Application of sheep-derived LGALS1 in inhibition of orf virus

Abstract

The invention discloses application of sheep-derived LGALS1 in inhibition of orf virus, and belongs to the field of genetic engineering. Research finds that the proliferation of OrfV can be inhibited by overexpressing LGALS1 gene in LT cells; the proliferation of OrfV can be promoted by interference expression of the LGALS1 gene. Therefore, the LGALS1 gene or a biological material containing the LGALS1 gene or the LGALS1 protein can be used for preparing a product for resisting the orf virus.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to application of sheep-derived LGALS1 in inhibiting sheep oropharyngeal canker sore virus. Background Art

[0002] With the in-depth study of the mechanism of viral infection, more and more host proteins have been found to be involved in the replication and spread of viruses. Understanding the role of these host proteins and their interaction mechanisms with viruses will help develop therapeutic strategies for viral infections. Galectin-1 (LGALS1) is a highly conserved β-galactoside binding protein that is widely present in a variety of organisms. LGALS1 is not only involved in a variety of biological processes, such as cell proliferation, differentiation and apoptosis, but also plays an important role in immune response, inflammation regulation and tissue repair. LGALS1 plays a key role in cell-cell and cell-matrix interactions by recognizing and binding to glycosylated proteins on the cell surface.

[0003] Recent studies have shown that during viral infection of host cells, N-linked glycans on viral glycoproteins are recognized by various endogenous host lectins. This interaction can facilitate viral entry or lead to viral clearance. Studies have reported that the LGALS1 protein binds to viral surface glycoproteins, promoting or inhibiting viral infection. For example, LGALS1 protein binds to West Nile virus, dengue virus, and human immunodeficiency virus, promoting viral attachment and infection; it binds to the envelope glycoproteins of influenza virus, Nipah virus, Ebola virus, Marburg virus, Newcastle disease virus, and vesicular stomatitis virus, inhibiting viral infection. Furthermore, duck-derived LGALS1 protein can inhibit the early in vitro replication of duck plague virus. These studies confirm that cellular LGALS1 protein is involved in the replication and proliferation of various viruses.

[0004] Orf disease is an acute, contact, epithelial infectious disease caused by the Orf virus (OrfV). Infected sheep mainly show severe ulcers and scabs on the lips, which makes it difficult for them to eat and insufficient nutrition intake, greatly reducing the production performance of adult goats and the growth and development of lambs. Orf disease is one of the most widespread viral diseases in the world. It mainly infects small ruminants such as goats and sheep. Surveys have found that the incidence of orf disease in lambs is as high as 100%, and the mortality rate caused by secondary infection is as high as 15%. So far, the high incidence and mortality rates have brought huge economic losses to the sheep industry. However, due to the unique immune escape mechanism of the orf virus, the repeated infection characteristics and the lack of suitable therapeutic drugs on the current market, the prevention and treatment of orf disease remains a problem worldwide. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide application of LGALS1 gene in inhibiting oropharyngeal canker sore virus.

[0006] The technical solution of the present invention is: use of the LGALS1 gene or a biological material containing the LGALS1 gene or the LGALS1 protein in preparing a product against oropharyngeal canker sore virus, wherein the LGALS1 gene is a gene that can encode the LGALS1 protein shown in SEQ ID No. 1.

[0007] Furthermore, the biological material containing the LGALS1 gene is an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the LGALS1 gene.

[0008] Furthermore, the nucleotide sequence of the LGALS1 gene is shown in SEQ ID No. 2.

[0009] Furthermore, the anti-stomatitis virus product refers to a drug that inhibits the proliferation of thestomatitis virus or a drug that prevents or treats diseases caused by thestomatitis virus infection.

[0010] The experimental data of the present invention show that LGALS1 plays an important inhibitory role in the OrfV infection process. First, through the LGALS1 overexpression experiment, it was found that the gene expression and viral titer of OrfV were significantly reduced. This shows that the overexpression of LGALS1 can effectively inhibit the replication and proliferation of OrfV. RT-qPCR results showed that the mRNA level of the OrfV B2L gene was significantly decreased in the LGALS1 overexpression group, and WB analysis showed that the expression of B2L protein was also significantly reduced. These results indicate that LGALS1 may inhibit the spread of the virus by reducing the synthesis of viral RNA and protein. In the LGALS1 silencing experiment, we observed a significant increase in the replication and proliferation of OrfV, further supporting the inhibitory effect of LGALS1. After silencing LGALS1, the expression levels of the OrfVB2L gene and protein were significantly increased compared with the control group, and the viral titer also increased significantly. This shows that when LGALS1 expression is restricted, the virus can proliferate more effectively in host cells. In experiments with small molecule inhibitors, we used OTX008 (CAS No. 286936-40-1) to inhibit the function of LGALS1 and observed a significant enhancement of OrfV replication. In cells treated with OTX008, both the mRNA and protein levels of the OrfV B2L gene increased significantly, and viral titers also increased accordingly. This result is consistent with the results of experiments silencing LGALS1 with siRNA, indicating that chemical inhibition of LGALS1 function can also promote viral replication. This suggests that the inhibitory effect of LGALS1 may be achieved through its biological activity, rather than relying solely on its expression level.

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

[0012] The present invention found that overexpressing the LGALS1 gene in LT cells can inhibit the proliferation of OrfV, while interfering with the expression of the LGALS1 gene can promote the proliferation of OrfV. Therefore, the LGALS1 gene, biomaterials containing the LGALS1 gene, or LGALS1 protein can be used to prepare products against OrfV. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 PCR amplification results of LGALS1 gene; M:DL2 000DNA Marker: 1-3:pCMV-HA-LGALS1 plasmid.

[0014] Figure 2 Identification of LGALS1 eukaryotic plasmid expression; M: protein marker; 1: pCMV-HA; 2-4: pCMV-HA-LGALS1.

[0015] Figure 3 Cell viability assay results.

[0016] Figure 4 RT-qPCR was used to detect the effect of LGALS1 overexpression on the expression level of OrfV B2L mRNA.

[0017] Figure 5 WB was used to detect the effect of LGALS1 overexpression on the expression level of OrfV B2L protein.

[0018] Figure 6 Grayscale analysis histogram of OrfV B2L protein.

[0019] Figure 7 TCID 50 Methods Virus titer in cell supernatant was measured.

[0020] Figure 8 Screening and validation of siRNA; A: RT-qPCR detection of siRNA silencing efficiency; B: WB detection of siRNA silencing efficiency.

[0021] Figure 9 Cell viability assay results.

[0022] Figure 10 RT-qPCR was used to detect the effect of interfering with LGALS1 on the expression level of OrfV B2L mRNA.

[0023] Figure 11 WB detection was used to detect the effect of interfering with LGALS1 on the expression level of OrfV B2L protein.

[0024] Figure 12 Grayscale analysis histogram of OrfV B2L protein.

[0025] Figure 13 TCID 50 Methods Virus titer in cell supernatant was measured.

[0026] Figure 14 Effects of different inhibitor concentrations on cell viability.

[0027] Figure 15 RT-qPCR was used to detect the effects of inhibitors on the expression of OrfV B2L mRNA levels.

[0028] Figure 16 Western blotting was used to detect the effects of inhibitors on the expression level of OrfV B2L protein.

[0029] Figure 17 Grayscale analysis histogram of OrfV B2L protein.

[0030] Figure 18 TCID 50 The virus titer in the cell supernatant was measured by the method. DETAILED DESCRIPTION

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0032] Main materials and reagents

[0033] The eukaryotic expression vector pCMV-HA and rabbit anti-OrfV B2L protein polyclonal antibody were prepared and stored by the Preventive Veterinary Laboratory of Guizhou University.

[0034] Endotoxin-free plasmid mini-preparation kit (Y1809) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; DNA transfection reagent Lipofectamine TM 3000 Transfection Reagent (L3000015) was purchased from Thermo Fisher; siRNA transfection reagent RNA TransMate (E607402) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; LGALS1-specific inhibitor OTX008 (product number: O797638-1 mL) was purchased from MacLean Chemical Reagent Co., Ltd.; Cell Counting Kit-8 (CCK-8) cell proliferation detection kit (C6005M) was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.

[0035] Example 1 Effect of overexpression of LGALS1 on OrfV proliferation

[0036] 1. Primer design

[0037] According to the gene sequences of OrfV B2L (GenBank accession number: GU320351.1) and LGALS1 (GenBank accession number: XM_018048739.1), RT-qPCR amplification primers for the OrfVB2L gene and conventional PCR amplification primers for LGALS1 were designed using the NCBI online primer designing tool. The designed primers were synthesized by Shanghai Bioengineering Co., Ltd. The primer information is shown in Table 1.

[0038] Table 1 Primer information

[0039]

[0040] Note: The underlined parts are the restriction enzyme sites Sal I and Kpn I

[0041] 2. Construction and expression of pCMV-HA-LGALS1

[0042] Using pET-32a-LGALS1 plasmid as template, high-fidelity enzyme The LGALS1 gene was amplified by HSDNA polymerase. After gel recovery, the target gene and the empty vector pCMV-HA were cut with restriction endonucleases Sal I and Kpn I, respectively. The target gene and the linearized vector were separated by agarose gel electrophoresis. After gel recovery, ligation, transformation, and plating were performed. Single clones were picked for colony PCR and sequencing identification.

[0043] The results are as follows Figure 1 As shown, the target band appeared between 250bp and 500bp, which was consistent with expectations. The sequencing results sent to the company also showed that pCMV-HA-LGALS1 was successfully constructed.

[0044] 3. Transfection of LT cells with pCMV-HA-LGALS1

[0045] The transfection reagent for the recombinant plasmid is Lipofectamine TM 3000 Reagent. The specific transfection method is as follows:

[0046] (1) After digestion of the monolayer of LT cells (primary sheep testicular cells), the cells were counted and plated in a 6-well plate at a density of 600,000 cells per well. The cells were transfected after 6 h of growth.

[0047] (2) Using Opti-MEM TM 125 μL of culture medium to dilute Lipofectamine TM7 μL of 3000 reagent was mixed thoroughly and allowed to stand at room temperature for 5 min;

[0048] (3) Using Opti-MEM TM Prepare a DNA premix by diluting 2.5 μg of plasmid DNA in 125 μL of culture medium, and then add 5 μL of P3000 TM Reagents, mix thoroughly and let stand for 5 minutes;

[0049] (4) In each tube of diluted Lipofectamine TM Add diluted DNA to the 3000 reagent (1:1 ratio), mix thoroughly, and incubate for 15 minutes;

[0050] (5) Add the DNA-lipid complex to a six-well plate and incubate at 37°C for 2-4 days before proceeding to the next step.

[0051] LT cells were transfected with pCMV-HA-LGALS1, and total cell protein was collected. The primary antibody was mouse HA monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-mouse IgG. WB was used to verify the expression of pCMV-HA-LGALS1. The results are as follows: Figure 2 As shown, LGALS1 protein can be expressed at a high level.

[0052] 4. Effect of pCMV-HA-LGALS1 transfection on LT cell activity

[0053] Perform cell viability analysis on cells transfected with the plasmid according to the instructions of the Cell Counting Kit-8 (CCK-8) cell proliferation kit. The specific steps are as follows:

[0054] (1) The cells not transfected with the plasmid and the cells transfected with pCMV-HA-LGALS1 (2.5 μg) and pCMV-HA (2.5 μg) in the 6-well plate were digested and counted, and the cells were seeded into 96-well plates at a density of 10,000 cells per well. Three replicate wells were set for each cell type, and a blank control was set.

[0055] (2) After the cells have grown overnight and become confluent, add 10 μL of CCK-8 reagent to each well and incubate the culture plate in an incubator for 4 h in the dark.

[0056] (3) Measure the absorbance of the cells at 450 nm using a microplate reader and calculate the cell viability according to the formula: Cell viability = [(As - Ab) / (Ac - Ab)] × 100%, where As is the test well (well containing pCMV-HA-LGALS1 and pCMV-HA cells); Ac is the control well (well containing cells not transfected with the plasmid); Ab is the blank well (culture medium).

[0057] The results are as follows Figure 3 As shown, there was no significant difference between the pCMV-HA-LGALS1 transfection group and the pCMV-HA transfection group and the blank control group (P>0.05), indicating that plasmid transfection did not affect cell activity.

[0058] 5. RT-qPCR detection of OrfV B2L gene mRNA transcription levels in LGALS1-overexpressing cells

[0059] LT cells were transfected with pCMV-HA-LGALS1 and pCMV-HA, respectively. Viruses were inoculated 48 h later. Total cellular RNA was collected 8, 16, and 24 h after inoculation, and the mRNA level of the OrfV B2L gene was detected by RT-qPCR.

[0060] (6) Western blot detection of OrfV B2L protein expression levels in LGALS1-overexpressing cells

[0061] LT cells were transfected with pCMV-HA-LGALS1 and pCMV-HA, respectively. Viruses were inoculated 48 hours later. Total cell proteins were collected 8, 16, and 24 hours after inoculation, and the protein expression level of the OrfV B2L gene was detected by WB.

[0062] (7) TCID of OrfV in LGALS1-overexpressing cells 50 Determination

[0063] pCMV-HA-LGALS1 and pCMV-HA were transfected into LT cells respectively. Viruses were inoculated 48 hours later. Cell culture supernatants were collected at 8, 16, and 24 hours after inoculation to determine the TCID of the virus. 50 value.

[0064] The results are as follows Figure 4 、 5 As shown in Figures 6 and 7, compared with the pCMV-HA transfection group, the OrfV B2L mRNA level in the pCMV-HA-LGALS1 transfection group was significantly decreased at 8h, 16h, and 24h, with extremely significant differences at 8h and 24h (P<0.01) and a significant difference at 16h (P<0.05). The OrfV B2L protein expression level was significantly decreased, with extremely significant differences at 16h and 24h (P<0.01). TCID 50 The values showed that the viral titer of the pCMV-HA-LGALS1 group was significantly lower than that of the pCMV-HA group, indicating that overexpression of the LGALS1 gene can significantly inhibit the proliferation of OrfV.

[0065] Example 2 Effect of Interference with LGALS1 on OrfV Proliferation

[0066] 1. siRNA sequence design

[0067] The LGALS1 gene sequence (GenBank accession number: XM_018048739.1) was submitted to Shanghai Genetech, which designed and synthesized four siRNA interference sequences and one negative control NC. The interference sequence information is shown in Table 2.

[0068] Table 2 Interference sequences

[0069]

[0070] 2. Screening and verification of siRNA sequences

[0071] The four interference sequences and the NC sequence were transfected into LT cells respectively. After culturing for 24 hours, the total RNA of the cells was extracted for RT-qPCR detection. After culturing for 48 hours, WB detection was performed to screen sequences with better interference effects.

[0072] The results are as follows Figure 8 As shown, the four interference sequences can silence the expression of LGALS1 gene in LT cells, and si-1 was selected for subsequent experiments.

[0073] 3. siRNA transfection into LT cells

[0074] siRNA was transfected using RNATransMate transfection reagent from Sangon Biotech (Shanghai) Co., Ltd. The specific steps are as follows:

[0075] (1) First, add 1OD of siRNA dry powder to 125μL of DEPC water to prepare a 20μM stock solution. Add 5μL to each well of a 6-well plate to make the final siRNA concentration in each well 50nM (the amount recommended in the instructions);

[0076] (2) After digesting the LT cells that have grown into a monolayer, count the cells and plate them in a 6-well plate at a rate of 600,000 cells per well. Transfection can be performed after the cells have grown for 6 hours.

[0077] (3) Dilute 4 μL of RNATransMate reagent with 100 μL of serum-free DMEM medium and mix thoroughly;

[0078] (4) Dilute siRNA in 100 μL of serum-free DMEM medium and mix thoroughly;

[0079] (5) Mix the two tubes and let them stand at room temperature for 10 min to form the RNA / RNATransMate complex mixture;

[0080] (6) Add the RNA / RNATransMate complex mixture dropwise to a six-well plate and proceed to the next step after 48 hours.

[0081] 4. Effect of siRNA transfection on LT cell activity

[0082] The cell viability of cells transfected with siRNA was analyzed according to the instructions of the Cell Counting Kit-8 (CCK-8) cell proliferation kit.

[0083] The results are as follows Figure 9 As shown in the figure, there was no significant difference between the siRNA (50 nM) transfection group, the NC (50 nM) group and the blank control group (MOCK) ((P>0.05), indicating that siRNA transfection did not affect cell activity.

[0084] 5. RT-qPCR detection of OrfV B2L gene transcription level, protein expression level and TCID in LT cells transfected with siRNA 50 Determination

[0085] LT cells were transfected with siRNA and NC, respectively, and the virus was inoculated 48 hours later. Total cellular RNA was collected 8 hours, 16 hours, and 24 hours after inoculation, and the transcription level of viral mRNA was detected by RT-qPCR.

[0086] LT cells were transfected with siRNA and NC, respectively, and the virus was inoculated 48 hours later. Total cell protein was collected 8 hours, 16 hours, and 24 hours after virus inoculation, and the expression level of OrfV B2L protein was detected by WB.

[0087] LT cells were transfected with siRNA and NC, and the virus was inoculated 48 hours later. The cell culture supernatant was collected 8, 16, and 24 hours after virus inoculation, and the TCID of the virus was determined. 50 value.

[0088] The results are as follows Figure 10 、 11 As shown in Figures 12 and 13, compared with those transfected with NC, the OrfV B2L mRNA level in the si-1 transfection group was significantly increased at 8h, 16h, and 24h, with extremely significant differences at 8h and 16h (P<0.01) and extremely significant differences at 24h (P<0.001). The OrfVB2L protein expression level was significantly increased, with extremely significant differences at 16h and 24h (P<0.01). TCID 50 The values showed that the virus titer in the si-1 group was higher than that in the NC group, indicating that interfering with the LGALS1 gene could significantly promote the proliferation of OrfV.

[0089] Example 3 Effect of the small molecule inhibitor OTX008 on the inhibition of LGALS1 function on OrfV proliferation

[0090] 1. Safe concentration screening of small molecule inhibitor OTX008

[0091] Cells were treated with the LGALS1-specific small molecule inhibitor OTX008, and the CCK-8 kit was used to screen the safe concentration of the inhibitor. The specific steps are as follows:

[0092] (1) After digestion of the confluent monolayer of LT cells, plate them. After counting the cells, add 10,000 cells per well to a 96-well plate with 100 μL of culture medium per well and culture overnight.

[0093] (2) When the cells were confluent monolayer, they were washed twice with PBS and different concentrations of OTX008 (100, 80, 40, 20, 10, 5, 2.5, and 1.25 μM) were added, with three replicates per well. A negative control (DMSO) and a blank control were also set up.

[0094] (3) After 24 h, add 10 μL of CCK-8 reagent to each well in the dark and incubate at 37°C for 4 h;

[0095] (4) Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability according to the formula: Cell viability = [(As - Ab) / (Ac - Ab)] × 100%, where As: test wells (wells containing different concentrations of OTX008 cells); Ac: control wells (wells containing cells but not OTX008 cells); Ab: blank wells (culture medium).

[0096] The results are as follows Figure 14 As shown in the results, the inhibitor concentration did not affect cell activity when it was 1.25μM to 10μM, but had a very significant effect on cell activity when it exceeded 20μM (P<0.001). The inhibitor concentration between 1.25μM and 10μM was a safe concentration, and 10μM was selected as the inhibitor concentration.

[0097] 2. RT-qPCR detection of OrfV B2L gene transcription levels in OTX008-treated LT cells

[0098] LT cells were plated in 6-well plates in advance. After the cells grew to 80%-90%, they were washed twice with PBS. OTX008 was added to a final concentration of 10 μM in each well. The virus was inoculated after 12 hours of incubation. After virus inoculation, 10 μM OTX008 and DMSO were added to 2% FBS maintenance solution and continued to incubate. Total cell RNA was collected 8 hours, 16 hours, and 24 hours after virus inoculation, and the transcription levels of viral mRNA in the experimental and control groups were detected by RT-qPCR.

[0099] The results are as follows Figure 15 As shown, compared with the DMSO control group, the OrfV B2L mRNA level in the OTX008 group was significantly increased at 8h, 16h, and 24h, with extremely significant differences at 8h and 16h (P<0.01), and extremely significant differences at 24h (P<0.001).

[0100] 3. Western blot detection of OrfV B2L protein expression in OTX008-treated LT cells

[0101] LT cells were plated in 6-well plates in advance. After the cells grew to 80%-90%, they were washed twice with PBS. OTX008 was added to a final concentration of 10 μM in each well. After incubation for 12 h, the virus was inoculated. 10 μM OTX008 and DMSO were added to the maintenance medium containing 2% FBS and incubation was continued. Total cell protein was collected 8 h, 16 h, and 24 h after virus inoculation, and the expression levels of OrfV B2L protein in the experimental and control groups were detected by WB.

[0102] The results are as follows Figure 16 and 17 As shown in the figure, compared with the DMSO control group, the expression level of OrfV B2L protein in the OTX008 group was significantly increased at 8h, 16h, and 24h, with a significant difference at 16h (P<0.05) and extremely significant at 24h (P<0.01).

[0103] 4. TCID of OrfV in LT cells treated with OTX008 50 Determination

[0104] LT cells were plated in 6-well plates in advance. After the cells grew to 80%-90%, they were washed twice with PBS. OTX008 and DMSO were added to each well at a final concentration of 10 μM. After incubation for 12 hours, the virus was inoculated. 10 μM OTX008 and DMSO were added to the maintenance solution containing 2% FBS and continued to incubate. The cell culture supernatant was collected 8 hours, 16 hours, and 24 hours after virus inoculation, and the TCID of the virus was determined. 50 value.

[0105] The results are as follows Figure 18 As shown in Figure 3, the viral titer in the OTX008 group was higher than that in the DMSO control group, indicating that blocking LGALS1 function can significantly promote the proliferation of OrfV.

Claims

1. Use of the LGALS1 gene, or a biological material containing the LGALS1 gene, or the LGALS1 protein in the preparation of a product against oropharyngeal canker sore virus, wherein the LGALS1 gene is a gene that can encode the LGALS1 protein shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The biological material containing the LGALS1 gene is an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the LGALS1 gene.

3. The use according to claim 1 or 2, characterized in that The nucleotide sequence of the LGALS1 gene is shown in SEQ ID No.

2.

4. The use according to claim 1 or 2, characterized in that The anti-stomatitis virus product refers to a drug that inhibits the proliferation of thestomatitis virus or a drug that prevents or treats diseases caused by thestomatitis virus infection.

Images