Function and application of lasp1 gene in fish antiviral immune regulation

Abstract

This invention discloses the function and application of the fish LIM and SH3 domain protein 1 (LASP1) gene in the antiviral immune regulation of fish, specifically revealing that LASP1 acts as a negative immune regulator in crucian carp herpesvirus (LCV). Ca Molecular mechanisms and applications of HV infection. For the first time, experiments have experimentally demonstrated that fish LASP1 has a significant immunomodulatory function that inhibits host interferon production and can promote... Ca HV proliferation, while knocking down this gene can effectively enhance the host's antiviral response and inhibit viral replication. Ca HV infection. Therefore. lasp1 Genes can be used for prevention and treatment Ca Novel targets for HV infection, used to prepare antiviral or therapeutic agents. Ca HV infection agents, or those used for anti-HV treatment Ca HV-infected cell models or gene editing of new crucian carp varieties.

Description

Technical Field

[0001] This invention belongs to the field of fish disease resistance technology, specifically involving lasp1 The function of the gene in the antiviral immune regulation of fish and its application in the prevention and control of crucian carp herpesvirus. Background Technology

[0002] Viral diseases in fish, particularly crucian carp hematopoietic organ necrosis caused by Carassius auratus herpesvirus (CaHV), have resulted in severe economic losses for the aquaculture industry. Current prevention and control measures for these viral diseases, including vaccines and antiviral drugs, suffer from inconsistent efficacy and limited protection rates in practical applications. Against this backdrop, developing precision control technologies has become a key requirement for industrial transformation and upgrading, and genetically modifying and breeding new aquaculture varieties with innate disease resistance is widely recognized as the most promising fundamental solution. Traditional disease-resistant breeding techniques mainly rely on phenotypic screening and family selection. While some progress has been made, their inherent limitations, such as reliance on dominant phenotypic selection, long breeding cycles, and difficulty in achieving broad-spectrum resistance, have made it difficult to meet the demands of modern aquaculture for efficient breeding. In recent years, with the rapid development of molecular biology techniques, especially the mature application of gene editing technology, creating new disease-resistant varieties by precisely modifying host immune-related genes has become the most promising research direction in aquaculture breeding. This new strategy based on molecular design breeding is expected to break through the bottlenecks of traditional methods and provide new technological support for the sustainable development of aquaculture.

[0003] In recent years, domestic scholars have made significant progress in fish genomics, virus-host interaction mechanisms, and the regulation of innate immune signaling pathways, laying an important foundation for antiviral breeding. Studies have shown that during pathogen infection, negative regulators of innate immunity often become accomplices in viral pathogenesis; the loss or mutation of these factors can significantly enhance the host's antiviral capabilities. Targeted knockout or modification of negative regulators of innate immunity using gene editing technology can precisely breed new strains with strong disease resistance. This strategy is highly efficient and targeted, representing an important development direction in current aquatic disease-resistant breeding and will provide strong support for the sustainable development of crucian carp farming in my country.

[0004] LIM and SH3 domain protein 1 (LASP1) is an F-actin-binding protein initially discovered to be highly expressed in malignant tumor tissues in 1995. While the role of LASP1 as a multifunctional protein in tumorigenesis and embryonic development has been extensively studied, its function in fish antiviral immunity remains a blank area. Through systematic research on the negative regulatory mechanism of LASP1 during crucian carp virus infection, we discovered that it may participate in the viral replication cycle as a key host factor. Based on this finding, this invention innovatively proposes a strategy for targeting and regulating LASP1 expression using gene editing technology, providing a theoretical basis and technical pathway for breeding new crucian carp varieties resistant to crucian carp herpesvirus infection. Summary of the Invention

[0005] The first object of the present invention is to provide lasp1 The application of genes or their encoded proteins as negative regulators in suppressing interferon immune responses in fish. Specifically, overexpression of LASP1 protein significantly inhibits interferon production in fish, while knockdown... lasp1 The gene significantly enhances interferon expression.

[0006] The second object of the present invention is to provide lasp1 The use of gene or its encoded protein inhibitors in the preparation of agents for the prevention or treatment of crucian carp herpesvirus infection, wherein the inhibitor targets and reduces the risk of crucian carp herpesvirus infection. lasp1 The expression level of the gene or LASP1 protein is increased, thereby enhancing host interferon expression and inhibiting the proliferation of crucian carp herpesvirus.

[0007] The third objective of this invention is to provide a cell model or method for breeding crucian carp that resists herpesvirus infection, by targeted knockdown of crucian carp. lasp1 Gene expression is enhanced, host interferon expression is increased, and the proliferation of crucian carp herpesvirus is inhibited.

[0008] To achieve the above objectives, this invention adopts the following technical solution:

[0009] Firstly, under CaHV infection conditions, LASP1, which binds to the interferon signaling pathway adaptor molecule STING, was screened by mass spectrometry, and the formation of the LASP1-STING complex was confirmed by immunoprecipitation. (Fish) lasp1 Gene expression levels are closely related to interferon production. Specifically, overexpression of LASP1 protein triggers STING degradation mediated by the autophagy receptor p62, thereby inhibiting interferon expression. Meanwhile, shRNA knockdown... lasp1 Following gene expression, interferon expression significantly increases. Therefore, LASP1 negatively regulates the interferon signaling pathway in fish through autophagy.

[0010] Secondly, the present invention discovers lasp1The gene is associated with CaHV replication and proliferation. lasp1 Gene overexpression promotes CaHV replication and proliferation, significantly enhancing viral cytopathic effects, while knockdown... lasp1 Gene expression does not affect the cell's own proliferative capacity, but it can significantly inhibit CaHV replication and proliferation, thereby enhancing the fish's resistance to CaHV infection. Therefore, lasp1 Genes can serve as novel targets for the prevention and treatment of CaHV infection, and can be used to prepare agents for the prevention or treatment of crucian carp herpesvirus infection, or for gene editing of cell models of crucian carp herpesvirus infection or new crucian carp varieties.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention provides lasp1 The novel function of the gene in the antiviral immune response of fish and its application in the prevention and control of CaHV were investigated. A negative regulator of the fish interferon signaling pathway was selected as the target, and knockdown was demonstrated for the first time. lasp1 Fish cells expressing this gene exhibit significant anti-CaHV effects, breaking through the existing breeding paradigm that only focuses on positive regulatory factors. This is achieved by directly knocking out or downregulating the gene using gene editing technology. lasp1 Compared with traditional methods such as hybridization, population selection, and mutation breeding, target genes have stronger targeting capabilities and can precisely regulate key factors in the interferon signaling pathway, providing an innovative technical path for cultivating new crucian carp strains with strong disease resistance using gene editing technology. Attached Figure Description

[0013] Figure 1 This is a volcano diagram of LASP1, which was screened by mass spectrometry in Example 1. In the diagram, the blue marker represents the decoy protein STING, and the green marker represents the target protein LASP1.

[0014] Figure 2 This is the result of the immunoprecipitation assay in Example 1 to detect the binding of LASP1 and STING.

[0015] Figure 3 Knockdown in Example 2 lasp1 Gene effect assessment. sh-NC represents the negative control group, sh- lasp1 -1 indicates sh- lasp1 -1 Targeted knockdown of CiCB cells lasp1 The experimental group of genes, sh- lasp1 -2 indicates sh- lasp1 -2 Targeted knockdown of EPC cells lasp1 The experimental group for the gene is the same as below.

[0016] Figure 4 In Example 3, quantitative real-time PCR was used to assess overexpression or knockdown. lasp1Genes on interferon ifn and its downstream genes vig1 The effect of LASP1 protein overexpression on interferon. A and B represent the effect of LASP1 protein overexpression on interferon. ifn and its downstream genes vig1 The effects of expression are indicated by vector, which represents the control group transfected with the empty vector (pCMV-Myc), LASP1-Myc-2, which represents the experimental group overexpressing LASP1 protein, Myc, which represents transfection with the pCMV-Myc plasmid, and STING-Myc-2, which represents transfection with the STING-Myc-2 plasmid (the same applies below). C and D represent knockdown. lasp1 Genes on interferon ifn and its downstream vig1 The impact of expression.

[0017] Figure 5 Example 4 illustrates the use of Western blotting to detect the effect of LASP on STING protein expression. A shows the Western blotting results of STING protein in LASP1-overexpressing cells; B shows the knockdown... lasp1 Immunoblotting results of STING protein in cells; C is the autophagy receptor. p62 Immunoblotting results of the effect of LASP1 protein overexpression on STING protein expression in gene knockdown cells; D represents the effect of autophagy receptor... p62 After gene knockdown, LASP1 inhibits STING-induced interferon. ifn The effect of quantitative real-time PCR on expression. sh- p62 This indicates targeted knockdown of EPC cells. p62 In the gene experimental group, Vector+STING indicates transfection with the empty vector pCMV-Myc+STING-Flag-2 plasmid, and LASP1+STING indicates simultaneous transfection with STING-Flag-2 and LASP1-Myc-2 plasmids.

[0018] Figure 6 This section describes the effect of LASP1 protein overexpression on CaHV proliferation in Example 5. A shows the lesioning degree of LASP1-overexpressing GiCB cells after CaHV infection; B shows the statistical results of viral titer in LASP1-overexpressing GiCB cells after CaHV infection; C shows the viral gene count in LASP1-overexpressing GiCB cells after CaHV infection. orf72 mRNA levels.

[0019] Figure 7 Knockdown in Example 6 lasp1 The degree of lesion in GiCB cells following CaHV infection.

[0020] Figure 8 Knockdown in Example 6 lasp1 Statistical results of viral titers in GiCB cells after CaHV infection.

[0021] Figure 9 Knockdown in Example 6 lasp1 GiCB cells with viral genes after CaHV infection orf72 mRNA levels.

[0022] Figure 10 Knockdown in Example 7 lasp1 The effect of genes on cell proliferation. GiCB cells transiently transfected with sh-NC and sh- lasp1 -1 plasmid was used to detect cell proliferation at different time points (24, 30, 36, 48, 72 h) after transfection using the CCK-8 assay kit. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods well known to those skilled in the art. All materials and reagents used in the following examples are commercially available unless otherwise specified.

[0024] Plasmid source and construction description: The vectors pCMV-Myc, pCMV-Tag2C, and PLKO.1 used in this example are all commercially available vectors. GiCB cells were obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). lasp1 Gene (XM_052552823.1) and sting The open reading frame sequence of the gene (XM_052614353.1) was amplified by PCR and digested with restriction enzymes, then ligated into the pCMV-Myc and pCMV-Tag2C vectors, respectively, to construct the LASP1-Myc-1 and STING-Flag-1 plasmids used in GiCB cells. EPC cells were obtained from the ZFIN website (https: / / zfin.org / ). lasp1 The open reading frame sequence of the gene (ZDB-GENE-030131-1936) and sting The open reading frame sequence of the gene (ZDB-GENE-120921-1) was amplified by PCR and digested with two restriction enzymes, and then... asp1 The plasmid LASP1-Myc-2, ligated into the pCMV-Myc vector, was constructed for use in EPC cells. stingThe plasmids STING-Myc-2 and STING-Flag-2, respectively, were constructed by ligating them into the pCMV-Myc and pCMV-Tag2C vectors for use in EPC cells. Software was then used to design targeting agents in EPC cells. p62 The shRNA sequence of the coding region (ZDBGENE-040426-2204) was used. Primers were annealed to form double-stranded DNA, which was then ligated into the PLKO.1 vector after double enzyme digestion to construct shRNA. p62 Plasmid.

[0025] Example 1: Mass spectrometry screening and verification of the binding of LASP1 and STING by immunoprecipitation.

[0026] 1.1 Mass spectrometry screening of target proteins

[0027] GiCB cells in good growth condition were passaged and seeded into 10 cm culture dishes. After being cultured overnight at 28°C, they were transiently transfected with the empty vector (pCMV-Myc) and the STING-Myc-1 plasmid, respectively. 24 hours after transfection, CaHV (MOI=100) was inoculated. 24 hours after infection, cells were lysed using freshly prepared RIPA lysis buffer (containing PMSF, Na3VO4 and a protease inhibitor cocktail), and the supernatant was collected. The supernatant was mixed with anti-Myc-tagged agarose gel beads and incubated overnight. After washing three times with PBS, mass spectrometry was performed.

[0028] The results show that ( Figure 1 Under CaHV infection conditions, LASP1 can bind to STING, a key molecule in the interferon signaling pathway, indicating that LASP1 is involved in regulating the interferon signaling pathway.

[0029] 1.2 Immunoprecipitation to verify the binding of LASP1 and STING

[0030] GiCB cells in good growth condition were passaged and seeded into 10 cm culture dishes and cultured overnight at 28°C. The control group was transfected with the empty vector (pCMV-Tag2C) + LASP1-Myc-1 plasmid, while the experimental group was transfected with the STING-Flag-1 + LASP1-Myc-1 plasmid. Twenty-four hours after transfection, cells were inoculated with CaHV (MOI=100). Twenty-four hours after infection, cells were lysed, and the supernatant was collected and incubated overnight with anti-Flag-tagged agarose gel beads. After washing three times with RIPA lysis buffer, the binding of LASP1 and STING was detected by Western blotting. The results showed that LASP1 could form a LASP1-STING complex with STING (…). Figure 2 This further confirms the combination between the two.

[0031] Example 2: Construction and knockdown of shRNA plasmids lasp1 Gene effect evaluation

[0032] 2.1 Construction of shRNA plasmid

[0033] Software design targets CiCB cells and EPC cells lasp1 Two shRNA sequences in the gene coding region, of which sh- lasp1 -1 targets CiCB cells lasp1 Gene, sh- lasp1 -2 targets EPC cells lasp1 Gene. The sequence is as follows: sh- lasp1 -1-F: 5'-CCGGGTGCTCTACAAGGAGGAATTTCTCGAGAAATTCCTCCTTGTAGAGCACTTTTTG-3',sh- lasp1 -1-R:5'-AATTCAAAAAGTGCTCTACAAGGAGGAATTTCTCGAGAAATTCCTCCTTGTAGAGCAC-3';sh- lasp1 -2-F: 5'-CCGGGCACACTATCCGAAGACGTCACTCGAGTGACGTCTTCGGATAGTGTGCTTTTTG-3',sh- lasp1 -2-R: 5'-AATTCAAAAAGCACACTATCCGAAGACGTCACTCGAGTGACGTCTTCGGATAGTGTGC-3'. Will sh- lasp1 -1 and sh- lasp1 The two primers, one forward and one reverse, were annealed to form double-stranded DNA. Simultaneously, the PLKO.1 vector was double-digested with Age I and EcoRI, and the digested vector was purified by gel extraction. Then, the double-stranded DNA was ligated into the PLKO.1 vector using ligase. The following day, E. coli were transformed with the plasmid, and single clones were selected and sequenced to identify the correct ligation plasmid.

[0034] 2.2 Knockdown lasp1 Gene effect evaluation

[0035] GiCB / EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28°C. Transient transfection with sh-NC (control) and sh- lasp1 -1 was introduced into CiCB cells, and sh-NC (control) and sh- lasp1 Cells were injected into EPC cells at -2°C, and collected 24 h later. Total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and detected by quantitative real-time PCR. lasp1The mRNA levels were determined using the following primers: gicb-lasp1-F: 5'-CATCTCATAACTATCACTATG-3', gicb-lasp1-R: 5'-ACGACACCTCGTCCTCATCAG-3', epc-lasp1-F: 5'-CTATCACTATGAGCCTGAGCCCG-3', epc-lasp1-R: 5'-CCCCCTGCTGGCCAGTGCGCT-3'.

[0036] The results are as follows Figure 3 As shown, compared with the sh-NC control group, sh- lasp1 -1 (CiCB cells) and sh- lasp1 -2 (EPC cells) can significantly reduce lasp1 The transcriptional level of the genes indicated that both shRNAs could effectively knock down CiCB / EPC cells, respectively. lasp1 Gene expression.

[0037] Example 3: Evaluation of overexpression or knockdown lasp1 Genes on interferon ifn and its downstream genes vig1 The impact of expression

[0038] 3.1 Overexpression of LASP1 protein on interferon ifn and its stimulating genes vig1 The impact of expression

[0039] EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28°C. Four experimental groups were set up, transfected with pCMV-Myc+ pCMV-Myc plasmid, pCMV-Myc+LASP1-Myc-2 plasmid, STING-Myc-2+ pCMV-Myc plasmid, and STING-Myc-2+LASP1-Myc-2 plasmid, respectively. Cells were collected 24 h after transfection, and total RNA was extracted using the TRIzol method. After reverse transcription into cDNA, the cDNA was detected by quantitative real-time PCR. ifn as well as vig1 The mRNA levels were detected using the following primers: epc-ifn-F: 5'-ATGAAAACTCAAATGTGGACGTA-3', epc-ifn-R: 5'-GATAGTTTCCACCCTTTCCTTAA-3'; epc-vig1-F: 5'-AGCGAGGCTTACGACTTCTG-3', epc-vig1-R: 5'-GCACCAACTCTCCCAGAAAA-3'.

[0040] 3.2 Knockdown lasp1Genes on interferon ifn and its stimulating genes vig1 The impact of expression

[0041] EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28 °C. Four experimental groups were set up, each transfected with pCMV-Myc+sh-NC plasmid, and the remaining cells were transfected with pCMV-Myc+sh-NC plasmid, respectively. lasp1 -2 plasmid, STING-Myc-2+sh-NC plasmid and STING-Myc-2+sh- lasp1 Cells were collected 24 h after transfection with plasmid -2, and total RNA was extracted using the TRIzol method. The RNA was then reverse transcribed into cDNA and detected by quantitative real-time PCR. ifn as well as vig1 Transcription level.

[0042] The above results are as follows Figure 4 As shown, overexpression of LASP1 protein can significantly inhibit STING-induced [therapeutic effects]. ifn and vig1 mRNA level of genes ( Figure 4 (A and B), while knockdown lasp1 Genes significantly enhance STING-induced ifn and vig1 Gene expression ( Figure 4 (C and D) The above results indicate that LASP1 is an immune negative regulator of the interferon signaling pathway.

[0043] Example 4: Detection of the effect of LASP1 on STING protein expression using Western blotting assay

[0044] 4.1 Effect of LASP1 overexpression on STING protein expression level

[0045] EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28°C. The cells were then transfected with pCMV-Myc and LASP1-Myc-2 plasmids, respectively. Cells were collected 24 h after transfection, lysed with RIPA lysis buffer, and changes in endogenous STING expression were detected by Western blotting.

[0046] 4.2 Knockdown lasp1 Effect of genes on STING protein expression levels

[0047] EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28°C. They were then transfected with sh-NC (negative control) and sh-... lasp1 -2 (targeted knockdown) plasmid, and the changes in endogenous STING expression were detected by immunoblotting.

[0048] 4.3 The role of autophagy receptor p62 in LASP1-mediated STING degradation

[0049] EPC cells in good growth condition were passaged and seeded into 6-well plates and cultured overnight at 28°C. A total of 4 experimental groups were set up, including 2 control groups and 2 experimental groups: non-knockdown cells... p62 The control group was transfected with sh-NC+STING-Flag-2+ pCMV-Myc plasmid, while the experimental group was transfected with sh-NC+STING-Flag-2+LASP1-Myc-2 plasmid; knockdown p62 Group, control group transfected with sh- p62 +STING-Flag-2+ pCMV-Myc plasmid, experimental group transfected with sh- p62 The plasmid STING-Flag-2+LASP1-Myc-2 was transfected, and STING expression was detected by Western blotting 24 h later. Specifically, it targeted EPC cells. p62 The primer sequences for shRNA are as follows: sh- p62 -F:5'-CCGGGGACTCACCTGAGCTCTAAAGCTCGAGCTTTAGAGCTCAGGTGAGTCCTTTTTG-3',sh- p62 -R: 5'-AATTCAAAAAGGACTCACCTGAGCTCTAAAGCTCGAGCTTTAGAGCTCAGGTGAGTCC-3'.

[0050] 4.4 Effect of autophagy receptor p62 on LASP1's inhibition of STING-induced interferon expression

[0051] Cell culture and transfection were performed according to method 4.3. Cells were collected after 24 h, total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and detected by real-time PCR. ifn mRNA levels.

[0052] like Figure 5 As shown in Figures A and B, overexpression of LASP1 protein significantly reduced endogenous STING protein levels, while knockdown... lasp1 Following gene sequencing, STING protein expression significantly increased; for example Figure 5 As shown in C and D, knockdown p62 The gene can inhibit LASP1's degradation of STING, while simultaneously rescuing LASP1-induced STING-mediated degradation. ifn Changes in mRNA levels. These results indicate that LASP1 regulates STING through the autophagy pathway, and this degradation depends on the autophagy receptor p62, thereby affecting interferon production.

[0053] Example 5: Effect of LASP1 protein overexpression on CaHV proliferation

[0054] 5.1 Observe cytopathic effects (CPE)

[0055] GiCB cells in good growth condition were passaged and seeded into 24-well plates and cultured overnight at 28°C. Transiently transfected with pCMV-Myc and LASP1-Myc-1 plasmids, respectively, and inoculated with CaHV (MOI=100) 24 h later. After 48 h of infection, the supernatant was collected, stored at -20°C, and infected with TCID45. 50 Virus titers were calculated using a method; cells in 24-well plates were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed for recording.

[0056] 5.2 Determination of viral titer (TCID) 50 )

[0057] GiCB cells were seeded into 96-well plates and cultured at 28°C for 24 h until the cells filled the wells. The CaHV infection supernatant collected in step 5.1 was serially diluted 3-fold or 10-fold using M199 medium. The medium was aspirated from the 96-well plates, and 100 μL of CaHV diluent was added to each well. The plates were then incubated at 28°C. After 2-3 days, once the CPE (Cellular Precipitation) level remained unchanged, the medium was discarded, and the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The viral titer (TCID) was calculated using the Reed-Muench method. 50。

[0058] 5.3 Detection of viral gene mRNA

[0059] Cell culture and transfection were performed according to method 5.1. Cells were collected 24 h after inoculation with CaHV (MOI=100), total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and then amplified and detected by real-time PCR. orf72 (CaHV) mRNA level, primer sequences are as follows: orf72-F: 5'-ATCGGTGGAGGCTTCAAAGG-3', orf72-R: 5'-TCGGGCCATAACGTGTTTCA-3'.

[0060] After cell transfection, in the uninfected CaHV group, cells remained intact without any cytopathic effects (CPE); however, in the CaHV-infected group, compared to the control group, the group overexpressing LASP1 protein showed more CPE. Figure 6 (A) This suggests that LASP significantly promotes CaHV proliferation. Supernatant from the CaHV-infected group was collected and serially diluted in 96-well plates to infect healthy cells. Cells were then analyzed using TCID50. 50Viral titers in cell culture medium were measured. Results showed that the viral titer in the LASP1 protein overexpression group was significantly higher than that in the control group, with TCID... 50 This represents an increase of approximately 15,130-fold, consistent with the results from CPE, which indicate that LASP1 promotes CaHV proliferation. Figure 6 (B) In addition, orf72 The mRNA level was significantly increased in the LASP1 overexpression group ( Figure 6 (C). Observation of CPE, viral titer determination, and mRNA detection of viral genes all confirmed that overexpression of LASP1 protein promoted the replication and proliferation of CaHV.

[0061] Example 6 Knockdown lasp1 Effects of genes on CaHV proliferation

[0062] 6.1 Observe cytopathic effects

[0063] As in method 5.1, transfect sh-NC and sh- lasp1 -1 plasmid was inoculated with CaHV virus, and cytopathic effects were observed.

[0064] 6.2 Determination of viral titer

[0065] As in 5.2, the knockdown was measured. lasp1 The influence of genes on CaHV viral titer.

[0066] 6.3 Detection of viral gene transcripts

[0067] Cell culture and transfection were performed according to method 6.1. Cells were collected 24 h after inoculation with CaHV (MOI=100), total RNA was extracted using the TRIzol method, reverse transcribed into cDNA, and then amplified and detected by real-time PCR. orf72 (CaHV) mRNA levels.

[0068] like Figure 7 As shown, after infection with CaHV, compared with the control group, knockdown... lasp1 The presence of fewer CPEs in the genome suggests knockdown. lasp1 The gene significantly inhibited CaHV proliferation. Supernatant from the CaHV-infected group was analyzed using TCID50. 50 The viral titer in the cell culture medium was measured, and the results showed that knockdown... lasp1 After gene therapy, the CaHV titer was significantly lower than that in the control group, and TCID was also significantly lower. 50 Reduced by approximately 1479 times ( Figure 8 ).also, orf72 mRNA levels at knockdown lasp1 The genome was significantly reduced ( Figure 9 All of the above results confirm that knockdown lasp1The gene significantly inhibited the replication and proliferation of CaHV.

[0069] Example 7 Knockdown lasp1 The influence of genes on cell proliferation

[0070] GiCB cells in good growth condition were passaged and seeded into 24-well plates and cultured overnight at 28°C. Transient transfection with sh-NC and sh- lasp1 Cell viability was detected using the CCK-8 assay kit at different time points (24, 30, 36, 48, and 72 h) after transfection with the -1 plasmid.

[0071] like Figure 10 As shown, sh- lasp1 There was no significant difference in cell viability between the -1 group and the sh-NC group at any time point, indicating that knockdown... lasp1 After gene therapy, the proliferation capacity of GiCB cells was not affected.

Claims

1. lasp1 The application of gene inhibitors in the preparation of drugs for the prevention or treatment of crucian carp herpesvirus infection is characterized by, The inhibitor reduces crucian carp lasp1 The expression level of the gene or LASP1 protein, wherein the inhibitor is a targeted knockdown of crucian carp. lasp1 The shRNAs expressed by the gene; the shRNA sequences for targeted knockdown of the lasp1 gene in GiCB cells are shown in SEQ ID.1 and 2.

2. The application according to claim 1, characterized in that, The inhibitor enhances host interferon expression and inhibits the proliferation of crucian carp herpesvirus.

3. A method for breeding crucian carp resistant to herpesvirus infection for non-disease treatment purposes, characterized in that, Targeted knockdown of crucian carp using RNA interference lasp1 Gene expression; the shRNA sequences for targeted knockdown of the lasp1 gene expression in GiCB cells are shown in SEQ ID.1 and 2.

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