Micropterus salmoides iridovirus, vaccine prepared from Micropterus salmoides iridovirus and application of Micropterus salmoides

Abstract

The invention provides a largemouth bass frog iridovirus, a vaccine prepared from the largemouth bass frog iridovirus and application of the largemouth bass frog iridovirus, and belongs to the technical field of biological The largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth largemouth The strain and the vaccine prepared from the strain can effectively prevent attack of the largemouth bass frog iridovirus, and have relatively good protection force on the largemouth bass frog iridovirus disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biological vaccines, and in particular to a largemouth bass frog iris virus and a vaccine prepared therefrom and applications thereof. Background Art

[0002] LMBV (Largemouth Bass Ranavirus) is a large double-stranded DNA virus belonging to the genus Ranavirus of the family Iridoviridae. It is one of the main pathogens that cause serious economic losses in the aquaculture of largemouth bass (Micropterus salmoides). Since LMBV infection often does not present characteristic clinical symptoms, or even obvious symptoms, clinical diagnosis is difficult. However, epidemiological data show that the incidence of largemouth bass infected with LMBV can reach 20% to 30%, even exceeding 40% in some years, and the mortality rate in the affected ponds is usually between 20% and 40%, and in extreme cases the cumulative mortality rate even exceeds 80%. The water temperature range of LMBV is between 25℃ and 32℃, with the peak period from July to August. Largemouth bass of all sizes can be infected and the disease can occur. The acute onset of the disease is only 3 to 5 days, and the entire disease process can last for about 1 month.

[0003] LMBV has a wide host range. In addition to largemouth black bass, it can also infect other fish such as mandarin fish (Siniperca chuatsi) and black snakehead (Channa argus). Especially in small-sized largemouth black bass at different growth stages, LMBV shows higher pathogenicity, and the difference in its virulence is closely related to the difference in strains. In the epidemiological surveys conducted in the past three years, the project team found that LMBV also has a high detection rate in mandarin fish in Jiangsu, Zhejiang and Anhui, and the incidence of mandarin fish frog iridovirus disease caused by LMBV has been increasing year by year. The whole genome sequence homology of LMBV strains isolated from mandarin fish and largemouth black bass is 99.0% to 100.0%, indicating that their genomes are highly similar between different hosts.

[0004] At present, vaccination is the most effective means to prevent and control the iridovirus disease of largemouth bass frog. Although some new strains have been isolated, the prevention and treatment effects of existing vaccines are not ideal due to the large variation between LMBV strains at the genetic level and the large difference between the prevalent strains and the vaccine strains. Although there are some LMBV vaccine patent applications CN118703447A, CN111135295A, and CN117625557A, which disclose the inactivated vaccine, attenuated vaccine strain and its application of largemouth bass frog iridovirus, none of them clearly defines the important virulence gene characteristics of the strains.

[0005] Therefore, there is still an urgent need to develop a attenuated vaccine strain with a clear background for largemouth bass frog iridovirus disease, especially a vaccine strain with a clear background of virulence genes that has higher protection against largemouth bass frog iridovirus disease, so as to further improve the effectiveness of vaccines in preventing and controlling LMBV. Summary of the invention

[0006] The purpose of the present invention is to provide a weak strain of largemouth black bass frog iridovirus and a vaccine prepared therefrom, which can effectively prevent the attack of largemouth black bass frog iridovirus and have good protection against largemouth black bass frog iridovirus disease.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a largemouth black bass frog iridovirus LMBV-ZJDSS-F110. The largemouth black bass frog iridovirus LMBV-ZJDSS-F110 is deposited in China Center for Type Culture Collection, with an address of Wuhan University, Wuhan, China. The deposit date is September 20, 2024, and the deposit number is CCTCCNO: V202485.

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

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

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

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

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

[0014] The present invention also provides an expression bacterium containing the expression plasmid, wherein the expression bacterium comprises 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 expression bacteria, and the amino acid sequence of the ORF81 protein 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 the LMBV-ZJDSS-F110 strain;

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

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

[0021] (4) using the recombinant transfer plasmid pFASTF110-ORF81 to construct the recombinant baculovirus rAcJDSS110-ORF81;

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

[0023] (6) expressing the recombinant baculovirus recombinant ORF81 protein of the black bass frog iridovirus, harvesting and purifying the black bass frog iridovirus recombinant ORF81 protein;

[0024] (7) The recombinant ORF81 protein of the black bass frog iridovirus is assisted with an adjuvant and emulsified to obtain a black bass frog iridovirus subunit vaccine.

[0025] The present invention also provides the use of the largemouth bass frog iridovirus LMBV-ZJDSS-F110 or the largemouth bass frog iridovirus attenuated vaccine or the ORF81 gene or the expression plasmid or the expression bacteria or the ORF81 protein or the subunit vaccine or the subunit vaccine obtained according to the preparation method in preventing largemouth bass frog iridovirus disease.

[0026] The ORF81 gene of the mutant attenuated strain of the largemouth bass frog iridovirus provided by the present invention is a gene that mutates during the evolution of the virus, and the polypeptide encoded by the gene can stimulate the body to produce neutralizing antibodies. The largemouth bass frog iridovirus vaccine composition or subunit vaccine prepared from the mutant attenuated strain of the largemouth bass frog iridovirus containing the mutant ORF81 gene can effectively prevent and treat the attack of LMBV, especially having good protection against largemouth bass frog iridovirus disease. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 The results of the pathogenicity of LMBV-ZJDSS viruses of different generations to largemouth bass in Example 2; A. The virus titer is 10 8 TCID 50 / mL survival rate of different generations; B. Virus titer is 10 7 TCID 50 / mL survival rate of different generations; C. Virus titer is 10 6 TCID 50 / mL survival rate of different generations;

[0029] Figure 3 The results of the immune protection rate of LMBV-ZJDSS-F110 against largemouth bass in Example 2; the "8" in the ordinate refers to the 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 It is the PCR result of the identification of the recombinant plasmid in Example 4; M is a 100 bp DNA marker, and 1 is the amplified product of the ORF81 gene.

[0031] Strain preservation

[0032] Largemouth bass ranavirus LMBV-ZJDSS-F110 Largemouth bass ranavirus is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is September 20, 2024, and the deposit number is CCTCC NO: V202485. DETAILED DESCRIPTION

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

[0034] Example 1

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

[0036] The strong strain of LMBV is the ZJDSS strain of frog iris virus of largemouth bass. The Fat Head Minnow cell (FHM cell line), a non-virus sensitive host cell, was selected to reduce the auxiliary effect of largemouth bass host cell components on the pathogenicity of the virus. The strong strain LMBV-ZJDSS strain was continuously attenuated by continuous passage. The temperature was induced by temperature mutagenesis culture at a temperature 6°C higher than the normal culture temperature (24°C) to promote virus mutation. During each passage, a low virus concentration (10 5 TCID 50 / mL) to increase the duration of virus replication during a passage period. After continuous passage for more than 120 generations, strains of different generations were selected for attenuation effect evaluation. The F5, F45, F90, and F110 strains were selected and injected into largemouth bass (12g±2g) respectively. 8 TCID 50 / mL, 10 7 TCID 50 / mL, 10 6 TCID 50 / mL of virus solution, 0.1ml was injected into the abdominal cavity of each fish. The results of the virus challenge showed that no disease or death was found in the largemouth bass infected with the F110 virus, indicating that the F110 virus of the strain has been completely attenuated and has good safety for largemouth bass. 8 TCID 50 / mL was injected with 0.1mL to immunize largemouth bass. 28 days after immunization, the homologous strong toxin was used for 10 7 TCID 50 / mL, injected 0.1mL / tail, and challenged with the virus. It was found that the largemouth bass in the immunized group were in good condition and had no symptoms. The control group had obvious disease, and about 90% (17-18 / 20) of the largemouth bass died after the challenge. The above experiment was repeated twice and consistent results were obtained, indicating that the attenuated strain has good immune protection. The F110 virus was selected as the candidate strain for vaccine development.

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

[0038] Example 2

[0039] Cultivation, genetic stability and immune efficacy of attenuated vaccine strain of Rana nigromaculata iridovirus ZJDSS-F110

[0040] Materials and methods

[0041] 2.1.1 Virus strain ZJDSS attenuated by passage

[0042] Use FHM cells to continuously pass down the ZJDSS strain to attenuate it. Inoculate 3 bottles of FHM cells for each generation, inject 0.1 ml into each tail, and culture at 24℃ for 72h. Observe the FHM cells twice a day. If CPE such as cell rounding and falling off occurs, store at -80℃. Select the cytopathic fluid with typical lesions for passaging.

[0043] 2.1.2 Virus titer determination

[0044] Take the virus solutions of F5, F45, F90, and F110 generations and make 10-fold serial dilutions respectively; select 4 dilution multiples (10 4 ~10 7 or 10 5 ~10 8 ) were inoculated into FHM cells and cultured at 24°C. The FHM cells were observed twice a day, and the cells with CPE within 1 week were recorded. The TCID50 was calculated according to the Reed-Muench method.

[0045] 2.1.3 Sterility test and mycoplasma test

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

[0047] 2.1.4 Exogenous virus test

[0048] The F5, F45, F90, and F110 generations of the ZJDSS strain were tested for exogenous viruses.

[0049] 2.1.5 Evaluation of attenuation of ZJDSS strain

[0050] Healthy largemouth bass (about 12g) were randomly divided into 10 groups (20 in each group), and the 10 groups of largemouth bass were raised in 10 breeding barrels. Groups 1 to 9 were treated with different generations of ZJDSS 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 ) were inoculated, with 0.1 ml injected into the abdominal cavity of each fish; the 10th group of largemouth bass served as the control group, with 0.1 ml PBS injected into each fish. From the day of the challenge, the incidence and mortality of largemouth bass were observed and recorded daily, and the dead largemouth bass were dissected to observe the pathological changes of organs and tissues.

[0051] 2.1.6 Evaluation of the immune efficacy of the attenuated strain of ZJDSS

[0052] 2.1.5 Largemouth bass that survived infection, on the 28th day after infection, serum was collected from each group for specific antibody detection, and the specific method was carried out according to the instructions of the Largemouth bass frog iridovirus antibody detection kit; at the same time, the homologous strong virus ZJDSS was used, 10 7 TCID 50 / mL, and 0.1mL / fish was injected for poison challenge; after poison challenge, the morbidity and mortality of largemouth black bass were observed and recorded daily, the dead largemouth black bass were dissected, the pathological changes of target organs and tissues were observed, and the immune protection rate was calculated.

[0053] 2.2 Results

[0054] 2.2.1 Attenuation of ZJDSS strain by subculture

[0055] We used the strong LMBV-ZJDSS strain to continuously pass on FHM cells for 110 generations and cultured a weak LMBV strain LMBV-ZJDSS-F110 with significantly reduced virulence and good immunogenicity. The experiment confirmed that the virulence of LMBV-ZJDSS F110 generation was significantly reduced. 8.0 TCID 50 Infected healthy largemouth bass, no adverse reactions. LMBV-ZJDSS F110 virus was continuously passed through healthy largemouth bass for 5 generations, and no virulence was found. 8 TCID 50 Among the largemouth bass weighing about 12 g, 14 / 20 were protected after infection, while 19 / 20 in the control group became ill.

[0056] 2.2.2 Virus titer determination results

[0057] The ZJDSS strain is highly adaptable to FHM cells. Figure 1 As shown, the virus titer gradually increased after passage from F5 to F110, and the TCID 50 For 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, F90~F110 generations tend to be stable, and the titer is about 10 9.0 TCID 50 / ml or so.

[0058] 2.2.3 Sterility test and mycoplasma test

[0059] The F5, F45, F90 and F110 generations of largemouth bass frog iris virus LMBV-ZJDSS during the subculture process were tested and found to be free of bacteria, mold and mycoplasma contamination.

[0060] 2.2.4 Exogenous virus test

[0061] During the subculture process of largemouth bass frog iris virus LMBV-ZJDSS, the F5, F45, F90 and F110 generations of viruses were tested and found to be free of exogenous virus contamination.

[0062] 2.2.5 Evaluation of the attenuation of different generations of ZJDSS strains

[0063] After continuous passage on FHM cells, the pathogenicity of ZJDSS strain to healthy largemouth bass weighing about 12 g gradually weakened. No mortality occurred in the F110 generation of ZJDSS strain. Figure 2 .

[0064] 2.2.6 Preliminary evaluation of the immune efficacy of the ZJDSS strain

[0065] After immunization with the F110 virulent strain of ZJDSS, serum samples of largemouth bass were collected 28 days after immunization. The indirect ELISA kit was used to test that all serum samples inoculated with the ZJDSS strain turned positive for LMBV antibodies, while the serum samples of the control group were all negative. After attacking the immunized group and the control group with the homologous virulent ZJDSS and observing for 10 days, it was found that the F110 different immune concentration groups showed a good immune protection rate, 10 8 TCID 50 / mL immunization group protection rate was 70% ( Figure 3 ); while about 90% (18 / 20) of the largemouth bass in the control group died after the infection.

[0066] Example 3

[0067] Vaccine preparation

[0068] 3.1 Preparation of attenuated live vaccine of Rana iridovirus (ZJDSS-F110 strain)

[0069] Take the ZJDSS-F110 strain virus (preservation number is CCTCC NO: V202485), dilute it 100 times with sterile saline and then add it to the mixture for about 10 6 TCID 50 / mL concentration was inoculated into FHM cells, maintenance solution was added, and the cells were incubated at 24°C and observed daily. When more than 80% of the cells showed pathological changes, the virus solution was harvested, all virus solutions were mixed into one group, placed in a sterile bottle, and stored at 6°C. At the same time, a sterility test was performed, and the virus content was measured to be 10 9.0 TCID 50 / ml. After the virus solution that has passed the sterility test and virus content determination is mixed, it is mixed with sucrose gelatin protective agent at a volume ratio of 1:1 (freeze-dried protective agent is 8% (W / W) gelatin, 40% (W / W) sucrose protective agent, sterilized at 115℃ for 40min, stored at 4℃, and used up within 72h). During the addition of freeze-dried protective agent, the virus solution should be shaken continuously, and after being fully mixed, it is the vaccine stock solution. The vaccine stock solution is aseptically packaged in quantitative form, quickly freeze-dried in a vacuum, and sealed with a cover. The attenuated vaccine of largemouth black bass frog iris virus is obtained.

[0070] 3.2 Active Immunization Test

[0071] Healthy largemouth bass weighing about 12 g were divided into 4 groups, with 20 fish in each group. Groups 1 to 3 were vaccinated with 3 batches of vaccine respectively. Each fish was intraperitoneally injected with 1 dose (i.e. 10 5.0 TCID 50 ). Each healthy largemouth bass in group 4, weighing about 12 g, was intraperitoneally injected with sterile saline. 28 days after immunization, each largemouth bass in group 4 was challenged with ZJDSS (10 7.0 TCID 50 )0.1ml. Observe for 10 days and record the incidence and mortality.

[0072] 3.3 Results

[0073] After 28 days of immunization, the immunized group was challenged with strong toxin, and all the normal saline control group died, while the protection rate of the immunized group was 80% (16 / 20).

[0074] The experimental results showed that the active immune protection rate was 80%, indicating that the attenuated strain ZJDSS-F110 of the present invention has a good immune protection effect on the largemouth bass frog iris virus.

[0075] Example 4

[0076] Preparation and efficacy test of subunit vaccine of ZJDSS strain of Rana iridovirus

[0077] Materials and methods

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

[0079] First, design primers

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

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

[0082] The ORF81 gene of LMBV-ZJDSS-F110 strain was amplified by PCR using the genomic DNA of LMBV-ZJDSS-F110 strain as a template, 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 transposable plasmid

[0084] The recombinant plasmid ZJDSS110-ORF81 and the transfer vector pFASTBac HTa were digested with BamH I and Sal I respectively, and the ZJDSS110-ORF81 fragment and the vector fragment of the LMBV-ZJDSS-F110 strain were recovered, connected with T4 DNA Ligase, transformed into Escherichia coli DH5a, coated on LB plates containing gentamicin and ampicillin, and cultured. After bacterial selection and enrichment, plasmid DNA was extracted, and plasmids with higher molecular weight were preliminarily screened by electrophoresis. Then, single and double enzyme digestion were performed to identify the insertion and connection 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 Escherichia coli DH10Bac competent cells (purchased from Invitrogen) containing the viral shuttle plasmid Bacmid to obtain the recombinant shuttle plasmid rBacmidZJDSS110-ORF81; PCR screening, extraction of positive plasmids, and preparation of positive recombinant plasmid rBacmidZJDSS110-ORF81 DNA. Reagent was used to transfect Sf9 cells (purchased from Invitrogen), and after the cells showed pathological changes, recombinant virus plaque purification and virus amplification were performed, and then PCR was performed to verify the target recombinant ORF81 gene using universal primers M13F and M13R. The recombinant baculovirus was purified and named rAcZJDSS110-ORF81. The recombinant baculovirus rAcZJDSS110-ORF81 was amplified as a seed virus, and the titer was titrated by plaque method and stored at 4°C for later use.

[0087] 4.1.4 Expression and identification of recombinant ORF81 protein

[0088] rAcZJDSS110-ORF81 recombinant baculovirus was used to infect suspension-cultured insect High Five™ (purchased from Invitrogen). High Five™ cells were aseptically cultured in suspension in a 500 ml spinner flask containing 200-350 ml of EXPRESS FIVE SFM medium, with an inoculation cell density of 0.3-0.6×10 6 cells / ml. When the cell density reaches 1×10 6 When the concentration of cells / ml was 1.547 W / mL, each roller bottle was inoculated with the recombinant virus rAcZJDSS110-ORF81. The recombinant baculovirus inoculated into each roller bottle had different multiplicity of infection (MOI), namely 0.01, 0.1, and 1. After inoculation of the recombinant baculovirus, the culture was carried out at 26-28°C and a rotation speed of 100 rpm for 4 days.

[0089] Each bottle was sampled every 12 hours after inoculation, i.e., 48h, 60h, 72h, 84h and 96h. Each sample was centrifuged at 10000g for 20min to separate the supernatant and the precipitate. The supernatant was filtered through a 1um filter membrane, and after filtration, SDS-PAGE electrophoresis and UV spectrophotometer were used to determine the recombinant ORF81 protein of the largemouth bass frog iridovirus in the supernatant and its content.

[0090] Western blotting was used to identify the expressed ORF81 protein of the Rana iridovirus. High Five TM cells infected with the recombinant virus were collected and centrifuged at 3000 r / min for 10 min to obtain the supernatant, which was then subjected to SDS-PAGE electrophoresis.

[0091] 4.1.5 Harvesting and Purifying the Recombinant ORF81 Protein of the Rana iridovirus

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

[0093] 4.1.6 Preparation of subunit vaccine of Rana iridovirus

[0094] Prepare different concentrations of antigen dilutions: dilute the recombinant ORF81 protein with PBS (PH7.4) to 0.5 μg / ml, 1 μg / ml, 2 μg / ml, and 4 μg / ml.

[0095] The above antigen dilutions of different concentrations were mixed and emulsified with the oil adjuvant ISA206. The ISA206 adjuvant was sterilized by high pressure wet heat at 121°C for 60 minutes, and mixed and emulsified in a volume ratio of 45:55 between antigen and adjuvant to obtain a subunit vaccine.

[0096] 4.1.7 Efficacy testing of the ORF81 subunit vaccine of the largemouth bass frog iridovirus

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

[0098] The experimental fish were healthy largemouth bass weighing about 12g. An immunization group and a control group were set up and immunized with 0.1ml of subunit vaccine by intraperitoneal injection. 28 days after injection, blood was collected and serum was separated to determine the ORF81 antibody titer.

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

[0100] The immunized largemouth bass and the control group in item "a" above were intraperitoneally injected with the strong strain of ZJDSS for 10 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 size is about 669 bp. The results are attached. Figure 4, where M is a 100 bp DNA marker and 1 is the amplified product of LMBV ORF81. The PCR product was sequenced, and the results of electrophoresis and sequencing showed that the recombinant vector pMD-ZJDSS110-ORF81 contained the ORF81 gene sequence.

[0104] The ORF81 gene sequence of the mutant attenuated strain of the largemouth bass frog 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 transposable plasmids

[0120] Enzyme digestion analysis showed that the insertion direction of ORF81 gene was correct and the size of the digested fragment was consistent with the expected result.

[0121] 4.2.3 Transfection of insect cells with shuttle vector

[0122] The transfer vector containing the ORF81 gene of the largemouth bass frog iridovirus was transformed into the competent cells of Escherichia coli containing the shuttle vector, and the shuttle vector containing the ORF81 gene of the largemouth bass frog iridovirus LMBV-ZJDSS-F110 strain was obtained. The shuttle vector was then transfected into insect cells (purchased from Invitrogen). Cells began to die 24 hours after transfection, and thereafter the cells had a tendency to continue to proliferate. After 72 hours, the cells began to have lesions, the nuclei were enlarged, and the lesions gradually increased. After 96 hours, the cells began to die, and after 1 week, almost all of them died.

[0123] 4.2.4 Expression and identification of recombinant ORF81 protein

[0124] The recombinant protein content in the supernatant reached 50 μg / ml 72 hours after the recombinant virus infected the cells, indicating that after the recombinant virus infected the cells, the culture supernatant was recovered after culturing for at least 72 hours, which could significantly increase the yield of the recombinant ORF81 protein of the iridovirus disease of the largemouth bass frog.

[0125] The specific band of about 26KDa was observed in the SDS-PAGE protein electrophoresis gel, which is the ORF81 protein of the recombinant largemouth bass frog iridovirus ZJDSS-F110 strain. The target protein was sequenced, and the electrophoresis and sequencing results showed that the sequence of the recombinant largemouth bass frog iridovirus ZJDSS-F110 strain ORF81 protein was as follows:

[0126] MAFTMNLKVCGKSVVIYGQLGLTFDTICKSAYAFIDEPFDDSVSVKA

[0127] VTAQGLFGCGDSVIRFDAGVYVCMKTDGTSAPVILFGRHLDKTPAIKFTT

[0128] VKKTVQTAECGTQTDSPALPTLFNPSQSSTSLFGPATPPQPSTSLFGPGPKP

[0129] AETQQPSTSAAQASLSGSLRQVNFAPAEANNNGYVCPFAAHRQALRTASPQ

[0130] GGLNQDLALQEPLPAGIIKYSIK(SEQ ID NO:2)

[0131] 4.2.5 Harvest and purification of recombinant protein: The content of purified protein was measured by UV spectrophotometer and reached 62 μg / mL.

[0132] 4.2.6 Inspection of subunit vaccines: After the prepared vaccines are tested for viscosity, centrifugation, dosage form and stability, all indicators meet the requirements. The vaccines are stored at 4°C.

[0133] The protein antigen produced by the method of the present invention can also be mixed with other oily adjuvants, aqueous adjuvants or other adjuvants, such as white oil adjuvants, aluminum gel adjuvants, Freund's adjuvants and the like commonly used in the art.

[0134] 4.2.7 Results of efficacy tests of subunit vaccines

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

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

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A largemouth bass frog iridovirus LMBV-ZJDSS-F110, characterized in that: The largemouth bass frog iridovirus LMBV-ZJDSS-F110 is deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is September 20, 2024, and the deposit number is CCTCC NO: V202485.

2. A largemouth bass frog iridovirus attenuated vaccine comprising the largemouth bass frog iridovirus LMBV-ZJDSS-F110 according to claim 1, characterized in that: The largemouth bass frog iris virus LMBV-ZJDSS-F110 and the protective agent are prepared in a volume ratio of 1: (1-1.1).

3. An ORF81 gene of the largemouth bass frog iris virus LMBV-ZJDSS-F110 according to claim 1, characterized in that: The nucleotide sequence of the ORF81 gene is shown in SEQ ID NO.

1.

4. A primer pair for amplifying the ORF81 gene according to claim 3, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NO.3 and SEQ ID NO.

4.

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

6. An expression bacterium comprising the expression plasmid according to claim 5, 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.

7. An ORF81 protein encoded by the ORF81 gene of claim 3 or produced by the expression bacteria of claim 6, characterized in that: The amino acid sequence of the ORF81 protein is shown in SEQ ID NO.

2.

8. A subunit vaccine prepared from the ORF81 protein according to claim 7.

9. A method for preparing the subunit vaccine according to claim 8, characterized in that: The steps include: (1) Cloning the gene encoding the ORF81 protein of the LMBV-ZJDSS-F110 strain; (2) cloning the ORF81 gene sequence into the pMD-18T vector to obtain the recombinant plasmid pMD-ZJDSS110-ORF81; (3) Construction of recombinant transfer plasmid pFASTZJDSS110-ORF81 containing the ORF81 gene; (4) using the recombinant transfer plasmid pFASTF110-ORF81 to construct the recombinant baculovirus rAcJDSS110-ORF81; (5) infecting insect cells with the recombinant baculovirus rAcZJDSS110-ORF81 and culturing the insect cells; (6) expressing the recombinant baculovirus recombinant ORF81 protein of the black bass frog iridovirus, harvesting and purifying the black bass frog iridovirus recombinant ORF81 protein; (7) The recombinant ORF81 protein of the black bass frog iridovirus is assisted with an adjuvant and emulsified to obtain a black bass frog iridovirus subunit vaccine.

10. Use of the largemouth bass frog iridovirus LMBV-ZJDSS-F110 described in claim 1 or the largemouth bass frog iridovirus attenuated vaccine described in claim 2 or the ORF81 gene described in claim 3 or the expression plasmid described in claim 5 or the expression bacteria described in claim 6 or the ORF81 protein described in claim 7 or the subunit vaccine described in claim 8 or the subunit vaccine obtained by the preparation method according to claim 9 in preventing largemouth bass frog iridovirus disease.

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