Polyclonal antibody of grouper iridovirus SGIV VP101 gene and application thereof

Abstract

The invention discloses a polyclonal antibody of a grouper iridovirus SGIV VP101 gene and application of the polyclonal antibody. The polyclonal antibody is prepared from an SGIVVP101 gene, and the nucleotide sequence of the VP101 gene of an optimized codon is as shown in SEQ ID NO. 1. The polyclonal antibody of the SGIV VP101 gene recombinant expression protein prepared by the invention has good specificity, not only can specifically recognize a prokaryotic expression product of the SGIV VP101, but also can be used for quantitative and positioning analysis of the SGIV VP101 in vivo and in vitro. The successful preparation of the polyclonal antibody of the recombinant expression protein of the SGIV VP101 gene lays a foundation for the establishment of an SGIV immunological detection method, the quantitative and positioning analysis of the protein of the VP101 gene and the analysis of gene functions.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary technical field of immunology and virology, and particularly relates to a polyclonal antibody against the grouper iridovirus SGIVVP101 gene and an application thereof. Background Art

[0002] Iridovirus and viral nervous necrosis (VNN) are two important viral pathogens of grouper, causing significant economic losses to the grouper aquaculture industry. Iridoviruses are a class of double-stranded DNA viruses with regular icosahedral structures belonging to the Iridoviridae family. The Iridoviridae family is divided into six genera: Ranavirus, Cytomegalovirus, Lymphocystisvirus, Iridovirus, Chloriovirus, and Decapodavirus. In recent years, members of the Ranavirus, Cytomegalovirus, and Lymphocystisvirus genera have frequently broken out and become epidemics in lower aquatic vertebrates, including fish, amphibians, and reptiles, severely impacting ecological security and causing significant economic losses.

[0003] Singapore grouper iridovirus (SGIV) is a major epidemic strain causing grouper iridovirus disease and belongs to a new species of the genus Ranavirus in the family Iridoviridae. SGIV primarily infects grouper (Epinephelus spp.) larvae and juveniles, with a mortality rate exceeding 90%. Based on the identification of its physicochemical properties, omics analyses of the viral genome, transcriptome, proteins, and miRNAs were completed. Bioinformatics predictions revealed that the SGIV genome contains 162 open reading frames (ORFs), 77 of which share homology with known viral genes, and 30 of which match predicted or verified iridovirus functional proteins, implicating them in DNA replication and transcription, nucleotide metabolism, and cell signaling. Bioinformatics predictions for over half of the ORFs indicate no homology and are therefore considered unknown genes. Whether these predicted ORFs are expressed and translated into proteins during viral infection can only be verified by prokaryotic expression of the recombinant proteins and subsequent antibody production. Once the ORFs are confirmed to be transcribed and translated into the corresponding proteins during viral infection, further investigation of their function will provide insights into the molecular mechanisms of viral pathogenicity. Furthermore, viral-specific proteins may serve as ideal targets for pathogen detection technologies. Application of ORF prediction tools to the SGIV genome revealed that VP101 (ORF101 in the genome) is a potential viral gene. Whether this gene expresses the corresponding protein during viral infection and its localization in infected cells remains unknown. Therefore, polyclonal antibodies prepared from the purified prokaryotically expressed proteins can specifically recognize VP101 in infected cells. Furthermore, the generated polyclonal antibodies can also be used for quantification and localization of SGIV VP101 in infected cells in vitro. Summary of the Invention

[0004] The first object of the present invention is to provide a grouper iridovirus SGIV VP101 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] The second object of the present invention is to provide the use of the above gene in preparing polyclonal antibodies to the grouper iridovirus SGIV VP101 gene.

[0006] A third object of the present invention is to provide a method for preparing a polyclonal antibody, comprising the following steps: constructing a recombinant expression vector containing the above-mentioned gene, transforming the recombinant expression vector into Escherichia coli host cells, adding IPTG to the culture medium to induce expression of the target protein to obtain a recombinant protein; using the recombinant protein as an antigen, immunizing Japanese large-eared white rabbits four times to prepare polyclonal antibodies; the four immunization injections include mixing the antigen with Freund's complete adjuvant once and mixing the antigen with Freund's incomplete adjuvant three times.

[0007] Preferably, the recombinant expression vector is a gene inserted into the initial vector pET-B2M, the Escherichia coli is Escherichia coli BL21, and the final IPTG concentration is 0.5 mmol / L. The four immunizations are: first immunization: each rabbit is subcutaneously injected with 500 μg of purified recombinant SGIV VP101 protein in Freund's complete adjuvant, and the immunization period is 2-3 weeks; second immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIV VP101 protein in Freund's incomplete adjuvant, and the immunization period is 2 weeks; third immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIV VP101 protein in Freund's incomplete adjuvant, and the immunization period is 1 week; fourth immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIV VP101 protein in Freund's incomplete adjuvant, and the immunization period is 2 weeks.

[0008] The fourth object of the present invention is to provide a polyclonal antibody prepared by the above method.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned polyclonal antibody in detecting the expression of SGIV VP101 protein in cells.

[0010] The sixth object of the present invention is to provide the use of the above-mentioned polyclonal antibody in detecting the localization of SGIV VP101 protein in cells.

[0011] The seventh object of the present invention is to provide the use of the above-mentioned polyclonal antibody in detecting SGIV for non-disease diagnosis purposes.

[0012] The eighth object of the present invention is to provide the use of the above-mentioned polyclonal antibody in the preparation of a reagent for detecting SGIVVP101 protein antigen for non-disease diagnosis purposes.

[0013] Preferably, the reagent is a colloidal gold immunoassay kit, an enzyme-linked immunosorbent assay kit or an immunoassay kit.

[0014] Advantages of the present invention:

[0015] The polyclonal antibodies against the recombinantly expressed SGIV VP101 protein prepared in the present invention can specifically recognize not only the prokaryotically expressed SGIV VP101 protein but also SGIV VP101 in infected cells. The successful preparation of polyclonal antibodies against the recombinantly expressed SGIV VP101 gene in the present invention provides an important detection tool for quantitative and localized analysis of SGIV VP101 in vitro and in vivo, as well as for related functional studies.

[0016] The polyclonal antibodies against the recombinantly expressed SGIV VP101 gene produced in this invention exhibit excellent specificity. They not only specifically recognize the prokaryotic expression product of SGIV VP101 but can also be used for quantitative and localization analysis of SGIV VP101 in vivo and in vitro. The successful preparation of polyclonal antibodies against the recombinantly expressed SGIV VP101 gene in this invention lays the foundation for the development of immunological detection methods for SGIV, quantitative and localization analysis of the VP101 gene, and analysis of gene function. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1: SDS-PAGE electrophoresis analysis of purified recombinant SGIV VP101 protein. (a) Distribution of expressed recombinant SGIV VP101 protein in total bacterial cells (lane 3), bacterial supernatant (lane 2), or precipitate (lane 1). M: protein molecular weight standard; lane 4: uninduced bacterial cells. (b) Purified recombinant SGIV VP101 protein. M: protein molecular weight standard; lane 1: purified recombinant SGIV VP101 protein.

[0018] Figure 2 This is an SDS-PAGE electrophoresis analysis of the purified polyclonal antibody against the recombinantly expressed protein of SGIV VP101. M: protein molecular weight standard; Lane 1: purified polyclonal antibody.

[0019] Figure 3 Immunoblotting analysis of purified polyclonal antibodies against SGIV VP101 recombinant protein. M: protein molecular weight standard; Lane 1: SGIV VP101 recombinant protein.

[0020] Figure 4 To quantitatively analyze the expression of SGIV VP101 in infected cells by immunoblotting using purified polyclonal antibodies.

[0021] Figure 5 Purified polyclonal antibodies were used to observe the localization of VP101 in SGIV-infected cells by indirect immunofluorescence technique.

[0022] Figure 6Purified polyclonal antibodies were used to specifically detect virus recognition in infected cells by immunoblotting. SGIV: Singapore grouper iridovirus; LMBV: Largemouth bass iridovirus; SDDV (ZH-04 / 2020): Yellowfin sea bream iridovirus SDDV isolate (ZH-04 / 2020); Mock: mock infection. DETAILED DESCRIPTION

[0023] In order to more clearly and concisely demonstrate the technical solutions, objectives and advantages of the present invention, the present invention is further described in detail below in conjunction with specific embodiments. The following embodiments are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0024] Example 1: Construction of SGIV VP101 prokaryotic expression recombinant plasmid and protein expression

[0025] (1) Based on the information of ORF101L (VP101) sequence in the whole genome of SGIV, the codon sequence was optimized and the full-length gene was synthesized at Jinkairui Biotechnology Co., Ltd. (its nucleotide sequence is shown in SEQ ID NO. 1). Primers F containing vector homology arms were designed: F: TCCACTGGGTTCTCGGACTATGAGCGTTATTAGCAGCGAACG TCGTACCGCAA ATGTTA, and R: GTGGTGCTCGAGTGCGGCCTTATTACATTGCCAGTTTGGTAATATTTGCTTCAATGGCA. The synthesized fragment of SEQ ID NO. 1 was amplified by PCR.

[0026] The pET-B2M vector was digested with the restriction endonuclease NotI. The VP101 gene PCR product and the linearized pET-B2M vector were homologously recombined and ligated using Exnase II ligase. The product was then transformed into the top10 cloning strain, and positive bacteria were screened by colony PCR and verified by sequencing to obtain the recombinant plasmid pET-B2M-VP101. The amino acid sequence of the recombinantly expressed protein of the SGIV VP101 gene is shown in SEQ ID NO. 2. The recombinant plasmid pET-B2M-VP101 was then transformed into Escherichia coli BL21 competent cells, and positive bacteria were screened by colony PCR and verified by sequencing.

[0027] (2) Place the recombinant plasmid pET-B2M-VP101-positive bacteria with the correct sequencing and the Escherichia coli BL21 (DE3) with the pET-B2M empty vector in LB liquid medium containing 50 μg / mL kanamycin and culture at 37°C overnight with shaking. The next day, dilute the overnight culture solution at a volume ratio of 1:100 and continue to culture until the OD 600When the p-value was about 0.6, isopropyl β-D-thiogalactoside (IPTG) was added at a final concentration of 0.5 mmol / L to induce protein expression. The bacterial solution without IPTG induction was used as the control. After 6 h of induction culture, the bacteria were collected by centrifugation and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0028] (3) The results showed that the induced recombinant bacterial protein had a distinct band at the molecular weight Mr = 52 kDa, while the uninduced bacterial protein had no corresponding band. The theoretical molecular weight of SGIV VP101 protein is 35 kDa, and the molecular weight of the tag protein is 17 kDa. Therefore, the predicted molecular weight of the recombinant protein is consistent with the size of the target protein shown by SDS-PAGE electrophoresis ( Figure 1 a). Ultrasonic disruption of the cells (parameters set to 200W power, 3s working, 3s pause, 10min), centrifugation at 16000rpm and 4°C for 50min, collection of the supernatant and precipitate. A small amount of supernatant and precipitate were taken for SDS-PAGE analysis of protein distribution. The results showed that the induced protein was mainly expressed in the precipitate, with only a very small amount of expression product in the supernatant ( Figure 1 a).

[0029] (4) Inclusion body protein purification. Protein purification was performed according to the inclusion body protein purification method. The specific procedures are as follows: ① Collect the bacterial solution, resuspend the bacterial cell pellet in 50 mL of STET buffer, and add DTT to a final concentration of 1 mM. ② Ultrasound promotes the dissolution of impurities, with the same parameters as above; centrifuge at 10,000 rpm and 4°C for 10 min, remove the supernatant; repeat the above steps ① to ② until the supernatant is transparent. ③ Resuspend the pellet in 1× PBS, sonicate, with the parameters set to power 200 W, work 3 s, pause 3 s, time 5 min; centrifuge at 16,000 rpm and 4°C for 10 min, remove the supernatant. ④ Resuspend the inclusion bodies in 4 mL of 6 M guanidine hydrochloride, add DTT to a final concentration of 5 mM; shake at 220 rpm and 37°C for 3 h until the inclusion bodies are completely dissolved; centrifuge at 10,000 rpm and 4°C for 10 min, and take 10 μL of the supernatant for SDS-PAGE electrophoresis. The results showed that the SGIV VP101 recombinant protein after inclusion body purification had a target band with a molecular weight of 52 kDa, a protein concentration of about 6 mg / mL, and a purity of about 85% ( Figure 1 b).

[0030] Example 2: Preparation of rabbit polyclonal antibodies against SGIV VP101 gene recombinant protein

[0031] (1) Immunity

[0032] Two Japanese white rabbits were immunized with purified SGIV VP101 recombinant protein as an antigen. The immunization was performed in four times, all of which were subcutaneously injected: ① one immunization: each rabbit was subcutaneously injected with 500 μg of purified recombinant SGIV VP101 protein and Freund's complete adjuvant (0.5 mL of 1 mg / mL recombinant SGIV VP101 protein + 0.5 mL of Freund's complete adjuvant, a total of 1 mL for immunization), and the immunization time was 2 weeks; ② two immunizations: each rabbit was subcutaneously injected with 300 μg of purified recombinant SGIV VP101 protein and Freund's incomplete adjuvant (0.5 mL of 0.6 mg / mL recombinant SGIV VP101 protein + 0.5 mL of Freund's incomplete adjuvant, a total of 1 mL for immunization), and the immunization time was 2 weeks; ③ three immunizations: each rabbit was subcutaneously injected with 300 μg of purified recombinant SGIV VP101 protein and Freund's incomplete adjuvant (0.5 mL of 0.6 mg / mL recombinant SGIV ④ Quadruple immunization: Each rabbit received a subcutaneous injection of 300 μg of purified recombinant SGIV VP101 protein and Freund's incomplete adjuvant (0.5 mL of 0.6 mg / mL recombinant SGIV VP101 protein plus 0.5 mL of Freund's incomplete adjuvant, for a total of 1 mL), 2 weeks after the third immunization. Serum titers were measured by ELISA.

[0033] (2) Antibody preparation and purification

[0034] Whole blood was collected from rabbits via the carotid artery, and serum was separated. The collected serum was purified using a Protein G-agarose affinity chromatography column as follows: ① Column pretreatment: Rinse the column five times with 10× column volume of deionized water at a flow rate of 1 mL / min, and then rinse the column five times with 10× column volume of a mixture of 0.02 M PB and 0.3 M NaCl at a flow rate of 1 mL / min. ② Sample loading: Serum was diluted with 0.02 M PB at a volume ratio of 1:5, filtered through a 0.22 μM filter, and loaded at a flow rate of 0.6 mL / min. ③ Washing: Rinse with 0.02 M PB until no protein eluted (G250 did not turn blue) at a flow rate of 1.5 mL / min. ④ Antibody elution: Elution with 0.1 M glycine, pH 3.0, and collection of the eluate. Detection of the eluted product with G250 was performed until it did not turn blue. ⑤ pH value adjustment: saturated sodium carbonate is used to adjust the pH of the eluted product to neutral. ⑥ Sample concentration: 10kDa ultrafiltration tube, ultrafiltration concentration to about 1-5mL. ⑦ Dialysis: 5L, 0.01M, pH=7.4PBS dialyzed overnight, and the liquid was changed once on the second day. After overnight dialysis, the SGIV VP101 gene recombinant protein polyclonal antibody was obtained and stored at -80℃. ⑧ Purity and activity identification: The purified polyclonal antibody was identified for its purity by SDS-PAGE. In this embodiment, a total of 8mL of serum and 2mL of purified antibody were obtained. The results of the purity detection of the purified antibody by SDS-PAGE showed that there were two protein bands with molecular weights of approximately 55kDa and 25kDa ( Figure 2 ), the antibody purity was above 90%, indicating that the purified antibodies were of high purity.

[0035] (3) Determination of titer and concentration of purified antibodies

[0036] The titer of the purified polyclonal antibody was determined by indirect ELISA. The specific steps are as follows: ① Antigen coating: The plate was coated with purified SGIV VP101 recombinant protein at a concentration of 1 μg / mL and incubated at 4°C overnight; ② The next day, the coating solution was discarded and the plate was washed once with 200 μL / well of washing solution. ③ Blocking: Block with 5% skim milk powder at 37°C for 2 hours, discard the coating solution, and wash three times with 200 μL / well of washing solution. ④ 100 μL of serially diluted purified antibody was added to each well (gradient dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000) and incubated at 37°C for 1 hour. PBS was used as a blank control. ⑤ Discard the sample to be tested, wash 3 times with 200μL / well of TBST washing solution, add horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:10000 dilution), 100μL / well, and place at 37℃ for 40min. ⑥ Discard the enzyme-labeled secondary antibody, wash 5 times with 200μL / well of TBST washing solution. ⑦ Color development: Add 90μL / well of freshly prepared TMB color development solution, incubate in the dark at 37℃ for 15min. ⑧ Stop the reaction and colorimetry: After adding 50μL / well of stop solution, measure the OD with a microplate reader 450 The absorbance value of each well at nm was 1:128,000. The ELISA results showed that the titer of the purified antibody was 1:128,000 and the antibody concentration was 10 mg / mL.

[0037] Preparation of 1×TBST washing solution for 5L system: Take 200mL 25×PBS, 2.5mL Tween 20, add pure water to dilute to 5L, and store at room temperature until use.

[0038] Preparation of 1L stop solution: Use a pipette to slowly add 28mL of concentrated sulfuric acid to 900mL of pure water, then adjust the volume to 1L and store at room temperature until use.

[0039] (4) Specific recognition and identification of purified antibodies

[0040] Western blotting was used to test the recognition of the purified antibody against the SGIV VP101 recombinant protein. The specific steps of the immunoblotting method are as follows: SGIV VP101 recombinant protein was separated by SDS-PAGE (5% stacking gel and 12% separating gel). The proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane using an electroporator and blocked with 1% casein in TBS for 1 hour at room temperature. Diluted purified antibody (1:2000) or an equal volume of unimmunized mouse serum (negative control) was added to the membrane and incubated for 2 hours at room temperature. After three rinses with TBST (0.05% Tween-20), the membrane was incubated with a secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG antibody, 1:10,000 dilution) at room temperature for another 2 hours. The membrane was then rinsed three times with TBST. ECL A and B luminescent solutions were mixed in equal volumes and incubated in the dark for 1 minute. The gel was developed and photographed in a gel scanner. The results showed that no bands appeared when the serum of unimmunized mice hybridized with the purified SGIV VP101 gene recombinant protein, while a band at 50 kDa appeared after the purified antibody hybridized with the SGIV VP101 gene recombinant protein, indicating that the purified antibody can specifically recognize and identify the SGIV VP101 prokaryotic expression protein ( Figure 3 ).

[0041] Example 3: Application of antibodies against SGIV VP101 recombinant protein

[0042] (1) Antibodies against SGIV VP101 recombinant protein for quantitative analysis of VP101 expression in in vitro infected cells

[0043] The antibody against the SGIV VP101 recombinant protein of the present invention was used for quantitative analysis of VP101 expression in in vitro infected cells by immunoblotting according to step (4) of Example 2. Lysates of grouper spleen (GS) cells at different time points of SGIV infection (0h, 6h, 12h, 18h, 24h and 48h) were used as samples to be tested, and a culture medium infection was set up as a mock infection as a negative control. Subsequently, equal amounts of protein samples were used to detect protein expression in the samples to be tested according to the immunoblotting method of step (4) of Example 2, wherein the antibody against the SGIV VP101 recombinant protein was used as the primary antibody and β-tubulin was used as the internal reference gene. The protein grayscale value was analyzed by Image J software. The expression of endogenous VP101 in infected cells was quantitatively analyzed by comparison with the grayscale value of the β-tubulin band. The results showed that no band was detected in cells infected at 0h and 6h, and a weak band was detected in cells infected at 12h. The band gradually increased with the delay of infection time. The expression level of β-tubulin in cells at different infection time points showed almost no difference. Comparison with the grayscale value of the β-tubulin band showed that the specific band intensity of VP101 in cells infected at 24h and 48h was 0.64 times and 0.83 times that of tubulin, respectively, indicating that the expression level of VP101 in infected cells was gradually increasing ( Figure 4 ).

[0044] (2) Antibodies against SGIV VP101 recombinant protein are used to localize VP101 in infected cells in vitro

[0045] The antibody of the present invention is used to observe the localization of endogenous VP101 in in vitro infected cells by indirect immunofluorescence experiment, and the specific steps are as follows: ① Grouper spleen cells (GS) are inoculated into a glass-bottomed culture dish for overnight culture, and SGIV with an infection index (MOI) of 2 is inoculated. At the same time, cells infected in the culture medium are used as mock-infected cells as a negative control; ② 24 hours after infection, the cells are fixed with 4% paraformaldehyde at 4°C for 1 hour; ③ The cells are permeabilized with 0.1% TritonX-100 for 10 minutes, rinsed with PBS, and then an antibody against VP101 recombinant protein (1:200) is added and incubated at room temperature for 2 hours; ④ After rinsing with PBS, a secondary antibody (1:500) of fluorescein (FITC)-labeled goat anti-rabbit IgG is added and incubated at room temperature for 2 hours; ⑤ After rinsing with PBS, the cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes, and the fluorescence signal is observed under an inverted fluorescence microscope. The results showed that the antibody of the present invention showed a strong green fluorescence signal when infected with SGIV. In cells infected for 24 hours, most of the green fluorescence was concentrated in the virus processing plant, with a small amount of green fluorescence diffusely located in the nucleus and cytoplasm. No green fluorescence signal was observed in the mock-infected cells (negative control group cells). Figure 5 ). This indicates that the antibody against SGIVVP101 recombinant protein can recognize VP101 in virus-infected cells, thereby indicating the localization of the protein in the cells.

[0046] (3) Antibodies against SGIV VP101 recombinant protein are used to specifically detect viral infection

[0047] The indirect immunoblotting method is used to determine the antibodies against the SGIV VP101 recombinant protein in the present invention for the specific detection of viral infection. Lysates of cells infected with viruses such as SGIV, LMBV and SDDV were used as samples to be tested, specifically SGIV-infected GS cells, LMBV-infected MsF cells and SDDV-infected yellowfin seabream kidney cell line (yellowfin seabream Kindey cell line, YSK) cells. At the same time, cells infected with culture medium were set up as a simulated infection group as a negative control. Subsequently, samples of equal amounts of protein were subjected to the immunoblotting method according to step (4) of Example 2 to detect whether there were specific bands in the samples to be tested, wherein the antibody against the SGIV VP101 recombinant protein was used as the primary antibody. The test results showed that the antibody against the SGIV VP101 recombinant protein only showed a clear positive reaction with the SGIV-infected cell sample to detect specific bands. However, no specific bands were detected in the other two virus-infected cells and the simulated-infected negative control cell samples ( Figure 6 ), indicating that the prepared antibody against SGIVVP101 recombinant protein can be specifically used for the detection of SGIV.

[0048] The above embodiments only represent several implementation methods of the present invention, and should not be construed as limiting the scope of the invention. Those skilled in the art should understand that any changes in the form and concept of the present invention fall within the scope of protection of the present invention.

[0049] SEQ ID NO.1

[0050] ATGAGCGTTATTAGCAGCGAACGTCGTACCGCAAATGTTATGGGCCTGTTTGATCGTGGTGGCATTCTGATTGATGCCCCGATGAAAGCAATTCGCCTGTATGCCCAGGATGTGGGTGGCATTAGCCTGGTGTGCACCCGTGATACCGAAAGCCTGTGGCGTCGCGAACTGCGCGAATTTCCGGAAATTACCGTGTGTAATACCATTGCCTTTATTATGCCGCATACCAAAATTGTTGTGCTGGAAACCCCGAGTGTGCTGACCGCAATGCTGAGCTATGATAAACTGATTGATGCGAGCATTATTTGGATTCGCAGCCCGAGTGTTCAGAATGTGCGTCGTGCCGTTCCGCATGATGAAGTTCCGAATTTTCGCCGCAATATGGGTAGCAGCATTCCGATTGTGAGCCATAATAAAAAAGTTACCGGCGTTCTGGAACATCCGATTACCTATCTGGAACGTCAGATGTTTGTTGATAGCAATAAAATTCCGTTCAGCCTGAGTCATCCGGCCCTGGCATATGCAAAAATGCTGATGGATCCGAGTCTGCGCACCCGCATTACCCAGCGCACCATGAGTCGCGCAGAAGAAACCTTTGGTGATGATAGCTATACCAGCAGCCAGATTAAAGCAATGCGCAGTGTTGTGGCCAGCGATGAAGATAGCTGGGCAATTAGTGTTGCAGATATTGGTCCGATGAAACAGCTGTTTACCCAGACCTTTCCGGGTGTTACCATTATTGATAATAGCCAGATTGCAAGTTGCGGCAAACCGCGTATTCTGGTGCATCAGAGTGTTGTTGAATTTGGCGTTCTGCGTACCAGCGAAGAAATTAGTCAGCTGCTGCTGCTGGAAGGCCAGATGGAAAGCCTGGAAATTTATGTTGTGGAACCGCTGGATCGCAGTGCCATTGAAGCAAATATTACCAAACTGGCAATGTAA

[0051] SEQ ID NO.2

[0052] MSVISSERRTANVMGLFDRGGILIDAPMKAIRLYAQDVGGISLVCTRDTESLWRRELREFPEITVCNTIAFIMPHTKIVVLETPSVLTAMLSYDKLIDASIIWIRSPSVQNVRRAVPHDEVPNFRRNMGSSIPIVSHNKKVTGVLEHPITYLERQMFVDSNKIPFSLSHPALAYAKMLMDPSLRTRITQRTMSRAEETFGDDSYTSSQIKAMRSVVASDEDSWAISVADIGPMKQLFTQTFPGVTIIDNSQIASCGKPRILVHQSVVEFGVLRTSEEISQLLLLEGQMESLEIYVVEPLDRSAIEANITKLAM。

Claims

1. Grouper iridovirus SGIVVP101 gene, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of the gene according to claim 1 in preparing polyclonal antibodies against the grouper iridovirus SGIVVP101 gene.

3. A method for preparing a polyclonal antibody, characterized in that: The method comprises the following steps: constructing a recombinant expression vector containing the gene according to claim 1, transforming the recombinant expression vector into an Escherichia coli host cell, adding IPTG to the culture medium to induce the expression of the target protein, and obtaining the recombinant protein; using the recombinant protein as an antigen, immunizing Japanese large-eared white rabbits four times to prepare polyclonal antibodies; and the injection reagents for the four immunizations comprise mixing the antigen with Freund's complete adjuvant once, and mixing the antigen with Freund's incomplete adjuvant three times.

4. The method according to claim 3, characterized in that The recombinant expression vector is the gene described in claim 1 inserted into the initial vector pET-B2M, the Escherichia coli is Escherichia coli BL21, the final IPTG concentration is 0.5 mmol / L, and the four immunizations are: one immunization: each rabbit is subcutaneously injected with 500 μg of purified recombinant SGIVVP101 protein and Freund's complete adjuvant, and the immunization time is 2-3 weeks; the second immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIVVP101 protein and Freund's incomplete adjuvant, and the immunization time is 2 weeks; the third immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIVVP101 protein and Freund's incomplete adjuvant, and the immunization time is 1 week; the fourth immunization: each rabbit is subcutaneously injected with 300 μg of purified recombinant SGIVVP101 protein and Freund's incomplete adjuvant, and the immunization time is 2 weeks.

5. The polyclonal antibody prepared by the method according to claim 3 or 4.

6. Use of the polyclonal antibody according to claim 5 in detecting the expression of SGIVVP101 protein in cells.

7. Use of the polyclonal antibody according to claim 5 in detecting the localization of SGIVVP101 protein in cells.

8. Use of the polyclonal antibody according to claim 5 in detecting SGIV for non-disease diagnosis purposes.

9. Use of the polyclonal antibody according to claim 5 in preparing a reagent for detecting SGIV VP101 protein antigen for non-disease diagnosis purposes.

10. The use according to claim 9, characterized in that The reagent is a colloidal gold immunoassay kit, an enzyme-linked immunosorbent assay kit or an immunoassay kit.

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