibodies in the immunity test of the H9N2 subtype avian influenza vaccine supplemented with a bursin pentapeptide fusion peptide of the present invention;
FIG. 3 is a diagram showing the variation of antibodies in an immune test of an H9N2 subtype avian influenza vaccine of the present invention with astragalus polysaccharide;
FIG. 4 is a diagram showing the variation of antibodies in an immunoassay of an H5N1 subtype avian influenza vaccine of the present invention with astragalus polysaccharide and a bursin pentapeptide fusion peptide;
FIG. 5 is a diagram of recombination (BP 5) 5 SDS-PAGE electrophoresis of expression of fusion peptides in E.coli.
Detailed Description
The invention is described below in connection with specific embodiments: it is to be understood that these specific embodiments are merely illustrative of the invention and are not limiting thereof. Modifications of the specific embodiments or technical features of the present invention will be apparent to those skilled in the art from the teachings of the present invention, and such modified or alternative embodiments fall within the scope of the present invention.
Example 1
H9N2 subtype avian influenza vaccine
The components of the 500-feather vaccine specifically comprise:
3 parts of oil phase (90 ml), 2 parts of water phase (60 ml);
the oil phase contained 84.6ml white oil (EXonMobil Co.) and 5.4ml span-80 (Zhaoqing Utility Co., ltd.);
the aqueous phase contained 52.6ml of H9N2 embryo solution inactivating antigen (10) 7 EID 50 0.1mL, zhaoqing Dahua agricultural chemicals Co., ltd.), 2.4mL Tween-80 (Zhaoqing super energy industries Co., ltd.), 5mL immunopotentiator.
The aqueous phase contains 10 μl/plume of immunopotentiator, and comprises the following components: 1wt% of a capsulorhein; 15wt% of astragalus polysaccharide; the balance of water.
The preparation method of the immunopotentiator comprises the following steps:
step 1: constructing a gene of a bursin pentapeptide;
5'-TGCAAACGCGTGTAC-3' is taken as BP5 gene, ecoRI and HindIII restriction enzyme sites and protective bases are respectively added before and after the fragment, the fragment length is 93BP, and the fragment is synthesized by the biological engineering (Shanghai) Co., ltd.
Fragment sequence F:
reverse complement sequence R:
step 2: connecting the gene of the bursin pentapeptide to an expression vector pET-32a to obtain a recombinant plasmid pET-32a- (BP 5) 5 ；
1. Mu.g of the nucleic acid fragment was dissolved in 50. Mu.L of TE buffer (10 mM Tris-HCl;1mM EDTA), digested with EcoRI and HindIII enzymes, and treated with the same enzymatic digestion to obtain pET-32a vector; mixing the digested fragments with a carrier, and connecting the fragments by using T4 DNA Ligase; transforming the connection product into E.coli DH5 alpha, verifying positive colonies by using a vector sequencing primer, and sequencing the positive colonies; recombinant plasmid pET-32a- (BP 5) with correct preservation sequence 5 。
Step 3: recombinant plasmid pET-32a- (BP 5) 5 Is introduced into escherichia coli for induction expression to obtainAn expression product;
plasmid introduction: taking 5 mu L of recombinant plasmid pET-32a- (BP 5) 5 Adding into 100 μl engineering bacteria Rosetta (DE 3) of Escherichia coli, mixing thoroughly on ice for 30min, standing for 3min after taking out in water bath at 42deg.C for 90s, and completing plasmid transformation.
Induction of expression: 0.1mL (OD) of seed bacterial liquid positive for PCR identification is taken 600 =0.1), inoculated into 5mL of LB liquid medium, shake-cultured at 37 ℃ to OD 600 When=0.4 to 0.6, IPTG was added to a final concentration of 1mM and induced to express at 25 ℃ for 6h. After centrifugation, the supernatant and pellet were collected and identified by SDS-PAGE electrophoresis, and the product was present in the culture supernatant, mainly in soluble expression, and the results are shown in FIG. 5. The meaning of each symbol in fig. 5 is: m: protein molecular weight standard; 1: culturing the supernatant; 2: sedimentation of thalli; 3: a negative control;
step 4: purifying and drying the expression product to obtain the capsaicine pentapeptide;
and (3) purifying a product: filling a Ni column, and slowly adding culture supernatant after Binding buffer balancing; after all samples flow through the Ni column, washing with a proper amount of Binding buffer to remove the impurity protein, slowly adding the washing buffer, and collecting the eluent at the peak value; the eluent is subjected to concentration gradient dialysis renaturation to obtain soluble BP 5-thioredoxin with the size of about 22kDa; adding enterokinase to the purified product, and removing thioredoxin to obtain (BP 5) 5 Fusion peptides; subjecting the above product to Ni column chromatography and dialysis renaturation again to obtain high purity (BP 5) 5 Fusion peptides; the product is freeze-dried to obtain the capsaicine pentapeptide used in the invention.
Step 5: mixing and dissolving the capsaicine pentapeptide and astragalus polysaccharide in water for injection.
BP5 used in the following examples, comparative examples and immunoassays was the bursin pentapeptide prepared in step 4 of this example.
Example 2
An H9N2 subtype avian influenza vaccine substantially as in example 1, immunopotentiator composition: 1wt% of a capsulorhein; astragalus polysaccharide 5wt%; the balance of water. The immunopotentiator in the vaccine was 15 μl/plume.
Example 3
An H9N2 subtype avian influenza vaccine substantially as described in example 1. The immunopotentiator comprises the following components: 1wt% of a capsulorhein; 20wt% of astragalus polysaccharide; the balance of water. The immunopotentiator in the vaccine was 5 μl/plume.
Example 4
H5N1 subtype avian influenza vaccine
The components of the 500-feather vaccine specifically comprise:
3 parts of oil phase (90 ml), 2 parts of water phase (60 ml);
the oil phase contained 84.6ml white oil (EXonMobil Co.) and 5.4ml span-80 (Zhaoqing Utility Co., ltd.);
the aqueous phase contained 52.6ml of h5n1 embryo liquid inactivating antigen (10 8 EID 50 0.1mL, guangdong Wen Shida Huanong Biotechnology Co., ltd.), 2.4mL Tween-80 (Zhaoqing Utility Co., ltd.), 5mL immunopotentiator.
The aqueous phase contains 10 μl/plume of immunopotentiator, and comprises the following components: 1wt% of a capsulorhein; 15wt% of astragalus polysaccharide; the balance of water. The immunopotentiator was prepared in the same manner as in example 1.
Comparative example 1
An H9N2 subtype avian influenza vaccine substantially as in example 1, wherein the immunopotentiator does not contain astragalus polysaccharide, and comprises the following components: 1wt% of a capsulorhein; the balance of water.
Comparative example 2
An H9N2 subtype avian influenza vaccine substantially as described in example 1. The immunopotentiator does not contain the bursin pentapeptide, and comprises the following components: the components are as follows: 15wt% of astragalus polysaccharide; the balance of water.
1. H9N2 subtype avian influenza vaccine immunoassay
1.1 animal Experimental groups
40 SPF chickens of 7 days of age were randomly divided into four groups of 10: a: conventional H9N2 subtype inactivated avian influenza vaccine is subcutaneously injected at the nape of the neck, 0.3 ml/feather; b: inactivated vaccine + APS-BP5 (example 1); c: APS-BP5 control group (10. Mu.l/0.3 ml/feather, APS-BP5 concentration content same as in example 1); d: PBS control (0.3 ml/plume per immunization).
1.2 sample collection
Blood was collected 1mL per chicken wing vein every week at 1-10 weeks before and after immunization, and serum was separated by centrifugation at 3000rpm for 10min and frozen at-20 ℃.
1.3 results
Antibody level determination. Specific antibody levels were tested by the hemagglutination inhibition assay (HI assay), as shown in fig. 1, and the avian influenza virus (H9) antibody levels of the APS-BP5 vaccine group began to be significantly higher than that of the conventional vaccine group (p < 0.01) 3 weeks after immunization, and continued until the end of the assay. The APS-BP5 vaccine group had an antibody level consistently above 3 titers in the conventional vaccine group and 7 titers in the fourth week from 3 weeks post immunization to the end of the test, with a peak antibody titer of greater than 11 (log 2).
2. H9N2 subtype avian influenza vaccine bursin pentapeptide adjuvant immunoassay
2.1 animal Experimental groups
40 SPF chickens of 7 days of age were randomly divided into four groups of 10: a: conventional H9N2 subtype inactivated avian influenza vaccine is subcutaneously injected at the nape of the neck, 0.3 ml/feather; b: inactivated vaccine + BP5 (comparative example 1); c: BP5 control group (10. Mu.l/0.3 ml/feather, BP5 concentration content same as comparative example 1); d: PBS control (0.3 ml/plume per immunization).
2.2 sample collection
Blood was collected 1mL per chicken wing vein every week at 1-10 weeks before and after immunization, and serum was separated by centrifugation at 3000rpm for 10min and frozen at-20 ℃.
2.3 results
Antibody level determination. Specific antibody levels were tested by the hemagglutination inhibition assay (HI assay), as shown in figure 2, with the avian influenza virus (H9) antibody levels of the bursin pentapeptide adjuvant vaccine group beginning progressively higher than the conventional vaccine group (p < 0.01) 2 weeks after immunization and continuing until the end of the assay. The antibody levels of the bursin pentapeptide vaccine group were consistently higher than the conventional vaccine group by about 2 titers from 3 weeks after immunization to the end of the trial.
3. H9N2 subtype avian influenza vaccine astragalus polysaccharide adjuvant immunity test
3.1 animal Experimental group
40 SPF chickens of 7 days of age were randomly divided into four groups of 10: a: conventional H9N2 subtype inactivated avian influenza vaccine is subcutaneously injected at the nape of the neck, 0.3 ml/feather; b: inactivated vaccine + APS (comparative example 2); c: APS control group10μl0.3 ml/feather, APS concentration content as in comparative example 2); d: PBS control (0.3 ml/plume per immunization).
3.2 sample collection
Blood was collected 1mL per chicken wing vein every week at 1-10 weeks before and after immunization, and serum was separated by centrifugation at 3000rpm for 10min and frozen at-20 ℃.
3.3 results
Antibody level determination. The specific antibody level was detected by the hemagglutination inhibition assay (HI assay), as shown in fig. 3, and 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 was continued until the end of the assay.
4. H5N1 subtype avian influenza vaccine immunoassay
4.1 animal Experimental groups
40 SPF chickens of 7 days of age were randomly divided into four groups of 10: a: conventional H5N1 subtype inactivated avian influenza vaccine is subcutaneously injected at the nape of the neck, 0.3 ml/feather; b: H5N1 inactivated vaccine + APS-BP5 (example 4); c: APS-BP5 control group (10. Mu.l/0.3 ml/feather, APS-BP5 concentration content same as in example 4); d: PBS control group (0.3 ml/Yu for each immunization)
4.2 sample collection
Blood was collected 1mL per chicken wing vein every week at 1-10 weeks before and after immunization, and serum was separated by centrifugation at 3000rpm for 10min and frozen at-20 ℃.
4.3 results
Antibody level determination. Specific antibody levels were detected by the hemagglutination inhibition assay (HI assay), as shown in fig. 4, avian influenza virus (H5) antibody levels of the APS-BP5 vaccine group began to be significantly higher than that of the conventional vaccine group (p < 0.01) 2 weeks after immunization, and continued until the end of the assay. The antibody level of the APS-BP5 vaccine group was consistently higher than 3 titers of the conventional vaccine group from 3 weeks after immunization to the end of the trial.
By means of the above described immunoassay we can conclude that:
bursin (BP) is a small molecule skin substance found in the bursa of Fabricius of birds and has a close relationship with immune function. The bursin pentapeptide (BP 5) is one of bursin families, and has the functions of inducing the differentiation and proliferation of B lymphocyte precursors of poultry, improving the antibody level and enhancing humoral immunity; promoting the transformation activity of T lymphocytes, thereby enhancing the effect of cellular immunity.
Polysaccharide compounds are good immunomodulators, and are becoming more and more important in improving and improving animal immune functions. Astragalus polysaccharides are the main bioactive components in the Astragalus root extract. Research shows that astragalus polysaccharide can stimulate macrophage activity, promote T cell proliferation, promote the expression of surface antigen in lymphocyte, induce powerful humoral and cellular immune response and play important role in non-specific and specific immune response. In general, astragalus polysaccharides have various functions of regulating immunity, improving immune response, relieving immune dysfunction caused by environmental stress, and the like.
The combined use of the two immunopotentiators plays a synergistic effect in effect, and can obtain ideal immune effect when being matched with the avian influenza vaccine.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> Zhaoqingda Hua agricultural and biological medicine Co., ltd
Huanong (Zhaoqing) Biological Industry Technology Research Institute Co., Ltd.
<120> an immunopotentiator, preparation method, avian influenza vaccine and application
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agcgaattct gcaaacgcgt gtactgcaaa cgcgtgtact gcaaacgcgt gtactgcaaa 60
cgcgtgtact gcaaacgcgt gtacaagctt cac 93
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<212> DNA
<213> Artificial sequence ()
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gtgaagcttg tacacgcgtt tgcagtacac gcgtttgcag tacacgcgtt tgcagtacac 60
gcgtttgcag tacacgcgtt tgcagaattc gct 93
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<213> Artificial sequence ()
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Glu Phe Cys Lys Arg Val Tyr Cys Lys Arg Val Tyr Cys Lys Arg Val
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Tyr Cys Lys Arg Val Tyr Cys Lys Arg Val Tyr Lys Leu
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Claims (8)
1. An immunopotentiator, characterized in that: comprises a cyst element pentapeptide fusion peptide and astragalus polysaccharide, wherein the weight ratio of the cyst element pentapeptide fusion peptide to the astragalus polysaccharide is 1:15, and the amino acid sequence of the cyst element pentapeptide fusion peptide is 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-NH 2 。
2. The immunopotentiator of claim 1, wherein: the composite material consists of the following components in percentage by weight:
1% of a bursin pentapeptide fusion peptide;
15% of astragalus polysaccharide;
the balance of water.
3. An avian influenza vaccine, characterized in that: the vaccine comprises the immunopotentiator of any one of claims 1-2; the dosage of the immunopotentiator in the avian influenza vaccine is 10 μl/serving.
4. The avian influenza vaccine of claim 3 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.
5. A method of preparing an immunopotentiator according to any one of claims 1 to 2, characterized in that: the capsulorhein pentapeptide fusion peptide and astragalus polysaccharide are mixed and dissolved in water.
6. The method for producing an immunopotentiator according to claim 5, comprising the steps of:
step 1: constructing a gene of a bursin pentapeptide;
step 2: connecting the gene of the bursin pentapeptide to an expression vector pET-32a to obtain a recombinant plasmid pET-32a- (BP 5) 5 ；
Step 3: recombinant plasmid pET-32a- (BP 5) 5 Introducing into escherichia coli for induction expression to obtain an expression product;
step 4: purifying and drying the expression product to obtain the bursin pentapeptide fusion peptide;
step 5: mixing and dissolving the bursin pentapeptide fusion peptide and astragalus polysaccharide in water for injection.
7. The method for preparing an immunopotentiator according to claim 6, wherein the steps 1 and 2 are specifically:
step 1: 5'-TGCAAACGCGTGTAC-3' is taken as BP5 gene, ecoRI and HindIII restriction enzyme sites and protective bases are respectively added before and after the fragment to obtain fragment sequence F, and the fragment length is 93BP;
step 2: 1 mug of the nucleic acid fragment is dissolved in 50 mug of TE buffer solution and digested with EcoRI and HindIII enzymes to obtain the digested fragment;
treating the pET-32a vector with EcoRI and HindIII enzyme digestions;
mixing the digested fragment with the digested pET-32a vector, and connecting the digested fragment with the digested pET-32a vector by using T4 DNA enzyme to obtain a connection product;
transforming the connection product into E.coli DH5 alpha, verifying positive colonies by using a vector sequencing primer, and sequencing the positive colonies; recombinant plasmid pET-32a- (BP 5) with correct preservation sequence 5 。
8. The method for preparing an immunopotentiator according to claim 7, wherein the step 3 is specifically:
plasmid introduction: recombinant plasmid pET-32a- (BP 5) is taken 5 Adding into escherichia coli Rosetta, fully mixing on ice, placing the mixture in a water bath at 42 ℃, taking out, rapidly standing on ice, and finishing plasmid transformation to obtain seed bacterial liquid;
induction of expression: inoculating seed bacterial liquid positive to PCR identification into culture medium, and culturing to OD 600 When the expression is=0.4-0.6, adding IPTG, and carrying out induction expression at 25 ℃; the product was identified to be present in the culture supernatant, predominantly in soluble form.
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| CN101255189A (en) * | 2008-04-07 | 2008-09-03 | 广东大华农动物保健品有限公司 | Animal vaccine immunopotentiator and production method thereof |
| CN101434650A (en) * | 2008-12-23 | 2009-05-20 | 李德元 | Bursa pentapeptide, deriving peptide thereof and use thereof |
| CN102000332A (en) * | 2010-11-16 | 2011-04-06 | 南京农业大学 | Composite adjuvant for duck bird flu oral mucosal immunization |
| CN103145854A (en) * | 2013-03-05 | 2013-06-12 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
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| CN101255189A (en) * | 2008-04-07 | 2008-09-03 | 广东大华农动物保健品有限公司 | Animal vaccine immunopotentiator and production method thereof |
| CN101434650A (en) * | 2008-12-23 | 2009-05-20 | 李德元 | Bursa pentapeptide, deriving peptide thereof and use thereof |
| CN102000332A (en) * | 2010-11-16 | 2011-04-06 | 南京农业大学 | Composite adjuvant for duck bird flu oral mucosal immunization |
| CN103145854A (en) * | 2013-03-05 | 2013-06-12 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
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