CN116606366B - Grouper interleukin 15 and preparation method and application thereof - Google Patents
Grouper interleukin 15 and preparation method and application thereof Download PDFInfo
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- CN116606366B CN116606366B CN202310553657.XA CN202310553657A CN116606366B CN 116606366 B CN116606366 B CN 116606366B CN 202310553657 A CN202310553657 A CN 202310553657A CN 116606366 B CN116606366 B CN 116606366B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5443—IL-15
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A61K2039/552—Veterinary vaccine
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A61K2039/55527—Interleukins
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- C12N2710/00034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention discloses a grouper interleukin 15 and a preparation method and application thereof. The amino acid sequence of the grouper interleukin 15 is shown as SEQ ID No: 1. The synthetic nucleotide sequence is shown as SEQ ID No:2, inserting the gene into an expression vector to obtain a recombinant vector, transforming the recombinant vector into prokaryotic expression bacteria, culturing, and screening out prokaryotic expression bacteria successfully transformed; inducing and expressing the prokaryotic expression bacteria which are successfully transformed; purifying by using the protein of induced expression to obtain the recombinant protein of the grouper interleukin 15. As a result of evaluating the SGIV immune protection effect of the EcIL-15L protein and the SGIV subunit vaccine on the garrupa, the result shows that the EcIL-15L protein can be used as a cytokine adjuvant to improve the MCP specific antibody response induced by the SGIV subunit vaccine, improve the expression of immune related genes, and improve the immune protection efficiency of the SGIV subunit vaccine by 81.5%. The invention can be applied to the prevention and control of SGIV virus diseases in the breeding process of garrupa.
Description
Technical Field
The invention belongs to the fields of molecular biology and immunology, and particularly relates to an grouper interleukin 15 (EcIL-15L), a preparation method thereof and application of the grouper interleukin 15 as a vaccine adjuvant.
Background
Interlukin (IL) 15 is a member of the family of cytokines yc, which also includes IL-2, IL-4, IL-7, IL-9, IL-13 and IL-21, most of which bind to receptors containing the IL-2 Rgamma chain. In 1994, IL-15 was first found as a cytokine with antitumor effect in the supernatant of monkey kidney endothelial cell line CV-1/EB-NA. The nucleotide homology of the cDNA coding region of human IL-15 and the cDNA coding region of monkey IL-15 is up to 97%, and the nucleotide homology with the mouse homolog is 73%. IL-15 mature peptides contain two intramolecular disulfide bonds, and four alpha helices formed by amino acid residues of four regions are typical features of the IL-15 domain. IL-15 is a pro-inflammatory cytokine, and mRNA for IL-15 is widely expressed in immune cells, including cardiac muscle, lung, liver, kidney, brain, pancreas, placenta, and skeletal muscle, with the highest levels in skeletal muscle and placenta sites. IL-15 has a structure similar to IL-2, and the β and yc subunits of the IL-15receptor (IL-15 receptor) are also part of the IL-2 receptor, and therefore IL-15 is thought to have similar biological activity to IL-2. IL-15 has strong effect of promoting cell proliferation on T cells and NK cells, can participate in immune response of the T cells, stimulates the proliferation of the T cells, promotes the differentiation and maturation of the T cells, and protects the activation of the T cells.
IL-15L gene was originally found in teleosts such as Fugu rubripes, zebra fish, etc. With intensive research, this gene has also been found in cartilaginous fish such as sharks, reptiles, and some mammals including cattle, horses, pigs, cats, mice, foxes, monkeys, rabbits, and hedgehog. IL-15L is functional in fish. In isolated trout spleen cells and in vivo experiments showed that purified recombinant IL-15L+IL-15Rα molecules induced expression of IL-4 and IL-13 homologs, which are markers for type II immunity. In contrast, trout IL-15 stimulated type I immune markers. Thus, IL-15 and IL-15L from trout may have opposite functions. In comparison to IL-15, trout IL-15L is more dependent on the "trans" expression of the receptor chain IL-15Rα and stimulates CD4-CD8- (IgM-) lymphocytes of the thymus and spleen. Thus, trout IL-15L may have an important role in the early stages of the type II immune cytokine cascade. Currently, in zebra and rainbow trout, two spliced forms of IL-15L have been found, designated IL-15La and IL-15Lb, which have different functions. IL-15L is inactive in humans and mice.
The groupers are important seawater economic fishes in China and southeast Asia, and have extremely high economic value. However, the frequent outbreaks and epidemic viral diseases severely restrict the healthy and sustainable development of the grouper industry, wherein the grouper iridovirus (Singapore grouper iridovirus, SGIV) is an important viral pathogen. Early studies on the SGIV and yc families found that SGIV infection induced up-regulation of Interleukin 2enhancer binding factor 2 (Intereukin-2enhancer binding factor 2,EcILF2) expression. In addition, IL-15 has been used as an inflammatory factor index after SGIV infection, but no study has been made about the role of grouper IL-15L in fish viral infection. The research on the function of the garrupa cytokine, especially the cytokine with the immune adjuvant characteristic, not only lays the foundation for developing the adjuvant of the virus subunit vaccine, but also provides important information for preparing the antiviral functional product.
Cytokines that have been identified to date include lymphokines, monokines, interferons, growth factors, and the like, and cytokines with adjuvant effects are among many lymphokines, monokines, and interferons. IL-1, IL-2, IL-3, IL-4, IL-6, IL-12, IL-15, IL-18, IFN-gamma, IFN-beta have been found to have various degrees of adjuvant effect. Studies have demonstrated that direct injection of purified cytokines as adjuvants with vaccines or antigens can result in an effective immune response in either the vaccine or antigen low responders or non responders. The adjuvant effect of cytokines is somewhat limited, and co-expression of cytokines with antigens is critical to the functioning of the cytokine adjuvant effect. Some cytokines must be co-located with the antigen to exert an adjuvant effect. By adopting the method of combined injection or fusion co-expression of antigen genes and cytokine plasmids, the efficiency of inducing immune response of organisms can be improved, and the immune effect can be enhanced. Researchers use porcine IL-15 or IL-18 as an adjuvant and add its coding gene directly into the expression plasmid of porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndromevirus, PRRSV) as an adjuvant for expression. The recombinant MLV virus vaccine expressing the membrane-bound IL-15 cytokine remarkably enhances the response capacity of NK cells and T cells and has better protection effect on PRRSV infection.
Subunit vaccines are favored by more and more researchers due to their high safety and low side effects. The characteristics of being beneficial to oral administration and the suitability for the research and development of vaccines widely applied to the field of aquatic products are extremely high. In recent years, subunit vaccines for preventing fish viruses have proven to be less and effective in side effects. Among the subunit vaccines of iridovirus in groupers, there are currently the Main Capsid Protein (MCP) subunit vaccine and the SGIV subunit vaccine of the taiwan grouper iridovirus (grouper iridovirus of Taiwan, china, TGIV). TGIV MCP is the major structural protein in iridovirus particles, estimated to account for 45% of all virion proteins in infected cells. As structural proteins of viruses, MCP is capable of eliciting a strong immune response to viral infection, and is considered as an important candidate antigen for subunit vaccines for preventing iridovirus infection. However, subunit vaccines are generally not highly immunogenic, and therefore, adjuvants are required to boost the immunogenicity of the vaccine. On the basis of defining the function of EcIL-15L, the recombinant DNA is compatible with SGIV MCP subunit vaccine, and the effect of the recombinant DNA as cytokine adjuvant on enhancing the immunity of the fish subunit vaccine is evaluated.
Disclosure of Invention
The invention aims to provide the garrupa interleukin 15 (EcIL-15L) which is simple and convenient, has low cost, high stability and convenient preservation, and can improve the immune protection efficiency of the iridovirus adata vaccine, a preparation method thereof and application of the garrupa interleukin 15 (EcIL-15L) as a vaccine adjuvant.
The first object of the invention is to provide a novel grouper interleukin 15 (EcIL-15L), wherein the amino acid sequence of the EcIL-15L is shown as SEQ ID No: 1.
The second object of the invention is to provide a novel coding gene of the grouper interleukin 15, the nucleotide sequence of which is shown as SEQ ID No: 2.
A third object of the present invention is to provide a recombinant expression vector characterized in that: it comprises the nucleotide sequence shown in SEQ ID NO. 2.
The fourth object of the present invention is to provide a recombinant bacterium characterized in that: it comprises the recombinant expression vector.
A fifth object of the present invention is to provide a recombinant protein characterized in that: the recombinant strain is used for inducing expression, purified and dialyzed, and fish experiments show that the recombinant strain has activity and can be used as a cytokine. The protein of the invention adopts the traditional production and purification mode of protein, and can be produced in large scale.
The sixth object of the invention is to provide the use of the recombinant protein as a cytokine adjuvant in the preparation of subunit vaccines of garrupa iridovirus.
Preferably, the novel grouper interleukin 15 is a medicament for activating antiviral immunity of fish bodies.
A seventh object of the present invention is to provide a method for preparing the novel grouper interleukin 15, which is characterized by comprising the steps of:
1) Construction of recombinant plasmids: obtaining a nucleotide sequence shown as SEQ ID No:2, inserting the gene into an expression vector to obtain a recombinant vector, transforming the recombinant vector into prokaryotic expression bacteria, culturing, and screening out prokaryotic expression bacteria successfully transformed;
2) Inducing and expressing the prokaryotic expression bacteria which are successfully transformed;
3) And (3) purifying the prokaryotic expression bacteria for induced expression, and dialyzing to obtain the recombinant protein of the grouper interleukin 15.
The eighth object of the invention is to provide an application of the novel grouper interleukin 15 recombinant protein as a cytokine adjuvant in preparation of the MCP subunit vaccine for improving the protection efficiency of the grouper iridovirus.
The invention has the following advantages:
1. the grouper interleukin 15 of the invention has the capability of resisting SGIV virus at the cellular level.
2. The recombinant protein of the grouper interleukin 15 is simple and convenient to prepare, low in cost, safe in product, nontoxic, pollution-free and diffusion-free, can be produced in a large scale, and is convenient to store.
3. The grouper interleukin 15 can improve the specificity antibody titer induced by co-injected subunit vaccine, can improve the expression of antiviral immune genes of the fish body, and improves the immune protection efficiency of the grouper iridovirus subunit vaccine by 81.5%.
Drawings
The result of PCR amplification electrophoresis of the EcIL-15L gene shown in FIG. 1A; b in FIG. 1 is the nucleotide and amino acid sequence of EcIL-15L; in FIG. 1, C is a schematic representation of the three-dimensional structure of the EcIL-15L protein.
EcIL-15L over-expression of EcIL-15L in cells and protein levels shown in FIG. 2A; in FIG. 2B, the overexpression of EcIL-15L reduced SGIV-induced CPE changes. White arrows indicate SGIV-induced cell rounding and cell aggregation; in FIG. 2, (C, D) is the inhibition of SGIV replication by overexpressing EcIL-15L.
The intracellular levels of transcriptional expression of EcIL-15L after knocking down EcIL-15L are shown in FIG. 3A; FIG. 3B shows that knocking down EcIL-15L promotes SGIV-induced CPE changes; in FIG. 3 (C, D) the knockdown of EcIL-15L promotes SGIV replication.
SDS-PAGE analysis of the pET-32a-EcIL-15L recombinant plasmid induced expression in E.coli is shown in FIG. 4A. Lane M: protein molecular weight standard Marker; lane 2: induced pET-32a-EcIL-15L recombinant bacteria whole-cell protein; FIG. 4B is the WB identification of recombinant protein 32 a-EcIL-15L; FIG. 4C shows SDS-PAGE analysis of recombinant protein 32a-EcIL-15L after PBS dialysis. FIG. 4D shows SDS-PAGE analysis of recombinant protein 32a-MCP after PBS dialysis. Lane M: protein molecular weight standard Marker; lane 1: PBS-solubilized supernatant after pET-32a no-load induction; lane 2: PBS dissolved and precipitated after pET-32a no-load induction; lane 3: the method is free; lane 4: SDS-PAGE analysis of recombinant protein 32a-MCP after PBS dialysis.
Fig. 5a shows a statistical chart of the survival rate of the garrupa after toxin attack; FIG. 5B shows ELISA analysis of each group of groupers SGIV-MCP specific antibodies.
FIG. 6 shows the expression level of the immune-related genes in the head and kidney of groupers after vaccine immunization.
Detailed Description
The invention is further illustrated below with reference to examples. The examples are intended to illustrate the invention and not to limit it in any way.
The conventional experimental methods and experimental materials involved in the examples of the present invention are as follows:
1. cell lines and viruses: the cell line was a Grouper Spleen (GS) cell line, and was cultured at 28 ℃ in leibevitz L15 (Sigma) medium containing 10% fetal bovine serum (FBS, gibco). The virus used in the experiment was SGIV stored in the laboratory, and GS cells were infected with SGIV and subjected to freeze thawing lysis 3 times, and collected and stored at-80 ℃. Infection dose method (TCID) with half of tissue culture 50 ) The titer of the virus was determined to be 3.73X10 8 TCID 50 /mL。
2. Experimental fish: the groupers are purchased from a grouper farm in Hainan province in China, are bred in a sea water culture system in Yanshan area of agricultural university in south China, and are of the variety of gold tiger (the groupers are the female parent of the groupers in brown spots and the female parent of the groupers in blue bodies). The weight is 3-4g, and the body length is 6.0-6.5cm. And randomly extracting part of groupers to extract tissue RNA before the test, carrying out reverse transcription to cDNA, and detecting the virus carrying condition by PCR, thereby confirming that the groupers do not carry SGIV.
3. Eukaryotic expression bacteria: coli strain DH5 alpha, a product of Optimago, was stored in a-80℃refrigerator.
4. Prokaryotic expression bacteria: coli BL21 is a product of Norwegian corporation and stored in a refrigerator at-80 ℃.
5. Experimental reagent: IPTG is a product of Sigma company, and is prepared into 1M concentration, and the IPTG is preserved at-20 ℃ for standby; leibovitz' sL-15 medium is Sigma company product; trypsin (amerco, 0458); FBS is purchased from GIBCO company; antibodies to CP were prepared by the laboratory itself and used at a concentration ratio of 1:3000; cell culture fluid: leibovitz' sL-15 culture medium of 10% FBS, and preserving at 4 ℃ for standby; LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride (20 g agar was added when plates were prepared)Powder), dissolved in ddH 2 O, regulating the pH value to 7.4 by 1mol/L NaOH, fixing the volume to 1000mL, sterilizing by steam at 120 ℃ under high pressure for 20min, and preserving at 4 ℃ for later use. 5 XSDS-PAGE running buffer: gly 94.0g,Tris 15.1g,SDS 5g was weighed and dissolved in 800mL ddH 2 In O, fully and uniformly stirring, then adding water to a constant volume of 1,000 mL, and preserving at room temperature for later use; membrane transfer buffer: gly 2.9g,Tris 5.8g,SDS 0.37g was added to 800mL ddH 2 In O, fully and uniformly stirring, and adding 200mL of methanol before use; TBST: trisHCl (1M, pH 7.5) 50mL, naCl 8g, KCl 0.2g, tween 0.5mL, distilled water to 1L;5% skim milk: 5g of skim milk was taken and added to 100mL TBST, and after sufficient dissolution, the mixture was stored at 4 ℃.
6. Cell transfection: GS cells are digested by 0.25% trypsin digestion solution, transferred into a 24-hole cell culture plate, placed in a 28 ℃ incubator for culturing for about 18 hours, observed under a microscope, and transfected after the cells are fully spread with a monolayer. Lipofectamine is adopted in the experiment TM The transfection of the plasmid with 2000 reagent (Invitrogen) was performed, with the specific steps being performed as required.
7. Total cell RNA extraction: cellular RNA was extracted using the SV Total RNA Isolation Kit (Promega) kit, and RNA extraction was performed according to the protocol.
8. Real-time fluorescent quantitative PCR (quantitative Real-time PCR, qRT-PCR) experiments were performed using a2 XSYBR Green Real-time PCR Mix kit (TOYOBO, japan) to prepare the reaction system.
9. Serum antibody titers were detected using enzyme-linked immunosorbent assay (ELISA), purified CP antigen and primary antibody to CP (1:2000 use) were both prepared by the present laboratory, secondary antibody (Abcam, USA), 96-well microtiter plates (BIOFIL, guangzhou, china) and colorimetric substrate TMB (Tiangen, beijing, china) were purchased from the corresponding commercial sources. According to the operation instruction. Finally, the results were read with a multifunctional microplate reader (Thermo, usa).
SDS-PAGE analysis and Western blotting were performed using protein gel prepared using Kaiki Bio Inc kit, and experimental procedures were performed according to SDS-PAGE and Western blotting protocols. Finally, photographing by a Tanon 5200 (Shanghai China) photo-adhesive instrument.
EXAMPLE 1 antiviral function study of EcIL-15L
1) Construction of eukaryotic recombinant plasmids
Construction of plasmid pcDNA3.1-Flag-EcIL-15L: according to the EST sequences of the grouper transcriptome, the gene of the grouper interleukin 15 (EcIL-15L) is obtained through PCR amplification (figure 1A, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2). The gene sequence (FIG. 1B) was then inserted into a plasmid pcDNA3.1-Flag (Invitrogen, shanghai) by homologous recombination (primer sequence for gene amplification: ecIL-15L-3.1-Flag-F: CTTGGTACCGAGCTCATGCTGAGAGGGAGGTTAGCT; ecIL-15L-3.1-Flag-R: GTGCTGGAT ATCTGCGTTGCAGTTACTAAAGTTCATCATCTCAAGAA), and pcDNA3.1-Flag-EcIL-15L was constructed. The plasmid was subsequently transformed into E.coli DH 5. Alpha. And cultured on LB solid medium containing ampicillin (using a concentration of 50. Mu.g/mL), and the transformed strain was subsequently selected by means of bacterial liquid PCR and sequencing.
2) Synthesis of EcIL-15L interfering RNA
Three pairs of EcIL-15L interfering RNAs were synthesized by Ji Ma Bio Inc. (IL-15L-siRNA-1-F: GGUUGAACUGCA CAUUGUATT, IL-15L-siRNA-1-R: UACAAUGUGCAGUUCAACCTT; IL-15L-siRNA-2-F: GAAAUGUCUUGCUGACGAATT, IL-15L-siRNA-2-R: UUCCGUCAG-CAAGACAUUUCTT; IL-15L-siRNA-3-F: GCUGUUCCUUAACGAUCUUTT, IL-15L-siRNA-3-R: AAGAUCGUUA AGGAACAGCTT). A pair of siRNA 2-EcIL-15L (IL-15L-siRN A-2-F: GAAAUGUCUUGCUGACGAATT, IL-15L-siRNA-2-R: UUCGGUCAG-CAAGACAUU UCTT) with the best interference effect was determined by qPCR for subsequent experiments.
3) Antiviral function study of EcIL-15L
To determine the role of ecll-15L during fish viral infection, the effect of over-expression of ecll-15L on viral gene transcription and protein expression was examined by qPCR and WB, respectively.
GS cells are digested by 0.25% trypsin digestion solution, transferred into a 24-hole cell culture plate, placed in a 28 ℃ incubator for culturing for about 18 hours, observed under a microscope, and transfected after the cells are fully spread with a monolayer. The experiment adopts Lipofectamine TM Transfection of pcDNA3.1-Flag-EcIL-15L (Flag-EcIL in FIG. 2) with 2000 reagent (Invitrogen)-15L) or si-RNA2-EcIL-15L, the specific steps being carried out according to the operating requirements. The different experiments were controlled with empty vector pcDNA3.1-Flag or NC. Wherein the siRNA experiments were all performed using RNase-free assay consumables.
In EcIL-15L transfected cells, transcription and protein levels of EcIL-15L were significantly up-regulated compared to the empty vector pcDNA3.1-Flag, indicating successful overexpression of EcIL-15L in transfected cells (FIG. 2A). From the view of virus-induced cytopathy, overexpression of ecll-15L delayed SGIV-induced CPE (fig. 2B). ecll-15L overexpression significantly reduced the transcription of SGIV MCP and VP19 and MCP protein expression levels (fig. 2C and D).
The effect of knockdown ecll-15L on SGIV replication was subsequently studied. As will be seen from FIG. 3A, three specific siRNAs targeting EcIL-15L inhibited expression of EcIL-15L with knockdown efficiencies of 61%, 45% and 68%, respectively, compared to control NC (FIG. 3A). Thus, si-RNA2-EcIL-15L was selected for subsequent experiments. Transfected si-RNA2-EcIL-15L cells were exacerbated by CPE induced by SGIV infection compared to control cells (FIG. 3B). Knocking down ecll-15L significantly promoted transcription of SGIV MCP and VP19 and MCP protein expression levels (fig. 3C and D).
In conclusion, ecIL-15L may exert antiviral effects in fish DNA virus infection.
Example 2 preparation of cytokine EcIL-15L protein
1) Construction of prokaryotic recombinant plasmid
Construction of plasmid pET-32 a-EcIL-15L: according to the EST sequences of the grouper transcriptome, the grouper IL-15L (EcIL-15L) gene (shown in figure 1A, the nucleotide sequence of which is shown as SEQ ID NO.1, and the coded amino acid sequence of which is shown as SEQ ID NO. 2) is obtained through PCR amplification. Then the gene sequence (FIG. 1B) was inserted into the plasmid pET-32a by homologous recombination (the amplified primer sequence of the gene is EcIL-15L-32a-F: CTTGTCGACGGAGCTATGCTGAGAGGGAG GTTAGCT, ecIL-15L-32a-R: AGGCCATGGCTGATAGTTGCAGTTACTAAAGTTCATCATCT CAAGAA), and pET-32a-EcIL-15L was constructed. Then, the plasmid was transformed into E.coli DH 5. Alpha. And cultured on LB solid medium containing ampicillin (using 50. Mu.g/mL), and the transformed strain was subsequently selected by means of bacterial liquid PCR and sequencing, and the plasmid pET-32a-EcIL-15L was extracted and transformed into E.coli BL21 to obtain host strain BL21 containing recombinant plasmid pET-32a-EcIL-15L.
2) Inducible expression of proteins
Expression and purification of EcIL-15L protein: host bacterium BL21 containing recombinant plasmid pET-32a-EcIL-15L is induced by 1mM IPTG at 37 ℃, then the whole bacterium is added with a proper proportion of protein loading buffer solution, boiled for 5min, directly analyzed by 12% SDS-PAGE electrophoresis (figure 4A), and the WB identification is carried out. The results show that: the protein expression product was about 21kDa in size (FIG. 4B), the uninduced sample did not have protein in large amounts at the corresponding location; since the induced EcIL-15L protein was insoluble, we dissolved inclusion bodies washed with 1% Triton X-100 and then with 1M urea in the experiment in 6M urea, dialyzed gradient into 2M urea solution, followed by purification with His-Ni-NTA resin, the purified protein strips were clear, and then imidazole eluted proteins were dialyzed with a large to small concentration gradient of imidazole solution until dialysis with PBS was completed. The concentration of protein dialyzed against PBS was 0.65mg/mL, and the amount of the target protein was about 90% by BandScan 5.0 analysis (FIG. 4C).
Expression and purification of MCP protein: according to the existing research results in the laboratory, the capsid protein MCP (SEQ ID NO. 3) of SGIV is selected as a subunit vaccine candidate protein, the encoding gene of the capsid protein MCP is transferred into pET-32a to obtain pET-32a-MCP, and then transferred into escherichia coli BL21 to obtain pET-32a-MCP prokaryotic expression bacteria, and the prokaryotic expression bacteria are stored after a large amount of culture induction protein expression is performed through a fermentation tank. In the experiment, the preserved induced expression bacteria are directly used for protein purification to prepare the SGIV subunit vaccine. SDS-PAGE analysis shows that the target protein expressed by pET-32a-MCP (SGIV-MCP protein) has a size of about 60kDa and exists mainly in inclusion bodies of cells. The preparation method comprises the following steps: the inclusion bodies of SGIV-MCP protein are washed by 1% Triton X-100 and then washed by urea with different concentrations, and 2.0M urea can dissolve part of the impurity protein and retain almost all target proteins. Since the induced SGIV-MCP protein is insoluble protein, the inclusion body washed in the previous step is dissolved in 6M urea, and dialyzed in urea solution from high to low gradient until the dialysis with PBS is completed. SGIV-MCP protein concentration was 1.05mg/mL dialyzed against PBS and the amount of target protein was about 85% as analyzed by BandScan 5.0 (FIG. 4D).
EXAMPLE 3 application of EcIL-15L protein as cytokine adjuvant in SGIV-MCP subunit vaccine
To determine the adjuvant effect of EcIL-15L protein, we mixed EcIL-15L protein with SGIV subunit vaccine at a mass ratio of 1:5 and injected the fish. Injecting 100 mu L of vaccine adjuvant mixed solution (50 mu g SGIV-MCP protein mixed with 10 mu g grouper EcIL-15L protein) into each tail fish abdominal cavity; BL21 (DE 3) expressing bacteria containing pET-32a plasmid washed protein (60. Mu.g/tail) served as Control. The groupers are purchased from a grouper farm in Hainan province in China, are bred in a sea water culture system in Yanshan area of agricultural university in south China, and are of the variety of gold tiger (the groupers are the female parent of the groupers in brown spots and the female parent of the groupers in blue bodies). The weight is 3-4g, and the body length is 6.0-6.5cm. In experiments to verify the adjuvant effect of ecll-15L in SGIV subunit vaccine, three groups, control group (Control), were divided; MCP groups were injected alone (60 μg/tail); mcp+ecil-15L group; for each group of 50 fish, 35 were used for statistical mortality and the remainder were used for sampling. The sampling and toxin attacking treatment modes are as follows: liver, spleen, head and kidney and serum were collected at 7d and 14d after immunization. On day 15 of immunization, 100. Mu.L of SGIV virus solution (LD) was intraperitoneally injected 80 )(1.0×10 6 TCID 50 fish -1 ) The number of dead fish was counted daily for 14 days. 3 tail fish liver, spleen and head and kidney samples are mixed into 1 sample; serum from 5 fish was pooled into 1 sample. The separation mode of serum: the grouper takes blood from the heart, the collected blood is flatly placed in a 1.5mL EP tube and kept at room temperature, the cover is opened to contact with air, the grouper is put into a refrigerator at 4 ℃ for standing overnight after 2 hours, the next day, 800 Xg is carried out, the centrifugation is carried out for 5min, and the supernatant is sucked out for preservation at-80 ℃. The experimental results are shown in FIG. 5A, where SGIV infection resulted in final survival rates of 51.4% and 77.1% for the MCP group and the MCP+EcIL-15L group, respectively, and 20.0% for the Control group. The calculated relative protection rate is shown in Table 1, the relative protection rate of the MCP+EcIL-15L group is 71.25%, and the relative protection rate of the subunit vaccine of the single expressed MCP protein group is 39.25%. The results of the relative protection rate calculation for the vaccine are shown in table 1. At the same time, MCP antibody titer water in serumThe MCP + EcIL-15L vaccine group was shown to be significantly improved over the MCP vaccine group (as shown in fig. 5B). Furthermore, the fluorescence quantification result showed that the MCP+EcIL-15L group had a better promoting effect on the immune-related genes (FIG. 6).
TABLE 1
Nucleotide sequence of EcIL-15L (SEQ ID NO. 1)
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MLRGRLALASVYLCFVCLLGLTLQLPAKPCANDIFAKLEALVGEVHNLEGLNCTLYTPTTEDFKNCPKSTLKCLADEVKVLTEELEAFNVPGMHEFPLIKNLRRLAVRIKKTESNCPRCELLIEKDAKLFLNDLHTILEMMNFSNCNSGIV-nucleotide sequence of MCP (SEQ ID NO. 3)
ATGACTTGTACAACGGGTGCTGGCGTAACCAGTGGCTTCATAGATTTGGCCACGTACGACAATCTCGACAGAGCGTTATACGGCGGAAAGGACGCCACTACGTACTTTATCAAAGAGCATTATCCCGTAGGATGGTTCACCAAGCTTCCCACCATGGCCACCAGAGTGTCTGGAAACCCAGCGTTCGGGCAAGAGTTTTCGGTCGGGGTTCCCAGGTCGGGCGATTACGTGCTCAACGCCTGGCTCACTCTTAAAACCCCCGAAATTAAACTGCTCGAAACAAATAGGCTCGGCGCAAACGGTACCGTGAGGTGGACCAAAAACCTAATGCACAACGCTGTAGAGCACGCTTCTCTCACCTTCAACGACATTTGCGCGCAGCAGTTTAACACAGCGTATTTAGACGCTTGGACACAGTTTAACATGTGCGAAGGTAAACGCATAGGTTACGACAACATGATCGGGAACACCAGCGACATGACCAACCCCACTCCCGCTCAGGGTCAGGACGGCGCAAGGACACTACCTTCCAAAAATTTAGTGCTCCCGTTGCCGTTCTTTTTCAGCAGAGACTGCGGATTGGCTCTGCCCACCGTAGTGTTGCCCTATAATGAAATCAGAATCAACATTAAACTGAGGTCGCTTCAGGAGCTTTTAGTGTTTCAGAACAAAGACACCGGAAATGTGATTCCTATCTCTGCTACCGACATAGCCGGCGGGCTCGCCGACACCGTGGAAGCTTACGTGTATATGACCGTGGGTCTCGTTTCCAACGTGGAAAGGTGCGCCATGGCAGGGACCGTCAGGGATATGGTCGTAGAACAAATGCAGGCCGCCCCCACACACATCGTTAACCCTCAAAACACAAATAACGTCCACGTAGACATGAGGTTCTCGCACGCCGTGAAAGCCCTCTTTTTCATGGTGCAAAACGTCACTTATAAATCTGTGGGTTCAAATTATACGTGTGTAACACCAGTTAACGGTCCGGGCAACACCGTGATGGAGCCCGCCATGTCCGTTGATCCCATCAAAAGCGCCAGCCTCACGTACGAAAATACGACCAGGTTGGCAAATATGGGTGTAGAGTATTACTCTCTGGTACAACCTTGGTATTTTTCAGCCTCCATTCCAGTGTACACCGGATACCACATGTATTCATACGCCCTAAACGTGGGCAGCGTTCATCCTTCGGGGTCTACCAATTACGGAAGATTGACCAACGCTAGCATCACTGTAACAATGTCCCCCGAGTCTGTCGTCGCCGCAGCAGGAGGAGGTAACAATAATTCTGGTTACAACGAACCTCAGAGGTTCGCGTTGGTAGTGATCGCCGTAAACCATAACGTCATCAGAATCAT
GAACGGTTCCATGGGGTTCCCTATCTTGTAA。
Claims (8)
1. The grouper interleukin 15 is characterized in that the amino acid sequence of the grouper interleukin 15 is shown as SEQ ID No: 2. as shown.
2. A gene encoding grouper interleukin 15 according to claim 1, characterized in that the nucleotide sequence is as set forth in SEQ ID No: 1.
3. A eukaryotic expression vector, characterized in that: it comprises the nucleotide sequence shown in SEQ ID NO. 1.
4. A prokaryotic recombinant expression vector, characterized in that: it comprises the nucleotide sequence shown in SEQ ID NO. 1.
5. A recombinant bacterium, characterized in that: comprising the prokaryotic recombinant expression vector of claim 4.
6. A recombinant protein, characterized in that: expressed by the recombinant bacterium of claim 5.
7. The preparation method of the grouper interleukin 15 recombinant protein is characterized by comprising the following steps:
1) Construction of recombinant plasmids: the synthetic nucleotide sequence is shown as SEQ ID No:1, inserting the gene into an expression vector to obtain a recombinant expression vector, transforming the recombinant expression vector into prokaryotic expression bacteria, culturing, and screening out the prokaryotic expression bacteria successfully transformed;
2) Inducing and expressing the prokaryotic expression bacteria which are successfully transformed;
3) Purifying and verifying functional activity by using the protein expressed by induction to obtain the grouper interleukin 15 recombinant protein.
8. Application of a recombinant protein of grouper interleukin 15 as a cytokine adjuvant in preparation of a grouper iridovirus MCP subunit vaccine, wherein the amino acid sequence of the recombinant protein of grouper interleukin 15 is shown as SEQ ID No: 2. as shown.
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| CN108079287A (en) * | 2017-12-19 | 2018-05-29 | 华南农业大学 | A kind of subunit vaccine of grouper irido virus and its preparation method and application |
| CN108273053A (en) * | 2018-01-04 | 2018-07-13 | 华南农业大学 | A kind of recombinant envelope protein vaccine of grouper irido virus and its application |
| CN111363758A (en) * | 2020-03-26 | 2020-07-03 | 华南农业大学 | Preparation and application of polyclonal antibody of major capsid protein of epinephelus neocalifornicus iridovirus |
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2023
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108079287A (en) * | 2017-12-19 | 2018-05-29 | 华南农业大学 | A kind of subunit vaccine of grouper irido virus and its preparation method and application |
| CN108273053A (en) * | 2018-01-04 | 2018-07-13 | 华南农业大学 | A kind of recombinant envelope protein vaccine of grouper irido virus and its application |
| CN111363758A (en) * | 2020-03-26 | 2020-07-03 | 华南农业大学 | Preparation and application of polyclonal antibody of major capsid protein of epinephelus neocalifornicus iridovirus |
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