Subunit oral vaccine for ricefield eel rhabdovirus and application
By screening and tandem expression of eel rosy virus G and N proteins, an efficient subunit oral vaccine was prepared, which solved the problem of eel's insufficient defense ability against the virus infection and achieved a high protection rate vaccine effect.
Patent Information
- Application Number
- CN202411744443.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-30
- Publication Date
- 2025-03-04
AI Technical Summary
There is currently a lack of oral vaccine for eel rosy virus subunits, which has led to a weak defense ability of eels against infection of the virus, especially the poor protection effect on seedlings.
By screening the B cell and T cell antigen epitope of the eel rhizovirus G protein, four protein sequences of 175-178aa length were designed, and combined with the N protein in tandem, the fusion protein was expressed through the baculovirus expression system, and finally the subunit oral vaccine was prepared by sodium alginate microspheres.
The vaccine immunized eels by oral administration, significantly improving the efficiency of its mask immune barrier, with a protection rate of more than 90%, effectively preventing eel rhizovirus infection.
Smart Images
Figure BDA0005163309490000031 
Figure BDA0005163309490000041 
Figure BDA0005163309490000042
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological gene engineering, and in particular relates to an oral vaccine for yellow eel rhabdovirus subunits and an application thereof. Technical Background
[0002] Chinese rice-field eels rhabdovirus (CrERV) infects farmed eels and causes viral hemorrhagic disease, which can not only cause the death of adult eels, but also cause more serious damage to eel fry, with a mortality rate of up to 90%. This virus belongs to the genus Perhabdovirus of the family Rhabdoviridae, and is a type of bullet-shaped or rod-shaped, enveloped, non-segmented single-stranded, negative-stranded RNA virus with a total length of 11545 nucleotides. The genome of CrERV consists of (from 3′-5′) nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large protein (L).
[0003] Based on bioinformatics analysis and previous research results, G protein is an important antigen of the Rhabdoviridae family. It produces host immune responses and induces the production of neutralizing antibodies to the virus. It has important immunogenicity and is often used in vaccine research. The most studied fish rhabdovirus vaccines are infectious hematopoietic necrosis virus (IHNV) and carp spring viremia virus (SVCV). Usually, the full-length G protein is used as the antigen of the subunit vaccine. Recent studies have shown that truncated IHNV G protein subunit vaccines also have good immune effects. However, due to the possibility of loss of immunogenicity after truncated expression, it is very necessary and critical to screen truncated proteins with good immunogenicity.
[0004] Although the N protein itself does not have an immune protection response of the body, it can enhance the immune protection induced by the G protein. The protection efficiency of dogs after combined immunization with rabies virus G protein and N protein is significantly improved. However, there are no reports on the tandem expression of G and N proteins, and it is not clear whether the protein conformation will change after tandem expression, thereby affecting the immunogenicity of G protein. There is no research on the yellow eel rhabdovirus vaccine, and the research on tandem protein subunit vaccine is even blank. Therefore, the establishment of an expression system that can efficiently express the yellow eel rhabdovirus tandem protein is of great significance to the development of yellow eel rhabdovirus vaccine and the effective prevention of yellow eel viral hemorrhagic disease.
[0005] The baculovirus expression system has been widely used as a powerful expression vector for producing recombinant proteins and developing subunit vaccines in insect cells, and is used to express a variety of viral structural proteins. The development of tandem protein subunit vaccines can enhance protection efficiency, reduce the number of vaccinations, and simplify the immunization procedure. Compared with injection immunization, oral immunization is the most natural immunization route for fish disease prevention, which is less harmful to the fish body and easy to implement. With the development and application of microspherization technology, oral vaccine antigens are less susceptible to enzymatic hydrolysis by the digestive system in the body, becoming a hot topic in aquatic vaccine research.
[0006] In response to the problem that there is currently no subunit oral vaccine for yellow eel rhabdovirus, the applicant used different algorithms to predict the B cell antigen epitopes and T cell antigen epitopes in the yellow eel rhabdovirus G protein, and finally screened out 4 protein sequences with a length of 175-178aa. The above-mentioned G protein truncated sequences were then combined in series with the N protein through a linker, and the fusion protein was expressed through a baculovirus expression system. The expression level and immunogenicity were screened, and the subunit oral vaccine was finally obtained after coating with sodium alginate microspheres. The yellow eel is immunized orally to improve the eel's facial mask immune barrier, thereby effectively preventing the yellow eel rhabdovirus infection. Summary of the invention
[0007] The object of the present invention is to provide an artificially synthesized fusion protein, wherein the protein is shown in SEQ ID NO.8.
[0008] Another object of the present invention is to provide the use of the above fusion protein in the preparation of an oral vaccine of the yellow eel rhabdovirus subunit.
[0009] In order to achieve the above object, the present invention adopts the following technical measures:
[0010] An artificially synthesized fusion protein, the protein is shown as SEQ ID NO.8.
[0011] A polynucleotide encoding the above fusion protein.
[0012] An expression vector for expressing the above fusion protein.
[0013] The expression vector described above is preferably a baculovirus expression vector.
[0014] A recombinant microorganism expressing the above fusion protein.
[0015] A complex containing the above fusion protein.
[0016] Application of the above fusion protein, polynucleotide, expression vector, recombinant microorganism or complex in the preparation of medicine for treating or preventing rice field eel rhabdovirus infection.
[0017] The application of the above fusion protein, polynucleotide, expression vector, recombinant microorganism or complex in the preparation of an oral vaccine of rice field rhabdovirus subunit.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1. The present invention provides for the first time an orally-administered rice field eel rhabdovirus vaccine. After oral administration of the vaccine, the vaccine is challenged with the virus, and the protection rate of the vaccine can reach more than 90% after 7 days of observation.
[0020] 2. The present invention cloned the G protein antigen sequence of the yellow eel rhabdovirus in full length, screened for truncation, and expressed it in tandem with the N protein, and selected two proteins that can be expressed at high levels in the baculovirus expression system. Further screening of immunogenicity found that G2-N had a better immune effect, and was ultimately used as an oral vaccine for yellow eel rhabdovirus.
[0021] 3. The vaccine prepared by the present invention has a simple immunization method, and oral mixing with feed is easy to implement on a large scale, avoiding the stress response caused by injection, and can effectively prevent the infection of the yellow eel rhabdovirus. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the nucleic acid electrophoresis diagram of the amplified fragment of the target gene of the yellow eel rhabdovirus.
[0023] Among them: A, M: DL1000, lane 1: G1, 2: G2, 3: G3, 4: G4; B, M: DL1000, lane 1: N.
[0024] Figure 2 This is a schematic diagram of the recombinant plasmid pFastBac-G1-N of the yellow eel rhabdovirus.
[0025] Figure 3 This is a schematic diagram of the blue-white screening recombinant Bacmid target plasmid.
[0026] Among them: A, Bacmid-G1-N; B, G2-N; C, G3-N; D, G4-N.
[0027] Figure 4 This is a schematic diagram comparing normal Sf9 cells and recombinant baculovirus-infected Sf9 cells.
[0028] Among them: A, normal Sf9 cells; B, Bacmid-G1-N infected Sf9 cells; C, Bacmid-G2-N infected Sf9 cells; D, Bacmid-G3-N infected Sf9 cells; E, Bacmid-G4-N infected Sf9 cells.
[0029] Figure 5 This is a schematic diagram of PCR detection of target genes in baculovirus.
[0030] Among them: M: DL1000, lane 1: G1-N, 2: G2-N, 3: GN 3, 4: G4-N, 5: negative control.
[0031] Figure 6 It is a schematic diagram of indirect immunofluorescence verification of protein expression.
[0032] Among them: A, normal Sf9 cells; B, Bacmid-G1-N infected Sf9 cells; C, Bacmid-G2-N infected Sf9 cells; D, Bacmid-G1-N infected Sf9 cells; E, Bacmid-G1-N infected Sf9 cells. DETAILED DESCRIPTION
[0033] In order to make the present invention easier to understand, the embodiments of the present invention will be further described below. The present invention is further described and demonstrated in conjunction with the implementation. However, this embodiment is not a limitation of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials, unless otherwise specified, are all derived from commercial channels.
[0034] Example 1: Obtaining recombinant baculovirus
[0035] 1. Design of key antigens of yellow eel rhabdovirus and construction of shuttle plasmid Bacmid
[0036] 1.1 Target gene cloning and cloning vector construction
[0037] The applicant screened out four antigenic regions from the G protein of the genomic sequence of the eel rhabdovirus (MH319839), which were named G1, G2, G3 and G4. The total RNA of the eel kidney cell line infected with the eel rhabdovirus was extracted by the TRIzol method, and reverse transcribed into cDNA using a reverse transcription kit and used as a template to perform PCR amplification of the G (G1, G2, G3 and G4) and N genes, respectively, wherein six His tags were added to the 3' end of the N gene. The primers for each target gene are shown in Table 1. The PCR products were detected by 1% agarose gel electrophoresis, and the results were as follows: Figure 1 As shown, the target fragments were recovered and purified using a PCR product purification kit.
[0038] Table 1 Target gene amplification primer sequence information
[0039]
[0040]
[0041] 1.2 Construction of pFastBac-GN recombinant baculovirus vector
[0042] The pFastBac vector was double-digested with BamHI / EcoR to recover the vector backbone; G1, G2, G3 or G4 and N were inserted into the vector by homologous recombination. Specifically, a 20ul recombination reaction system was prepared:
[0043]
[0044] Ice bath for 10 minutes, immediately add to DH5α competent cells, ice bath for 20 minutes, heat shock at 42℃ for 90s, add SOB liquid medium and place on a shaker for 45 minutes, then spread on LB solid medium containing 100ug / ml ampicillin antibiotics; amplify and identify positive clones containing the target gene with universal primers, and identify the correct transformants and name them as: pFastBac-G1-N (such as Figure 1 ), pFastBac-G2-N, pFastBac-G3-N, pFastBac-G4-N, and the schematic diagrams of the other three transformants are Figure 1 Replace G1 with G2, G3 or G4.
[0045] 1.3 Construction and identification of recombinant plasmid Bacmid-GN
[0046] Refer to the instructions of Bac-to-Bac baculovirus expression system, transform 1ng of the correctly constructed recombinant plasmid (pFastBac-G1-N, pFastBac-G2-N, pFastBac-G3-N, pFastBac-G4-N) into DH10Bac competent cells containing baculovirus backbone plasmid Ba cmid. Mix well and place on ice for 30min. Heat shock at 42℃ for 45s, then immediately place on ice for 2min. Add 900μL of room temperature SOC medium and culture at 37℃, 225 rpm for 4 hours. Use SOC medium at 10 -1 , 10 -2 , 10 -3 After diluting the competent cells, take 100 μL of the diluted resuscitated bacterial solution and spread it on LB plates containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL Biuo-gal, and 40 μg / mL IPTG, and culture it upside down at 37°C for 48 hours. With the assistance of the Helper plasmid, the recombinant transfer vector pFastBac and the baculovirus backbone plasmid Bacmid undergo homologous recombination, and the exogenous target gene is integrated into the baculovirus backbone plasmid to form a recombinant Bacmid plasmid. Blue-white colonies can be seen, such as Figure 3 shown.
[0047] Pick 10 white positive monoclonal colonies each, re-streak them on fresh LB plates containing 50μg / mL kanamycin, 7μg / mL gentamicin, 10μg / mL tetracycline, 100μg / mL Biuo-gal, and 40μg / mL IPTG, and culture them at 37°C overnight. After three rounds of blue-white screening, purify the positive colonies. The screened white positive monoclones were inoculated into 2mL LB medium containing 50μg / mL kanamycin, 7μg / mL gentamicin, and 10μg / mL tetracycline, and cultured at 37°C in a shaker at 250-300rpm for more than 24h. Subsequently, the recombinant plasmid was extracted with a plasmid extraction kit, and the positive clones containing the target gene were amplified and identified with universal primers (PUC / M13-F; PUC / M13-R). The correctly identified plasmids were named: Bacmid-G1-N, Bacmid-G2-N, Bacmid-G3-N, and Bacmid-G4-N.
[0048] 2. Obtaining recombinant baculovirus
[0049] 2.1 Obtaining P1 recombinant baculovirus
[0050] According to Cellfection TM II Lipofectamine transfection kit instructions, well-growing SF9 cells were inoculated into a 6-well plate, with a cell density of about 8×10 5 100 μg recombinant plasmid and 6 μL cell culture medium were added. TM Ⅱ Reagent was added with 100 μL of double-antibody serum-free Sf-900 TM -ⅢSFM (1×), stand at room temperature for 30 minutes and mix well. After incubation at room temperature for 20 minutes, add the mixed solution to the 6-well plate and place it in a 27℃ constant temperature incubator for 4-6 hours. TM Ⅱ Reagent mixture of cell culture medium, add 2mL fresh Sf-900 TM -ⅢSFM (1×) medium, cultured in a 27℃ constant temperature incubator. After 72h, obvious cell lesions can be seen, such as Figure 4 As shown, the diseased cells and culture fluid were collected, centrifuged at 1000 r / min for 5 min, the supernatant was collected, and stored at 4° C. in the dark, which is the P1 generation recombinant baculovirus.
[0051] 2.2 Virus expansion and culture
[0052] SF9 cells (about 9×10 5The cells were cultured overnight in a T25 cell flask. The P1 virus was infected at a volume ratio of 5% in cells in the logarithmic growth phase. The cells were cultured in a 27°C constant temperature incubator for 3-5 days. When the cells showed obvious lesions, the cells were blown off with a pipette and divided into 15 mL centrifuge tubes. The tubes were centrifuged at 1000 r / min for 5 min, the supernatant was collected, and the tubes were stored at 4°C in the dark. The P2 virus was obtained. The P3 virus was collected in the same way and stored at 4°C in the dark, or stored at -80°C for a long time. After the viral DNA was extracted, the target gene was amplified with the primers in Table 1 and identified by 1% agarose gel electrophoresis. Figure 5 As shown, the amplified bands are consistent in size with the sum of the genes of G1 and N, G2 and N, G3 and N, and G4 and N, respectively, indicating that the target gene was successfully recombined in the baculovirus.
[0053] 2.3 Recombinant baculovirus titer (TCID 50 ) determination
[0054] SF9 cells with good growth status were plated in 96-well cell culture plates. When the cell density grew to 70%, the P3 generation recombinant baculovirus was taken and graded diluted according to the following ratios: 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , discard the culture medium in the cell culture wells, add 100 μL of virus dilutions of each gradient, repeat 8 wells for each gradient, make blank controls, observe the cell lesions every day and make statistics. 96 hours after infection, the Reed-Muench method (lgTCID 50 = logarithm of dilution higher than 50% + distance ratio × logarithm of dilution factor) to calculate virus titer. TCID50 = 10 -6.4 / 0.1mL (P3 generation virus).
[0055] Example 2: Recombinant baculovirus highly expressed tandem proteins
[0056] Cells in good growth condition were transferred to confocal dishes and cultured until the cell confluence reached 70%. The cell culture medium was removed, and the recombinant baculovirus was inoculated into each well at an inoculation volume of MOI = 1. After incubation with SF9 cells for 1 hour, the virus solution was discarded, 1 mL of fresh culture medium was added, and cultured at 27°C for 3 days. After washing with PBS three times, 4% paraformaldehyde was fixed at room temperature for 20 minutes; after washing with PBST three times, 0.2% TritonX-100 was permeabilized at room temperature for 10 minutes. After blocking with 5% BSA, anti-His-tagged mouse monoclonal antibody was incubated at room temperature for 2 hours. Washed with PBST three times, and incubated with FITC-labeled goat anti-mouse secondary antibody for 1 hour. After DAPI staining, the cells were observed under a confocal microscope. The Bacmid empty group was used as the control. The brighter the fluorescence displayed by the cells, the more efficient the recombinant virus was infecting SF9 cells and producing the target protein. The results showed that G2-N and G3-N had the highest fluorescence intensity, that is, the highest protein expression level, such as Figure 6 shown.
[0057] Based on the above results, the applicant selected G2-N (the protein is shown in SEQ ID NO.8, and the polynucleotide sequence encoding it is shown in SEQ ID NO.6) and G3-N (the polynucleotide encoding G3-N is SEQ ID NO.7) as candidate subunit oral vaccines.
[0058] Embodiment 3:
[0059] Screening for Linker Protein Codon Optimization
[0060] According to the method of Example 1, recombinant baculoviruses with different codon optimizations for Linker protein were constructed, and the most suitable Linker protein codon sequence was determined by evaluating the protein expression level as shown in Table 2.
[0061] Table 2 Linker protein codon optimization
[0062]
[0063] Note: Experimental verification shows that the optimized GGGGS*2(1) sequence is the best sequence, and this application uses this sequence.
[0064] Embodiment 4:
[0065] Preparation and immunological evaluation of oral vaccines of G2-N and G3-N proteins
[0066] SF9 cells (about 9×10 5The cells were subcultured to T25 cell flasks, and when the confluence of SF9 cells reached 70%, the recombinant baculovirus prepared in Example 1 of P3 generation was inoculated into each well at an inoculum MOI of 1, and the total cell protein was extracted. After purification by affinity chromatography, the concentrations of the recombinant target proteins G2-N and G3-N were determined to be 2 μg / mL and 1.8 μg / mL, respectively. The obtained recombinant proteins were freeze-dried and stored at -80°C.
[0067] Take 25mL of 2% sodium alginate solution, add 0.165g of CaCO3, powder, and then add 1mg / mL of recombinant target protein (the control group was prepared with PBS at pH = 7.4 instead of protein). Add 20mL of liquid paraffin (containing 1mL span-80) dropwise, stir at 800rpm for 15min at room temperature. Continue to add 10mL of liquid paraffin (containing 675μL of glacial acetic acid) dropwise, stir at 600rpm for 30min at room temperature. Add 100mL of 0.85% NaC1 solution (containing 1mL tween 80), stir at 600rpm for 10min at room temperature. Add the solution to a separatory funnel, let it stand for 2h, then remove the lower aqueous phase and store at 4℃. The vaccine encapsulation efficiency is 80%.
[0068] One week before the experiment, healthy and disease-free eels of the same batch with a body length of 13-18 g were selected and raised in water at 28° C. The eels were randomly divided into 5 groups, with 30 eels in each group.
[0069] The feeding conditions are as follows: normal feed (control group), G2-N (50 mL / kg, i.e. 40 mg G2-N protein / Kg feed, G2-NA group), G2-N (100 mL / kg, i.e. 80 mg G2-N protein / Kg feed, G2-NB group), G3-N (50 mL / kg, i.e. 40 mg G3-N protein / Kg feed, G3-NA group), G3-N (100 mL / kg, i.e. 80 mg G3-N protein / Kg feed, G3-NB group), mixed with feed and orally administered on the 1st, 3rd and 5th days each week, and immunized for a total of 28 days starting from the first day of feeding.
[0070] The eels in each group were given 100 μL of 10 7 TCID 50 / mL of yellow eel rhabdovirus.
[0071] The observation was carried out for 2 weeks, and the number of sick and dead fish was recorded daily. Table 3 shows the observation results on the 7th day of infection.
[0072] Table 3 Immune protection efficacy of oral vaccine 7 days after challenge
[0073] Group Number of experiments Vaccine content Protection ratio Oral immunization group 1G2-NA 30 50mL / kg 23 / 30 Oral immunization group 2G2-NB 30 100mL / kg 27 / 30 Oral immunization group 3G3-NA 30 50mL / kg 19 / 30 Oral immunization group 4G3-NB 30 100mL / kg 22 / 30 Control group 30 PBS 12 / 30
[0074] According to the results in the table, the survival rate of the G2-NB group was the highest, at 93.3%, indicating that immunization of grass carp with purified recombinant protein G2-N had a good immune protection against hemorrhagic disease.
Claims
1. An artificially synthesized fusion protein, the protein being shown in SEQ ID NO.
8.
2. A polynucleotide encoding the fusion protein of claim 1.
3. An expression vector for expressing the fusion protein according to claim 1. The expression vector according to claim 3 , which is a baculovirus expression vector.
5. A recombinant microorganism expressing the fusion protein of claim 1. A complex comprising the fusion protein according to claim 1.
7. Use of the fusion protein of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the recombinant microorganism of claim 5 or the complex of claim 6 in the preparation of a drug for treating or preventing rice field rhabdovirus infection.
8. Use of the fusion protein of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the recombinant microorganism of claim 5 or the complex of claim 6 in the preparation of an oral vaccine of the subunit of yellow eel rhabdovirus.