CN116790605B - Mutant of siRNA for inhibiting influenza virus and application thereof - Google Patents
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Abstract
The invention discloses a mutant of siRNA for inhibiting influenza virus and application thereof, belonging to the technical field of inhibiting influenza virus. In order to solve the technical problem that the siRNA is limited by the length and has no effect on inhibiting viruses. The invention provides a mutant of siRNA for inhibiting influenza virus, which is characterized in that the 2 nd, 3 rd, 5 th, 6 th or 7 th site base of a gene sequence shown in SEQ ID NO.9 is mutated to obtain a sense strand mutant, and the sequence of an antisense strand is complementary with the sense strand according to a gene complementary principle. Further perfecting the research of anti-influenza virus and simultaneously providing a new strategy and a new thought for the prevention and control of influenza virus.
Description
Technical Field
The invention belongs to the technical field of inhibiting influenza viruses, and particularly relates to a mutant of siRNA for inhibiting influenza viruses and application thereof.
Background
Influenza virus is an important pathogen that can cause large-scale acute respiratory infectious diseases, and is also an important zoonotic virus. The influenza virus has extremely strong transmission force, and can be transmitted mainly through spray, or can be directly or indirectly transmitted after being contacted with mucous membranes such as oral cavities, nasal cavities and the like of patients.
The influenza viruses are classified into four types A, B, C, D, because of their different types of matrix proteins and nucleoprotein surface antigens. Among them, influenza A Virus (IAV) has strong antigen variability and wide infection host range, and can cause serious respiratory diseases of human beings and other animals, and has serious harm to life health of human beings and animals and economic benefit of animal husbandry. Influenza B Virus (IBV) is commonly co-transmitted with IAV, with infection by IBV mostly occurring in children and young children, and several studies have shown that 20% -30% of established influenza viruses are caused by IBV. In addition, a serological investigation of pigs showed that pigs are also susceptible to IBV. Influenza C Virus (ICV) causes less symptoms in healthy adults, but can cause severe complications of lower respiratory tract infections in children, particularly children under 2 years of age. Influenza D Virus (IDV) is currently found to infect mainly pigs, cattle, etc., and has not been found to be capable of causing infection to humans.
Influenza virus is an important zoonotic virus, can cause serious respiratory tract infection of human beings and animals, and has serious harm to the life health of the human beings and the economic benefit of animal husbandry. Currently, methods for controlling influenza virus transmission are mainly divided into two aspects, namely vaccine prevention and drug treatment. Because of more influenza virus subtypes and strong variability, vaccines need to be updated continuously, but the prevention effect of the vaccines is still to be improved, and influenza virus treatment drugs including amantadine, oseltamivir and the like have certain defects and limitations. Thus, it is urgent and necessary to find new, flexible, and effective treatments for influenza viruses.
Compared with traditional anti-influenza virus medicines such as amantadine, oseltamivir and zanamivir, the novel siRNA medicine has the advantages of being high in designability, high in specificity, quick in response and the like in application of the anti-influenza virus medicines, and mainly has the advantages that the siRNA medicine can effectively inhibit influenza virus under the condition of not depending on host immune functions, but the effective siRNA medicine has a certain number of base lengths, the effect is difficult to maintain if the length of the siRNA needs to be reduced, once one base is reduced, the effect of the siRNA is possibly lost, and the technical problem that the siRNA is limited by the length needs to be solved.
Disclosure of Invention
The invention aims to find an siRNA drug for replacing an antiviral drug, and solves the technical problem that the siRNA is limited by the length and has no effect on inhibiting viruses.
The invention provides a mutant of siRNA for inhibiting influenza virus, which is characterized in that the 2 nd, 3 rd, 5 th, 6 th or 7 th site base of a gene sequence shown in SEQ ID NO.9 is mutated to obtain a sense strand mutant, and the sequence of an antisense strand is complementary with the sense strand according to a gene complementary principle.
Further, the mutant is any one of the following:
(1) The 2 nd G base in the gene sequence shown in SEQ ID NO.9 is mutated into a C base to be used as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 16;
(2) Mutating the 3 rd base A in the gene sequence shown in SEQ ID NO.9 into a U base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 18;
(3) Mutating the 5 th U base in the gene sequence shown in SEQ ID NO.9 into an A base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 22;
(4) Mutating the 6 th C base in the gene sequence shown in SEQ ID NO.9 into a G base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 24;
(5) Mutating the 7 th C base in the gene sequence shown in SEQ ID NO.9 into a G base as a sense strand; the sequence of the antisense strand is shown in SEQ ID NO. 26.
Further, the influenza virus is an influenza a virus, an influenza B virus, an influenza C virus or an influenza D virus.
The invention provides a drug for treating influenza, and an active ingredient of the drug is a mutant of siRNA for inhibiting influenza virus.
The invention provides application of the mutant of siRNA for inhibiting influenza virus in preparing a medicament for preventing or treating influenza virus infection.
The invention provides a recombinant vector containing the mutant of the siRNA for inhibiting influenza virus.
The invention provides a recombinant microbial cell containing the above-mentioned mutant encoding siRNA for inhibiting influenza virus.
Further, the microbial cells are a549 cells.
The invention provides an application of the recombinant microbial cells in preparing vaccines for preventing or treating influenza diseases.
The beneficial effects are that: mutation is carried out on the 2 nd, 3 rd, 5 th, 6 th or 7 th site base of the gene sequence shown in SEQ ID NO.9 to obtain a sense strand mutant, the sequence of an antisense strand is complementary with the sense strand according to a gene complementary principle, and the specific siRNA (the gene sequence shown in SEQ ID NO. 9) plays a role in inhibiting influenza virus of siRNA short-2 (the siRNA sequence of which the 5' -end is truncated by 2 bp); the siRNA sequence of the length after the 2 nd, 3 rd, 5 th, 6 th and 7 th positions of siRNA short-3 (the siRNA sequence after 3bp shortening at the 5' end of the specific siRNA) are replaced restores the inhibition effect of the siRNA of the original length, namely SEQ ID NO.9, on influenza viruses.
Drawings
FIG. 1 is a graph showing the results of the effect of specific siRNA expressing AASDHPPT; wherein, (A) PCR detects the result graph of the interference efficiency of the specific siRNA to AASDHPPT protein; (B) Western Blot detects the result graph of the interference efficiency of the specific siRNA to the AASDHPPT protein; (C) a graph of the effect of the specific siRNA on cell viability;
FIG. 2 is a graph showing the results of specific siRNA inhibiting replication of influenza virus WSN (influenza A virus A/WSN/33); wherein, (a) a graph of the effect of transfection-specific siRNA on proliferation of influenza virus WSN in cells; (B) A graph of the effect of transfection specific siRNA on influenza NP protein synthesis;
FIG. 3 is a graph showing the results of transfection of the specific siRNA for expression of influenza virus proteins;
FIG. 4 is a graph showing the results of specific siRNA inhibiting influenza virus vRNP complex nuclear entry;
FIG. 5 is a graph of the results of the effect of specific siRNA on VSV (vesicular stomatitis virus) replication; wherein, (A) Western Blot detects the result of transfection of the specific siRNA on VSV; (B) Observing a result graph of the effect of transfection of the specific siRNA on VSV by an inverted fluorescence microscope;
FIG. 6 is a graph of the results of the effect of specific siRNA on influenza virus on different cells; wherein, (A) Western Blot is used for detecting the effect of transfection of the specific siRNA on the NP protein of the influenza virus on 293T cells; (B) A plot of the results of plaque assay to detect the effect of transfection of the specific siRNA on replication of influenza virus WSN on 293T cells; (C) A result graph of Western Blot detection of the effect of transfection of the specific siRNA on the influenza virus NP protein on CEF cells; (D) A plot of the results of plaque assay to detect the effect of transfection of the specific siRNA on replication of influenza virus WSN on CEF cells;
FIG. 7 is a graph showing the effect of specific siRNA on replication of other subtypes of influenza virus; (A) A graph of the results of the effect of specific siRNA on H5N1 subtype influenza virus replication; (B) Results graphs of the effect of specific siRNA on H7N9 subtype influenza virus replication;
FIG. 8 is a graph showing the results of specific siRNA sequence alignment;
FIG. 9 is a graph of the results of the effect of specific siRNA in an AGO2 knockout cell line; wherein (a) protein levels verify the results of AGO2 knockouts; (B) a graph of results of gene level verification of AGO2 knockout; (C) Results of specific siRNA did not affect influenza virus WSN replication in AGO2 knockout cells;
FIG. 10 is a graph showing the results of inhibition of influenza virus by FAM fluorescent-labeled specific siRNA negative strand; wherein, (A) plaque assay detects the result of transfection antisense strand fluorescence labeling the effect of the specific siRNA on viral replication; (B) A result graph of the effect of the fluorescent labeling of the transfected antisense strand by the Western Blot on the NP; (C) A result graph of the effect of the fluorescence labeling of the transfected antisense strand of the specific siRNA on NP nuclear entry by laser confocal detection;
FIG. 11 is a graph showing the results of the inhibition of influenza virus by the sense strand of FAM fluorescent-labeled specific siRNA; wherein, (A) plaque assay detects the result of transfection sense strand fluorescence labeling the effect of the specific siRNA on viral replication; (B) A result graph of the effect of the fluorescent labeling of the transfected sense strand by the Western Blot on the NP; (C) A result graph of the effect of the fluorescent labeling of the transfected sense strand by the specific siRNA on the NP protein nuclear entering is detected by laser confocal;
FIG. 12 is a graph showing the effect of specific siRNA length on its function; wherein, (A) Western Blot is used for detecting the result graph of the influence of specific siRNA on NP after transfection and truncation; (B) A result diagram of the effect of specific siRNA on virus replication after transfection and truncation is detected by a plaque experiment;
FIG. 13 is a graph showing the effect of specific siRNA base on its function; wherein, (A) results of the effect of the siRNA on replication of influenza virus after base mutation at positions 1, 4, 8, 9, 10, 11, 12, 13, 14, 15, 16; (B) Results of the effect of the siRNA on influenza replication after mutation at positions 2, 3, 5, 6, 7.
Detailed Description
Documents 1 to 3 describe viruses involved in the present invention:
wang G#, zhao Y#, zhou Y#, jiang L, liang L, kong F, yan Y, wang X, wang Y, wen X, zeng X, tian G, deng G, shi J, liu L, chen H, li C.2022 PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virus, PLoS Pathogens, 18 (4): e1010446. Describe A/WSN/33 (H1N 1), A/Anhui/2/2005 (H5N 1) and A/Anhui/1/2013 (H7N 9);
wang X, jiang L, wang G, shi W, hu Y, wang B, zeng X, tian G, deng G, shi J, liu L, li C, chen H, 2022. Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α 7 is counteracted by TBK1-p62 axis-mediated autophagy, celluar & Molecular Mol Immunology 19 (10): 1168-1184. Describe A/WSN/33 (H1N 1), A/Anhui/2/2005 (H5N 1) and A/Anhui/1/2013 (H7N 9);
wang G, jiang L, wang J, zhang J, kong F, li Q, yan Y, huang S, zhao Y, liang L, li J, sun N, hu Y, shi W, deng G, chen P, liu L, zeng X, tian G, bu Z, chen H, li C.2020 The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus entry, journal of Virology 94:e 01707-19, describe A/WSN/33 (H1N 1), A/Anhui/2/2005 (H5N 1).
VSV is vesicular stomatitis virus; WSN is influenza A virus A/WSN/33 (H1N 1).
Example 1 specific siRNA was effective in inhibiting influenza Virus replication
1. siRNA of specific target RNA sequence and control Scramble siRNA were synthesized (Table 1).
2. And centrifuging the synthesized siRNA packaging tube in a high-speed centrifuge for 2 min at 10000 rpm. A corresponding volume of RNase Free H was added to the siRNA packaging tube at a final concentration of 20 pmol/. Mu.L 2 O, blowing and sucking, mixing uniformly, subpackaging in 1.5mL RNase Free EP tube, and storing in a refrigerator at-40 ℃ for later use. siRNA and RNAiMAX reagent according to 1:2 are added into a 1.5mL RNase Free EP tube containing Opti-MEM solution, blown and sucked evenly and kept stand for 25 min. During this time, cells were digested with pre-warmed pancreatin and collected in 15 mL centrifuge tubes and centrifuged at 1800 rpm for 2 min. The centrifuged cells were discarded and resuspended in 1mL of medium according to 1:1.5 (a 549 cells) or 1:3 (293T cells) are added into a 12-hole cell culture plate, 100 mu L of standing mixed liquid is added into the 12-hole cell culture plate, the mixture is uniformly shaken, and then the mixture is placed into a 37 ℃ cell culture box for culturing 36 h-48 h, and then a subsequent experiment can be carried out.
TABLE 1 siRNA sequences
Virus plaque titration experiments: the supernatants of 24H, 48H after A/WSN/33 (H1N 1) virus infection cells of different experimental groups were collected and stored at-80℃and 2 XMEM solutions were prepared before the experiment was started. Firstly, uniformly spreading MDCK cells in a 12-hole plate according to a certain proportion, and carrying out virus plaque titration experiments after the cells in the hole are full. Next, the prepared 2 XMEM solution was autoclaved for ddH 2 O was diluted into a 1 XMEM solution, the collected 24 XMEM solution was diluted 10 times with the collected 24 XMEM and 48h supernatant was diluted 4 gradients, the 24h supernatant was diluted 5 gradients, and the diluted sample was placed in an ice box. Then, the MDCK cell plate laid before was taken out, the medium was removed therefrom, filtered and washed twice with 1 XMEM solution, 100. Mu.L of diluted virus supernatant was added after the residual liquid was removed, and the mixture was slowly shaken uniformly and then placed in an incubator at 37℃for infection of 1h, each timeShake once every 15 min. During this time, the formulated 2% low melting point gum (the sea plaque reagent was added to the autoclave ddH in a 2% ratio 2 O), placing in a microwave oven for boiling, placing in a room temperature for cooling, and then placing in a water bath kettle at 55 ℃ for preservation. Near the end of viral infection, 2 xmem and 2% low melting glue were mixed according to 1:1, adding 0.5 mug/mL of TPCK pancreatin into the uniform liquid, and placing in a water bath kettle at 37 ℃ for preservation. After the virus infection is finished, the virus liquid is sucked and removed, 1mL low-melting-point glue containing 2% of TPCK pancreatin is added into each hole, the mixture is kept stand for 20 to 30 minutes, and after the low-melting-point glue in the holes is solidified, the 12-hole plate is reversely buckled and placed in a cell incubator at 37 ℃ for culture. After 48 and h culture, the 12-well plate was removed and fixed with formalin at room temperature for 8 h. After fixation, formalin solution and bottom gel were rinsed off with running water, and left to dry. Finally, plaques in 12-well plates were counted and viral titers were calculated according to the moi=pfu V/cell number formula. 2 XMEM was prepared in the following proportions.
10xMEM100 ml
Sodium Bicarbonate（7.5%）30 ml
50×MEM Amino Acids20 ml
MEM Vitamin10 ml
Glutamin10 ml
PenStrep10 ml
HEPES10 ml
BSA10 ml
ddH 2 OTo 500 ml。
Cell viability assay: siRNA in different groups is transferred into A549 cells, and the siRNA is blown and sucked uniformly. 100. Mu.L of the mixed liquid was pipetted into a 96-well plate (3 replicates per experimental group) and incubated in a 37℃cell incubator at 48 h. After the culture is finished, 100 mu L of cell viability detection reagent is added into each hole under the light-proof condition, and the mixture is incubated for 2 min at normal temperature and in a shaking table at 40 rpm. Subsequently with GLOMAX TM 96 microplate luminometer the instrument detects the cell viability value, derives and stores the result, and further calculates according to the formula.
Virus infection experiment:
viral maintenance is required to be formulated before the experimentThe virus maintenance solution was Opti-MEM mixture with a concentration of 0.125. Mu.g/mL for TPCK pancreatin when A549 cells were infected, and with a concentration of 0.05. Mu.g/mL for TPCK pancreatin when 293T cells were infected. According to experimental design, cells in a 12-well cell culture plate are filtered and washed twice with a virus maintenance solution, residual liquid is sucked, and 100 mu L of a solution containing 1X10 is added into each well 4 (moi=0.01) or 5×10 6 Virus solution of individual virus particles (moi=5) was infected in a cell culture incubator for 1h, and the cell plates were shaken once every 10 min. After the infection is finished, the cell plate is taken out, the virus liquid in the cell plate is discarded, and 1mL of virus maintenance liquid is added into the hole, and then the cell plate is cultured in a cell culture box.
Experimental effect: 1. specific siRNA significantly down-regulates expression of AASDHPPT protein without affecting cell viability
1. Specific siRNA fragments (experimental group) and control Scramble siRNA (control group) in Table 1 were transfected into A549 cells, the amount of transfection was 30pmol in each group, the transfection was repeated three times in each group, cells were lysed 36h after transfection, RNA thereof was extracted and cDNA was obtained by reverse transcription, and then fluorescent quantitative PCR (primer sequence see Table 2 below) was performed to detect the expression level of AASDHPPT, and as a result, as shown in FIG. 1, the specific siRNA was able to reduce the expression level of AASDHPPT to 3.166%. In addition, the transfected cells were lysed for Western Blot detection.
TABLE 2 fluorescent quantitative primers
Experimental results show that the specific siRNA can effectively reduce the expression of AASDHPPT in both gene and protein levels. Meanwhile, it was also verified that the siRNA was able to effectively reduce the expression of AASDHPPT without affecting the cell activity.
2. Specific siRNA significantly inhibited replication of influenza virus: to determine the effect of this specific siRNA on influenza virus, specific siRNA (experimental group) and control Scrambled siRNA (control group) in table 1 were transfected into a549 cells, respectively, the amount of transfection was 30pmol in each group, three groups were repeatedly transfected in each group, transfected cells were obtained, a/WSN/33 (H1N 1) (moi=0.01) virus was infected after 36H, respectively, and cell supernatants were collected at 24H, 48H, and virus plaque titration experiments were performed.
The results of virus plaque titration show that the specific siRNA (experimental group) has a remarkable inhibition effect on replication of H1N1 subtype influenza virus, and the inhibition times of 24H and 48H after virus infection are about 450 times and 600 times respectively (A in figure 2). Meanwhile, in order to verify the inhibition effect of the specific siRNA on H1N1 subtype influenza virus, we detected the influence of the specific siRNA on the expression of viral NP protein, and after infection, cells were lysed and Western Blot detection was performed on 2H, 4H, 6H and 8H (B in FIG. 2), and the expression level of NP protein was significantly reduced.
3. Specific siRNA inhibits expression of influenza virus proteins: on the basis that the specific siRNA has been determined to have a significant inhibitory effect on the NP protein expression of influenza virus, the effect of the specific siRNA on the expression level of other proteins of influenza virus is clarified. After transferring the specific siRNA in table 1 into a549 cells by the exogenous transfection technique, the amount of transfection was 30pmol, three groups were transfected repeatedly, WSN virus was infected at moi=5, and cell samples were lysed at 0 h, 3 h, 6h, 9 h after infection, and the expression of influenza virus PB1, PB2, M2, NS1, HA, PA proteins were detected.
Experimental results show that the siRNA has inhibition effect on protein expression of influenza virus after being transferred into A549 cells (figure 3).
4. Specific siRNA inhibits influenza vRNP complex entry: replication processes of influenza virus include adsorption, endocytosis, membrane fusion, capsid removal, nuclear entry, transcription, replication, assembly and release. To determine the effect of this specific siRNA on influenza virus life cycle, the siRNA in table 1 was first transferred into a549 cells by exogenous transfection technique in an amount of 30pmol, three groups were repeatedly transfected, WSN virus was infected at moi=5, and samples were fixed at 2 h, 4h, 6h, 8h after infection, and differences in the localization of NP proteins in the experimental and control groups in a549 cells were observed by laser confocal microscopy.
Experimental results show that at the time of infection with 2 h, the NP protein of the control group had entered the nucleus, while the NP transfected with the specific siRNA experimental group was still blocked outside the nucleus (fig. 4), indicating that the specific siRNA affected the stage of the influenza vRNP complex entering the nucleus or before the nucleus, and that specific stages were to be studied further.
Specific siRNA had no effect on VSV-GFP replication: to determine whether the specific siRNA has an inhibitory effect on other single-stranded negative strand RNA viruses, VSV-GFP (single-stranded negative strand RNA virus and enters cells after endocytosis) was selected as a representative strain for verification. The specific siRNA (experimental group) and control Scrambled siRNA (control group) transfected 293T cells were compared by an inverted fluorescence microscope, the amount of each group was 30pmol, three groups were repeatedly transfected, and after 12 h of the VSV-GFP virus infection, GFP fluorescence intensity was observed, and no significant difference was found between the fluorescent intensities of the experimental group and the control group. The results showed that this specific siRNA had no significant effect on VSV-GFP replication (FIG. 5).
6. Effect of specific siRNA on influenza virus on different cell lines: to determine whether this specific siRNA had significant inhibition of influenza virus on other cells, the specific siRNA (experimental group) in table 1 and the control Scrambled siRNA (control group) were transfected into 293T cells and CEF cells, each at 30pmol, three groups were repeatedly transfected, WSN virus was infected after 36h, western Blot experiments were performed on cells lysed after infection of 2 h, 4h, 6h, 8h, and virus plaque titration experiments were performed by pipetting supernatants from cells infected with viruses 24h and 48 h.
The experimental results showed that the specific siRNA drastically reduced the inhibition of influenza virus on 293T cells, the fold inhibition was reduced to about 15 fold (a in fig. 6, B in fig. 6), and no significant inhibition of replication of influenza virus on CEF cells was detected (C in fig. 6, D in fig. 6).
7. Effect of specific siRNA on replication of different subtypes of influenza virus: the specific siRNA has remarkable inhibiting effect on the replication of WSN (H1N 1), and in order to determine whether the specific siRNA has broad spectrum on the regulation and control effect of influenza viruses, two influenza strains with high pathogenicity, namely A/Anhui/2/2005 (H5N 1) and A/Anhui/1/2013 (H7N 9), are selected as the representative strains of other subtype influenza viruses. Specific siRNA (experimental group) and control Scrambled siRNA (control group) in table 1 were transfected into a549 cells in an amount of 30pmol each, three groups were repeatedly transfected, AH05 (H5N 1) and AH13 (H7N 9) viruses were infected at moi=0.1 after 36H, and cell supernatants were harvested after 24H, 48H for virus plaque titration experiments. The evaluation-related experiments were all completed in a biosafety class 3 laboratory.
The result shows that the specific siRNA has obvious inhibition effect on AH05 (H5N 1) and AH13 (H7N 9) viruses, and the inhibition times are respectively 400 times (H5N 1) and 145 times (H7N 9) of 24H after virus infection; the specific siRNAs were found to have broad spectrum in controlling the replication of different subtypes of influenza virus (FIG. 7) up to 330-fold (H5N 1), 400-fold (H7N 9) at 48 hours post-viral infection, respectively.
The target protein targeted by the specific siRNA sequence in the table 1 is AASDHPPT by BLAST comparison, and the comparison result is shown in figure 8. Then research is developed based on the AASDHPPT protein targeted by the specific siRNA, but after repeated verification, the specific siRNA has the effect of inhibiting the influenza virus function independently of the AASDHPPT protein, namely, only the siRNA which reduces the expression of the AASDHPPT protein has a remarkable inhibiting effect on the influenza virus, and other siRNA which can reduce the expression of the AASDHPPT protein and an AASDHPPT protein knockout cell line can not have the effect of inhibiting the influenza virus equally to the specific siRNA, so that the AASDHPPT overexpression cell line is constructed for further eliminating the influence of the AASDHPPT, and the result shows that the cell line has no obvious influence on the replication of the influenza virus. Then, the specific siRNA itself was examined and analyzed from the viewpoint of exerting an inhibitory action mechanism on influenza virus. In the study, firstly, the inhibition effect of the specific siRNA on influenza virus is clarified, and meanwhile, the obvious inhibition effect on the expression of various viral proteins of the influenza virus and the obvious inhibition of the nuclear progress of NP protein are found. Second, the effect of this specific siRNA on VSV-GFP was evaluated and found to have no significant effect on VSV-GFP replication. Meanwhile, the inhibition effect of the specific siRNA on replication of influenza viruses on different cells is evaluated, and the inhibition effect of the specific siRNA on replication of influenza viruses on 293T cells is obviously reduced, the inhibition multiple is about 15 times, and no obvious influence on replication of influenza viruses on CEF cells is detected.
In summary, the inhibition of influenza virus replication and various viral proteins by specific siRNA in table 1 was clarified as described above, while its effect on different strains and its effect on influenza virus replication on different cells was evaluated.
Example 2 sequence characterization of specific siRNA to inhibit influenza Virus
siRNA interference experiments: siRNAs for specific target RNA sequences and control Scramble siRNAs were designed and synthesized according to laboratory preliminary studies and experimental content (Table 3).
And centrifuging the synthesized siRNA packaging tube in a high-speed centrifuge for 2 min at 10000 rpm. A corresponding volume of RNase Free H was added to the siRNA packaging tube at a final concentration of 20 pmol/. Mu.L 2 And O, blowing and sucking, mixing uniformly, subpackaging the mixture in 1.5mL RNase Free EP pipes, and storing the mixture in a refrigerator at the temperature of minus 40 ℃ for later use. siRNA and RNAiMAX reagent according to 1:2 are added into a 1.5mL RNase Free EP tube containing Opti-MEM solution, blown and sucked evenly and kept stand for 25 min. During this time, cells were digested with pre-warmed pancreatin and collected in 15 mL centrifuge tubes and centrifuged at 1800 rpm for 2 min. The centrifuged cells were discarded and resuspended in 1mL of medium according to 1:1.5 (a 549 cells) or 1:3 (293T cells) are added into a 12-hole cell culture plate, 100 mu L of standing mixed liquid is added into the 12-hole cell culture plate, the mixture is uniformly shaken, and then the mixture is placed into a 37 ℃ cell culture box for culturing 36 h-48 h, and then a subsequent experiment can be carried out.
TABLE 3siRNA sequences
The last "TT" base of the SiRNA sequence is the protecting base.
Construction of AGO2 knockout cell line:
(1) First, sgrnas for different exons of AGO2 protein were designed (table 3) and the following system was configured:
10x T4 DNA Ligase Buffer1 μL
T4 PNK（1000 units/mL）1 μL
AGO2 sgRNA F（100 μm）1 μL
AGO2 sgRNA R（100 μm）1 μL
ddH 2 OTo 10 μL
the reaction procedure: 30 min at 37 deg.C and 5 min at 95 deg.C, and cooling to 25deg.C at 5 deg.C per minute
(2) Secondly, enzyme cutting PX458 carrier, selecting BbsI endonuclease to prepare the following system:
PX458 pSpCas91 μL
10X FastDigest Buffer2 μL
FastDigest BbsI （20000 units/mL）1 μL
FastAP1 μL
ddH 2 OTo 10 μL
the reaction is carried out according to the reaction procedure: 30 min at 37 ℃. And recycling the reacted system gel.
(3) Finally, the enzyme-cut PX458 carrier is connected with the annealed sgRNA to prepare the following system:
1. Mu.L of digested PX458 carrier (100 ng/. Mu.L)
After dilution sgRNA (50-fold dilution) 2. Mu.L
10XT4 DNA Ligase Buffer1 μL
T4 DNA Ligase400,000 units/ml1 μL
ddH 2 OTo 10 μL
The reaction is carried out according to the reaction procedure: 16℃6 h.
(4) And (3) transforming the well-connected vector, shaking bacteria, small extracting, sequencing, and carrying out intermediate extracting on a positive result.
(5) The positive plasmid is electrically transferred into A549 cells, after the electric transfer, 8h cells are cultured in a cell culture box at 37 ℃ and then are changed, single cell sorting is carried out after 24 hours h hours, and single cells after sorting are continuously expanded and cultured. And meanwhile, carrying out knockout verification on the separated cells at the protein and gene level.
TABLE 4 AGO2 primers used for knockout
Experimental effect: 1. specific siRNA has the action mode of siRNA classical pathway
The double-stranded siRNA is bound to a Dicer complex containing AGO2 protein, and the double-stranded siRNA is unwound into a single strand by the Dicer complex, and then target cleavage is performed on target mRNA. Thus, to verify whether the specific siRNA functions through the classical pathway of siRNA function, target cleavage of the mRNA of interest. This study established whether the specific siRNA still exerted an inhibitory effect on influenza virus in the absence of AGO2 by constructing an AGO2 knockout a549 cell line.
Specific siRNA (experimental group) and control Scrambled siRNA (control group) in table 1 were transfected into AGO2 knockout cell lines, each group transfected with three replicates according to the method of construction of AGO2 knockout cell lines. WSN virus was infected at moi=0.01 at 36h after exogenous transfection, and cell supernatants were collected 24h, 48h after infection for virus plaque titration experiments.
As a result, as shown in fig. 9, when AGO2 protein was knocked out, the specific siRNA was transfected exogenously, and then almost lost replication of influenza virus, i.e., the inhibition of influenza virus by the specific siRNA was a classical pathway requiring the presence of AGO2 protein and acting by siRNA.
2. Specific siRNA antisense strand marker fluorescence does not affect the inhibition of influenza virus: to clarify the dynamic process of entry of the specific siRNA into cells, carboxyfluorescein (FAM) labelling was used. FAM fluorescent marker is firstly marked on the antisense strand (UUCGGGUAAGGAUUCGAUGTT) of the specific siRNA, the dynamic process of the specific siRNA entering cells is observed, and meanwhile, whether the specific siRNA after FAM fluorescent marker has influence on virus replication is verified. The newly synthesized siRNA (FAM-UUCGGGUAAGGAUUCGAUGTT and CAUCGAAUCCUUACCCGAATT) experimental group and the control Scrambled siRNA (control group) in Table 1 were transfected into A549 cells in an amount of 30pmol each, and the transfection was repeated three times in each group, and after 36h, WSN virus was infected, and 24h, 48h cell supernatants were collected for plaque titration experiments. The experimental results show that the specific siRNA has no effect on the inhibition of influenza virus when fluorescence is labeled at the 5' end of the antisense strand (a in fig. 10). Western Blot experiments were performed to lyse cells after infection with H1N1 virus 2H, 4H, 6H, 8H, and samples were collected, and the experimental results showed that fluorescence labeling on the 5' -end of the antisense strand had no significant effect on the expression level of the influenza NP protein by the specific siRNA (B in FIG. 10). Meanwhile, the effect of the specific siRNA on the vRNP in the case of fluorescence labeling at the 5 '-end of the antisense strand was not changed as observed by laser confocal experiments, and the experimental results showed that the effect of the specific siRNA on the vRNP in the case of fluorescence labeling at the 5' -end of the antisense strand was not changed (C in FIG. 10).
3. Specific siRNA sense strand marker fluorescence affects its inhibition of influenza virus: similarly, FAM was fluorescently labeled on the sense strand of the specific siRNA (CAUCGAAUCCUUACCCGAATT), the dynamic process of its entry into the cell was observed and the effect of fluorescence labeling of the specific siRNA on influenza virus inhibition was determined, and fluorescence labeling was performed on the 5' end of the sense strand of the specific siRNA. The newly synthesized siRNA (FAM-CAUCGAAUCCUUACCCGAATT and UUCGGGUAAGGAUUCGAUGTT) experimental group and the control Scrambled siRNA (control group) in Table 1 were transfected into A549 cells at 30pmol each, three groups were repeatedly transfected in each group, after 36h, WSN virus was infected, and 24h, 48h cell supernatants were collected for virus plaque titration experiments.
The experimental results show that fluorescence labeling at the 5' end of the sense strand eliminates the inhibition of influenza virus by this specific siRNA (A in FIG. 11). Western Blot experiments were performed to lyse cells after infection with virus 2 h, 4h, 6h, 8h, and samples were collected, and the experimental results showed that fluorescence labeling on the 5' -end of the sense strand had no significant effect on the expression level of the specific siRNA on the influenza NP protein (B in FIG. 11). Meanwhile, the effect of the specific siRNA on the nuclear penetration of vRNP when the fluorescent marker is at the 5 'end of the sense strand is observed through a laser confocal experiment, and the experimental result shows that the effect of the specific siRNA on the nuclear penetration of vRNP when the fluorescent marker is at the 5' end of the sense strand is not observed (C in FIG. 11).
Example 3 application of truncated specific siRNA in inhibiting influenza Virus
Determining the shortest length of the specific siRNA exerting an inhibitory effect on influenza virus: to determine the effect of the base length of the specific siRNA on the inhibition of influenza virus, the original specific siRNAs (specific siRNA-sense and specific siRNA-anti in Table 3) were truncated 1bp each time from the 5 'end or the 3' end. First, a novel siRNA (specific siRNA short-1-sense and specific siRNA short-1-dose in Table 3) truncated 1bp from the 5' end was transfected into A549 cells with a control siRNA of 30pmol in each group, and after 36h, WSN virus was infected, western Blot experiments were performed on cells lysed 2 h, 4h, 6h, 8h after virus infection, and virus plaque titration experiments were performed by pipetting supernatants of 24h and 48h cells after virus infection. The specific siRNA was truncated by 2bp (specific siRNAs short-2-sense and specific siRNAs short-2-antisense), 3bp (specific siRNAs short-3-sense and specific siRNAs short-3-antisense) from the 5 'end in sequence, the original specific siRNA was truncated by 1bp (specific siRNAs short-4-sense and specific siRNAs short-4-antisense) from the 5' end, and the siRNA sequence was repeated. The experimental results are shown in FIG. 12, the truncated new siRNA sequences (siRNA short-1 and siRNA short-2) still have the equivalent inhibition effect on influenza virus compared with the original siRNA, the experimental results show that the truncated new siRNA sequences (siRNA short-1 and siRNA short-2) still have the equivalent inhibition effect on influenza virus compared with the original specific siRNA, the times of the detection inhibition of the growth of the siRNA short-1 after 24h and 48h of virus infection are about 190 times and 510 times respectively, the times of the inhibition of the siRNA short-2 after 24h and 48h of virus infection are about 140 times and 480 times, and the truncated new siRNA sequences (siRNA short-3 and siRNA short-4) have no inhibition effect on influenza virus. That is, if the inhibitory effect on influenza virus is maintained, two bases at most can be removed from the 5' -end. Next, 3 'ends of siRNA were truncated 1bp at a time from siRNA short-2 (the shortest length to maintain inhibition from 5' end). The novel siRNA with 1bp truncated from the 3' end and the control Scrambled siRNA are transfected into A549 cells, after 36h, viruses are infected, western Blot experiments are carried out on cells which are lysed after the viruses are infected by 2 h, 4h, 6h and 8h, and simultaneously, the cell supernatants of 24h and 48h of the viruses are sucked for plaque titration experiments. Experimental results show that siRNA short-2 (siRNA with 2bp truncated from 5 'end) has no inhibition effect on influenza virus after 1bp truncated from 3' end. In conclusion, the shortest length of the specific siRNA for inhibiting the influenza virus is siRNA short-2, namely after 2bp is removed from the 5' end.
Key bases of specific siRNA for inhibiting influenza virus: in order to determine the important base of the specific siRNA playing an inhibitory effect on influenza virus, a single base substitution method is adopted, the length of the non-inhibitory effect on influenza virus obtained in the last step is siRNA short-3, single base complementary substitution is sequentially carried out from the 5' end, the replaced new siRNA sequence and the control Scrambled siRNA are transfected into A549 cells, the amount of transfection in each group is 30pmol, the transfection is repeated three times in each group, after 36 hours, WSN viruses are infected with MOI=0.01, and cell supernatants after the infected viruses 24h and 48h are sucked for virus plaque titration experiments. As a result, as shown in FIG. 13, the number of spots in the plaque titration experiment after single complementary substitution was performed on the 1 st (specific siRNA mutation-1-sense), 4 th (specific siRNA mutation-4-sense), 8 th (specific siRNA mutation-8-sense), 9 th (specific siRNA mutation-9-sense), 10 th (specific siRNA mutation-10-sense), 11 th (specific siRNA mutation-11-sense), 12 th (specific siRNA mutation-12-sense), 13 th (specific siRNA mutation-13-sense), 14 th (specific siRNA mutation-14-sense), 15 th (specific siRNA mutation-15-sense), and 16 th (specific siRNA mutation-16-sense) site bases were approximately the same as the number of siRNAshort-3 siRNAs, but different from the original number. However, after single complementary substitutions of site bases 2 (specific siRNA mutation-2-sense), 3 (specific siRNA mutation-3-sense), 5 (specific siRNA mutation-5-sense), 6 (specific siRNA mutation-6-sense), 7 (specific siRNA mutation-7-sense), the number of spots in the plaque titration experiment is still about the same as the number of original siRNAs. Experimental results show that the 2 nd, 3 rd, 5 th, 6 th and 7 th sites of siRNAshort-3 are mutated to restore the inhibition effect on influenza virus, and the inhibition effect is basically the same as that of the original specific siRNA (specific siRNA-sense and specific siRNA-anti in table 3). In summary, the 2 nd, 3 rd, 5 th, 6 th or 7 th site base of the gene sequence shown in SEQ ID NO.9 is mutated to obtain a sense strand mutant, the sequence of the antisense strand is complementary with the sense strand according to the gene complementary principle, and the shortest sequence of the specific siRNA (the gene sequence shown in SEQ ID NO. 9) which has the effect of inhibiting the influenza virus is siRNA short-2 (the siRNA sequence of which the 5' -end is truncated by 2 bp); the siRNA sequence of the length after the 2 nd, 3 rd, 5 th, 6 th and 7 th positions of siRNA short-3 (the siRNA sequence after 3bp shortening of the 5' end of the specific siRNA) is replaced, and the inhibition effect of the siRNA of the original length, namely the specific siRNA-sense, on influenza viruses is restored.
Claims (7)
1. A mutant of siRNA for inhibiting influenza virus is characterized in that the 2 nd, 3 rd, 5 th, 6 th or 7 th site base of a gene sequence shown in SEQ ID NO.9 is mutated to obtain a sense strand mutant, and the sequence of an antisense strand is complementary with the sense strand according to a gene complementary principle;
the mutant is any one of the following:
(1) The 2 nd G base in the gene sequence shown in SEQ ID NO.9 is mutated into a C base to be used as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 16;
(2) Mutating the 3 rd base A in the gene sequence shown in SEQ ID NO.9 into a U base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 18;
(3) Mutating the 5 th U base in the gene sequence shown in SEQ ID NO.9 into an A base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 22;
(4) Mutating the 6 th C base in the gene sequence shown in SEQ ID NO.9 into a G base as a sense strand; the sequence of the antisense strand is shown as SEQ ID NO. 24;
(5) Mutating the 7 th C base in the gene sequence shown in SEQ ID NO.9 into a G base as a sense strand; the sequence of the antisense strand is shown in SEQ ID NO. 26.
2. The mutant of siRNA that inhibits influenza virus according to claim 1, wherein the influenza virus is influenza a virus.
3. A medicament for treating influenza, characterized in that the active ingredient of the medicament is the mutant of siRNA for inhibiting influenza virus according to claim 1 or 2.
4. Use of a mutant of siRNA that inhibits influenza virus according to claim 1 in the manufacture of a medicament for preventing or treating infection by influenza virus, wherein the influenza virus is influenza virus type a.
5. A recombinant vector comprising the mutant of the siRNA that inhibits influenza virus of claim 1.
6. A recombinant microbial cell comprising a mutant of the siRNA that inhibits influenza virus of claim 1, wherein the microbial cell is an a549 cell.
7. Use of the recombinant microbial cell of claim 6 for the preparation of a vaccine for the prevention or treatment of influenza disease, wherein the influenza virus is influenza a virus.
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| CN101275132A (en) * | 2008-03-06 | 2008-10-01 | 中山大学 | SiRNA capable of inhibiting influenza A virus replication and coding sequence thereof |
| CN106947763A (en) * | 2017-03-20 | 2017-07-14 | 中国医学科学院医药生物技术研究所 | The siRNA of the resisiting influenza virus infection of aerosolizable suction and its application |
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| CN101275132A (en) * | 2008-03-06 | 2008-10-01 | 中山大学 | SiRNA capable of inhibiting influenza A virus replication and coding sequence thereof |
| CN106947763A (en) * | 2017-03-20 | 2017-07-14 | 中国医学科学院医药生物技术研究所 | The siRNA of the resisiting influenza virus infection of aerosolizable suction and its application |
| CN115120608A (en) * | 2021-03-26 | 2022-09-30 | 圣诺生物医药技术(苏州)有限公司 | siRNA drug, drug composition, siRNA-small molecule drug conjugate and application thereof |
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