A target site inhibitor against zika virus, recombinant gene, expression vector and application

Abstract

The application discloses an anti-zika virus target point inhibitor, a recombinant gene, an expression vector and application, and belongs to the field of antiviral drugs and molecular targeting technology; the application discloses that, through structural analysis of ATP2A2b, it is revealed that the calcium ion regulatory protein ATP2A2b on the endoplasmic reticulum can be used as an action target point of the zika virus inhibitor; and a recombinant vector and an inhibitor are designed to accurately target ATP2A2b, avoid the potential of virus drug resistance, and reduce side effects on the host; the cyclopia acid as a small molecule compound of the ATP2A2b protein inhibitor can be used for inhibiting in-vitro infection of the zika virus; and the effectiveness and safety are improved through targeted treatment of the zika virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of antiviral drugs and molecular targeting technology, and particularly relates to a Zika virus-targeting inhibitor, a recombinant gene, an expression vector and application. BACKGROUND

[0002] Zika virus (ZIKV) is a flavivirus associated with neurological diseases, and infection of pregnant women with ZIKV can cause infant microcephaly or abnormal neuronal development, while adult infection can cause neurological diseases such as Guillain-Barre syndrome, neuritis or meningitis (1, 2). However, there is no specific vaccine or antiviral drug. Therefore, it is necessary to develop effective and safe antiviral drugs and vaccines for the treatment and prevention of ZIKV infection worldwide. Some small molecule compounds, such as 7-deaza-2'-C-methyladenosine (3) or epigallocatechin gallate (4), and drugs approved by the U.S. Food and Drug Administration, such as nicotinamide, nicotinamide, mefloquine, and bromocriptine (5-7), have been found to inhibit ZIKV infection. However, the safety of small molecule drugs for pregnant women needs to be more widely evaluated.

[0003] 2A10G is a murine antibody with broad flavivirus neutralizing activity by targeting ZIKV E protein, which can neutralize ZIKV infection in vitro and in vivo (8). However, its relatively low efficacy and humanization requirements hinder its further development. Viral NS2B-NS3 protease (9), RNA-dependent RNA polymerase (RdRp) (10), etc. are non-structural proteins against ZIKV, and are also targets for direct antiviral drugs, but lack the ability to penetrate the blood-brain barrier and target neural tissues. Therefore, treatment strategies targeting host factors have become a research hotspot. For example, AXL receptor (11) and ER membrane complex EMC (12) are host protein targets that inhibit infection by suppressing their expression, but only partially inhibit infection; RIG-I / MAVS pathway (13) and ZIKV NS5-STAT2 (14) mechanisms are regulated by host innate immunity, but IFN therapy can easily trigger excessive inflammatory response; inhibiting lipid metabolism in infected hosts can also reduce viral load, but affects normal cell function. More than 50% of ZIKV-interacting host proteins are unknown, so researchers in the field have been working on host factor targets for antiviral drugs.

[0004] REFERENCES

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[0010] 6. Chan JF, Chik KK, Yuan S, Yip CC, Zhu Z, Tee KM, Tsang JO, Chan CC, Poon VK, Lu G, Zhang AJ, Lai KK, Chan KH, Kao RY, Yuen KY. 2017. Novel antiviral activity and mechanism of bromocriptine as a Zika virus NS2B-NS3 protease inhibitor. Antiviral Research 141. http: / / doi.org / 10.1016 / j.antiviral.2017.02.002.

[0011] 7. Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G, Soto-Acosta R, Galarza- G, McGrath EL, Urrabaz-Garza R, Gao J, Wu P, Menon R, Saade G, Fernandez-Salas I, Rossi SL, Vasilakis N, Routh A, Bradrick SS, Garcia-Blanco MA. 2016. A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell Host Microbe 20:259-70. http: / / doi.org / 10.1016 / j.chom.2016.07.004

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[0013] 9. Nunes DAF, Santos F, da Fonseca STD, de Lima WG, Nizer W, Ferreira JMS, de JA-O. 2022. NS2B-NS3 protease inhibitors as promising compounds in the development of antivirals against Zika virus: A systematic review. J Med Virol 94:442-453. http: / / doi.org / 10.1002 / jmv.27386

[0014] 10. Zhou GF, Xie CQ, Xue JX, Wang JB, Yang YZ, Zheng CB, Luo RH, Yang RH, Chen W, Yang LM, Wang YP, Zhang HB, He YP, Zheng YT. 2022. Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3H)-ones targeting the ZIKV NS5 RNA dependent RNA polymerase. Front Chem 10: 1010547. http: / / doi.org / 10.3389 / fchem.2022.1010547

[0015] 11. Xie S, Zhang H, Liang Z, Yang X, Cao R. 2021. AXL, an Important Host Factor for DENV and ZIKV Replication. Front Cell Infect Microbiol 11: 575346. http: / / doi.org / 10.3389 / fcimb.2021.575346

[0016] 12. Ngo AM, Shurtleff MA-O, Popova KD, Kulsuptrakul J, Weissman JA-OX, Puschnik AA-O. 2019. The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins. Elife 8: e48469. http: / / doi.org / 10.7554 / eLife.48469

[0017] 13. Hu Y, Dong X, He Z, Wu Y, Zhang S, Lin J, Yang Y, Chen J, An S, Yin Y, Shen Z, Zeng G, Tian H, Cai J, Yang Y, Guan H, Wu J, Li M, Zhu X. 2019. Zika virus antagonizes interferon response in patients and disrupts RIG-I-MAVS interaction through its CARD-TM domains. Cell Biosci 9: 46. http: / / doi.org / 10.1186 / s13578-019-0308-9

[0018] 14. Shu J, Ma X, Zhang Y, Zou J, Yuan Z, Yi ZA-O. 2021. NS5-independent Ab lation of STAT2 by Zika virus to antagonize interferon signalling. Emerg Microbes Infect 10: 1609-1625. http: / / doi.org / 10.1080 / 22221751.2021.1964384. SUMMARY

[0019] The present application overcomes the deficiencies of the prior art, and provides an anti-Zika virus target inhibitor, a recombinant vector and an application, which improves effectiveness and safety through targeted treatment of Zika virus.

[0020] The present application is realized by the following technical solutions:

[0021] An anti-Zika virus recombinant gene, the recombinant gene is three shRNAs; the nucleotide sequences targeted by the three shRNAs are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

[0022] An expression vector, the expression vector is a p-GIPZ vector into which the recombinant gene of claim 1 is inserted.

[0023] Further, an expression cassette containing the expression of the recombinant gene.

[0024] The anti-Zika virus recombinant gene is applied to inhibit the replication and proliferation of Zika virus.

[0025] The application of the expression vector in inhibiting Zika virus replication and proliferation.

[0026] A target point inhibitor against Zika virus, the target point inhibitor is cyclopiazonic acid.

[0027] The application of the target point inhibitor against Zika virus in inhibiting Zika virus replication and proliferation.

[0028] The principles of the present application are:

[0029] When the host is infected with ZIKV, the endoplasmic reticulum (ER) is the central hub of flavivirus replication and assembly, involved in the synthesis, processing and immune escape of viral proteins, and involved in the whole life cycle of ZIKV, such as entry, replication, assembly and budding. 2+ ATPase, which maintains low cytoplasmic calcium levels and maintains calcium homeostasis. 2+ Abnormal function can cause intracellular Ca 2+ Stability imbalance, which ultimately leads to the death of nerve cells, indicating that intracellular Ca 2+ Stability imbalance caused by ZIKV infection is not corrected in time, suggesting that the function of ATP2A2b may be impaired. 2+ Stability imbalance, ER lumen Ca 2+ Depletion, which in turn mediates the occurrence of ER stress. 2+ Therefore, ATP2A2b plays a key role in cellular Ca

[0030] The beneficial effects of the present application relative to the prior art are:

[0031] The present application discloses that the calcium ion regulatory protein ATP2A2b on the endoplasmic reticulum can be used as a target point for ZIKV inhibitors through structural analysis of ATP2A2b.

[0032] The present application inhibits ZIKV infection by inducing host ER stress mechanism through ATP2A2b protein, and experiments prove that knocking down the expression of ATP2A2b can inhibit the replication and proliferation of ZIKV, and cell death is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Figure 1 shows the construction of shATP2A2b cell line, where (1a) is the ATP2A2b mRNA expression level after knockdown, and (1b) is the ATP2A2b protein expression level after knockdown;

[0034] Figure 2 Figure 2 shows the inhibition of virus proliferation by knockdown of ATP2A2b, where (2a) is the ATP2A2b mRNA expression level in shRNA and shATP2A2b cell lines after ZIKV infection, and (2b) is the virus mRNA expression level in shRNA and shATP2A2b cell lines after ZIKV infection;

[0035] Figure 3 Figure 3 shows the virus protein expression level in shRNA and shATP2A2b cell lines after ZIKV infection;

[0036] Figure 4 Figure 4 shows the antiviral activity of ATP2A2b knockdown cell line, where (4a) is the death of ATP2A2b knockdown cell line, and (4b) is the survival rate of shRNA and ATP2A2b shRNA cells under virus infection;

[0037] Figure 5 Figure 5 shows the construction of ATP2A2b overexpression cell line, where (5a) is the ATP2A2b mRNA expression level after overexpression, and (5b) is the ATP2A2b protein expression level after overexpression;

[0038] Figure 6 Figure 6 shows the promotion of virus proliferation by ATP2A2b at mRNA level, where (6a) is the ATP2A2b mRNA expression level in Vector and ATP2A2b cell lines after ZIKV infection, and (6b) is the virus mRNA expression level in Vector and ATP2A2b cell lines after ZIKV infection;

[0039] Figure 7 Figure 7 shows the virus protein expression level in Vector and ATP2A2b cell lines after ZIKV infection;

[0040] Figure 8 Figure 8 shows the antiviral activity of ATP2A2b cell line, where (8a) is the death of ATP2A2b cell line, and (8b) is the survival rate of Vector and ATP2A2b cells under virus infection;

[0041] Figure 9 Figure 9 shows the inhibition effect of cyclopianamic acid on ZIKV at cellular level, where (9a) is the virus mRNA expression level, and (9b) is the virus protein expression level. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear and explicit, the present application is further described in detail in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. The technical solutions of the present application are described in detail below in combination with embodiments and drawings, but the protection scope is not limited thereto.

[0043] Experimental materials

[0044] Vero (ATCC, #CCL-81), 293T (ATCC, #CRL-3216), U251 cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), and the cell growth solution was DMEM containing 10% FBS, and the cell maintenance solution was DMEM containing 2% FBS. ZIKV strain SZ01 / 2016 (GenBank No: KU866423) was isolated from a patient returning from Samoa. The virus was amplified in Vero cells for in vivo and in vitro experiments. The viral titer was determined by virus plaque assay, and diluted to 0.01 for in vitro experiments.

[0045] Example 1

[0046] Construction of shATP2A2b cell line

[0047] Experimental method:

[0048] 1) Three lentiviral vectors p-GIPZ expressing short hairpin RNA (shRNA) were used to specifically knock down the expression of ATP2A2b, respectively. The same lentiviral vector alone (empty vector) was used as a negative control. The cDNA of shRNA was cloned into the lentiviral vector p-GIPZ. The lentiviral vector plasmid encoding shRNA and packaging plasmids psPAX2 and pLP / VSVG were co-transfected into HEK 293T cells to package lentivirus using Vigofect transfection reagent (Vigorous Biotechnology);

[0049] The sequences of the three shRNAs are as follows:

[0050] shRNA target 1 sequence, SEQ ID NO. 1: CTGGTGATATTGTAGAAAT

[0051] shRNA target 2 sequence, SEQ ID NO. 2: AGAAAGTCAATGTCGGTTT

[0052] shRNA target 3 sequence, SEQ ID NO. 3: AGGTGATACTTGTTCCCTT

[0053] 2) 48h post transfection, collect the supernatant containing infectious lentivirus and add to U251 cells, 2h later, add complete medium, 24h later, change the medium.

[0054] 3) 48h later, screen the infected cells with 5 μg / ml puromycin (Selleck, Shanghai, China). The selected cell lines are resistant to puromycin and are cultured for expansion;

[0055] 3) 14 days later, collect the cell samples and verify the knockdown effect on the target gene ATP2A2b expression by Western blot and qPCR; the primers used for the genes are as follows:

[0056] SEQ ID NO. 4: ATP2A2b-F TCCGCTACCTCATCTCGTCCAA;

[0057] SEQ ID NO. 5: ATP2A2b-R CGTCAGCAGCAATGAACCACCA;

[0058] SEQ ID NO. 6: β-actin-F 5'-AAGGAGAAGCTGTGCTACGTCGC-3'

[0059] SEQ ID NO. 7: β-actin-R 5'-AGACAGCACTGTGTTGGCGTACA-3

[0060] Experimental results:

[0061] Results are shown in Figure 1 ; from the results of qPCR and WB, it can be shown that the shATP2A2b cell line is successfully constructed, and from the figure, it can be seen that the mRNA and protein expression of ATP2A2b in the shATP2A2b group is reduced compared with the control group shRNA cells.

[0062] Example 2

[0063] Inhibition effect of ATP2A2b knockdown on virus at mRNA level of cells

[0064] Experimental method:

[0065] 1) Take shATP2A2b cells in good growth state for digestion and subculture, inoculate in a 12-well plate, 2 mL / well, and place in a 37°C, 5% CO2 incubator for incubation for 24h;

[0066] 2) 24h later, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture at 37°C, 5% CO2.

[0067] 3) RNA samples were collected at 24h, 48h and 72h post-infection, respectively, and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Reverse transcription was performed using Prime Script TM RT reagent Kit with g DNA Eraser kit (Takara, Japan). The cDNA template was prepared using TB Green Premix Ex Taq TM II kit was prepared into 20uL system and real-time quantitative PCR reaction system was carried out in Quant Studio 6 PCR instrument (Thermo, USA).

[0068] The primers used are as follows:

[0069] SEQ ID NO. 4: ATP2A2b-F TCCGCTACCTCATCTCGTCCAA;

[0070] SEQ ID NO. 5: ATP2A2b-R CGTCAGCAGCAATGAACCACCA;

[0071] SEQ ID NO. 6: β-actin-F 5'-AAGGAGAAGCTGTGCTACGTCGC-3'

[0072] SEQ ID NO. 7: β-actin-R 5'-AGACAGCACTGTGTTGGCGTACA-3

[0073] SEQ ID NO. 8: ZIKV-E-F 5'-GGGTTGATGTTGTCTTGGAACAT-3'

[0074] SEQ ID NO. 9: ZIKV-E-R 5'-AGGCTTCACCTTGTGTTGGG-3'

[0075] Results of the experiment:

[0076] Results are shown in Figure 2 It can be seen from the qPCR result graph that the expression of ATP2A2b mRNA in the shATP2A2b cells infected with the virus is also inhibited compared with the virus group shRNA cells, and even the activity of Zika virus is significantly inhibited as the virus infection time increases. It is proved that knocking down ATP2A2b inhibits the proliferation of ZIKV.

[0077] Example 3

[0078] Inhibitory effect of ATP2A2b knockdown on virus at the cellular protein level

[0079] Experimental method:

[0080] 1) Take shATP2A2b cells in good growth state for digestion and subculture, inoculate in 6-well plates, 2 mL / well, and place in a 37°C, 5% CO2 incubator for 24 h;

[0081] 2) After 24 h, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and place in a 37°C, 5% CO2 incubator for continuous culture.

[0082] 3) Collect cell lysate samples at 24, 48 and 72 h after infection, centrifuge to obtain the supernatant, and run electrophoresis after BCA quantification. Use polyclonal ZIKV E rabbit antibody (1:1000, Gene Tex), anti-ZIKV NS1 mouse antibody (1:1000, Gene Tex), anti-ATP2A2b rabbit antibody (1:3000, Abeam) or monoclonal GAPDH rabbit antibody (1:1000, Protein Tech) as the primary antibody.

[0083] Horseradish peroxidase (HRP)-labeled anti-mouse IgG or rabbit IgG antibody (1:3000) obtained from Protein Tech (Wuhan, China) is used as the secondary antibody.

[0084] Experimental results:

[0085] The results are shown in Figure 3 . From the WB results, it can be seen that the ATP2A2b protein in the control group shRNA cells increases with the increase of infection time, and the ZIKV protein also increases, while the Zika virus in the shATP2A2b cells is significantly inhibited.

[0086] Example 4

[0087] Antiviral activity of ATP2A2b knockdown cell lines

[0088] Experimental method:

[0089] 1) Take shATP2A2b cells in good growth state for digestion and subculture, inoculate in 96-well plates, 100 μL / well, and place in a 37°C, 5% CO2 incubator for 24 h;

[0090] 2) After 24 h, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and place in a 37°C, 5% CO2 incubator for continuous culture.

[0091] 3) At 48h post-infection, the CPE was observed by microscope and the cell viability was detected by CCK8 at 24h, 48h and 72h respectively.

[0092] Results:

[0093] Results: Figure 4 From the microscope pictures, it can be observed that the shRNA infected cells have a large number of vacuoles and fuzzy boundaries, while the CPE of shATP2A2b cells is weakened. Meanwhile, the CCK8 results in the right panel show that the knockdown of ATP2A2b inhibits the proliferation of ZIKV and reduces cell death. Therefore, the knockdown of ATP2A2b inhibits the proliferation of ZIKV.

[0094] Example 5

[0095] Construction of ATP2A2b overexpression cell line

[0096] Experimental methods:

[0097] 1) CRISPR / dCas9 technology was used to co-transfect LentiMPHv2-hygro (Addgene), psPAX2 and PMD-2.G into HEK293T cells at a ratio of 2:3:4. 48h after transfection, the lentivirus was collected and used to infect U251 cells.

[0098] 2) The infected cells were selected by 250μg / mL hygromycin B (Beyotime, Shanghai) 48h after infection. Two weeks later, Western Blot and qPCR were used for verification.

[0099] 3) Then, three sgRNAs were constructed in the lentiSAMv2-Blasticidin plasmid, and the lentivirus packaged with the empty plasmid LentisAMv2-Blasticidin was set as the negative control for overexpression of ATP2A2b. A total of four plasmids were used.

[0100] 4) Then, the four plasmids constructed above were combined with psPAX2 and PMD-2.G to construct lentivirus and infect U251 cells stably expressing LentiMPH v2-hygro.

[0101] 5) The infected cells were selected by 250μg / mL hygromycin B and 1.5μg / mL blast (MCE) 48h after infection,

[0102] 6) The U251 cells expressing ATP2A2b were verified by Western Blot and qPCR 14 days later.

[0103] Results:

[0104] Results are shown in Figure 5 ATP2A2b cell line construction success was verified by qPCR and WB results. As can be seen from the figure, the mRNA and protein expression of ATP2A2b in the ATP2A2b group increased by about six times compared with the control group Vector. Therefore, the ATP2A2b overexpression cell line was successfully constructed.

[0105] Example 6

[0106] ATP2A2b overexpression promotes virus proliferation at the mRNA level of cells

[0107] Experimental method:

[0108] 1) Take well-grown ATP2A2b cells for digestion and subculture, inoculate in 12-well plates, 2 mL / well, and place in a 37°C, 5% CO2 incubator for 24 h;

[0109] 2) After 24 h, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture at 37°C, 5% CO2.

[0110] 3) Collect RNA samples at 24 h, 48 h and 72 h after infection, respectively. According to the manufacturer's instructions, use TRIzol reagent (Thermo Fisher Scientific) to extract total RNA from uninfected and infected cells at 24 h, 48 h and 72 h, respectively. Use Prime Script TM RT reagent Kit with g DNA Eraser kit (TaKaRa, Japan) reverse transcription, cDNA template uses TB Green Premix Ex Taq TM II kit to prepare 20 uL system for real-time quantitative PCR reaction system in Quant Studio 6 PCR instrument (Thermo, USA).

[0111] Experimental results:

[0112] Results are shown in Figure 6 From the qPCR data results, it can be seen that with the increase of infection time, compared with the control group Vector cells, the expression of ATP2A2b in ATP2A2b cells increased, especially at 48 h, and the ZIKV mRNA level reached a maximum at 72 h. It is shown that ATP2A2b promotes ZIKV replication at the mRNA level of cells.

[0113] Example 7

[0114] ATP2A2b overexpression promotes proliferation of virus at the level of cell protein

[0115] Experimental method:

[0116] 1) Take the well-grown ATP2A2b cells for digestion and subculture, inoculate in a 6-well plate, 2 mL / well, and place in a 37°C, 5% CO2 incubator for incubation;

[0117] 2) After 24 h, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture at 37°C, 5% CO2.

[0118] 3) Collect cell lysate samples at 24 h, 48 h and 72 h after infection, centrifuge to obtain the supernatant, run electrophoresis after BCA quantification, and use ZIKV E rabbit antibody, anti-ZIKV NS1 mouse antibody, anti-ATP2A2b rabbit antibody or GAPDH rabbit antibody as the primary antibody. HRP-labeled anti-mouse IgG or rabbit IgG antibody as the secondary antibody.

[0119] Experimental results:

[0120] The results are shown in Figure 7 . From the Western blot in the figure, it can be observed that compared with the Vector group, the expression of ATP2A2b gradually increases with the increase of infection time, and the protein expression of ZIKV also increases. Therefore, ATP2A2b promotes the replication and proliferation of ZIKV.

[0121] Example 8

[0122] Antiviral activity of ATP2A2b cell line

[0123] Experimental method:

[0124] 1) Take the well-grown ATP2A2b cells for digestion and subculture, inoculate in a 96-well plate, 100 μL / well, and place in a 37°C, 5% CO2 incubator for incubation for 24 h;

[0125] 2) After 24 h, discard the supernatant, treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture at 37°C, 5% CO2.

[0126] 3) At 48 h after infection, observe the cytopathic effect by taking pictures with a microscope, and at 24 h, 48 h and 72 h, use CCK8 reagent for cell viability detection.

[0127] Experimental results:

[0128] The results are shown in Figure 8From the microscope images, it can be seen that as the infection time increases, a large number of vacuoles appear in the infected cells, the boundary is blurred, and the CPE phenomenon of ATP2A2b cells is significantly enhanced. At the same time, the CCK8 results show that ATP2A2b promotes the proliferation of ZIKV, and the cell death increases.

[0129] Example 9

[0130] Inhibitory effect of cyclopamine on ZIKV at the cellular level

[0131] Experimental methods:

[0132] 1) In U251 cells with or without cyclopamine (CPA), the culture medium was collected at 18, 30 and 42 h after infection, respectively. Virus RNA was extracted using TIAN amp Virus RNA Kit (TIANGEN, China), and the copy number of virus RNA was quantified using One Step Prime Script RT-PCR Kit (TakaRa, Japan) and Quant Studio 6 PCR instrument (Thermo, USA).

[0133] The primers used are as follows:

[0134] SEQ ID NO. 10: ZIKV-E-F-Taq (5'-GGTCAGCGTCCTCTCTAATAAACG-3'),

[0135] SEQ ID NO. 11: ZIKV-E-R-Taq (5'-GCACCCTAGTGTCCACTTTTTCC-3'),

[0136] Probe (5'-6-FAM-AGCCATGACCGACACCACACCGT-BHQ1-3'). The standard curve was prepared using a plasmid encoding ZIKV E gene.

[0137] 2) In U251 cells with or without 5uMOL of cyclopamine (CPA), the culture medium was collected at 18, 30 and 42 h after infection, respectively. The supernatant was centrifuged, quantified by BCA and run electrophoresis, and ZIKV E rabbit antibody, anti-ZIKV NS1 mouse antibody and GAPDH rabbit antibody were used as primary antibody. HRP-labeled anti-mouse IgG or rabbit IgG antibody (1:3000) was used as secondary antibody.

[0138] Experimental results:

[0139] The results are shown in Figure 9The data of ZIKV copy number and ZIKV protein in the cell supernatant reflect the inhibitory effect of CPA on the proliferation of ZIKV. As can be seen from the figure, at 30h, the inhibitory effect of CPA on ZIKV is significant.

[0140] The amino acid sequence of the ATP2A2b protein SEQ ID NO. 12 is:

[0141] MENAHTKTVEEVLGHFGVNESTGLSLEQVKKLKERWGSNELPAE

[0142] EGKTLLELVIEQFEDLLVRILLLAACISFVLAWFEEGEETITAFVEPFVILLILVANAIVGVWQERNAENAIEALKEYEPEMGKVYRQDRKSVQRIKAKDIVPGDIVEIAVGDKVPADIRLTSIKSTTLRVDQSILTGESVSVIKHTDPVPDPRAVNQDKKNMLFSGTNIAAGKAMGVVVATGVNTEIGKIRDEMVATEQERTPLQQKLDEFGEQLSKVISLICIAVWIINIGHFNDPVHGGSWIRGAIYYFKIAVALAVAAIPEGLPAVITTCLALGTRRMAKKNAIVRSLPSVETLGCTSVICSDKTGTLTTNQMSVCRMFILDRVEGDTCSLNEFTITGSTYAPIGEVHKDDKPVNCHQYDGLVELATICALCNDSALDYNEAKGVYEKVGEATETALTCLVEKMNVFDTELKGLSKIERANACNSVIKQLMKKEFTLEFSRDRKSMSVYCTPNKPSRTSMSKMFVKGAPEGVIDRCTHIRVGSTKVPMTSGVKQKIMSVIREWGSGSDTLRCLALATHDNPLRREEMHLEDSANFIKYETNLTFVGCVGMLDPPRIEVASSVKLCRQAGIRVIMITGDNKGTAVAICRRIGIFGQDEDVTSKAFTGREFDELNPSAQRDACLNARCFARVEPSHKSKIVEFLQSFDEITAMTGDGVNDAPALKKAEIGIAMGSGTAVAKTASEMVLADDNFSTIVAAVEEGRAIYNNMKQFIRYLISSNVGEVVCIFLTAALGFPEALIPVQLLWVNLVTDGLPATALGFNPPDLDIMNKPPRNPKEPLISGWLFFRYLAIGCYVGAATVGAAAWWFIAADGGPRVSFYQLSHFLQCKEDNPDFEGVDCAIFESPYPMTMALSVLVTIEMCNALNSLSENQSLLRMPPWENIWLVGSICLSMSLHFLILYVEPLPLIFQITPLNVTQWLMVLKISLPVILMDETLKFVARNYLEPGKECVQPATKSCSFSACTDGISWPFVLLIMPLVIWVYSTDTNFSDMFWS

[0143] The above further describes the present application in detail in connection with specific preferred embodiments, and is not intended to limit the specific embodiments of the present application to only these, and for those skilled in the art of the present application, without departing from the present application, a number of simple deductions or substitutions can also be made, which should be considered to belong to the present application, and the scope of patent protection is determined by the claims submitted.

Claims

1. Use of shRNA in the preparation of a drug against Zika virus, characterized in that, The shRNA is three, the targeted nucleotide sequence is respectively as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.

3. 2.The application of the shRNA according to claim 1 in the preparation of an anti-Zika virus drug, characterized in that, The shRNA is inserted in a p-GIPZ vector.

Images