A recombinant siRNA against type I herpes simplex virus and its application

Abstract

This invention discloses recombinant siRNAs against type I herpes simplex virus. The recombinant siRNA sequences are one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10, or sequences with a similarity of more than 90% to one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10. Furthermore, this invention also discloses the applications of these recombinant siRNAs. The recombinant siRNAs of this invention exhibit excellent biological activity and can significantly reduce the titer of type I herpes simplex virus in vitro, significantly reduce the viral load on the skin surface in vivo, and improve animal survival rates. They also have advantages such as high yield, low cost, and good functionality.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant siRNA against type I herpes simplex virus and its application. Background Technology

[0002] Herpes simplex virus type 1 (HSV1) is a highly contagious human alpha herpesvirus. HSV1 infection primarily causes oral herpes, and in a small number of immunocompromised patients, it can lead to complications such as keratitis and encephalitis. The general population is susceptible (the seropositivity rate in adults is over 85%). The virus is mainly transmitted through oral-oral contact, and the vast majority of HSV1 infections occur in childhood and persist throughout life. Currently, HSV1 prevention and control remain highly challenging; there are no specific treatments or vaccines available.

[0003] RNAi (RNA interference) refers to gene repression mediated by RNA molecules. RNAi has wide applications in gene function research and disease treatment. Currently, several RNAi-based drugs have been approved by the FDA or have entered clinical trials. The increasing number of approved RNA drugs each year fully demonstrates the feasibility of RNA therapy and also indicates that RNA therapy is rapidly developing as a new generation of treatment options.

[0004] The development of RNA-based drugs and the study of RNA function have been hampered by the availability of RNA raw materials. Currently, RNA reagents used in ncRNA research are mainly synthesized chemically or through in vitro transcription. RNA produced by these methods is not only expensive but also has very low yields. Therefore, the development of RNA therapy requires the intervention of emerging biotechnologies to significantly reduce research and medical costs. The use of recombinant tRNA scaffolds to produce and express small RNA molecules in living cells has been applied in several research fields and has achieved good results. Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing recombinant siRNAs against type I herpes simplex virus (HSV-1). These recombinant siRNAs possess excellent biological activity and can significantly reduce HSV-1 viral titers in vitro and in vivo. In animal experiments, they can significantly reduce viral load on the skin surface, prolong animal survival, and have advantages such as high yield, low cost, and good functionality. Compared with traditional small molecule drugs, siRNA drugs can directly act on the viral genome, interfering with viral mRNA expression by utilizing the base complementarity principle, significantly inhibiting viral replication, and reducing viral load.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a recombinant siRNA against type I herpes simplex virus, characterized in that the sequence of the recombinant siRNA is one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10, or a sequence with a similarity of more than 90% to one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10.

[0007] The above-mentioned recombinant siRNAs against type I herpes simplex virus are characterized in that, during the production process of the recombinant siRNAs, small interfering RNAs are designed with different locations in the HSV1 genome as targets, and the sequences of the small interfering RNAs are shown in SEQ ID NO: 11 to SEQ ID NO: 20.

[0008] Furthermore, the present invention provides the application of the above-mentioned recombinant siRNAs in the preparation of genomic reagents, prodrugs, drugs, active pharmaceutical ingredients, or drug combinations that reduce the titer of type I herpes simplex virus in vitro / in vivo and prolong animal survival.

[0009] Furthermore, the present invention provides the use of the above-mentioned recombinant siRNAs in the preparation of reagents, prodrugs, drugs, active pharmaceutical ingredients, or drug combinations having anti-herpes simplex virus type I activity.

[0010] The method for producing recombinant siRNAs against type I herpes simplex virus of the present invention includes the following steps:

[0011] Step 1: Design and synthesize hsa-miR-34a precursor primers that contain chimeric siRNA sequences;

[0012] Step 2: Using the restriction enzyme sites at the anticodon loop of tRNA in the pBSKrnaSeph plasmid, insert the precursor sequence of recombinant siRNAs into the pBSKrnaSeph plasmid to construct the expression vector;

[0013] Step 3: Transform the expression vector containing the chimeric target sequence into competent E. coli;

[0014] Step 4: After E. coli culture and amplification, total RNA is extracted from the bacteria and the target recombinant siRNAs are isolated and purified by FPLC.

[0015] This invention uses human keratinocytes (HaCaT), human retinal pigment epithelial cells (ARPE19), and human neuroblastoma cells (SY5Y) as cell models to detect the anti-HSV1 activity of recombinant siRNAs, including:

[0016] I. The recombinant siRNAs expressed were transfected into HaCaT, ARPE19, and SY5Y, and the expression level of the mature recombinant siRNAs was detected by Stem loop qPCR.

[0017] 2. Recombinant siRNAs were transfected into HaCaT, ARPE19, and SY5Y and infected with HSV1 virus. The inhibitory effect of recombinant siRNAs on target genes was detected by qPCR.

[0018] III. Recombinant siRNAs were transfected into HaCaT, ARPE19, and SY5Y and infected with HSV1 virus. The antiviral effect of recombinant siRNAs was detected by plaque assay.

[0019] IV. In an animal model of herpetic dermatitis, HSV1 was dropped onto the skin of mice and recombinant siRNAs encapsulated in flexible liposomes were applied to the skin of mice for 5 consecutive days to evaluate the effects of recombinant siRNAs on viral load and protection rate in mice.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The biologically active siRNAs expressed using novel tRNA scaffolds in this invention have higher yields, lower prices, and higher activity and safety compared to chemically synthesized siRNAs.

[0022] 2. The recombinant siRNAs of this invention possess excellent biological activity and can significantly reduce HSV1 viral titers in vitro and in vivo. In animal experiments, they can significantly reduce viral load on the skin surface and prolong animal survival rates. They also offer advantages such as high yield, low cost, and good functionality. Compared to traditional small molecule drugs, siRNA drugs can directly act on the viral genome, interfering with viral mRNA expression by utilizing the base complementarity principle, significantly inhibiting viral replication, and reducing viral load.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 Example 1 of the present invention utilizes denaturing polyacrylamide gel electrophoresis to detect the expression of recombinant siRNA in Escherichia coli;

[0025] Figure 2 This is Example 2 of the present invention utilizing Bio-Rad NGC. TM The recombinant siRNA was purified using a Chromatography System, and the purity of the collected fractions was identified by denaturing polyacrylamide gel electrophoresis.

[0026] Figure 3 This is a graph showing the results of detecting the expression levels of HaCaT, ARPE19, and SY5Y in mature organisms using Stem loop qPCR technology in Example 3 of the present invention.

[0027] Figure 4 This is a diagram illustrating the inhibition of target genes by qPCR technology at the mRNA level in Example 4 of the present invention, after 24 hours of transfection with recombinant siRNA by HaCaT, ARPE19, and SY5Y and 48 hours of HSV1 virus infection.

[0028] Figure 5 This is a graph showing the effect of plaque assay on viral titer of HaCaT, ARPE19, and SY5Y transfected recombinant siRNA for 24 hours and infected with HSV1 virus for 48 hours, as described in Example 5 of the present invention.

[0029] Figure 6 This is a graph showing the results of Example 6 of the present invention, which uses immunofluorescence assay to detect the number of diseased cells in ARPE19 infected with HSV1 48 h after transfection with recombinant siRNA, in order to evaluate the antiviral effect of recombinant siRNA.

[0030] Figure 7 This is a graph showing the results of qPCR detection of viral load on the skin surface of HSV1-infected mice after treatment with recombinant SSA (a negative control of recombinant r / si-UL8-1), r / si-UL8-1, and acyclovir (ACV) in Example 7 of the present invention.

[0031] Figure 8 This is a graph showing the survival rate of mice after HSV1 infection and treatment with recombinant SSA and r / si-UL8-1 in Example 7 of the present invention. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Unless otherwise stated, the experimental methods disclosed in the present invention all adopt conventional techniques in this technical field.

[0033] The recombinant siRNAs of the present invention have a sequence of one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10, or a sequence that has a similarity of more than 90% to one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 10.

[0034] The sequences of the small interfering RNA of the present invention are shown in SEQ ID NO: 11 to SEQ ID NO: 20.

[0035] The method for producing recombinant siRNAs of the present invention includes:

[0036] Step 1: Ten pairs of siRNAs were designed for the HSV1 genome, and hsa-miR-34a precursor primers with chimeric siRNA sequences were synthesized.

[0037] Step 2: Using the restriction enzyme site at the anticodon loop of the tRNA in the pBSKrnaSeph plasmid, the precursor sequence of recombinant siRNAs is inserted into the pBSKrnaSeph plasmid to construct an expression vector; the tRNA scaffold sequence is designed based on the human serine tRNA sequence, and its tRNA portion is a sequence with more than 90% similarity to the human serine tRNA sequence, as shown in SEQ ID NO: 21; the precursor sequence of recombinant siRNAs is the hsa-miR-34a precursor sequence in which the mature sequence has been partially replaced, as shown in SEQ ID NO: 22;

[0038] Step 3: Transform the expression vector containing the chimeric target sequence into competent E. coli;

[0039] Step 4: After E. coli culture and amplification, total RNA is extracted from the bacteria and the target recombinant siRNAs are isolated and purified by FPLC.

[0040] The following section uses recombinant r / si-UL8-1 (SEQ ID NO: 1) as an example to specifically describe the production method of the recombinant siRNAs of this invention:

[0041] Example 1: A recombinant siRNA expression plasmid was constructed using the pBSKrnaSeph / has-mir-34a expression vector to express recombinant siRNAs.

[0042] (1) Based on the effective sequence of recombinant r / si-UL8-1 and the sequence on the pBSKrnaSeph / has-mir-34a expression vector, primers were designed and named mir-34a / recombinant r / si-UL8-1. At the same time, homologous sequences of 1-15 nt on both sides of the vector insertion site were added to both ends of the primers.

[0043] (2) Synthesis of inserted fragments

[0044] Using the two primers in Table 1 as templates, and leveraging the restriction enzyme site at the anticodon loop of the pBSKrnaSeph plasmid, the precursor sequence of recombinant r / si-UL8-1 was inserted into the pBSKrnaSeph plasmid to construct the expression vector. The reaction system is shown in Table 1, and the reaction process is shown in Table 2.

[0045] Table 1. Polymerase in vitro amplification chain reaction system (50 μL)

[0046]

[0047]

[0048] Table 2. Polymerase in vitro amplification chain reaction process

[0049]

[0050] (3) Double digestion of pBSKrnaSeph / has-mir-34a vector

[0051] Using Eagle I-HF TM The vector was digested with Sac II restriction endonuclease at 37°C. The reaction system is shown in Table 3.

[0052] Table 3 50μL double enzyme digestion system

[0053]

[0054] (4) Recovery and purification of enzyme-digested plasmids and PCR fragments

[0055] After identification by agarose gel electrophoresis, the PCR products and enzyme-digested plasmids were purified using the OMEGA Gel Extraction Kit. The DNA separation results after agarose gel electrophoresis were observed under 365 nm UV light in a gel imaging system. The gel containing the target DNA band was carefully cut off with a blade, removing as little gel as possible, and placed in a 1.5 mL EP centrifuge tube. The mass of the gel was weighed. Binding buffer was added to the centrifuge tube containing the agarose gel at a 1:1 volume ratio, and the mixture was incubated in a 60℃-65℃ water bath for 7 min, shaking every two to three minutes until the gel was completely melted. The melted solution was transferred to a DNA Mini Column centrifuge column, and the column was placed in a 2 mL Collection Tube. The column was centrifuged at 10000 rpm for 1 min. The maximum volume of solution for each centrifugation is 700 μL. The solution can be centrifuged multiple times until all solution is collected, discarding the filtrate in the collection tube and reusing the tube. Add 700 μL of SPW Wash Buffer (contained with anhydrous ethanol) to the centrifuge column. Centrifuge at 10,000 rpm for 1 min at room temperature, repeating this step once. Discard the filtrate and centrifuge the column at 13,000 rpm for 2 min at room temperature to completely remove ethanol from the purification column. Place the column in a new, clean centrifuge tube. Add 30–100 μL of Elution Buffer to the center of the column, allowing it to stand for 2 min to allow the DNA to completely dissolve in the elution buffer. Centrifuge at 13,000 rpm for 1 min at room temperature, collecting the elution buffer at the bottom of the tube. Perform DNA gel electrophoresis on a small amount of the elution buffer to determine if it is the target product. Store at -20°C.

[0056] (5) Connection of the inserted fragment to the carrier

[0057] Glue recycling fragments The Ligation-Free Cloning System was connected, and the reaction system is shown in Table 4.

[0058] Table 4 Seamlessly Connected Reaction System (20 μL)

[0059]

[0060] After mixing, incubate at 37°C for 30 min; transform Escherichia coli HST08 competent cells; screen cloned colonies for ampicillin resistance.

[0061] (6) DNA sequencing identification of the recombinant r / si-UL8-1 expression vector

[0062] Single colonies were picked and cultured in LB medium containing ampicillin for approximately 8 hours. 100 μL of the bacterial culture was then sent to Qingke Biotechnology Co., Ltd. for DNA sequencing identification using sequencing primers M13Fow-GTAAAACGACGGCCAGT and Rev-CAGGAAACAGCTATGAC.

[0063] (7) After transforming 200 ng of the recombinant r / si-UL8-1 expression plasmid into HST08 competent bacteria, 5 mL of LB medium was added and cultured overnight at 37°C with shaking at 200 rpm. After centrifuging the bacterial culture at 10000 g for 2 min, the precipitate was collected. 180 μL of 10 mM magnesium acetate-Tris·HCl solution was added to the precipitate for resuspending, followed by 200 μL of saturated phenol, and the mixture was shaken at room temperature for 20-60 min. After centrifuging at 10000 g for 10 min, the aqueous phase was collected, and 0.1 times the volume of the aqueous phase of 5 M NaCl was added to precipitate macromolecular impurities. Two volumes of anhydrous ethanol were added to the supernatant, and after centrifugation at 10000 g for 10 min, the supernatant was discarded. The residual ethanol was blotted dry with absorbent paper, and after the RNA was dried, DEPC water was added to dissolve the RNA, the concentration was determined, and the RNA was stored at -80°C.

[0064] (8) Identification by denaturing polyacrylamide gel electrophoresis

[0065] Mix 2 μg of RNA sample with 2×RNA loading buffer and add to the wells of the denaturing gel. Electrophoresis at 120–150 V for 40–60 min, then place the gel in a solution containing 0.5 μg / mL ethidium bromide and gently shake for 20–30 min. Observe the gel under a gel imaging system, take pictures and save them.

[0066] Figure 1 This embodiment utilizes denaturing polyacrylamide gel electrophoresis to detect the expression of recombinant r / si-UL8-1 in *E. coli*. Bacterial total RNA transformed with the recombinant r / si-UL8-1 expression plasmid showed an additional band at 150-300 nt. The results indicate that the recombinant siRNA expression plasmid can highly express recombinant r / si-UL8-1 in *E. coli*.

[0067] Example 2: FPLC purification of recombinant r / si-UL8-1

[0068] (1) Using Bio-Rad NGC TM Chromatography System, using ion exchange column (ENrich) TM Q10×100Column) Purification of recombinant r / si-UL8-1.

[0069] Mobile phase A: 10 mM NaH₂PO₄ solution, pH 7.0. Mobile phase B: 10 mM NaH₂PO₄ solution, 1 M NaCl solution, pH 7.0. Flow rate: 2.0 mL / min. The column was washed alternately with DEPC water, mobile phase A, and mobile phase B for approximately 1 h each time. Five column volumes were used for each wash.

[0070] Total RNA was separated using the following procedures: 0–8.9 min (0% B), 8.9–13.7 min (55% B), 13.7–53.7 min (55–75% B), 53.7–73.7 min (75–85% B), 73.7–83.7 min (100% B), and 83.7–93.7 min (0% B). RNA was detected at 260 nm absorbance, and the peak corresponding to recombinant RNA was collected. Purity was determined by denaturing polyacrylamide gel electrophoresis.

[0071] (2) RNA sample processing methods

[0072] The total RNA extraction procedure is the same as above. After centrifuging the extracted total RNA at 13,000 rpm for 10 min at 4℃, the supernatant was filtered through a 0.22 μm microporous membrane, and 5-10 mg was injected each time.

[0073] (3) FPLC component collection and concentration desalting

[0074] The purity of the collected fractions was determined by denaturing polyacrylamide gel electrophoresis. RNA was precipitated from the mixed fractions with two volumes of anhydrous ethanol and incubated at -80°C for approximately 1 hour. RNA was collected by centrifugation at 10000g at 4°C for 10 minutes. The RNA precipitate was dissolved in DEPC water and centrifuged at 7500g for 10 minutes at 4°C using tra-2 mL centrifugal filters. The filtrate was discarded. This step was repeated until all solutions were centrifuged. The filters were then inverted and centrifuged at 2000g for 2 minutes. The resulting solution was collected, its concentration determined, and it was stored at -80°C.

[0075] Figure 2 This embodiment utilizes Bio-Rad NGC TM Recombinant siRNAs (recombinant r / si-UL8-1) were purified using a chromatography system, and the purity of the collected fractions was identified by denaturing polyacrylamide gel electrophoresis. The results showed that high-purity recombinant r / si-UL8-1 could be obtained after FPLC purification.

[0076] Example 3: Intracellular processing and maturation of recombinant r / si-UL8-1

[0077] (1) Transfection with recombinant siRNAs

[0078] HaCaT, ARPE19, and SY5Y were used at a ratio of 1×10 5 The cells were seeded into 12-well plates. After the cells adhered and spread, the medium was replaced with blank DMEM. Recombinant r / si-UL8-1 was added to a certain amount of blank DMEM medium, and transfection reagent lipo2000 was added to a certain amount of blank DMEM medium. Each was incubated for 5 min. Then, the lipo2000 and DMEM incubation was added to the recombinant r / si-UL8-1 and DMEM incubation, mixed well, and allowed to stand for 20 min. The incubation was then added to the corresponding 12-well plates. The final concentration of recombinant r / si-UL8-1 was 10 nM. After 6 h, the medium was replaced with 10% FBS DMEM medium.

[0079] (2) RNA extraction

[0080] RNA was extracted according to the RNA extraction instructions, and the obtained RNA was then frozen and stored at -80°C.

[0081] (3) qPCR detection was used to detect the expression of recombinant r / si-UL8-1 in cells.

[0082] RNA was reverse transcribed using a reverse transcription kit, and the reverse transcription product was frozen at -20°C. The specific process is as follows:

[0083] a:gDNA digestion, prepare the following mixture in an RNase-free centrifuge tube, and gently mix with a pipette. Incubate at 42℃ for 2 min.

[0084] Table 5. Stem loop qPCR gDNA digestion reaction system

[0085]

[0086] b: Preparation of the reverse transcription reaction system (20 μL system)

[0087] Table 6. Stem loop qPCR cDNA reverse transcription reaction system

[0088]

[0089] The reverse-transcribed cDNA was diluted 5-fold. Using GAPDH as an internal control, the expression level of recombinant siRNA in cells was detected by stem loop qPCR. The qPCR reaction program was as follows: 95℃, 2 min; 95℃, 5 s, 60℃, 30 s, 39 cycles; 95℃, 5 s; 65℃, 5 s; 95℃, 50 s. The primer sequences used are as follows:

[0090] Table 7 Stem loop qPCR primers

[0091]

[0092]

[0093] Figure 3 This embodiment utilizes Stem loop qPCR technology to detect the processing and maturation of recombinant r / si-UL8-1 in ARPE19 cells (values ​​are expressed as mean ± standard deviation; significance between two groups was determined using Students' t test, ***P < 0.001). Compared with the negative control (SSA), the expression of mature si-UL8-1 was significantly increased, indicating that recombinant r / si-UL8-1 was processed into mature siRNA in ARPE19 cells.

[0094] Example 5: Regulatory effect of recombinant r / si-UL8-1 on the target gene (UL8)

[0095] Based on the HSV1 genome (X04370.1), siRNA sequences were designed to detect whether the expression of recombinant r / si-UL8-1 using a tRNA scaffold could specifically knock down the expression of target genes.

[0096] 1. qPCR detection of target gene knockdown effect

[0097] (1) Recombinant siRNA transfection and viral infection

[0098] HaCaT, ARPE19, and SY5Y were used at a ratio of 1×10 5 After the cells were seeded into 12-well plates and allowed to adhere and spread, the medium was replaced with blank DMEM. Recombinant r / si-UL8-1 was added to a certain amount of blank DMEM medium, and transfection reagent lipo2000 was added to a certain amount of blank DMEM medium. Each was incubated for 5 min. Then, the lipo2000 and DMEM incubation was added to the recombinant r / si-UL8-1 and DMEM incubation, mixed well, and allowed to stand for 20 min. The incubation was then added to the corresponding 12-well plates. The recombinant r / si-UL8-1 was 10 nM. After 6 h of transfection, the medium was replaced with 10% FBS DMEM medium. After 24 h, the medium was discarded and the cells were infected with HSV1 virus (moi = 0.1). After 2 h of infection, the cells were washed twice with DPBS and then added to DMEM medium containing 2% FBS.

[0099] (2) RNA extraction

[0100] RNA extraction instructions: Extract RNA and freeze the obtained RNA at -80°C.

[0101] (3) qPCR detection of target gene (UL8) expression

[0102] RNA was reverse transcribed using a reverse transcription kit, and the reverse transcription product was frozen at -20°C. The specific process is as follows:

[0103] a:gDNA digestion

[0104] Prepare the following mixture in an RNase-free centrifuge tube and gently mix by pipetting. Incubate at 42°C for 2 minutes.

[0105] Table 8 Real-time qPCR gDNA digestion reaction system

[0106]

[0107] b: Preparation of reverse transcription reaction system (20 μL system)

[0108] Table 9 Real-time qPCR cDNA reverse transcription reaction system

[0109]

[0110] The pre-transcribed cDNA was diluted 5-fold. Using 18S as an internal control, the expression level of the target gene (UL8) in cells was detected by qPCR. The qPCR reaction program was as follows: 95℃, 2 min; 95℃, 5 s, 60℃, 30 s, 39 cycles; 95℃, 5 s; 65℃, 5 s; 95℃, 50 s. The primer sequences used are as follows:

[0111] Table 10 Recombinant r / si-UL8-1 qPCR Primers

[0112]

[0113] Figure 4 In this embodiment, qPCR was used to detect the knockdown effect of recombinant r / si-UL8-1 on the target gene UL8 of type I herpes simplex virus (HSV1) (values ​​are expressed as mean ± standard deviation; significance between two groups was determined using Students's t test, **P < 0.01, ***P < 0.001). Compared with the negative control (SSA) group, recombinant r / si-UL8-1 significantly inhibited the expression of the target gene, indicating that recombinant r / si-UL8-1 has biological activity.

[0114] Experiments on other recombinant siRNAs of the present invention showed that they could also significantly inhibit the expression of target genes, indicating that the recombinant siRNAs of the present invention have biological activity.

[0115] Example 6: Evaluation of the in vitro antiviral efficacy of recombinant r / si-UL8-1

[0116] 1. Plaque assay for antiviral efficacy of recombinant r / si-UL8-1

[0117] (1) Recombinant r / si-UL8-1 transfection and viral infection

[0118] HaCaT, ARPE19, and SY5Y were used at a ratio of 1×10 5 The cells were seeded into 12-well plates. After cell adhesion and spread, the medium was replaced with blank DMEM. Recombinant r / si-UL8-1 and transfection reagent lipo2000 were added to a certain amount of blank DMEM medium and incubated for 5 min each. Then, the lipo2000 and DMEM incubation was added to the recombinant r / si-UL8-1 and DMEM incubation, mixed well and allowed to stand for 20 min. The incubation was then added to the corresponding 12-well plates. The final concentration of recombinant r / si-UL8-1 was 10 nM. After 6 h of transfection, the medium was changed to 10% FBS DMEM medium. After 24 h, the medium was discarded and the cells were infected with HSV1 virus (moi = 0.1 or 1). After 2 h of infection, the cells were washed twice with DPBS and then added to DMEM medium containing 2% FBS.

[0119] (2) Plaque detection

[0120] Vero E6 cells were seeded in 12 plates. When the cells reached a density of over 90%, the samples infected 24 hours prior to (1) were serially diluted (10⁻¹⁰). -1 Up to 10 -7 After infecting Vero E6 cells with the virus solution (to the power of 1), 2 hours later, discard the virus solution and add maintenance medium covering medium—1.6% methylcellulose-DMEM (2% FBS)—and incubate in a cell culture incubator for 3 days. Finally, discard the covering medium, wash 3-5 times with DPBS to remove any remaining covering medium, add crystal violet staining solution, and stain at room temperature for 10 minutes. Gently wash away any remaining crystal violet with tap water, air dry, count empty spots, and calculate the virus titer. Virus titer (PFU / mL) = number of spots in 12-well plate × virus dilution factor / amount of virus inoculated into 12-well plate (mL).

[0121] Figure 5 In this embodiment, the effect of recombinant r / si-UL8-1 on the titer of type I herpes simplex virus was detected using the plaque assay (values ​​are expressed as mean ± standard deviation; significance between two groups was determined using Students' t test, ***P<0.001, ****P<0.001). As shown in the figure, compared with the negative control SSA, recombinant r / si-UL8-1 significantly reduced the viral titer, indicating that recombinant r / si-UL8-1 has significant antiviral activity.

[0122] 2. Immunofluorescence (IF) assay to evaluate the antiviral efficacy of recombinant r / si-UL8-1

[0123] (1) Recombinant siRNA transfection and viral infection

[0124] ARPE19 at 1×10 5 The cells were seeded into 12-well plates. After cell adhesion and spread, the medium was changed to blank DMEM. Recombinant r / si-UL8-1 was added to a certain amount of blank DMEM medium, and transfection reagent lipo2000 was added to a certain amount of blank DMEM medium. Each was incubated for 5 min. Then, the lipo2000 and DMEM incubation was added to the recombinant r / si-UL8-1 and DMEM incubation, mixed well, and allowed to stand for 20 min. The incubation was then added to the corresponding 12-well plates. The final concentration of recombinant r / si-UL8-1 was 10 nM. After 6 h of transfection, the medium was changed to 10% FBS DMEM medium. After 24 h, the medium was discarded and the cells were infected with HSV1 virus (moi = 0.1). After 2 h of infection, the cells were washed twice with DPBS and then added to DMEM medium containing 2% FBS.

[0125] (2) Immunofluorescence staining

[0126] 24 hours after infection, discard the culture medium in the 12-well plate, wash twice with DPBS, fix with 4% paraformaldehyde for 30 min, wash twice with DPBS, break the membrane with 0.5% Triton-X100 at room temperature for 10 min, wash twice with DPBS, block with 3% BSA for 30 min (prepared immediately), wash twice with DPBS, add HSV1 gD primary antibody (1:100, Santa) and incubate overnight at 4°C. The next day, wash five times with DPBS, add Cy5-labeled mouse secondary antibody (1:500, CST) and incubate at room temperature in the dark for 1 h, discard the secondary antibody, wash five times with DPBS, add nuclear dye DAPI (1:10000) and incubate at room temperature for 5 min, discard the dye, wash five times with DPBS, and observe and photograph using a fluorescence inverted microscope (e.g., Figure 6 ).

[0127] Figure 6 In this embodiment, immunofluorescence (IF) was used to detect the effect of recombinant r / si-UL8-1 on the fluorescence quantity of type I herpes simplex virus (HSV1) (values ​​are expressed as mean ± standard deviation, and the significance between the two groups was determined by Students' t test, *P<0.05). As shown in the figure, compared with the negative control SSA, recombinant r / si-UL8-1 significantly reduced the viral fluorescence quantity, indicating that recombinant r / si-UL8-1 has significant antiviral activity.

[0128] Experiments on other recombinant siRNAs of this invention yielded similar results, indicating that these recombinant siRNAs also significantly reduced viral titers and viral fluorescence levels, demonstrating significant antiviral activity. The recombinant siRNAs of this invention can be used to prepare genomic reagents, prodrugs, drugs, active pharmaceutical ingredients (APIs), or drug combinations that reduce type I herpes simplex virus titers in vitro / in vivo and prolong animal survival; they can also be used to prepare reagents, prodrugs, drugs, APIs, or drug combinations with anti-type I herpes simplex virus activity.

[0129] Example 7: Evaluation of the in vivo antiviral effect of recombinant r / si-UL8-1

[0130] (1) Animal modeling

[0131] Female C57 / B6J mice, aged 6-8 weeks, were intraperitoneally injected with 100 μL of 8% sodium pentobarbital anesthetic. Hair was then removed from the left side of the mice using depilatory cream. Subsequently, 10 μL of HSV1 virus (2 × 10⁻⁶) was instilled lateral to the spine and above the spleen. 6 Use a 27G needle to scrape back and forth 20 times to promote virus absorption (pfu / each).

[0132] (2) Drug treatment

[0133] Six hours after viral challenge, 40 μg of SSA and r / si-UL8-1 encapsulated in flexible liposomes were first applied to the skin of mice. Subsequently, every 24 hours, SSA and r / si-UL8-1 encapsulated in flexible liposomes were applied to the skin of mice or mice were administered via gavage (acyclovir group, 6 mg / mouse). After 5 consecutive days of drug treatment, drug treatment was stopped, and the viral load, body weight, and survival rate of the mice were statistically analyzed.

[0134] (3) Viral load detection

[0135] 1) RNA extraction

[0136] After adding the above animal skin tissue samples to Trizol lysis buffer and magnetic beads, the samples were broken up and ground using a tissue homogenizer. RNA was then extracted using the Omega RNA extraction manual, and the obtained RNA was frozen and stored at -80°C.

[0137] 2) Viral load assay in skin tissue

[0138] RNA was reverse transcribed using a reverse transcription kit, and the reverse transcription product was frozen at -20°C. The specific process is as follows:

[0139] a:gDNA digestion

[0140] Prepare the following mixture in an RNase-free centrifuge tube and gently mix by pipetting. Incubate at 42°C for 2 minutes.

[0141] Table 8 Real-time qPCR gDNA digestion reaction system

[0142]

[0143] b: Preparation of reverse transcription reaction system (20 μL system)

[0144] Table 9 Real-time qPCR cDNA reverse transcription reaction system

[0145]

[0146] The pre-transcribed cDNA was diluted 5-fold. Using 18S as an internal control, the expression level of the target gene (UL8) in cells was detected by qPCR. The qPCR reaction program was as follows: 95℃, 2 min; 95℃, 5 s, 60℃, 30 s, 39 cycles; 95℃, 5 s; 65℃, 5 s; 95℃, 50 s. The primer sequences used are as follows:

[0147] Table 10 Recombinant r / si-UL8-1 qPCR Primers

[0148]

[0149] Figure 7 In this embodiment, qPCR technology was used to detect the effect of recombinant r / si-UL8-1 on HSV1 viral load (values ​​are expressed as mean ± standard deviation, and the significance between the two groups was determined by Students' t test, **P<0.01). Compared with the negative control (SSA) group, recombinant r / si-UL8-1 significantly reduced viral load, indicating that recombinant r / si-UL8-1 has biological activity.

[0150] (4) Effects on mouse body weight and survival rate

[0151] Mice were modeled and treated with drugs according to steps (1) and (2) above. The weight and survival of the mice were observed daily.

[0152] Figure 8 This is a statistical analysis of mouse survival rates in this embodiment. Compared with the blank control (SSA) group, recombinant r / si-UL8-1 significantly reduced viral load, indicating that recombinant r / si-UL8-1 has biological activity.

[0153] Experiments on other recombinant siRNAs of the present invention yielded similar results, indicating that these recombinant siRNAs significantly reduced viral load, demonstrating that the recombinant siRNAs of the present invention possess biological activity. The recombinant siRNAs of the present invention can be used to prepare reagents, prodrugs, drugs, active pharmaceutical ingredients, or drug combinations with activity against type I herpes simplex virus.

[0154] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A recombinant siRNA against type I herpes simplex virus, characterized in that, The sequence of the recombinant siRNA is SEQ ID NO:

1.

2. Use of the recombinant siRNA of claim 1 in the preparation of a medicament or a pharmaceutical composition having an activity against herpes simplex virus type I.

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