Application of tollip gene in fish antiviral immune regulation

Abstract

The invention discloses an application of a tollip gene in fish antiviral immune regulation, a negative regulation factor of a fish interferon signal channel is selected as a target spot for the first time, TOLLIP is proved to have an effect of remarkably promoting GCRV replication in fish cells, the GCRV inhibition capability can be remarkably improved by knocking out or knocking down the TOLLIP gene, and the TOLLIP gene can be used as a target spot for the first time. Therefore, the gene can be used as a novel genetic breeding target for fish anti-GCRV immune regulation and antiviral ability, and an important molecular target and theoretical support are provided for developing a novel genetic breeding technology for enhancing the antiviral ability of cultured fishes.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of fish virus prevention and control, in particular to tollip Application of a gene in fish antiviral immune regulation. BACKGROUND

[0002] Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is one of the most serious viral diseases in China's freshwater aquaculture industry, which often leads to large-scale fish death and causes huge economic losses. At present, the prevention and control measures for GCRV, such as inactivated vaccine and antiviral preparations, have problems such as short immune protection period, fast virus variation and uneven prevention and control effect in practical application. Under this background, developing an efficient and durable prevention and control strategy has become an urgent need for the development of the industry, and cultivating a new grass carp variety with natural disease resistance through genetic improvement is considered as the most sustainable solution. Traditional disease-resistant breeding methods mainly rely on hemorrhagic disease resistance phenotype screening and population selection, which has certain effect, but has inherent defects such as long breeding cycle, unstable resistance traits and difficulty in considering growth performance, which cannot meet the needs of modern aquaculture for efficient and precise breeding. In recent years, with the deep integration of aquatic virology and molecular genetics, especially the mature application of gene editing technology, the creation of new disease-resistant varieties through targeted regulation of host antiviral immune pathways or precise modification of host immune-related genes has become a frontier direction in the field of aquatic disease-resistant breeding. This new strategy based on molecular design breeding is expected to break the shackles of traditional breeding mode and inject new technical vitality into the sustainable and stable development of aquaculture industry.

[0003] In recent years, domestic scholars have made important breakthroughs in the fields of fish disease-resistant genetic mechanisms, virus invasion pathways and host immune regulation networks, providing a theoretical basis for disease-resistant breeding. Research has found that during the process of viral infection, negative regulatory factors of the innate immune pathway are often hijacked by viruses, becoming key targets for inhibiting host immune response. The loss of function or moderate inhibition of these negative regulatory factors can significantly enhance the antiviral immune response of grass carp. Targeted knockout or modification of natural negative regulatory factors using CRISPR / Cas9 and other gene editing technologies can precisely cultivate new disease-resistant strains. This strategy has the advantages of clear targeting and high efficiency, and has become an important development direction of current grass carp disease-resistant breeding, which is expected to provide a breakthrough solution for disease prevention and control in grass carp aquaculture industry.

[0004] TOLLIP is an important innate immune regulatory protein, which was first discovered in 2000 when studying the Toll-like receptor signaling pathway. As a multifunctional adaptor protein, TOLLIP plays a key role in inflammatory response and autophagy process, but its function mechanism in the immune response of aquatic fish is still lacking of systematic study. We found that TOLLIP may act as a key negative regulatory factor in the host's antiviral immune response by studying the immune regulation of TOLLIP in the process of GCRV infection. Based on this finding, this study innovatively proposes a strategy for targeted intervention of TOLLIP expression using gene editing technology, which provides an important molecular target and theoretical support for developing new genetic breeding techniques to enhance the antiviral ability of farmed fish. SUMMARY

[0005] The present application provides tollip The application of the gene in the immune regulation of fish against GCRV provides a molecular target and theoretical support for developing new genetic breeding techniques to enhance the antiviral ability of farmed fish.

[0006] Therefore, the present application provides the following solutions: The first aspect of the present application is to provide tollip The application of the gene as a target in the immune regulation of fish against GCRV.

[0007] Further, the application includes at least one of the following: a) By low expression of TOLLIP, the expression amount of interferon is improved; b) By low expression of TOLLIP, the inhibition ability of STING to GCRV is improved.

[0008] The second aspect of the present application is to provide tollip The application of the gene as a target in the preparation of a cell model, which includes a gene edited cell model against GCRV infection or a cell model for screening drugs for the prevention and treatment of hemorrhagic disease of hematopoietic organs.

[0009] Further, the gene edited cell model against GCRV infection is obtained based on low expression of TOLLIP in cells.

[0010] Preferably, the reagent for low expression of TOLLIP is an mRNA level inhibitor.

[0011] Preferably, the mRNA level inhibitor is shRNA, including two shRNA sequences as shown in SEQ ID NO: 1-4.

[0012] Further, the cell model for screening drugs for the prevention and treatment of hemorrhagic disease of hematopoietic organs is obtained based on overexpression of TOLLIP in cells. Further, the cell model for screening drugs for the prevention and treatment of hemorrhagic disease of hematopoietic organs is obtained based on overexpression of TOLLIP in cells.

[0013] Further, the cell model is constructed based on a carp epithelioma cell line.

[0014] It can be understood that the grass carp reovirus infection object includes various fish, mainly grass carp, and the carp epithelioma cell line is used as a main cell line for fish virus research. Different antiviral cell models or drug screening models can be obtained by gene editing of the carp epithelioma cell line, which are used for antiviral research and drug screening of grass carp.

[0015] The third aspect of the present application is to propose the application of TOLLIP inhibitors in the preparation of fish anti-GCRV immune regulation drugs or anti-GCRV breeding products.

[0016] Further, the TOLLIP inhibitor is shRNA, including two shRNA sequences as shown in SEQ ID NO: 1-4.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application provides tollip The new function of the gene in fish antiviral immune response and its application in virus disease prevention and control, for the first time, the negative regulator of fish interferon signaling pathway is selected as a target, and it is proved that TOLLIP has a significant effect on promoting GCRV replication in fish cells, and the knock-out or knock-down tollip The gene can significantly improve the GCRV inhibition ability, so it can be used as a new genetic breeding target for fish anti-GCRV immune regulation and antiviral ability, which provides an important molecular target and theoretical support for developing new genetic breeding technology to enhance the antiviral ability of farmed fish.

[0018] The present application breaks through the breeding paradigm of the prior art which only focuses on positive regulators; the gene can be directly knocked out or knocked down by gene editing technology tollip Compared with traditional hybrid breeding, population breeding and mutagenic breeding, the method has stronger targeting and can accurately regulate the hub factor of the interferon signaling pathway, which provides an innovative technical path for cultivating new fish strains with strong disease resistance by using gene editing technology. BRIEF DESCRIPTION OF DRAWINGS

[0019] figure 1 For the heat map of TOLLIP selected by transcriptome screening in Example 1, the red box in the figure is the up-regulated tollip Gene.

[0020] figure 2 For the effect evaluation result of knocking down the gene in Example 2. Figure A is the knocking down effect of mRNA level detected by fluorescence quantitative PCR, and Figure B is the knocking down effect of protein level detected by immunoblotting. tollip ​

[0021] figure 3 Results of the effect of overexpression of TOLLIP or knockdown of TOLLIP on GCRV-induced interferon tollip and its downstream genes expression in Example 3. Fig. A is the effect of overexpression of TOLLIP on interferon ifn and its downstream genes expression; Fig. B is the effect of knockdown of TOLLIP on interferon vig1 and its downstream genes expression. ifn vig1 tollip and its downstream genes expression in Example 3. Fig. A is the effect of overexpression of TOLLIP on interferon ifn and its downstream vig1 expression.

[0022] figure 4 Results of the effect of overexpression of TOLLIP on GCRV proliferation in Example 4. Fig. A is the results of the extent of cytopathic effect of cells overexpressing TOLLIP after GCRV infection; Fig. B is the statistical results of viral titers of cells overexpressing TOLLIP after GCRV infection; Fig. C is the results of fluorescence quantitative detection of mRNA levels of viral genes s6 and s9 of cells overexpressing TOLLIP after GCRV infection.

[0023] figure 5 Results of the detection of TOLLIP-STING complex by co-immunoprecipitation in Example 5.

[0024] figure 6 Results of the effect of TOLLIP on STING protein expression detected by Western blotting in Example 5. Fig. A is the effect of overexpression of TOLLIP on STING protein; Fig. B is the effect of gradient overexpression of TOLLIP on STING.

[0025] figure 7 Results of the effect of overexpression of TOLLIP on the antiviral function of STING in Example 6. Fig. A is the results of the extent of cytopathic effect of cells overexpressing TOLLIP and its control and STING after GCRV infection; Fig. B is the statistical results of viral titers of cells overexpressing TOLLIP and its control and STING after GCRV infection; Fig. C is the results of fluorescence quantitative detection of mRNA levels of viral genes s6 and s9 of cells overexpressing TOLLIP and its control and STING after GCRV infection. DETAILED DESCRIPTION

[0026] ​​The technical solutions of the present application will be clearly and completely described below in combination with preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0027] In one embodiment, a new target gene that plays an important breeding value in the prevention and control of grass carp viral disease is provided tollip . It is found through verification that overexpression of TOLLIP significantly inhibits the production of interferon, while knockdown of the gene significantly enhances the expression of interferon. tollip In addition, a new function of TOLLIP in the regulation of grass carp antiviral immunity is provided. TOLLIP can degrade the key molecule protein level of the interferon signaling pathway, thereby significantly inhibiting the production of interferon and promoting the replication of grass carp reovirus. tollip As a new target, the gene can be effectively applied to the prevention and control of grass carp hemorrhagic disease.

[0028] In the above embodiments, it is verified that under the condition of viral infection, the up-regulated autophagy gene is screened through transcriptome tollip , the expression of fish TOLLIP is related to the production of interferon, and the expression of interferon is obviously increased after knockdown of the gene tollip . A new way of TOLLIP negatively regulating fish interferon signaling pathway by degrading STING is provided. The protein degrades STING as an autophagy receptor, thereby inhibiting the expression of interferon. Further, the present application finds that TOLLIP has relevance with GCRV replication. TOLLIP weakens the ability of STING to inhibit GCRV infection, and significantly enhances the cytopathic effect caused by the virus. Subsequent detection of the amplification of virus-related genes in cells shows that the expression amounts of virus-related genes s6 and s9 in the cells are significantly increased.

[0029] In one embodiment, the application of TOLLIP as a target in the preparation of a cell model is disclosed. By designing an shRNA sequence targeting knockdown of the gene tollip , low expression of TOLLIP in the epithelioma of carp cell line (EPC) is realized, and a gene editing cell model resistant to GCRV infection is obtained. Based on the overexpression of TOLLIP in the epithelioma of carp cell line (EPC), a GCRV proliferation cell model is obtained, the virus titer is improved, which can be used to improve the sensitivity in the screening of anti-GCRV drugs and efficiently screen anti-GCRV drugs.

[0030] Embodiment 1: Screening of target gene by transcriptome tollip

[0031] The well-grown EPC cells were subcultured into six-well plates, inoculated with GCRV (MOI=0.001) after overnight culture at 28°C, and the cells were collected 24 hours after infection, total RNA was extracted by TRIzol and sent for transcriptome test. The results are shown in figure 1 As shown, the autophagy genes tollip were significantly up-regulated after GCRV infection.

[0032] Example 2 Construction of shRNA plasmid and evaluation of knockdown effect tollip of shRNA plasmid

[0033] 2.1 Construction of shRNA plasmid

[0034] Two shRNA sequences targeting the coding region of TOLLIP were designed by software, wherein sh- tollip #1 and sh- tollip #2 target the tollip gene in EPC cells. The sequences are as follows: sh- tollip #1-F: 5'-CCGGGCAACAGCAGGTACAGTTAGACTCGAGTCTAACTGTACCTGCTGTTGCTTTTTG-3' (SEQ ID NO: 1); sh- tollip #1-R: 5'-AATTCAAAAAGCAACAGCAGGTACAGTTAGACTCGAGTCTAACTGTACCTGCTGTTGC-3' (SEQ ID NO: 2); sh- tollip #2-F: 5'-CCGGGGACTCCTTCTACCTGGAAATCTCGAGGGACTCCTTCTACCTGGAAATTTTTTG-3' (SEQ ID NO: 3); sh- tollip #2-R: 5'-AATTCAAAAAGGACTCCTTCTACCTGGAAATCTCGAGATTTCCAGGTAGAAGGAGTCC-3' (SEQ ID NO: 4).

[0035] The two primers of sh- tollip #1 and sh- tollip #2 were annealed to form double-stranded DNA, and the PLKO.1 vector was double-digested with AgeI and EcoRI, the digested vector was recovered and purified, and then the double-stranded DNA was ligated to the PLKO.1 vector by ligase. The next day, after the E. coli was transformed with the plasmid, single colonies were picked and sent for sequencing to identify the correct ligated plasmid.

[0036] 2.2 Knockdown tollip Gene effect evaluation

[0037] 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- tollip #1、sh- tollip #2 cells were introduced into EPC cells, and cells were collected 24 h later for quantitative real-time PCR and immunoblotting. Total RNA was extracted with TRIzol, reverse transcribed into cDNA, and detected by quantitative real-time PCR. tollip The mRNA level was determined using the following primers: tollip -F:5'-CTGCAGGATTCGACTTGGGT-3' (SEQ ID NO: 5) tollip -R:5'-TGTCCATGCGATGCGATCAT-3' (SEQ ID NO: 6) Cells were lysed with RIPA lysis buffer (containing PMSF, Na3VO4 and the protease inhibitor Cocktail), and changes in endogenous TOLLIP expression were detected by Western blotting.

[0038] The results are as follows figure 2 As shown, compared with the sh-NC control group, sh- tollip #1 can reduce the transcriptional and protein expression levels of TOLLIP, indicating that sh- tollip #1 can effectively knock down tollip Gene expression.

[0039] Example 3: Evaluation of TOLLIP overexpression or knockdown tollip Genes on interferon ifn and its downstream genes vig1 The impact of expression

[0040] 3.1 Overexpression of TOLLIP 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. The control group was transfected with the Flag plasmid, and the experimental group was transfected with the TOLLIP-Flag plasmid. 24 h after transfection, cells were inoculated into GCRV (MOI = 0.001). Cells were collected 24 h after infection, total RNA was extracted with TRIzol, reverse transcribed into cDNA, and detected by quantitative real-time PCR. ifn as well as vig1 mRNA levels.

[0042] 3.2 Knockdown tollip Genes on interferon ifn and its stimulating genes vig1 The impact of expression

[0043] Cell culture was performed according to method 3.1. The control group was transfected with sh-NC plasmid, and the experimental group was transfected with sh- tollip #1 plasmid was transfected and inoculated into GCRV cells (MOI = 0.001) 24 h later. Cells were collected 24 h after infection, total RNA was extracted with TRIzol, reverse transcribed into cDNA, and detected by real-time PCR. ifn as well as vig1 Transcription level, primers as follows: ifn -F: 5'-CCGATACCAGCCATCACATAAG-3' (SEQ ID NO: 7), ifn -R: 5'-GATCTGCTCCCATGCTTGAG-3' (SEQ ID NO: 8), vig 1-F: 5'-TTCCACACTGCGAAGACCTC-3' (SEQ ID NO: 9), vig1 -R: 5'-CCATTACTAACGATGCTGACGC-3' (SEQ ID NO: 10).

[0044] like figure 3 As shown, overexpression of TOLLIP can significantly inhibit GCRV-induced [therapeutic effects]. ifn and vig1 mRNA level of genes ( figure 3 A and B), and knockdown tollip Genes significantly enhance GCRV-induced ifn and vig1 Gene expression ( figure 3 (C and D) The above results indicate that TOLLIP is an immune negative regulator of the interferon signaling pathway.

[0045] Example 4: Effect of TOLLIP overexpression on GCRV proliferation

[0046] 4.1 Observe cytopathic effects (CPE)

[0047] The EPC cells in good growth state were subcultured into 24-well plates and cultured at 28°C overnight. The Flag and TOLLIP-Flag plasmids were transiently transfected, and 24 h after transfection, GCRV (MOI = 0.001) virus was inoculated, 48 h after inoculation, the supernatant was transferred to CIK cells to observe the lesions, and the supernatant was collected at 24-48 h and stored at -80°C and used for TCID 50 The virus titer was calculated by TCID

[0048] 4.2 Determination of virus titer (TCID 50 )

[0049] The CIK cells were inoculated into 96-well plates and cultured at 28°C for 24 h until the cells covered the wells; 3-fold or 10-fold gradient dilution was performed with M199 medium; the culture medium in the 96-well plate was aspirated, 100 μL of the diluted liquid was added to each well, and the plate was placed in a 28°C incubator for further culture; after 2-3 d, when the CPE no longer changed, the culture medium was discarded, the cells were fixed with 4% polyethylene glycol and stained with crystal violet, and the virus titer TCID 50 .

[0050] 4.3 Detection of mRNA of virus gene

[0051] The cells were cultured and transfected according to the method of 4.1, the cells were collected 24 h after inoculation, total RNA was extracted by TRIzol, and cDNA was reverse transcribed, and the mRNA levels of s6 and s9 were detected by fluorescence quantitative PCR amplification, and the primers were as follows: s6- F: 5'-GTGTTGACCCTGGATGTGAG-3' (SEQ ID NO: 11); s6 R: 5'-GTTAGCAGCGGTAGTGACTTG-3' (SEQ ID NO: 12); s9 F: 5'-GCCGCTCGTGATTTGTTA-3' (SEQ ID NO: 13); s9 R: 5'-GGGTAGGTGTCGGGTAGTTC-3' (SEQ ID NO: 14).

[0052] The above results are shown in figure 4 , compared with the control group, more CPE appeared in the TOLLIP group after cell transfection and GCRV infection figure 4A), indicating that TOLLIP significantly promotes GCRV proliferation. The supernatant of the control group and the experimental group was collected and gradient diluted to infect healthy cells in a 96-well plate. The TCID 50 The virus titer in the cell culture fluid was determined. The results showed that the virus titer of the TOLLIP overexpression group was significantly higher than that of the control group, TCID 50 increased by about 100 times, which was consistent with the results of CPE, that is, TOLLIP promoted the proliferation of viruses ( figure 4 B). In addition, s6 and s9 the mRNA level of TOLLIP overexpression group was significantly increased ( figure 4 C). Through the observation of CPE, virus titer determination and mRNA detection of viral genes, it was confirmed that overexpression of TOLLIP promoted the replication and proliferation of GCRV virus.

[0053] Example 5 Co-immunoprecipitation verifies the binding of TOLLIP and STING and detects the influence of TOLLIP on the expression of STING protein

[0054] 5.1 Co-immunoprecipitation verifies the binding of LASP1 and STING

[0055] Well-grown EPC cells were subcultured into 10 cm culture dishes and cultured overnight at 28°C, then transiently transfected with corresponding plasmids, and 24 h later, the cells were lysed with RIPA lysis buffer and the supernatant was collected. After incubation with anti-HA agarose beads overnight, the RIPA lysis buffer was washed 3 times, and the binding of TOLLIP and STING was detected by immunoblotting.

[0056] 5.2 Immunoblotting detects the influence of TOLLIP on the expression of STING protein

[0057] Well-grown EPC cells were subcultured into six-well plates and cultured overnight at 28°C, then transiently transfected with corresponding plasmids, and 24 h later, the cells were lysed with RIPA lysis buffer and the supernatant was collected. The change of STING was detected by immunoblotting.

[0058] The above results show that TOLLIP can form a TOLLIP-STING complex with STING ( figure 5 ), overexpression of TOLLIP significantly reduces the level of endogenous STING protein ( figure 6 A), and after gradient overexpression of TOLLIP, STING shows a gradient degradation trend ( figure 6 B).

[0059] Example 6 Influence of overexpression of TOLLIP on the antiviral function of STING

[0060] 6.1 Observation of cytopathic effect

[0061] EPC cells in good growth state were subcultured into 24-well plates and cultured overnight at 28°C. The corresponding plasmids were transiently transfected, and 24 h after transfection, GCRV (MOI = 0.001) virus was inoculated, respectively. 48 h after inoculation, the supernatant was transferred to CIK cells to observe the lesions, and the supernatant was collected at 24-48 h and stored at -80°C and the virus titer was calculated by TCID 50 method; the CIK cells in the 24-well plate were fixed with 4% polyethylene glycol and stained with crystal violet and photographed.

[0062] 6.2 Determination of virus titer

[0063] CIK cells were inoculated into 96-well plates and cultured at 28°C for 24 h until the cells covered the wells; 3-fold or 10-fold gradient dilution was performed with M199 medium; the culture medium in the 96-well plate was aspirated, 100 μL of the diluted liquid was added to each well, and the culture was continued in a 28°C incubator; after 2-3 d, when the CPE no longer changed, the culture medium was discarded, the cells were fixed with 4% polyethylene glycol and stained with crystal violet, and the virus titer TCID 50 was calculated by the Reed-Muench method.

[0064] 6.3 Detection of viral gene transcripts

[0065] Cell culture and transfection were performed according to the method of 6.1, and 24 h after inoculation, the cells were collected, total RNA was extracted by TRIzol, and after reverse transcription into cDNA, the mRNA levels of s6 and s9 were detected by fluorescent quantitative PCR amplification.

[0066] The above results are shown in figure 7 A, after infection with GCRV, compared with overexpression of STING alone, the group of co-overexpression of TOLLIP and STING showed less CPE, indicating that TOLLIP significantly inhibited the antiviral function of STING and thus inhibited the proliferation of GCRV. The supernatant was taken and the virus titer in the cell culture fluid was determined by TCID 50 , the results showed that overexpression of TOLLIP rescued the inhibition of GCRV by STING, thus the virus titer of the group of co-transfection of TOLLIP and STING was about 74 times higher than that of the group of transfection of STING alone ( figure 7 B). In addition, s6 and s9 the mRNA levels of figure 7C). The above results all confirmed that TOLLIP inhibited the antiviral function of STING, thus rescuing the inhibition of GCRV proliferation by STING.

[0067] While embodiments of the application have been shown and described, it is to be understood that the application is not limited to the details of the embodiments described, since numerous further modifications and changes can be apparent to one skilled in the art without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. tollip Application of genes as targets in immune regulation against GCRV in fish.

2. Use according to claim 1, characterized in that, The application comprises at least one of the following: a) increasing interferon expression by low expression of TOLLIP; b) increasing the inhibitory ability of STING to GCRV by low expression of TOLLIP.

3. tollip The application of genes as targets in the preparation of cell models, characterized in that, The cell model comprises a genetically edited cell model against GCRV infection or a grass carp hemorrhagic disease prevention and treatment drug screening cell model.

4. Use according to claim 3, characterized in that, The genetically edited cell model against GCRV infection is obtained based on low expression of TOLLIP in cells.

5. Use according to claim 4, characterized in that, The reagent for low expression of TOLLIP is an mRNA level inhibitor.

6. Use according to claim 5, characterized in that, The mRNA level inhibitor is shRNA, comprising two shRNA sequences as shown in SEQ ID NO: 1-4.

7. Use according to claim 3, characterized in that, The hemorrhagic disease prevention and treatment drug screening cell model of hematopoietic organs is obtained based on overexpression of TOLLIP in cells.

8. Use according to claim 3, characterized in that, The cell model is constructed based on a carp epithelioma cell line.

9. Application of a TOLLIP inhibitor in the preparation of a fish immune regulation drug against GCRV or an anti-GCRV breeding product.

10. Use according to claim 9, characterized in that, The TOLLIP inhibitor is shRNA, comprising two shRNA sequences as shown in SEQ ID NO: 1-4.

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