CN116813795B - Recombinant AaLS-BSP fusion peptide, preparation method and application - Google Patents
Recombinant AaLS-BSP fusion peptide, preparation method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a recombinant AaLS-BSP fusion peptide, a preparation method and application thereof. The invention is formed by connecting AaLS and bursa active peptide BSP (TPSGLVY) in series through a flexible connecting peptide Linker gene by a genetic engineering method, expresses fusion peptide AaLS-BSP, and forms stable nano particles through self-assembly. The purified fusion peptide AaLS-BSP is added into an inactivated vaccine according to a certain dosage as an immunopotentiator, so that the cellular immune response and the humoral immune response of the organism to the antigen are enhanced, the protection rate of chicken flocks is improved, and the influence of the vaccine on the local inflammatory response and the growth performance of the organism is reduced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant AaLS-BSP fusion peptide, a preparation method and application thereof.
Background
The Lumazine synthase (AaLS) from the hyperthermophiles Aquifex aeolicus consists of 60 identical subunits, with spherical and hollow icosahedral capsid structures, capable of forming a self-assembled protein nanoparticle (Kim et al 2022;Zha ng et al 2001). The granular nature and repeated subunit organization of these assemblies were able to act as a display platform for antigen presentation (La montagne et al 2022) and produced higher levels of antibodies than the monomeric protein scaffold (ladenstein morganova 2020). Antigen display on self-assembled nanoparticles can stimulate a strong immune response, and in some clinical studies, hyperthermophiles Aquifex aeolicus (AaLS) has been used as a scaffold for nanoparticle immunogens.
The N-terminus of AaLS is exposed on the nanoparticle surface and thus can be used as antigen display. The C-terminus of AaLS may also be exposed on the nanoparticle surface and may be used to attach or display purification tags (Ba Reun Kim et al 2022). For different antigen display, modification of AaLS protein scaffold particles is required, for example Aebischer et al have established a durable and adaptable self-assembled polyprotein scaffold particle (LS-MPSP) using LS, and SpyTag/SpyCatcher mediated plug-in display technology was applied to pre-assembled particles of LS-MPSP to display both model antigens, resulting in nanoparticles with improved immunogenicity and vaccine efficacy (Aebischer et al 2021). Therefore, in the invention, in order to display the bursa of Fabricius polypeptide by using the AaLS protein scaffold particles and improve the expression quantity, amino acid mutation is carried out on the AaLS protein scaffold, and a His tag is added at a proper position so as to facilitate purification.
Bursa fabricius is a humoral central immune organ unique to birds and plays an important role in the induction of differentiation and maturation of T, B cells (Cai et al 2022). Bursa of heptapeptide BSP (TPSGLVY) active peptides derived from bursa of fabricius were effective in stimulating T, B cell differentiation and enhancing antibody production (Feng et al 2010). Zhang Xiugen and the like can ensure that the newcastle disease maternal antibody in serum can be maintained at a higher level for a longer time by jointly applying the artificially synthesized bursin and the newcastle disease attenuated vaccine to immunized chickens, and has a promotion effect on the induced antibody.
Because the bursa of Fabricius polypeptide can only be extracted from bursa of Fabricius tissues of chickens, the preparation difficulty is high, the content of hetero protein is high, and the mass purification and the application are difficult. The chemically synthesized bursa fabricius active peptide has high cost, is only suitable for laboratory research, and cannot be put into practical production and application. Therefore, the research by adopting the genetic engineering method has great significance.
Aiming at the problems, the invention is formed by connecting AaLS and bursa of Fabricius polypeptide BSP in series through a flexible connecting peptide Linker gene, expresses fusion peptide AaLS-BSP and forms stable nano particles through self-assembly. The purified fusion peptide AaLS-BSP is added into an inactivated vaccine according to a certain dosage, so that the cellular immune response and the humoral immune response of an organism to antigen are enhanced, and the protection rate of chicken flocks is improved. Further reducing the effect of the adjuvant and antigen on the local inflammatory reaction and growth performance of the organism.
Disclosure of Invention
The invention aims at providing a recombinant AaLS-BSP fusion peptide, and the amino acid sequence of the fusion peptide is shown as SEQ ID NO. 2.
The invention also aims at providing a microbial preparation method of the recombinant AaLS-BSP fusion peptide, which is simple and has high protein expression.
A final object of the present invention is to provide the use of recombinant AaLS-BSP fusion peptides for the preparation of immunopotentiators. In order to achieve the above object, the present invention adopts the following technical measures:
the amino acid sequence of the recombinant AaLS-BSP fusion peptide is shown as SEQ ID NO.2, and one of polynucleotides encoding the fusion peptide is shown as SEQ ID NO. 1.
A method of preparing a recombinant fusion peptide comprising:
1) Aiming at the recombinant AaLS-BSP fusion peptide gene, a primer F1 is designed: 5'-cgagcggcctggtgtatAGCAGCAG CGGGTCCGGA-3' and F2:5'-atacaccaggccgctcggggtCATCCATGGTATATCTCCTTCTTAAAG T-3', F3:5'-cgagcggcctggtgtatGGATCGGGCTCTCATCACCA-3', and F4:5'-atacaccaggccg ctcggggtGCCTCCTCCTGAGCCTCCA-3', followed by SOE-PCR amplification; recovering the polynucleotide product shown in SEQ ID NO. 1;
(2) The polynucleotide shown in SEQ ID NO.1 and a plasmid vector pET28a are subjected to enzyme digestion and connection, and the obtained plasmid is named pET28a-AaLS-BSP; the recombinant plasmid pET28a-AaLS-BSP is transformed into competent escherichia coli BL21 (DE 3), wherein the competent escherichia coli BL21 (DE 3) is derived from a indigenous organism, and the product number is EC1002.
The protection scope of the invention also comprises:
the application of recombinant AaLS-BSP fusion peptide or a gene encoding the fusion peptide in the preparation of immunopotentiator.
In the above application, preferably, the subject to which the immunopotentiator is applied is poultry.
Compared with the prior art, the invention has the following advantages:
1. the applicant carries out site-directed mutagenesis transformation on an AaLS framework aiming at the original AaLS protein, so as to improve the expression quantity of the BSP polypeptide.
2. The polypeptide is inserted into the expression sites at both sides of the AaLS skeleton, so that the stability of the skeleton plasmid is improved, and the expression quantity of the polypeptide is improved.
3. Successfully preparing the escherichia coli engineering bacteria for expressing the recombinant AaLS-BSP fusion peptide.
4. The expressed recombinant AaLS-BSP fusion peptide can self-assemble to form stable nano particles.
5. The recombinant AaLS-BSP fusion peptide serving as an immunopotentiator is added into an inactivated vaccine for poultry, so that the level of Ig G antibodies in the body of an immunized chicken can be obviously promoted, and the AaLS-BSP can simultaneously promote the production of Th1 type (IL-2, IL-12 and interferon-gamma) and Th2 type (IL-4) cytokines.
Drawings
FIG. 1 is a schematic representation of expression and purification of recombinant AaLS-BSP fusion peptides;
wherein A: lane 1: non-induced protein expression; lane 2: inducing the expression condition of the whole mycoprotein; lane 3: inducing the AaLS-BSP protein in the supernatant to a size of about 21kDa; lane 4: inducing protein expression in inclusion bodies.
B: as a result of Western immunoblotting after purification of AaLS-BSP, lane 1 was AaLS-BSP.
FIG. 2 is a negative EM image of AaLS-BSP nanoparticles after purification.
FIG. 3 is a schematic representation of cytokine levels and antibody levels after 21 days of vaccine immunization for different treatment groups;
wherein: th1 (IL-2, IL-12, interferon-gamma) and Th2 (IL-4) cytokine levels; b: igG antibody levels in each group after 21 days of immunization; c: peripheral blood lymphocyte proliferation.
Figure 4 is a schematic representation of weight gain for each group 21 days after immunization.
FIG. 5 is a graph showing cytokine detection results after 7 days of the second-day-free period;
wherein: a: IL-2 secretion levels; b: IL-4 secretion levels; c: IL-12 secretion levels; d: IFN-gamma secretion levels.
FIG. 6 is a schematic diagram showing the measurement of the proliferation potency of peripheral blood lymphocytes.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available.
Example 1:
screening and optimizing AaLS framework bearing protein:
applicants have discovered that the bursa of Fabricius active peptides BPP and BSP (shown as SEQ ID NO.6 and encoded by SEQ ID NO. 5) can be expressed when a plurality of groups of active peptides with immunity enhancement are combined with an AaLS framework;
further, the applicant finds that two epitopes in the AaLS skeleton can be expressed by inserting the same bursa of Fabricius active peptide and can not be expressed smoothly by inserting different bursa of Fabricius active peptides; the two antigen epitopes are inserted into the same bursa active peptide at the same time, so that the expression quantity is higher and more stable compared with the insertion of only one side;
in order to further increase the expression level of the AaLS-BSP fusion protein, the applicant mutates amino acids 34, 94 and 108 of the AaLS protein (shown as WP_010880027.1,SEQ I D NO.8 and encoded by polynucleotide shown as SEQ ID NO. 7), and the protein expression level is not obviously increased compared with that of the AaLS-BSP fusion protein; the 28 th, 79 th and 91 st amino acids of the AaLS protein (WP_ 010880027.1) are mutated, the mutated AaLS protein is shown as SEQ ID NO.4 (the polynucleotide for encoding the mutated AaLS protein is shown as SEQ ID NO. 3), and the expression quantity of the BSP protein is obviously improved and is 2.17 times of the unmutated expression quantity after mutation.
After mutation, the sequence of the obtained recombinant AaLS-BSP fusion peptide is shown as SEQ ID NO. 2.
Example 2:
expression and purification of AaLS-BSP
In order to enable the bursa fabricius polypeptide BSP to be expressed smoothly and improve the expression quantity, amino acids at positions 28, 79 and 91 of an AaLS protein skeleton are mutated, and a group of 6 XHis labels are added between a Linker gene sequence and the N end of the AaLS skeleton.
(1) Recombinant AaLS-BSP fusion peptide gene overlap extension PCR amplification
Designing a recombinant AaLS-BSP fusion peptide gene according to the escherichia coli preference codon, and designing a primer F1 aiming at the fusion peptide gene: 5'-cgagcggcctggtgtatAGCAGCAGCGGGTCCGGA-3' and F2:5'-atacaccaggccgctcggggtCA TCCATGGTATATCTCCTTCTTAAAGT-3', F3:5'-cgagcggcctggtgtatGGATCGGGCTCTCAT CACCA-3', and F4:5'-atacaccaggccgctcggggtGCCTCCTCCTGAGCCTCCA-3', followed by SOE-PCR amplification; and (3) identifying the PCR amplification product by agarose gel electrophoresis, cutting off a target band, and recovering, wherein the fragment size of the recovered product is 624bp, namely the recombinant AaLS-BSP fusion peptide gene.
(2) Construction of engineering bacteria carrying recombinant AaLS-BSP fusion peptide gene
Carrying out enzyme digestion connection on the recombinant AaLS-BSP fusion peptide gene and a plasmid vector pET28a, converting E.coli DH5 alpha by a connection product, extracting a plasmid, and identifying, wherein the correct plasmid is named pET28a-AaLS-BSP; the recombinant plasmid pET28a-AaLS-BSP is transformed into competent escherichia coli BL21 (DE 3) (unique organism, EC 1002), and BL21 (DE 3) competence provided by unique organism compared with other brand receptive peptides can be used for constructing various polypeptide and protein expression libraries, and has higher transformation efficiency. And screening positive clones to obtain engineering bacteria for expressing recombinant AaLS-BSP fusion peptide.
(3) Recombinant AaLS-BSP fusion peptide protein expression
The competent E.coli was inoculated into a medium of Luria-Bertani (LB) 1.0L containing 50mg/L kanamycin and cultured at 37 ℃. Under the condition that the OD600 nm light absorption density is 0.5-0.6, 400 mu M IPTG is used for inducing cells to express protein, and the temperature is 37 ℃ for 7 hours. Cells were collected, resuspended in 20mM Tris-HCl (pH=8.6), broken under high pressure, and the supernatant was centrifuged (12000 Xg, 30 min).
(4) Recombinant AaLS-BSP fusion peptide protein purification
And purifying the protein by adopting nickel column affinity chromatography to obtain a single recombinant AaLS-BSP fusion peptide. The operation steps are as follows:
before loading, a 10CV Binding Buffer was used to equilibrate the nickel column at a flow rate of 5 mL/min; loading the solution to an balanced Ni column through a constant flow pump at a flow rate of 1mL/min, eluting the Ni column with a gradient solution Buffer/Binding Buffer mixture (500 mM imidazole is determined by a pre-experiment), monitoring an OD280 value, collecting an eluent corresponding to a protein absorption peak, and determining the position of a target protein after SDS-PAGE detection; the Ni column was thoroughly eluted with a 10CV solution Buffer to remove residual column binding protein and then equilibrated with 5-10CV of 20% ethanol, removed and stored at room temperature.
The purified target protein can be further concentrated and dialyzed to obtain single high-concentration recombinant AaLS-BSP fusion peptide, and the concentration of the obtained protein is measured by using a Bradford protein quantitative kit (TIANGEN, beijing, china), and 31.2mg of recombinant AaLS-BSP fusion protein is expressed per 1L of culture medium through conversion.
Western immunoblotting revealed that AaLS-BSP was able to migrate between 21kDa and 23kDa as a single protein band, respectively, in the supernatant (FIG. 1A), B). AaLS-BSP was confirmed to have a spherical and hollow structure by transmission electron microscopy (FIG. 2). These results indicate that BSP fuses successfully with AaLS and can self-assemble.
In conclusion, the bursa fabricius active peptide BSP is constructed on an AaLS protein skeleton to form fusion protein, and then the fusion protein can be stably expressed by an escherichia coli expression system and further purified by means of a His tag, so that a stable single target protein is obtained.
Example 3:
immune enhancement effect of recombinant AaLS-BSP fusion peptide in inactivated vaccine for avian colibacillosis
(1) Preparation of vaccine
The AaLS-BSP fusion peptide after expression and purification and O78 type avian pathogenic escherichia coli inactivated antigen (Vac) are used as water phases and are emulsified with an oil phase HMT13 adjuvant for 15 minutes by adopting a low shear stirrer at 1000 revolutions per minute according to a proper volume ratio, the water in oil in water is detected as a dosage form, the property is stable, the viscosity is proper, and the finished vaccine (AaLS-BSP+Vac) is obtained and stored at 4 ℃.
Chemically synthesized bursa of Fabricius peptide BSP (Optimus, praeparata) was also vaccine prepared and stored by this method (BS P+Vac).
(2) Immunoprotection test
The standard is worn on each feather chicken before immunization, and the chickens are weighed. Each group of SPF chickens with 14 days of age has 10 feathers and 0.3 ml/feather
The experiments were divided into 4 groups in total:
PBS control group: PBS was emulsified as an aqueous phase with HMT13 adjuvant and used at 0.3 ml/plume.
Vaccine group (avian pathogenic escherichia coli inactivated Vaccine immunization group): when the vaccine is used, the O78 type avian pathogenic escherichia coli inactivated antigen in the vaccine is 1.5X10 9 CFU/plume.
Bsp+vac group: when the vaccine is used, the BSP in the vaccine is 100 mug/feather, and the Vac (namely O78 type avian pathogenic escherichia coli inactivated antigen) is 1.5X10 9 CFU/plume.
AaLS-bsp+vac group: when the vaccine is used, aaLS-BSP in the vaccine is 100 mug/feather, and Vac (namely O78 type avian pathogenic escherichia coli inactivated antigen) is 1.5X10 9 CFU/plume.
Serum was collected from the subwing veins after 21 days of immunization, and the production of Th 1-type (IL-2, IL-12, interferon-gamma) and Th 2-type (IL-4) cytokines and their effects on lymphocyte proliferation were detected by a double antibody one-step sandwich ELISA. The serum was collected from the subwing vein 21 days after immunization and assayed for IgG antibody levels by indirect ELISA.
The absorption was checked by weighing 21 days of immunization and passed through the chestPartial intramuscular injection of bacterial solution for detoxification (avian pathogenic E.coli O78 type strain) 2X 10 10 CFU/feather, protection rate was calculated, protection rate = number of non-dead chicken feathers/total number of tested chicken feathers in the group.
The results showed that after 21 days of immunization, the injection site absorbed well, the weight gain was normal, and there was no inhibition on the growth of the chicken flock (table 1).
21 days after immunization, the AaLS-BSP-added group significantly promoted an increase in IgG antibody levels in the immunized chickens (FIG. 3B). AaLS-BSP was able to promote both Th1 (IL-2, IL-12, interferon-gamma) and Th2 (IL-4) cytokine production (FIG. 3A). To study the effect on lymphocyte proliferation, the chickens were immunized for 21 days and peripheral blood lymphocytes were collected, and lymphocyte single cell suspensions were prepared, and ConA was used as a positive control, and the results showed that the AaLS-BSP immune group significantly improved the proliferation capacity of peripheral blood lymphocytes (C in FIG. 3).
The immunization was followed by detoxification 21 days later (strain was O78 type avian pathogenic E.coli, 2.0X10) 10 CFU/feather), the results show: the protection rate of the immune group added with the AaLS-BSP fusion peptide to chicken is not lower than 90 percent, which is obviously higher than the protection rate of PBS (0/10) and vaccine immune control group (6/10) (table 1), and after the AaLS-BSP fusion peptide is formed by the bursa-fabricius active peptide BSP and the AaLS framework, the protection effect, the antibody level and the induction cytokine production are better than those of the vaccine and chemical synthesis bursa-fabricius active peptide BSP immune group. These results confirm that the AaLS-BSP fusion peptide has immunomodulatory activity in chicken immunization.
TABLE 1 protective effects of inactivated vaccine against avian colibacillosis and different immunopotentiator groups after 21 days of immunization
| Grouping | viscosity/cP | Absorption conditions | Weight gain/g | Protection rate |
| PBS | / | Complete absorption | 202.73±24.5 | 0/10 |
| Vaccine | 51.0 | Good absorption | 207.46±25.6 | 6/10 |
| BSP+Vac | 57.2 | Good absorption | 205.11±18.1 | 7/10 |
| AaLS-BSP+Vac | 56.8 | Good absorption | 220.92±28.2 | 9/10 |
In conclusion, the recombinant AaLS-BSP fusion peptide is added into the water phase of the avian colibacillosis vaccine according to a proper proportion and is prepared into the vaccine with the oil phase HMT13, after the chicken is subjected to an immunity test, compared with each control group, the cell immunity and the humoral immunity level can be remarkably improved, the protection rate is improved, and further the AaLS-BSP fusion peptide has an immunity regulation activity in chicken immunity and has a wide application prospect as an immunity enhancer.
Example 4:
immunopotentiation effect of recombinant AaLS-BSP fusion peptide in newcastle disease-avian influenza (H9 subtype) -adenovirus (I group, 4 type) triple inactivated vaccine
(1) Preparation of vaccine
The purified AaLS-BSP and the inactivated antigen of the newcastle disease-avian influenza (H9 subtype) -adenovirus (I group, 4 type) are used as water phase and are emulsified with an oil phase HMT13 adjuvant for 15 minutes by a low shear stirrer at 1000 revolutions per minute according to a proper volume ratio, and then the finished vaccine (AaLS-BSP+Vac) is obtained and stored at 4 ℃.
Chemically synthesized bursa of Fabricius peptide BSP (Optimus, praeparata) was also vaccine prepared and stored by this method (BS P+Vac).
(2) Immunoprotection test
The standard is worn on each feather chicken before immunization, and the chickens are weighed. Each group immunized 7-day-old SPF chicken 10 feathers, 0.3 ml/feather.
The immunization procedure was: one immunization was performed at 7 days of age, and a second immunization was performed 21 days after one immunization.
The experiments were divided into 4 groups:
PBS control group: PBS as aqueous phase and HMT13 adjuvant, and when used, 0.3 ml/feather
Vaccine group (avian triple inactivated Vaccine immunization group): when the vaccine is used, the inactivated antigen of newcastle disease in the vaccine is 10 8 EID 50 Inactivated antigen of avian influenza (H9 subtype) 10 6 EID 50 The amount of the recombinant rFi ber2 protein of adenovirus (group I, type 4) was 30. Mu.g/serving.
Bsp+vac group: when the vaccine is used, the BSP in the vaccine is 100 mug/feather, and the inactivated antigen of the newcastle disease in the Vac is 10 8 EID 50 Inactivated antigen of avian influenza (H9 subtype) 10 6 EID 50 The amount of recombinant rFiber2 protein of adenovirus (group I, type 4) was 30. Mu.g/serving.
AaLS-bsp+vac group: when the vaccine is used, aaLS-BSP in the vaccine is 100 mug/feather, and the inactivated antigen of newcastle disease in Vac is 10 8 EID 50 Feather/featherThe inactivated antigen of the avian influenza (H9 subtype) is 10 6 EID 50 The amount of recombinant rFiber2 protein of adenovirus (group I, type 4) was 30. Mu.g/serving.
After 14 days of the second immunization, all test chickens were challenged with newcastle disease virus (CVCC AV1611 strain, 10 5 ELD 50 ) Intramuscular injection of 0.2ml of virus solution, and intravenous injection of 0.2ml (2×10) of avian influenza virus (subtype H9, CVCC AV 1554) 6 ELD 50 ) Viral solution adenovirus (group 1, type 4, CVCC AV211 strain) was injected intramuscularly with 0.2ml (2X 10) 5 TCID 50 )。
The serum was collected from the subwing vein 21 days after the first immunization and assayed for newcastle disease virus, avian influenza virus HI antibody titers, and the level of resistance to avian adenoviruses by ELISA. Serum was collected from the subwing vein 7 days after the second immunization, and the production of Th1 type (IL-2, IL-12, interferon-gamma) and Th2 type (IL-4) cytokines and their effect on lymphocyte proliferation were detected by a double antibody one-step sandwich ELISA.
The results showed that 21 days after the first immunization, the injection site absorbed well and the weight gain was normal (fig. 4). Compared with the vaccine immune group, the AaLS-BSP fusion peptide added group can obviously promote the increase of the antibody titer of Newcastle disease and avian influenza HI of immunized chickens and the level of adenovirus antibody, and the level of antibody produced by the BSP immune group added with the chemical synthesis method bursa peptide is lower than that of the fusion peptide immune group (Table 2). The cytokine detection results after 7 days of the second immunization showed that AaLS-BSP significantly regulated Th1 (IL-2, IL-12, interferon-gamma) and Th2 (IL-4) cytokine production (FIG. 5), and that the AaLS-BSP immune group significantly improved peripheral blood lymphocyte proliferation capacity (FIG. 6).
After 14 days of secondary immunization, all the test chickens were challenged, after the newcastle disease virus was challenged, all the control test chickens died, the vaccine immunization group was 5/10) protected by 50%, the chemical synthesis bursa peptide BSP immunization group (8/10) was 80% protected by the test chickens, and the AaLS-BSP fusion peptide immunization group was 90% (Table 2). After the virus attack of the avian influenza virus, the PBS control group is positive in virus separation, the protection rate is 0%, the vaccine immune group is 6/10 negative in virus separation, the chemical synthesis method bursa peptide BSP immune group is 8/10 negative in virus separation, and the protection rate is 80%; aaLS-BSP fusion peptide immune group, 9-feather test chicken virus separation is negative, which shows that the protection force of the fusion peptide immune group on chicken flock is 90%. After adenovirus (group 1, type 4) challenge, the protection rate of AaL S-BSP fusion peptide immune group was 100% higher than that of each control group. The result shows that the AaLS-BSP fusion peptide can be added into the triple inactivated vaccine for immunization, so that the chicken flock has good immune efficacy and potential of becoming an immune enhancer.
Table 2 antibody levels and protective efficiency after immunization of avian triple inactivated vaccine
In conclusion, the recombinant AaLS-BSP fusion peptide is added into the water phase of the triple inactivated vaccine of the fowl according to a proper proportion and is prepared into the vaccine with the oil phase HMT13, and after the chicken is subjected to an immune test, compared with each control group, the antibody titer of each virus of the immune chicken can be obviously improved, and the secretion level of Th1 type and Th2 type cytokines and the proliferation capacity of peripheral blood lymphocytes can be obviously improved. The protection rate of the AaLS-BSP fusion peptide on immunized chickens is not lower than 90%, which is obviously higher than the protection level of other control groups, and further shows that the AaLS-BSP fusion peptide has immunoregulatory activity in chicken immunization and has wide application prospect as an immunopotentiator.
Claims (5)
1. An artificially synthesized recombinant AaLS-BSP fusion peptide, wherein the fusion peptide is shown as SEQ ID NO. 2.
2. A polynucleotide encoding the fusion peptide of claim 1.
3. The polynucleotide according to claim 2, which is set forth in SEQ ID No. 1.
4. The method for producing a fusion peptide according to claim 1, comprising:
1) Aiming at the recombinant AaLS-BSP fusion peptide gene, a primer F1 is designed: 5'-cgagcggcctggtgtatAGCAGCAGCGGGTCCGGA-3' and F2: 5'-atacaccaggccgctcggggtCATCCATGGTATATCTCCTTCTTAAAGT-3', F3:5'-cgagcggcctggtgtatGGATCGGGCTCTCATCACCA-3', and F4:5'-atacaccaggccgctcggggtGCCTCCTCCTGAGCCTCCA-3', followed by SOE-PCR amplification; recovering the polynucleotide product shown in SEQ ID NO. 1;
2) The polynucleotide shown in SEQ ID NO.1 and a plasmid vector pET28a are subjected to enzyme digestion and connection, and the obtained plasmid is named pET28a-AaLS-BSP; the recombinant plasmid pET28a-AaLS-BSP was transformed into competent E.coli BL21 (DE 3).
5. The recombinant AaLS-BSP fusion peptide or the gene encoding the fusion peptide is applied to the preparation of an immunopotentiator, wherein the fusion peptide is shown in SEQ ID NO.2, and the application object of the immunopotentiator is poultry.
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