Use of a BmDPAGT1 gene inhibitor in preparation of a drug for resisting BmNPV

Abstract

The application discloses application of a BmDPAGT1 gene inhibitor in preparation of an anti-BmNPV drug, and proves that BmDpagt1 gene is necessary for BmNPV proliferation by knocking out and overexpressing BmDpagt1 gene in BmN cells; meanwhile, budding and horizontal diffusion of BVs can be inhibited by constructing a cell line with BmDpagt1 gene knocked out or by inhibiting expression of BmDpagt1 by means of tunicamycin; these results show that BmDpagt1 gene and its inhibitor have an important role in proliferation of BmNPV, and can affect mutual recognition of virus invasion into a host, and the application provides a new target for BmNPV prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of BmDPAGT1 gene inhibitors in the preparation of anti-BmNPV drugs. Background Technology

[0002] N-glycosylation of eukaryotic proteins is initiated by dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1). The initial metabolite required for N-glycosylation is N-acetylglucosamine pyrophosphate polyphenol (GlcNAc-PP-dolichol), which is synthesized by DPAGT1. Mutations in DPAGT1 lead to a rare congenital glycosylation disorder known as DPAGT1-congenital glycosylation disorder. Protein N-glycosylation plays a crucial role in development and homeostasis by affecting a variety of cellular functions. N-glycosylation is important in protein folding, targeting, secretion, protein-protein interactions, and cell signal transduction. Dysregulation of N-glycosylation is associated with various diseases.

[0003] In recent years, the functions of glycosylation modification in viruses have been continuously explored and widely applied in virus control, disease prevention, and treatment. Some viral proteins can exert important biological functions through glycosylation modification, mediating recognition, binding, and invasion of host cells during infection, or helping viruses evade clearance by the host's immune system.

[0004] Baculoviruses are a class of double-stranded DNA (dsDNA) viruses with genome sizes ranging from 80 to 180 kbp, encoding 90 to 180 viral proteins. Baculoviruses infect only invertebrates and are widely used in insect control and eukaryotic expression systems, and also have potential applications in gene therapy. Baculoviruses undergo glycosylation modification during infection. Specifically, the envelope proteins GP64, EGT, GP41, GP37, F protein, v-CATH, and chitinase in *AcMnopterus mongolicus* nucleopolyhedrovirus (AcMNPV), and GP64, FGF, EGT, GP41, and v-CATH in *BmNPV*, all exhibit glycosylation modification. Glycosylation modification of baculovirus-encoded proteins can affect their structure, physicochemical properties, intracellular transport, and function.

[0005] Glycoproteins participate in protein structure formation and play a crucial role in membrane interactions, such as membrane fusion, nucleocapsid budding, and OB release. Studies have shown that compared to susceptible BmNPV strains, resistant BmNPV strains exhibit significantly increased levels of glycosylation-related enzymes, which may exert their effects by altering the N-linked glycans of viral GP64, thereby regulating viral replication. Furthermore, N-glycosylation of AcMNPV GP64 is critical for its intracellular transport, pathogenicity, and BV infectivity. Therefore, investigating the role and regulatory mechanism of the key glycosylation modification gene Dpagt1 in viral invasion is of great significance for the prevention and treatment of BmNPV. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide the application of a BmDPAGT1 gene inhibitor in the preparation of anti-BmNPV drugs.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. Application of BmDPAGT1 gene inhibitors in the preparation of anti-BmNPV drugs.

[0009] Preferably, the BmDPAGT1 gene inhibitor of the present invention is a BmDpagt1 gene knockout vector constructed based on the CRISPR / Cas9 system.

[0010] Preferably, the nucleotide sequence of the BmDpagt1 gene is shown in SEQ ID No. 1.

[0011] Preferably, the BmDpagt1 knockout vector constructed based on the CRISPR / Cas9 system comprises Cas9 expressed by the IE1 promoter and sgRNA expressed by the U6 promoter.

[0012] Preferably, the guide sequence of the sgRNA is shown in SEQ ID No. 2 and SEQ ID No. 3.

[0013] Preferably, the BmDPAGT1 gene inhibitor of the present invention is tunicamycin.

[0014] Preferably, the concentration of tunicamycin is 10 μg / mL.

[0015] The beneficial effects of this invention are as follows: This invention provides the application of the glycosyltransferase gene BmDpagt1 and its inhibitor in the preparation of anti-BmNPV drugs. Knockout and overexpression of the BmDpagt1 gene in BmN cells demonstrate that the BmDpagt1 gene is essential for BmNPV proliferation. Simultaneously, by constructing a BmDpagt1 gene knockout cell line or inhibiting BmDpagt1 expression with tunicamycin, the glycosylation level of BmNPV GP64 is reduced, thereby inhibiting BV budding and horizontal diffusion. This invention lays the foundation for understanding the interaction mechanism between the virus and the host, and provides target genes for elucidating the antiviral mechanism of silkworms and for virus prevention, control, and treatment. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0017] Figure 1 Construction of BmDpagt1 knockout cell lines (A: Schematic diagram of BmDpagt1 gene knockout vector construction; B: Fluorescence microscopy observation of BmDpagt1 gene knockout cell line construction; C: Transcriptional analysis of BmDpagt1 expression.)

[0018] Figure 2 To investigate the effect of BmDpagt1 gene knockout on BmNPV proliferation (A: Effect of BmDpagt1 knockout cell lines on the transcriptional level of viral replication-related genes; B: Flow cytometry analysis and statistical analysis of the efficiency of BmDpagt1 in inhibiting viral proliferation; C: Western Blot analysis of the effect of BmDpagt1 on BmNPV VP39 protein expression).

[0019] Figure 3 Effects of BmDpagt1 overexpression on BmNPV proliferation and replication (A: Schematic diagram of BmDpagt1 gene overexpression vector construction; qRT-PCR detection of BmDpagt1 gene overexpression; B: Effect of BmDpagt1 overexpression on viral genome copy number; C: Effect of BmDpagt1 overexpression on transcriptional levels of BmNPV proliferation and replication-related genes; D: Effect of BmDpagt1 overexpression on BmNPV VP39 protein expression level.)

[0020] Figure 4 To investigate the effect of DPAGT1 inhibition on BmNPV replication (A: Effect of DPAGT1 inhibition on viral genome copy number; B: Viral fluorescence analysis after DPAGT1 inhibition; C: Flow cytometry analysis of the effect of DPAGT1 inhibition on BmNPV replication);

[0021] Figure 5 To regulate the horizontal transmission ability of BV using DPAGT1 (A: Fluorescence observation of the green fluorescence intensity of BV after different treatment groups; B: TCID50 detection of BV virulence, the right figure shows the statistical analysis of fluorescence results). Detailed Implementation

[0022] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Test methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the reagent manufacturer. It should be particularly noted that the embodiments described in this specification are merely for the purpose of aiding understanding the invention and are not intended to limit it in any way; that is, the present invention may have other embodiments besides those described. Therefore, any technical solution formed by equivalent substitution or equivalent transformation is within the scope of protection of this invention.

[0023] Example 1: BmDpagt1 gene regulates BmNPV proliferation and replication

[0024] To determine the role of the BmDpagt1 gene (BMSK0007119, SEQ ID No. 1) in the replication and proliferation of BmNPV and to identify its regulatory function on viral replication, this study constructed the BmDpagt1 knockout vector Puro-mCherry-Cas9-U6 using the sequences shown in SEQ ID No. 2 and SEQ ID No. 3 as guide sequences based on the CRISPR / Cas9 system. prm -sgDpagt1-TTTTTT,Puro-mCherry-Cas9-U6 prm The construction process of the -sgDpagt1-TTTTTT vector is as follows:

[0025] 1) The pSL1180-IE1-Cas9-SV40 vector (patent number: ZL201510630939.0) was digested with BglII enzyme, and sgDpagt1 with BglII restriction site was annealed and ligated into the vector to obtain pSL1180-IE1-Cas9-U6-sgDpagt1.

[0026] 2) The pSL1180-IE1-Cas9-U6-sgDpagt1 fragment was digested with Asc I to obtain the IE1-Cas9-U6-sgDpagt1 fragment, which was then ligated into the Asc I-digested puro-OpIE2prm-mCherry-PA vector (patent number: ZL202110682610.4) to obtain Puro-mCherry-Cas9-U6prm-sgDpagt1-TTTTTT. Figure 1 A).

[0027] After transfection, puromycin was used as a selection marker to screen BmN cells transfected with the knockout vector using antibiotics until all cells could elicit red fluorescence under a fluorescence microscope. Figure 1 As shown in B; then, RT-PCR analysis was performed using primers shown in SEQ ID No. 4 and 5 to analyze the expression of the BmDpagt1 gene in the knockout cell line. The results showed that the transcription of the BmDpagt1 gene was significantly inhibited, thus successfully obtaining the BmDpagt1 knockout cell line, as shown in B. Figure 1 As shown in C.

[0028] BmDpagt1-CDS:ATGCTACTTCTTGGATTTGCTGATGATGTGTTAGATCTTAGATGGA (SEQ ID No.1)

[0029] sgDPAGT1 / F:AAGTGGCATCATTGCCATTACTTG(SEQ ID No.2)

[0030] sgDPAGT1 / R:AAACCAAGTAATGGCAATGATGCC(SEQ ID No.3)

[0031] RT-BmDpagt1 / F:GCGGCGGTATGTACAGGAAG(SEQ ID No.4)

[0032] RT-BmDpagt1 / R:TGCCAGCAAACGGACAGAG(SEQ ID No.5)

[0033] To identify the effect of the BmDPAGT1 gene on BmNPV proliferation, this study analyzed the proliferation characteristics of BmNPV in BmDpagt1 knockout cells using RT-PCR, flow cytometry, and Western blotting. The results showed that the transcriptional levels of BmNPV replication-related immediate early gene ie1, early gene gp64, late gene vp39, and very late gene poly were significantly downregulated in BmDpagt1 knockout cells. Figure 2 As shown in Figure A (SEQ ID No. 6 to No. 15). Flow cytometry results showed that the fluorescence count in BmDpagt1 knockout cells decreased after BmNPV infection, as shown in Figure A. Figure 2 As shown in Figure B. Western blot analysis showed that the viral nucleocapsid protein VP39 was also downregulated, as... Figure 2 As shown in Figure C, the above results indicate that BmDpagt1 knockout significantly inhibits the proliferation of BmNPV.

[0034] RT-vp39-F:TGTCAAAAATGTTATTCAGCC(SEQ ID No.6)

[0035] RT-vp39-R:TTTCCGTAAAGAGTCAGTTCC(SEQ ID No.7)

[0036] RT-gp64-F:ATGCTAAGAACGCCAACAGAAG(SEQ ID No.8)

[0037] RT-gp64-R:TTGTAACAAATCCATGCCCAC(SEQ ID No.9)

[0038] RT-ie1-F:AAGAAGGAGGACGGCAGCAT(SEQ ID No.10)

[0039] RT-ie1-R:ATCTCGCCAGAAATCCAATAAAAC(SEQ ID No.11)

[0040] RT-poly-F:TTAATCGTCAACTGGAGCGG(SEQ ID No.12)

[0041] RT-poly-R:TGAGCGAGGAACTTGTAGCAC(SEQ ID No.13)

[0042] RT-SW22934-F:TTCGTACTGGCTCTTCTCGT(SEQ ID No.14)

[0043] RT-SW22934-R:CAAAGTTGATAGCAATTCCCT (Internal reference gene, SEQ ID No. 15)

[0044] Example 2: Overexpression of the BmDpagt1 gene promotes the proliferation and replication of BmNPV.

[0045] To determine the effect of BmDPAGT1 on BmNPV proliferation, we constructed a BmDpagt1 overexpression vector (plZ-OpIE2). prm -EGFP-Dpagtl-OpIE2 Pa-Zeocin, Figure 3 A) Construction of the BmDpagt1 overexpression vector: The commercial vector pIZ-V5 / His (Thermo Fisher Scientific Inc.) was digested with KpnI and BamHI, and the EGFP fragment was ligated to obtain pIZ-OpIE2prm-EGFP-OpIE2 Pa-Zeocin (constructed and preserved in our laboratory). Then, pIZ-OpIE2prm-EGFP-OpIE2 Pa-Zeocin was digested with EcoRI and XbaI, and ligated with the target fragment of Dpagt1 (containing the Flag tag, EcoRI and XbaI restriction sites) to obtain plZ-OpIE2prm-EGFP-Dpagtl-OpIE2Pa-Zeocin.

[0046] As shown in the figure, after overexpression of BmDpagt1, RT-PCR analysis using primers shown in SEQ ID No. 16 and 17 showed a highly significant increase in BmDpagt1 gene expression. In this study, RT-PCR using primers shown in SEQ ID No. 18 and No. 19 was used to detect the proliferation characteristics of BmNPV in BmDpagt1-overexpressing cells, showing a significant increase in viral genome copy number in BmDpagt1-overexpressing cells. Figure 3 As shown in Figure B, the transcriptional levels of both the early gene gp64 and the late gene vp39, which are related to BmNPV proliferation and replication, were significantly increased. Figure 3 As shown in C (SEQ ID No. 6 to No. 9). Western blot analysis also showed upregulated expression of the viral nucleocapsid protein VP39, as... Figure 3 As shown in Figure D, the above results indicate that overexpression of BmDpagt1 significantly promotes the proliferation of BmNPV.

[0047] BmDpagt1-F: cggaattcATGGATTACAAGGATGACGACGATAAGCTACTTCTTGGATTTGC (SEQ ID No. 16)

[0048] BmDpagt1-R:

[0049] tttatagcggccgcTTATTTATCATAAAAATAAGTTGCTA(SEQ ID No.17)

[0050] RT-gp41-F:ATGTTGATGTGCGGAAAGC(SEQ ID No.18)

[0051] RT-gp41-R:GTGGCGGAATCGGTGA(SEQ ID No.19)

[0052] Example 3: DPAGT1 inhibitor - tunicamycin inhibits the proliferation and replication of BmNPV.

[0053] To verify whether tunicamycin, a DPAGT1-specific inhibitor, affects the proliferation and replication of BmNPV, tunicamycin was first prepared to a concentration of 10 μg / mL using DMSO and used to pretreat BmN cells for 48 h. After BmNPV infection of cells at different time points, the proliferation characteristics of BmNPV in tunicamycin-pretreated BmN cells were analyzed by RT-PCR, fluorescence observation, and flow cytometry. The results showed that inhibition of DPAGT1 significantly reduced the viral genome copy number, such as... Figure 4As shown in Figure A (SEQ ID No. 17 and No. 18). Compared to the control group, the proportion of fluorescent cells in the experimental group was significantly reduced. Figure 4 As shown in Figure B. Simultaneously, flow cytometry results showed that inhibition of DPAGT1 also reduced the fluorescence count after BmNPV infection, as shown in Figure B. Figure 4 As shown in C.

[0054] Example 4: Chlamydiaein affects horizontal transmission of BV

[0055] To confirm whether DPAGT1 inhibition affects the virulence of progeny viruses, this study infected BmNPV with tunicamycin-treated, knockout, and control cells for 120 hours, collecting cell supernatants to obtain progeny viruses. These were then used to re-infect normal BmN cells, and fluorescence imaging was performed at different time points. The results showed that green fluorescence was observed in both the negative control and experimental groups, but the intensity of green fluorescence was significantly weakened in the tunicamycin-treated and knockout groups. In the negative control group, the green fluorescence gradually increased over time, such as... Figure 5 As shown in Figure A. TCID50 results showed that viral particles were detected in the tunicamycin-treated, knockout, and control groups, but the number of viral particles in the tunicamycin-treated and knockout groups was significantly lower than that in the control group. Figure 5 As shown in Figure B. In summary, knocking out BmDpagt1 significantly weakens the horizontal transmission ability of BV, indicating that BmDPAGT1 can regulate the proliferation of BmNPV by affecting secondary infection of BV.

[0056] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Application of BmDPAGT1 gene inhibitor in the preparation of anti-BmNPV drugs, wherein the BmDPAGT1 gene inhibitor is a BmDpagt1 gene knockout vector constructed based on the CRISPR / Cas9 system, the nucleotide sequence of the BmDpagt1 gene is shown in SEQ ID No. 1, and the BmDpagt1 gene knockout vector contains Cas9 expressed by the IE1 promoter and sgRNA expressed by the U6 promoter, the guide sequence of the sgRNA is shown in SEQ ID No. 2 and SEQ ID No. 3.

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