Anti-Zika virus target inhibitor, recombinant gene, expression vector and application

Abstract

The invention discloses an anti-Zika virus target inhibitor, a recombinant gene, an expression vector and application, and belongs to the technical field of antiviral drugs and molecular targeting. 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 of a Zika virus inhibitor; a recombinant vector and an inhibitor are designed to accurately target ATP2A2b, so that the potential of virus drug resistance is avoided, and the side effect on a host is reduced; the cyclopiazonic acid serving as a small molecule compound of the ATP2A2b protein inhibitor can be used for inhibiting in-vitro infection of Zika virus; effectiveness and safety are improved through targeted therapy of the Zika virus.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drugs and molecular targeting, and particularly relates to a target inhibitor, recombinant gene, expression vector and application for anti-Zika virus. Background Art

[0002] Zika virus (ZIKV) is a flavivirus associated with neurological diseases. Pregnant women infected with ZIKV can lead to microcephaly or abnormal neuronal development in infants, while adult infection can lead to neurological diseases such as Guillain-Barré 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), as well as drugs approved by the US Food and Drug Administration, such as nicotinamide, nicotinamide, mefloquine, bromocriptine (5-7), etc., have been found to inhibit ZIKV infection. However, the safety of small molecule drugs for pregnant women requires more extensive evaluation.

[0003] 2A10G is a murine antibody that has broad flavivirus neutralizing activity by targeting the ZIKV E protein and 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 they lack the ability to penetrate the blood-brain barrier and cannot target nerve tissues. Therefore, treatment strategies targeting host factors have become a research hotspot. For example, AXL receptor (11), ER membrane complex EMC (12) are host protein targets, and inhibiting their expression can inhibit infection, but only partially inhibit the infection effect; RIG-I / MAVS pathway (13), ZIKV NS5-STAT2 (14) mechanism regulates through host innate immunity, but IFN treatment is prone to cause excessive inflammatory reactions; inhibiting the lipid metabolism of infected hosts can also reduce the viral load, but it affects the functions of normal cells. More than 50% of the functions of ZIKV-interacting host proteins are unknown, so those skilled in the art have been committed to studying host factor targets for antiviral treatment.

[0004] References:

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[0007] 3. Zmurko J, Marques RE, Schols D, Verbeken E, Kaptein SJ, Neyts J. 2016. The Viral Polymerase Inhibitor 7 - Deaza - 2'-C - Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model. PLoS Negl Trop Dis 10:e0004695. http: / / doi.org / 10.1371 / journal.pntd.0004695

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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

[0012] 8. Dai L, Song J, Lu X, Deng YQ, Musyoki AM, Cheng H, Zhang Y, Yuan Y, Song H, Haywood J, Xiao H, Yan J, Shi Y, Qin CF, Qi J, Gao GF. 2016. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host Microbe 19:696-704. http: / / doi.org / 10.1016 / j.chom.2016.04.013

[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 Ablation of STAT2 by Zika virus to antagonize interferon signalling. Emerg Microbes Infect 10:1609-1625. http: / / doi.org / 10.1080 / 22221751.2021.1964384. Summary of the Invention

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

[0020] The present invention is achieved by the following technical solutions:

[0021] A recombinant gene against Zika virus, which 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 respectively.

[0022] An expression vector, which is a p-GIPZ vector inserted with the recombinant gene as described in claim 1.

[0023] Furthermore, it contains an expression cassette for expressing the recombinant gene.

[0024] Application of the recombinant gene against Zika virus in inhibiting the replication and proliferation of Zika virus.

[0025] Application of the described expression vector in inhibiting Zika virus replication and proliferation.

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

[0027] Application of the described target inhibitor against Zika virus in inhibiting Zika virus replication and proliferation.

[0028] The principle of the present invention is as follows:

[0029] When the host is infected with ZIKV, the endoplasmic reticulum (ER) is the central hub for flavivirus replication and assembly, participating in the synthesis, processing and immune escape of viral proteins, and involving the entire life cycle of ZIKV, including entry, replication, assembly and budding. Among them, ATP2A2b is a sarcoplasmic / endoplasmic reticulum Ca 2+ -ATPase, which maintains a low cytoplasmic calcium level and calcium ion homeostasis. Abnormal function of it can lead to imbalance of intracellular Ca 2+ homeostasis, causing ER stress imbalance and further leading to disease occurrence. ZIKV infection leads to an increase in intracellular Ca 2+ , ultimately resulting in neuronal cell death, indicating that the imbalance of intracellular Ca 2+ homeostasis caused by ZIKV infection is not corrected in time, suggesting that the function of ATP2A2b may be impaired. Impaired function of ATP2A2b can lead to Ca 2+ homeostasis imbalance and depletion of Ca 2+ in the ER lumen, thereby mediating the occurrence of ER stress. Therefore, ATP2A2b plays a key role in cellular Ca 2+ homeostasis, and ATP2A2b can be used as an important target for the design of anti-Zika virus drugs.

[0030] The beneficial effects of the present invention compared with the prior art are as follows:

[0031] Through the structural analysis of ATP2A2b, the present invention reveals that the calcium ion regulatory protein ATP2A2b on the endoplasmic reticulum can be used as a target for ZIKV inhibitors; and recombinant vectors and inhibitors are designed to precisely target ATP2A2b, avoiding the potential of virus drug resistance and reducing side effects on the host.

[0032] The present invention inhibits ZIKV infection through the ATP2A2b protein-induced host ER stress mechanism. Experiments have shown that knocking down the expression of ATP2A2b will inhibit the replication and proliferation of ZIKV and reduce cell death; an endoplasmic reticulum (RSV) inhibitor: cyclopiazonic acid, a small molecule compound as an ATP2A2b protein inhibitor, can be used to inhibit ZIKV in vitro infection. Brief Description of the Drawings

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

[0034] Figure 2 To inhibit virus proliferation by knocking down ATP2A2b, where (2a) is the expression level of ATP2A2b mRNA in the shRNA and shATP2A2b cell lines after ZIKV infection, and (2b) is the expression level of viral mRNA in the shRNA and shATP2A2b cell lines after ZIKV infection;

[0035] Figure 3 Is the expression level of viral protein in the shRNA and shATP2A2b cell lines after ZIKV infection;

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

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

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

[0039] Figure 7 Is the expression level of viral protein in the Vector and ATP2A2b cell lines after ZIKV infection;

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

[0041] Figure 9 Is the inhibitory effect diagram of cyclopiazonic acid on ZIKV at the cellular level, where (9a) is the expression level of viral mRNA, and (9b) is the expression level of viral protein. Specific implementation mode

[0042] To make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clear and understandable, the present invention will be 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 invention and are not used to limit the present invention. The technical solutions of the present invention will be described in detail below in combination with embodiments and drawings, but the protection scope is not limited by this.

[0043] Experimental materials

[0044] Vero (ATCC, #CCL-81), 293T (ATCC, #CRL-3216), and U251 cells were all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell growth medium was DMEM containing 10% FBS, and the cell maintenance medium was DMEM containing 2% FBS. The ZIKV strain SZ01 / 2016 (GenBank accession number: KU866423) was isolated from a patient returning from Samoa. The virus was amplified in Vero cells and used for in vitro and in vivo experiments. The original virus titer was determined by the 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) The expression of ATP2A2b was specifically knocked down using three lentiviral vectors p-GIPZ expressing short hairpin RNA (shRNA). 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 the packaging plasmids psPAX2 and pLP / VSVG were co-transfected into HEK 293T cells using Vigofect transfection reagent (Vigorous Biotechnology) to package lentivirus;

[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) At 48 h after transfection, the supernatant containing infectious lentivirus was collected and added to U251 cells. After 2 h, complete medium was supplemented, and the medium was changed 24 h later.

[0054] 3) Subsequently, after 48 h, the infected cells were screened with 5 μg / ml puromycin (Selleck, Shanghai, China). The selected cell line was resistant to puromycin and was cultured for amplification;

[0055] 3) After 14 days, cell samples were collected, and the knockdown effect on the expression of the target gene ATP2A2b was verified by Western blot and qPCR; the gene primers used were 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] The results are shown in Figure 1 ; it can be shown by the qPCR and WB results that the shATP2A2b cell line was successfully constructed. As can be seen from the figure, compared with the control group of shRNA cells, the mRNA and protein expressions of ATP2A2b in the shATP2A2b group were reduced.

[0062] Example 2

[0063] Inhibitory effect of ATP2A2b knockdown on virus at the cell mRNA level

[0064] Experimental method:

[0065] 1) Take shATP2A2b cells with good growth status for digestion and subculture, inoculate them in a 12-well plate at 2 mL / well, and incubate them in an incubator at 37 °C and 5% CO2 for 24 h;

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

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

[0068] The primers used were 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] Experimental results:

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

[0077] Example 3

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

[0079] Experimental method:

[0080] 1) Digest and passage the shATP2A2b cells in good growth state, inoculate them in a 6-well plate at 2 mL / well, and incubate them in an incubator at 37 °C with 5% CO2 for 24 h;

[0081] 2) After 24 h, discard the supernatant, and treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture them at 37 °C with 5% carbon dioxide.

[0082] 3) Collect cell lysate samples at 24, 48, and 72 h after infection respectively, centrifuge to obtain the supernatant, perform electrophoresis after BCA quantification, and 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, Abcam), or monoclonal GAPDH rabbit antibody (1:1000, Protein Tech) as the primary antibody.

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

[0084] Experimental results:

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

[0086] Example 4

[0087] Antiviral activity of ATP2A2b knockdown cell line

[0088] Experimental method:

[0089] 1) Digest and passage the shATP2A2b cells in good growth state, inoculate them in a 96-well plate at 100 μL / well, and incubate them in an incubator at 37 °C with 5% CO2 for 24 h;

[0090] 2) After 24 h, discard the supernatant, and treat the cells with 0.1 MOI of ZIKV / SZ01 or 2% FBS, and continue to culture them at 37 °C with 5% CO2.

[0091] 3) At 48 h post-infection, microscopic photographs were taken to observe the cytopathic effect, and at 24 h, 48 h, and 72 h respectively, the CCK8 reagent was used for cell viability detection.

[0092] Experimental results:

[0093] The results are shown in Figure 4 . It can be observed from the microscopic pictures in the figure that a large number of vacuolizations appeared in the infected shRNA cells, with blurred boundaries, while the CPE phenomenon of the shATP2A2b cells was weakened. At the same time, the CCK8 results in the right figure showed that after knocking down ATP2A2b, virus proliferation was inhibited and cell death decreased. Thus, it can be seen that knocking down ATP2A2b inhibits ZIKV proliferation.

[0094] Example 5

[0095] Construction of ATP2A2b overexpression cell line

[0096] Experimental method:

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

[0098] 2) At 48 h after infection, the infected cells were screened with 250 μg / mL hygromycin B (Beyotime, Shanghai). After two weeks, verification was performed by Western Blot and qPCR.

[0099] 3) Subsequently, 3 sgRNAs were constructed into the lentiSAMv2-Blasticidin plasmid, and the lentivirus packaged with the empty plasmid LentisAMv2-Blasticidin was set as the negative control for overexpressing ATP2A2b. There were a total of four plasmids.

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

[0101] 5) At 48 h after infection, the infected cells were screened with 250 μg / mL hygromycin B and 1.5 μg / mL blast (MCE).

[0102] 6) After 14 days, the U251 cells expressing ATP2A2b were verified by Western Blot and qPCR.

[0103] Experimental results:

[0104] The results are shown in Figure 5 Figure 5 . The successful construction of the ATP2A2b cell line was verified by qPCR and WB results. It can be seen from the figure that the mRNA and protein expressions of ATP2A2b in the ATP2A2b group increased, about six times that of the control group Vector. Therefore, the overexpression cell line of ATP2A2b was successfully constructed.

[0105] Example 6

[0106] Overexpression of ATP2A2b promotes virus proliferation at the cellular mRNA level

[0107] Experimental method:

[0108] 1) Digest and passage the ATP2A2b cells with good growth status, inoculate them in a 12-well plate, 2 mL / well, and incubate them in an incubator at 37 °C and 5% CO2 for 24 h;

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

[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 gDNA Eraser kit (TaKaRa, Japan) for reverse transcription, and the cDNA template is prepared into a 20 μL system with TB Green Premix Ex Taq TM II kit and perform real-time quantitative PCR reaction system on a Quant Studio 6 PCR instrument (Thermo, USA).

[0111] Experimental results:

[0112] The results are shown in Figure 6 Figure 6 . It can be seen from the qPCR data results that as the infection time increases, compared with the control group Vector cells, the expression of ATP2A2b in ATP2A2b cells increases, especially at 48 h, and the ZIKV mRNA level reaches the maximum at 72 h. This shows that ATP2A2b promotes ZIKV replication at the cellular mRNA level.

[0113] Example 7

[0114] Overexpression of ATP2A2b promotes the proliferation of the virus at the cellular protein level

[0115] Experimental method:

[0116] 1) Digest and passage the ATP2A2b cells in good growth state, inoculate them in a 6-well plate at 2 mL / well, and incubate them in an incubator at 37 °C and 5% CO₂;

[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 them at 37 °C and 5% CO₂.

[0118] 3) Collect the cell lysate samples at 24 h, 48 h, and 72 h after infection respectively, centrifuge to obtain the supernatant, perform 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 is used as the secondary antibody.

[0119] Experimental results:

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

[0121] Example 8

[0122] Antiviral activity of the ATP2A2b cell line

[0123] Experimental method:

[0124] 1) Digest and passage the ATP2A2b cells in good growth state, inoculate them in a 96-well plate at 100 μL / well, and incubate them in an incubator at 37 °C and 5% CO₂ 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 them at 37 °C and 5% carbon dioxide.

[0126] 3) At 48 h after infection, use a microscope to take pictures to observe the cytopathic effect, and at 24 h, 48 h, and 72 h respectively, use the CCK8 reagent to detect the cell viability.

[0127] Experimental results:

[0128] The results are shown in Figure 8As can be seen from the microscopic images, with the increase of the infection time, a large number of vacuolizations appeared in the infected cells, and the boundaries were blurred, while the CPE phenomenon of ATP2A2b cells was significantly enhanced. At the same time, the CCK8 results showed that ATP2A2b promoted the proliferation of ZIKV and increased cell death.

[0129] Example 9

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

[0131] Experimental method:

[0132] 1) In U251 cells with or without cyclopiazonic acid (CPA), the culture supernatants were collected at 18, 30, and 42 h post-infection, respectively. Viral RNA was extracted using the TIANamp Virus RNA Kit (TIANGEN, China), and the viral RNA copy number was quantified using the One Step Prime Script RT-PCR Kit (TakaRa, Japan) and the Quant Studio 6 PCR instrument (Thermo, USA).

[0133] The primers used were 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'). A standard curve was prepared using the plasmid encoding the ZIKV E gene.

[0137] 2) In U251 cells with or without 5 μM of cyclopiazonic acid (CPA), the culture supernatants were collected at 18, 30, and 42 h post-infection, respectively. After centrifugation, the supernatants were taken, quantified by BCA and then electrophoresed. Rabbit anti-ZIKV E antibody, mouse anti-ZIKV NS1 antibody, and rabbit anti-GAPDH antibody were used as primary antibodies. HRP-labeled anti-mouse IgG or rabbit IgG antibody (1:3000) was used as the secondary antibody.

[0138] Experimental results:

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

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

[0141] MENAHTKTVEEVLGHFGVNESTGLSLEQVKKLKERWGSNELPAE

[0142] EGKTLLELVIEQFEDLLVRILLLAACISFVLAWFEEGEETITAFVEPFVILLILVANAIVGVWQERNAENAIEALKEYEPEMGKVYRQDRKSVQRIKAKDIVPGDIVEIAVGDKVPADIRLTSIKSTTLRVDQSILTGESVSVIKHTDPVPDPRAVNQDKKNMLFSGTNIAAGKAMGVVVATGVNTEIGKIRDEMVATEQERTPLQQKLDEFGEQLSKVISLICIAVWIINIGHFNDPVHGGSWIRGAIYYFKIAVALAVAAIPEGLPAVITTCLALGTRRMAKKNAIVRSLPSVETLGCTSVICSDKTGTLTTNQMSVCRMFILDRVEGDTCSLNEFTITGSTYAPIGEVHKDDKPVNCHQYDGLVELATICALCNDSALDYNEAKGVYEKVGEATETALTCLVEKMNVFDTELKGLSKIERANACNSVIKQLMKKEFTLEFSRDRKSMSVYCTPNKPSRTSMSKMFVKGAPEGVIDRCTHIRVGSTKVPMTSGVKQKIMSVIREWGSGSDTLRCLALATHDNPLRREEMHLEDSANFIKYETNLTFVGCVGMLDPPRIEVASSVKLCRQAGIRVIMITGDNKGTAVAICRRIGIFGQDEDVTSKAFTGREFDELNPSAQRDACLNARCFARVEPSHKSKIVEFLQSFDEITAMTGDGVNDAPALKKAEIGIAMGSGTAVAKTASEMVLADDNFSTIVAAVEEGRAIYNNMKQFIRYLISSNVGEVVCIFLTAALGFPEALIPVQLLWVNLVTDGLPATALGFNPPDLDIMNKPPRNPKEPLISGWLFFRYLAIGCYVGAATVGAAAWWFIAADGGPRVSFYQLSHFLQCKEDNPDFEGVDCAIFESPYPMTMALSVLVTIEMCNALNSLSENQSLLRMPPWENIWLVGSICLSMSLHFLILYVEPLPLIFQITPLNVTQWLMVLKISLPVILMDETLKFVARNYLEPGKECVQPATKSCSFSACTDGISWPFVLLIMPLVIWVYSTDTNFSDMFWS

[0143] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific embodiments of the present invention are limited thereto. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the present invention, several simple deductions or substitutions can still be made, and all should be regarded as falling within the patent protection scope determined by the claims submitted for the present invention.

Claims

1. A recombinant gene against Zika virus, characterized in that, 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 respectively.

2. An expression vector, characterized in that, The expression vector is a p-GIPZ vector into which the recombinant gene as described in claim 1 is inserted.

3. An expression vector according to claim 2, characterized in that, It contains an expression cassette for expressing the recombinant gene.

4. Use of a recombinant gene against Zika virus as described in claim 1 in inhibiting the replication and proliferation of Zika virus.

5. Use of an expression vector as described in claim 2 or 3 in inhibiting the replication and proliferation of Zika virus.

6. A target inhibitor against Zika virus, characterized in that, The target inhibitor is cyclopiazonic acid.

7. Use of a target inhibitor against Zika virus as described in claim 6 in inhibiting the replication and proliferation of Zika virus.

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