A largemouth bass iridovirus, its prepared vaccine, and its applications.

Abstract

This invention provides a largemouth bass iridovirus, its prepared vaccine, and its applications, belonging to the field of biological vaccine technology. The largemouth bass iridovirus LMBV-ZJDSS-F110 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on September 20, 2024, with accession number CCTCC NO: V202485. This strain and the vaccine prepared from it can effectively prevent largemouth bass iridovirus infection and provide good protection against largemouth bass iridovirus disease.

Description

Technical Field

[0001] This invention relates to the field of biological vaccine technology, and in particular to a largemouth bass iridovirus, a vaccine prepared from it, and its application. Background Technology

[0002] LMBV (Largemouth Bass Ranavirus) is a large double-stranded DNA virus belonging to the genus Ranavirus in the family Iridoviridae. It is one of the main pathogens causing severe economic losses in the largemouth bass (Micropterus salmoides) aquaculture industry. Because LMBV infection often does not present characteristic clinical symptoms, or even obvious signs, clinical diagnosis is difficult. However, epidemiological data show that the morbidity rate of largemouth bass infected with LMBV can reach 20% to 30%, and in some years even exceeds 40%. The mortality rate in affected ponds is usually between 20% and 40%, and in extreme cases, the cumulative mortality rate can even exceed 80%. The prevalence of LMBV is in the water temperature range of 25°C to 32°C, with the peak incidence period from July to August. Largemouth bass of all sizes can be infected, with an acute onset mortality period of only 3 to 5 days, and the entire disease process can last for about one month.

[0003] LMBV has a wide host range, infecting not only largemouth bass but also other fish such as mandarin fish (Siniperca chuatsi) and snakehead (Channa argus). Especially in smaller largemouth bass at different growth stages, LMBV exhibits higher pathogenicity, and its virulence varies closely with different strains. Epidemiological surveys conducted by the project team over the past three years have found a high detection rate of LMBV in mandarin fish in the Jiangsu, Zhejiang, and Anhui regions, and the incidence of LMBV-induced iridovirus disease in mandarin frogs has been increasing year by year. The whole genome sequence homology of LMBV strains isolated from mandarin fish and largemouth bass ranges from 99.0% to 100.0%, indicating a high degree of similarity in their genomes across different hosts.

[0004] Currently, immunization is the most effective means of preventing and controlling largemouth bass iridovirus disease (LMBV). Although some new strains have been isolated, the significant genetic variations among LMBV strains and the large differences between circulating strains and vaccine strains have resulted in unsatisfactory efficacy of existing vaccines. While some LMBV vaccine patent applications (CN118703447A, CN111135295A, CN117625557A) disclose inactivated LMBV vaccines, attenuated vaccine strains, and their applications, none of them have clearly defined the key virulence gene characteristics of the strains.

[0005] Therefore, there is still an urgent need to develop a low-virulence vaccine strain with a clear background against largemouth bass iridovirus disease, especially a vaccine strain with a clear virulence gene background that provides higher protection against largemouth bass iridovirus disease, in order to further improve the effectiveness of vaccine in controlling LMBV. Summary of the Invention

[0006] The purpose of this invention is to provide a weakened strain of largemouth bass iridovirus and a vaccine prepared therefrom. This weakened strain and the vaccine prepared therefrom can effectively prevent attacks by largemouth bass iridovirus and have good protective efficacy against largemouth bass iridovirus disease.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a largemouth bass iridovirus LMBV-ZJDSS-F110, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on September 20, 2024, with accession number CCTCCNO: V202485.

[0009] The present invention also provides a method comprising the aforementioned largemouth bass iridovirus.

[0010] The largemouth bass iris virus attenuated vaccine LMBV-ZJDSS-F110 is prepared by mixing the largemouth bass iris virus LMBV-ZJDSS-F110 with a protectant at a volume ratio of 1:(1-1.1).

[0011] The present invention also provides an ORF81 gene of the largemouth bass iridovirus LMBV-ZJDSS-F110, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] The present invention also provides a primer pair for amplifying the ORF81 gene, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] The present invention also provides an expression plasmid containing the ORF81 gene, the expression plasmid comprising recombinant plasmid pDM-ZJDSS110-ORF81, recombinant transfer plasmid pFASTF110-ORF81 and recombinant shuttle plasmid rBacmidZJDSS110-ORF81.

[0014] The present invention also provides an expression bacterium containing the expression plasmid, the expression bacterium comprising Escherichia coli DH5a containing the recombinant transfer plasmid pFASTF110-ORF81 and Escherichia coli DH10Bac containing the recombinant shuttle plasmid rBacmidZJDSS110-ORF81.

[0015] The present invention also provides an ORF81 protein encoded by the ORF81 gene or produced by the expressing bacteria, the amino acid sequence of which is shown in SEQ ID NO.2.

[0016] The present invention also provides a subunit vaccine prepared from the ORF81 protein.

[0017] The present invention also provides a method for preparing the subunit vaccine, comprising the following steps:

[0018] (1) Cloning the gene encoding the ORF81 protein of LMBV-ZJDSS-F110 strain;

[0019] (2) The ORF81 gene sequence was cloned into the pMD-18T vector to obtain the recombinant plasmid pMD-ZJDSS110-ORF81;

[0020] (3) Construct a recombinant transfer plasmid pFASTZJDSS110-ORF81 containing the ORF81 gene;

[0021] (4) Recombinant baculovirus rAcJDSS110-ORF81 was constructed using the recombinant transfer plasmid pFASTF110-ORF81;

[0022] (5) Infect insect cells with recombinant baculovirus rAcZJDSS110-ORF81 and culture the insect cells;

[0023] (6) The recombinant baculovirus was used to express the recombinant ORF81 protein of the largemouth bass iris virus, and the recombinant ORF81 protein of the largemouth bass iris virus was harvested and purified.

[0024] (7) The recombinant ORF81 protein of the largemouth bass iris virus was supplemented with an adjuvant and emulsified to obtain a largemouth bass iris virus subunit vaccine.

[0025] This invention also provides the application of the largemouth bass iris virus LMBV-ZJDSS-F110, or the largemouth bass iris virus attenuated vaccine, or the ORF81 gene, or the expression plasmid, or the expression bacterium, or the ORF81 protein, or the subunit vaccine, or the subunit vaccine prepared according to the method described, in the prevention of largemouth bass iris virus disease.

[0026] The ORF81 gene of the largemouth bass iris virus mutant attenuated strain provided by this invention is a gene that has mutated during viral evolution, and its encoded polypeptide can stimulate the body to produce neutralizing antibodies. Largemouth bass iris virus vaccine compositions or subunit vaccines prepared from the largemouth bass iris virus mutant attenuated strain containing the mutant ORF81 gene can effectively prevent LMBV attacks, and are particularly effective against largemouth bass iris virus disease. Attached Figure Description

[0027] Figure 1 The results show the viral titers of LMBV-ZJDSS at different generations of infection in Example 2;

[0028] Figure 2 The results of the pathogenicity of different generations of LMBV-ZJDSS virus against largemouth bass in Example 2; A. Virus titer of 10 8 TCID 50 Survival rates at different passages at / mL; B. Virus titer of 10 7 TCID 50 C. Virus titer of 10 at different passages; 6 TCID 50 Survival rates at different passages at / mL;

[0029] Figure 3 The results show the immunoprotective effect of LMBV-ZJDSS-F110 on largemouth bass in Example 2; the "8" on the vertical axis refers to a concentration of 10. 8 TCID 50 / mL, "7" refers to 10 7 TCID 50 / mL, "6" refers to 10 6 TCID 50 / mL;

[0030] Figure 4 The PCR results for the identification of recombinant plasmids in Example 4 are shown; M is a 100bp DNA Marker, and 1 is the ORF81 gene amplification product.

[0031] strain preservation

[0032] Largemouth bass ranavirus LMBV-ZJDSS-F110 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on September 20, 2024, with accession number CCTCC NO: V202485. Detailed Implementation

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Preparation of a live attenuated vaccine strain of largemouth bass iridovirus (LMBV)

[0036] The virulent LMBV strain is the ZJDSS strain of largemouth bass iridovirus. A non-virus-sensitive host cell line, the fathead minnow cell (FHM cell line), was selected to reduce the auxiliary effect of largemouth bass host cell components on viral pathogenicity. The virulent LMBV-ZJDSS strain was continuously passaged to attenuate it. Temperature-induced mutagenesis was performed at 24°C (6°C higher than normal) to promote viral mutation. At each passage, a low viral concentration (10T) was used. 5 TCID 50 The virus was passaged at a concentration of 10 μg / mL to increase the duration of viral replication within a passage period. After passage for more than 120 generations, strains from different passage intervals were selected for attenuation effect evaluation experiments. F5, F45, F90, and F110 strains were selected and injected into largemouth bass (12g ± 2g) for challenge, using three challenge concentrations of 10 μg / mL. 8 TCID 50 / mL, 10 7 TCID 50 / mL, 10 6 TCID 50 At a viral load of / mL, 0.1ml was injected intraperitoneally into each fish. The challenge results showed that no disease or death was observed in the largemouth bass infected with the F110 progeny, indicating that the F110 progeny of this strain was completely attenuated and has good safety for largemouth bass. Simultaneously, the attenuated strain F110 of this invention was used at 10... 8 TCID 50 Largemouth bass were immunized with 0.1 mL / mL injection, and 28 days post-immunization, they were treated with a homologous potent virus. 7 TCID 50 At a rate of 0.1 mL / fish, the largemouth bass in the immunized group were challenged with the attenuated virus. The results showed that the immunized group remained healthy and showed no symptoms of disease. In contrast, the control group exhibited significant disease progression, with approximately 90% (17-18 / 20) of the largemouth bass dying after challenge. The experiment was repeated twice, yielding consistent results, indicating that the attenuated virus strain provides good immunoprotection. Therefore, the F110 substitute virus was selected as a candidate strain for vaccine development.

[0037] For the specific experimental procedure, please refer to Example 2.

[0038] Example 2

[0039] Culture, gene stability and immunogenicity of the attenuated vaccine strain ZJDSS-F110 for largemouth bass iridovirus

[0040] 2.1. Materials and Methods

[0041] 2.1.1 Attenuation of virus strain ZJDSS through passage

[0042] The ZJDSS strain was continuously attenuated by passage using FHM cells. Three flasks of FHM cells were inoculated into each passage, with 0.1 ml injected per cell. The cells were incubated at 24°C for 72 hours. FHM cells were observed twice daily. If cell morphology (CPE) was observed (e.g., cell rounding and detachment), the cells were stored at -80°C. Cellular pathogenesis media with typical cytopathic effects were selected for passage.

[0043] 2.1.2 Virus titer determination

[0044] Take viral fluids from generations F5, F45, F90, and F110, and perform serial 10-fold dilutions respectively; select four dilution factors (10... 4 ~10 7 Or 10 5 ~10 8 The viral suspension was inoculated into FHM cells and cultured at 24°C. FHM cells were observed twice daily, and cells that developed CPE within one week were recorded. TCID50 was calculated using the Reed-Muench method.

[0045] 2.1.3 Sterility test and mycoplasma test

[0046] The F5, F45, F90, and F110 generations of ZJDSS strain were subjected to sterility and mycoplasma testing according to the appendix of the current "Veterinary Pharmacopoeia of the People's Republic of China".

[0047] 2.1.4 Exogenous virus testing

[0048] Exogenous virus detection was performed on the F5, F45, F90, and F110 generations of ZJDSS strain.

[0049] 2.1.5 Attenuation evaluation of ZJDSS strain

[0050] Healthy largemouth bass (approximately 12g) were randomly divided into 10 groups (20 bass per group), and each group was housed in one of 10 rearing tanks. Groups 1-9 were treated with different generations of ZJDSS poison F45 (10... 8.0 TCID 50 F45 (10) 7.0 TCID 50 F45 (10) 6.0 TCID 50 ), F90(10 8.0 TCID 50), F90(10 7.0 TCID 50 ), F90(10 6.0 TCID 50 ), F110(10 8.0 TCID 50 ), F110(10 7.0 TCID 50 ), F110(10 6.0 TCID 50 Group 10, serving as the control group, received 0.1 ml of PBS per fish via intraperitoneal injection. From the start of the challenge, the incidence and mortality of the largemouth bass population were observed and recorded daily. Autopsies were performed on the dead largemouth bass to observe pathological changes in organs and tissues.

[0051] 2.1.6 Evaluation of the immunogenicity of the attenuated ZJDSS strain

[0052] 2.1.5 For largemouth bass that survived infection, serum samples were collected from each group on day 28 for specific antibody testing, following the instructions for the largemouth bass frog iridovirus antibody detection kit; simultaneously, homologous potent virus ZJDSS was used. 7 TCID 50 / mL, inject 0.1mL / tail for challenge; after challenge, observe and record the morbidity and mortality of largemouth bass daily, perform necropsy on dead largemouth bass, observe the pathological changes of target organs and tissues, and calculate the immune protection rate.

[0053] 2.2 Results

[0054] 2.2.1 Passage attenuation of ZJDSS strain

[0055] We cultured a significantly weakened LMBV strain, LMBV-ZJDSS-F110, by passaged the virulent LMBV-ZJDSS strain 110 times in FHM cells, resulting in a strain with significantly reduced virulence and good immunogenicity. Experiments confirmed that the virulence of LMBV-ZJDSS F110 was significantly reduced, with a 10-generation passage ratio. 8.0 TCID 50 Infection of healthy largemouth bass showed no adverse reactions. The LMBV-ZJDSS F110 substitute was passaged five times in healthy largemouth bass without any reversion to virulence. At 10 8 TCID 50 Largemouth bass immunized with approximately 12g of virus were protected 14 / 20 after challenge, while the control group developed the disease 19 / 20.

[0056] 2.2.2 Virus titer determination results

[0057] The ZJDSS strain is highly adaptable to FHM cells. Figure 1 As shown, the viral titer gradually increased after passages from F5 to F110, with the TCID of the F5 generation being the highest. 50 10 8.0 / ml, F45 is 10 8.5 TCID 50 / ml, F90 is 10 8.8 TCID 50 / ml, F110 is 10 9.1 TCID 50 / ml, the titer tends to stabilize after generations F90 to F110, with a titer of approximately 10. 9.0 TCID 50 Approximately / ml.

[0058] 2.2.3 Sterility test and mycoplasma test

[0059] The F5, F45, F90, and F110 generations of LMBV-ZJDSS iridovirus from the largemouth bass frog were tested and found to be free of bacterial, fungal, and mycoplasma contamination.

[0060] 2.2.4 Exogenous virus testing

[0061] The F5, F45, F90, and F110 generations of LMBV-ZJDSS iridovirus from the largemouth bass frog were tested and found to be free of exogenous virus contamination.

[0062] 2.2.5 Evaluation of attenuation of ZJDSS strain at different generations

[0063] The pathogenicity of the ZJDSS strain to healthy largemouth bass weighing approximately 12g gradually decreased after successive passages in FHM cells. No mortality was observed in the F110 generation of the ZJDSS strain. (See results below.) Figure 2 .

[0064] 2.2.6 Preliminary evaluation of the immunogenicity of the ZJDSS strain

[0065] Following immunization with ZJDSS strain F110 progeny, serum samples were collected from largemouth bass 28 days post-immunization. Indirect ELISA testing revealed that all serum samples inoculated with the ZJDSS strain were positive for LMBV antibodies, while all serum samples from the control group were negative. After challenging the immunized and control groups with the homologous potent ZJDSS virus for 10 days, different immunization concentrations of F110 showed good immunoprotection rates. 8 TCID 50 The protection rate of the immunization group was 70% ( / mL). Figure 3 In contrast, approximately 90% (18 / 20) of the largemouth bass in the control group died after being challenged with the virus.

[0066] Example 3

[0067] vaccine preparation

[0068] 3.1 Preparation of live attenuated vaccine against largemouth bass iris virus (ZJDSS-F110 strain)

[0069] Take strain ZJDSS-F110 (preservation number CCTCC NO: V202485), dilute it 100 times with sterile physiological saline, and then... 6 TCID 50 Inoculate FHM cells at a concentration of / mL, add maintenance medium, and incubate at 24℃. Observe daily. When more than 80% of cells show cytopathic effects, harvest the virus solution, mix all virus solutions together, place in a sterile bottle, and store at 6℃. Perform sterility testing simultaneously, and determine the virus content to be 10. 9.0 TCID 50 / ml. Virus solutions that have passed sterility testing and virus content determination are mixed and then combined with sucrose-gelatin protectant at a 1:1 (volume ratio) to prepare the vaccine (the freeze-drying protectant is 8% (w / w) gelatin and 40% (w / w) sucrose protectant, sterilized at 115℃ for 40 min, stored at 4℃, and used within 72 hours). During the addition of the freeze-drying protectant, the virus solution should be continuously shaken to ensure thorough mixing; this is the vaccine stock solution. The vaccine stock solution is aseptically aliquoted, rapidly freeze-dried under vacuum, and sealed. This yields a largemouth bass iridovirus attenuated vaccine.

[0070] 3.2 Active immunization test

[0071] Healthy largemouth bass weighing approximately 12g were divided into 4 groups of 20 fish each. Groups 1-3 were vaccinated with 3 batches of vaccine, with each fish receiving an intraperitoneal injection of one treatment dose (i.e., 10). 5.0 TCID 50 Group 4 consisted of healthy largemouth bass weighing approximately 12g each, injected intraperitoneally with sterile saline. 28 days post-immunization, all four groups of largemouth bass were challenged intraperitoneally with ZJDSS potent venom (10...). 7.0 TCID 50 0.1ml. Observe for 10 days and record the onset and mortality.

[0072] 3.3 Results

[0073] Twenty-eight days after immunization, the immunized group was challenged with a potent virus. All members of the saline control group died. The immunized group, however, had a protection rate of 80% (16 / 20).

[0074] Experimental results show that the active immunization protection rate is 80%, indicating that the attenuated strain ZJDSS-F110 of this invention has good immunoprotective efficacy against the largemouth bass iridovirus.

[0075] Example 4

[0076] Preparation and efficacy testing of subunit vaccine against ZJDSS strain of iridovirus from largemouth bass.

[0077] 4.1. Materials and Methods

[0078] 4.1.1 Sequencing of LMBV-ZJDSS-F110 strain and construction of pMD-ZJDSS110-ORF81 recombinant plasmid

[0079] First, primers were designed.

[0080] LMBV81-F(5'-ATGGCATTTACTATGAACCTCAAA-3')SEQ ID NO:3

[0081] LMBV81-R(5'-TTATACTATATTTTATTATACCTGCTGGGAG-3')SEQ ID NO:4

[0082] Using the genomic DNA of LMBV-ZJDSS-F110 strain as a template, the ORF81 gene of LMBV-ZJDSS-F110 strain was amplified by PCR, and the amplified ORF81 gene sequence was cloned into the pMD-18T vector to obtain the recombinant plasmid pMD-ZJDSS110-ORF81.

[0083] 4.1.2 Construction of Recombinant Transposon Plasmids

[0084] The recombinant plasmid ZJDSS110-ORF81 and the transfer vector pFASTBac HTa were digested with BamHI and SalI, respectively. The ZJDSS110-ORF81 fragment and the vector fragment of LMBV-ZJDSS-F110 strain were recovered, ligated with T4 DNA Ligase, and transformed into E. coli DH5α. The transformed cells were plated on LB agar plates containing gentamicin and ampicillin and cultured. After bacterial selection and enrichment, plasmid DNA was extracted. Plasmids with higher molecular weight were preliminarily screened by electrophoresis. Then, they were identified by single and double enzyme digestion to determine the insertion and ligation direction of the target gene, and the recombinant transfer plasmid pFASTF110-ORF81 was obtained.

[0085] 4.1.3 Obtaining recombinant baculovirus rZJDSS110-ORF81

[0086] The pFASTF110ORF81 obtained above was transformed into *E. coli* DH10Bac competent cells (purchased from Invitrogen) containing the viral shuttle plasmid Bacmid, yielding the recombinant shuttle plasmid rBacmidZJDSS110-ORF81. PCR screening was performed, and positive plasmids were extracted to prepare the positive recombinant plasmid rBacmidZJDSS110-ORF81 DNA. Sf9 cells (purchased from Invitrogen) were transfected with Reagent. After the cells developed cytopathic effects, recombinant viral plaque purification and viral amplification were performed. The target recombinant ORF81 gene was then verified by PCR using universal primers M13F and M13R. The purified recombinant baculovirus was named rAcZJDSS110-ORF81. The recombinant baculovirus rAcZJDSS110-ORF81 was amplified and used as a seed virus. After titration using the plaque assay, it was stored at 4°C for later use.

[0087] 4.1.4 Expression and Identification of Recombinant ORF81 Protein

[0088] High Five™ insects (purchased from Invitrogen) were infected with recombinant rAcZJDSS110-ORF81 baculovirus during suspension culture. High Five™ cells were aseptically suspended in 500 ml roller flasks containing 200–350 ml of Express Five SFM medium, with a cell density of 0.3–0.6 × 10⁶ cells / ml. 6 Cells / ml. When the cell density reaches 1×10⁻⁶ cells / ml. 6 At a cell / ml ratio, each roller bottle was inoculated with recombinant virus rAcZJDSS110-ORF81. The recombinant baculovirus inoculated into each roller bottle had different multiples of infection (MOIs), which were 0.01, 0.1, and 1, respectively. After inoculation with recombinant baculovirus, the bottles were cultured at 26–28°C and 100 rpm for 4 days.

[0089] Samples were taken from each vial every 12 hours after inoculation, specifically at 48h, 60h, 72h, 84h, and 96h. Each sample was centrifuged at 10000g for 20min to separate the supernatant and precipitate. The supernatant was filtered through a 1µm filter membrane, and the resulting solution was subjected to SDS-PAGE electrophoresis and UV spectrophotometry to determine the recombinant ORF81 protein of the largemouth bass iridovirus and its concentration.

[0090] Western blotting was used to identify the expression of recombinant ORF81 protein from the largemouth bass iridovirus. High Five™ cells infected with the recombinant virus were collected, centrifuged at 3000 rpm for 10 min, and the supernatant was collected for SDS-PAGE electrophoresis.

[0091] 4.1.5 Harvesting and Purification of Recombinant ORF81 Protein from Largemouth Bass Iridovirus

[0092] The culture was centrifuged at 10000g for 20min to separate the precipitate and supernatant. The supernatant was filtered through a 1µm filter membrane and then concentrated and purified by ultrafiltration. The ultrafiltration membrane had a pore size of 10000 (molecular weight cutoff). The purified sample solution was inactivated with 7mM diethyleneimine (BEI) to neutralize the recombinant baculovirus vector. After 48 hours, an equal amount of sodium thiosulfate was added for neutralization.

[0093] 4.1.6 Preparation of Largemouth Bass Iridovirus Subunit Vaccine

[0094] Prepare antigen diluents of different concentrations: Dilute recombinant ORF81 protein with PBS (pH 7.4) to 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL.

[0095] The antigen diluents of different concentrations were mixed and emulsified with oil adjuvant ISA206. ISA206 adjuvant was then autoclaved at 121°C for 60 minutes and emulsified with the mixture at a volume ratio of antigen to adjuvant of 45:55 to obtain the subunit vaccine.

[0096] 4.1.7 Potency testing of the ORF81 subunit vaccine against the iridovirus of the largemouth bass.

[0097] a. Detection of immune response and antibody levels of subunit vaccine in healthy largemouth bass

[0098] The experimental fish were healthy largemouth bass weighing approximately 12g. An immunization group and a control group were established. Immunization was performed via intraperitoneal injection of 0.1ml of subunit vaccine. Twenty-eight days post-injection, blood samples were collected, serum was separated, and ORF81 antibody titers were measured.

[0099] b. LMBV virulent challenge test after subunit vaccine immunization of largemouth bass

[0100] The immunized largemouth bass and control group in item "a" above were injected intraperitoneally with the ZJDSS virulent strain 28 days after immunization. 7.0 TCID 50 / ml 0.1ml, observe for 10 days.

[0101] 4.2 Results

[0102] 4.2.1 Construction and Identification of Recombinant Plasmids

[0103] The PCR product is approximately 669 bp in size. See attached image for the results. Figure 4In this context, M represents a 100bp DNA marker, and 1 represents the LMBV ORF81 amplification product. Sequencing of the PCR product revealed that the recombinant vector pMD-ZJDSS110-ORF81 contains the ORF81 gene sequence, as shown by electrophoresis and sequencing results.

[0104] The gene sequence of the attenuated variant strain ORF81 of the largemouth bass iris virus is as follows:

[0105] ATGGCATTTACTATGAACCTCAAAGTTTTGTGGCAAATCAGTTGTCA

[0106] TTTACGGGCAACTTGGACTTACCTTTGACACAATTTGCAAAAGCGCTTA

[0107] CGCGTTTATAGACGAACCGTTTGACGATTCCGTATCCGTCAAAGCTGTA

[0108] ACTGCGCAAGGTTTGTTTGGCTGCGGAGACTCTGTCATAAGGTTTGAC

[0109] GCTGGCGTGTACGTTTGCATGAAGACAGATGGTACCAGCGCCCCCGTC

[0110] ATTCTATTTGGACGTCATTTGGACAAAACTCCTGCAATAAAGTTTACCA

[0111] CCGTAAAGAAACGGTACAAACTGCCGAATGCGGTACGCAGACAGAT

[0112] TCACCTGCTTTGCCAACGCTGTTTAACCCTTCTCAGTCTTCCACTTCTC

[0113] TGTTTGGCCCCGCAACCCCTCCTCAGCCATCCACTTCTCTGTTTGGTCC

[0114] TGGCCCAAAACCAGCCGAGACGCAGCAACCCTCCACCTCTGCAGCGC

[0115] AGGCTTCCCTCTCTGGCAGTTTACGTCAAGTAAACTTTGCCCCAGCTG

[0116] AGGCAAACAATGGTTATGTTTGCCCATTTGCCGCACATAGGCAAGCTTT

[0117] AAGAACTGCATCACCTCAAGGTGGCCTTAACCAAGACTTGGCCCTGCA

[0118] AGAGCCACTCCCAGCAGGTATAATAAAATATAGTATAAAATAA(SEQ ID NO:1)

[0119] 4.2.2 Identification of Recombinant Transposon Plasmids

[0120] Enzyme digestion confirmed that the ORF81 gene was inserted in the correct orientation and the size of the digested fragments was consistent with the expected results.

[0121] 4.2.3 Transfection of insect cells using shuttle vectors

[0122] A transfer vector containing the ORF81 gene of the largemouth bass iris virus was transformed into competent E. coli cells containing a shuttle vector to obtain a shuttle vector containing the ORF81 gene of the largemouth bass iris virus strain LMBV-ZJDSS-F110. This shuttle vector was then transfected into insect cells (purchased from Invitrogen). Cell death began to occur 24 hours after transfection, and the cells continued to proliferate thereafter. After 72 hours, lesions began to appear in the cells, with enlarged nuclei, and the lesions gradually increased. Cell death began after 96 hours, and almost all cells died after one week.

[0123] 4.2.4 Expression and identification of recombinant ORF81 protein

[0124] The recombinant protein content in the supernatant reached 50 μg / mL 72 h after cell infection with the recombinant virus. This indicates that culturing cells with the recombinant virus for at least 72 h and then recovering the culture supernatant can significantly improve the yield of recombinant ORF81 protein from the largemouth bass iridovirus.

[0125] SDS-PAGE protein electrophoresis gel revealed a specific band of approximately 26 kDa, which is the ORF81 protein of the recombinant largemouth bass iridovirus strain ZJDSS-F110. Sequencing of the target protein, based on the electrophoresis and sequencing results, revealed the following sequence of the ORF81 protein of the recombinant largemouth bass iridovirus strain ZJDSS-F110:

[0126] MAFTMNLKVCGKSVVIYGQLGLTFDTICKSAYAFIDEPFDDSVSVKA

[0127] VTAQGLFGCGDSVIRFDAGVYVCMKTDGTSAPVILFGRHLDKTPAIKFTT

[0128] VKKTVQTAECGTQTDSPALPTLFNPSQSSTSLFGPATPPQPSTSLFGPGPKP

[0129] AETQQPSTSAAQASLSGSLRQVNFAPAEANNNGYVCPFAAHRQALRTASPQ

[0130] GGLNQDLALQEPLPAGIIKYSIK(SEQ ID NO:2)

[0131] 4.2.5 Harvesting and purification of recombinant protein: The content of purified protein was determined by ultraviolet spectrophotometer, reaching 62 μg / mL.

[0132] 4.2.6 Testing of Subunit Vaccines: After viscosity testing, centrifugation testing, dosage form testing, and stability testing, all indicators of the prepared vaccine met the requirements. The vaccine was stored at 4℃.

[0133] The protein antigen produced by the method of this invention can also be used in combination with other oily adjuvants, aqueous adjuvants, or other adjuvants, such as white oil adjuvants, aluminum glue adjuvants, Freund's adjuvants, etc., which are commonly used in the art.

[0134] 4.2.7 Results of subunit vaccine potency testing

[0135] Antibody titer was tested after subunit vaccine immunization. The antibody was coated with ORF81 antigen and the antibody titer was 1:256.

[0136] In the challenge protection trial of the subunit vaccine, the mortality rate in the control challenge group was 19 / 20, the protection rate in the immunized group reached 90%, and the normal control group, the largemouth bass, showed no clinical symptoms.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The ORF81 gene of a largemouth bass iridovirus LMBV-ZJDSS-F110, characterized in that, The nucleotide sequence of the ORF81 gene is shown in SEQ ID NO.1; the largemouth bass iridovirus LMBV-ZJDSS-F110 is deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on September 20, 2024, with accession number CCTCC NO: V202485.

2. A primer pair for amplifying the ORF81 gene as described in claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. An expression plasmid comprising the ORF81 gene of claim 1, characterized in that, The expression plasmids include recombinant plasmid pDM-ZJDSS110-ORF81, recombinant transfer plasmid pFASTF110-ORF81, and recombinant shuttle plasmid rBacmidZJDSS110-ORF81.

4. An expression bacterium comprising the expression plasmid of claim 3, characterized in that, The expression bacteria include Escherichia coli DH5a containing the recombinant transfer plasmid pFASTF110-ORF81 and Escherichia coli DH10Bac containing the recombinant shuttle plasmid rBacmidZJDSS110-ORF81.

5. An ORF81 protein encoded by the ORF81 gene of claim 1 or produced by the expression bacteria of claim 4, characterized in that, The amino acid sequence of the ORF81 protein is shown in SEQ ID NO.

2.

6. A subunit vaccine prepared from the ORF81 protein of claim 5.

7. A method for preparing the subunit vaccine according to claim 6, characterized in that, Includes the following steps: (1) Cloning the gene encoding the ORF81 protein of LMBV-ZJDSS-F110 strain; (2) The ORF81 gene sequence was cloned into the pMD-18T vector to obtain the recombinant plasmid pMD-ZJDSS110-ORF81; (3) Construct a recombinant transfer plasmid pFASTZJDSS110-ORF81 containing the ORF81 gene; (4) Recombinant baculovirus rAcJDSS110-ORF81 was constructed using the recombinant transfer plasmid pFASTF110-ORF81; (5) Infect insect cells with recombinant baculovirus rAcZJDSS110-ORF81 and culture the insect cells; (6) The recombinant baculovirus was used to express the recombinant ORF81 protein of the largemouth bass iris virus, and the recombinant ORF81 protein of the largemouth bass iris virus was harvested and purified. (7) The recombinant ORF81 protein of the largemouth bass iris virus was supplemented with an adjuvant and emulsified to obtain a largemouth bass iris virus subunit vaccine.

8. The use of the ORF81 gene of claim 1, the expression plasmid of claim 3, the expression bacterium of claim 4, or the ORF81 protein of claim 5 in the preparation of a subunit vaccine for preventing iridovirus disease in largemouth bass.

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