WO2020237909A1 - 一种可用于埃博拉病毒病治疗的靶点 - Google Patents

一种可用于埃博拉病毒病治疗的靶点 Download PDF

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WO2020237909A1
WO2020237909A1 PCT/CN2019/105871 CN2019105871W WO2020237909A1 WO 2020237909 A1 WO2020237909 A1 WO 2020237909A1 CN 2019105871 W CN2019105871 W CN 2019105871W WO 2020237909 A1 WO2020237909 A1 WO 2020237909A1
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akip1
ebola virus
creb
expression
pka
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French (fr)
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曹诚
杨莎莎
朱林
靳彦文
高婷
刘萱
张部昌
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中国人民解放军军事科学院军事医学研究院
安徽大学
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • A61P31/14—Antivirals for RNA viruses
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention relates to the field of biomedicine, in particular to a target that can be used for the treatment of Ebola virus disease.
  • Ebola virus disease is also known as Ebola hemorrhagic fever.
  • the pathogen of the disease is Ebola virus.
  • Ebola virus is a filovirus, a single-stranded negative-stranded RNA virus with an envelope that can cause acute hemorrhagic infectious diseases in humans and non-human primates, with a mortality rate of about 50%-90%.
  • the 2013-2016 Ebola epidemic caused 28,000 infections and 11,000 deaths worldwide.
  • the Ebola epidemic broke out in West Africa again. So far, 1206 infections and 764 deaths have been reported, with a mortality rate of about 60% (as of April 10, 2019).
  • my country and West African countries have increasingly close cooperation and exchanges. In order to protect the health and safety of entry-exit personnel and reduce the risk of importing Ebola cases, it is very necessary to stock up on the prevention and treatment of Ebola virus.
  • the sequence of the Ebola virus genome is the 3'non-coding region-NP-VP35-VP40-GP-VP30-VP24-L-5' non-coding region, which can encode nucleoprotein NP, viral particle protein VP35, matrix protein VP40, There are 7 structural proteins in glycoprotein GP, VP30, VP24, RNA-dependent RNA polymerase L.
  • Ebola virus initially targets macrophages and dendritic cells, it can eventually infect all types of cells except lymphocytes.
  • VP35 participates in virus replication and immune evasion in many ways. Recent studies have shown that VP35 can inhibit the production of type I interferon, destroy the RNA silencing effect, and play an important role in virus replication.
  • kinase interacting protein 1 is a protein originally found in breast and prostate cancer cell lines. AKIP1 consists of 210 amino acids encoded by five exons and has a molecular weight of 23kDa. AKIP1 is a protein that regulates the PKA signaling pathway. It acts as an adaptor or structural intracellular protein, and promotes the introduction of the PKA catalytic subunit into the nucleus by interacting with the amino terminus of the human protein kinase A (PKA) catalytic subunit. PKA, also known as cAMP-dependent protein kinase A, is an effector protein of cAMP signal, which is activated after cAMP stimulation.
  • PKA also known as cAMP-dependent protein kinase A
  • PKA activation can lead to phosphorylation of a series of substrates.
  • phosphorylation of CREB can promote transcription such as Bcl-2, CyclinA&D, IL-2, IL-6, etc., thereby regulating cell growth, A series of cell activities such as metabolism and immunity.
  • the purpose of the present invention is to provide a target that can be used for the treatment of Ebola virus disease.
  • the present invention claims the application of PKA, AKIP1 and/or CREB as targets in any of the following:
  • the present invention claims the application of PKA, AKIP1 and/or CREB as targets in screening candidate drugs for the prevention and treatment of Ebola virus disease.
  • the present invention claims the application of substances capable of inhibiting PKA expression, substances capable of inhibiting AKIP1 expression, and/or substances capable of inhibiting CREB expression in any of the following:
  • the substance capable of inhibiting the expression of PKA can be any substance capable of inhibiting the expression of PKA.
  • the substance capable of inhibiting the expression of PKA may be a PKA inhibitor.
  • the substance capable of inhibiting the expression of PKA is specifically a PKA inhibitor-H89.
  • H89 2HCl which is an effective PKA inhibitor.
  • the chemical formula is C 20 H 20 BrN 3 O 2 S. 2HCl, the molecular weight is 519.28, and the structural formula is shown in Formula I.
  • the substance capable of inhibiting the expression of AKIP1 may be any substance capable of inhibiting the expression of AKIP1.
  • the substance capable of inhibiting the expression of AKIP1 may be a substance that knocks down the expression of AKIP1 or a substance that knocks down the expression of AKIP1.
  • the substance that knocks down AKIP1 expression may be AKIP1 siRNA.
  • the AKIP1 siRNA is specifically an siRNA formed by annealing of two single strands shown in SEQ ID No. 1 and SEQ ID No. 2.
  • the substance for knocking out AKIP1 expression can be a gene editing tool for knocking out AKIP1 expression.
  • the gene editing tool is CRISPR/Cas9 nuclease
  • the target sequence for specific cleavage is specifically SEQ ID No. 3 or SEQ ID No. 4.
  • the substance that can inhibit the expression of CREB can be any substance that can inhibit the expression of CREB.
  • the substance capable of inhibiting the expression of CREB may be a CREB inhibitor.
  • the substance capable of inhibiting the expression of CREB is specifically a CREB inhibitor-666-15 or KG-501.
  • 666-15 is a potent and selective CREB inhibitor with an IC 50 value of 81 nM.
  • the structural formula is shown in formula II.
  • the structural formula is shown in formula III.
  • the PKA may be specifically the protein shown in SEQ ID No. 5.
  • the AKIP1 may be specifically the protein shown in SEQ ID No. 6.
  • the CREB may be specifically the protein shown in SEQ ID No. 7.
  • the present invention claims any of the following methods:
  • Method A A method to treat Ebola virus infection, using PKA, AKIP1 and/or CREB as targets to treat Ebola virus infection;
  • Method B A method for the treatment of Ebola virus disease, using PKA, AKIP1 and/or CREB as targets to treat Ebola virus disease;
  • Method C A method of inhibiting Ebola virus replication, using PKA, AKIP1 and/or CREB as targets to inhibit Ebola virus replication;
  • Method D A method for inhibiting the proliferation of Ebola virus in cells, using PKA, AKIP1 and/or CREB as targets to inhibit the proliferation of Ebola virus in cells;
  • Method E A method for screening candidate drugs for the prevention and treatment of Ebola virus disease, using PKA, AKIP1 and/or CREB as targets to screen candidate drugs for the prevention and treatment of Ebola virus disease.
  • the method includes the following steps: administering a substance capable of inhibiting the expression of PKA, a substance capable of inhibiting the expression of AKIP1 and/ Or substances that can inhibit the expression of CREB.
  • the substance capable of inhibiting the expression of PKA the substance capable of inhibiting the expression of AKIP1
  • the substance capable of inhibiting the expression of CREB are further defined in the foregoing.
  • Figure 1 shows the interaction between VP35 and AKIP1 by immunoprecipitation and immunoblotting.
  • lysate refers to the whole cell lysate, the cell product before IP.
  • Figure 2 shows the immunofluorescence staining of VP35 to promote the nuclear localization of PKA.
  • Figure 3 shows the Western blot detection of VP35 promoting CREB phosphorylation.
  • Figure 4 shows the dual luciferase reporter system detecting the level of VP35 promoted CREB transcription.
  • Figure 5 shows that H89 significantly inhibits the proliferation of Ebola virus-like particles trVLP.
  • a and B are PKA inhibitor H89 added to p1 and p2 cells to detect virus proliferation.
  • Figure 6 shows that knocking down or knocking out AKIP1 will significantly inhibit the proliferation of Ebola virus-like particles trVLP.
  • A is the verification of the effect of knocking down AKIP1 with AKIP1 siRNA;
  • B is the identification of Ebola virus proliferation in cells after knocking down AKIP1 with AKIP1 siRNA;
  • C and D are the endogenous AKIP1 knockout cell lines AKIP1KO1 and AKIP1KO2 of HepG2, respectively Transfect the minimal genome system related plasmids to detect the proliferation of the virus in p1 and p0 cells.
  • Figure 7 shows that 666-15 and KG-501 significantly inhibit the proliferation of Ebola virus-like particles trVLP.
  • a and B are the addition of CREB inhibitor 666-15 to p0 and p1 cells to detect virus proliferation.
  • C is the addition of CREB inhibitor KG-501 to p1 cells to detect virus proliferation.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.
  • amino acid sequence of the PKA protein involved in the following examples is shown in SEQ ID No. 5; the amino acid sequence of the AKIP1 protein is shown in SEQ ID No. 6; the amino acid sequence of the CREB protein is shown in SEQ ID No. 7.
  • HEK293 cells, HeLa cells and HepG2 cells are all preserved in our laboratory. Plasmids pcDNA3.0-Flag-VP35, pEGFP-VP35, pCMV-Myc-AKIP1, pGL3-CRE-Luc are all constructed in this study, pcDNA3.0-Flag-VP35, pEGFP-VP35, pCMV-Myc-AKIP1 and pGL3-CRE -Luc are all constructed in this study. The nucleotide sequence of VP35 is shown in SEQ ID No. 8.
  • the sequence together with the Flag tag was cloned into the pcDNA3.0 vector through the BamH I and EcoR I restriction sites to obtain the recombinant vector pcDNA3.0- Flag-VP35.
  • the VP35 sequence (SEQ ID No. 8) was cloned into the pEGFP-C1 vector through EcoRI and BamH I restriction sites to obtain the recombinant vector pEGFP-VP35.
  • the nucleotide sequence of AKIP1 is shown in SEQ ID No. 9. The sequence was cloned into pCMV-Myc vector through restriction sites Xho I and Not I, and then the recombinant vector pCMV-Myc-AKIP1 was obtained.
  • the adenovirus Ad-VP35 is a sequence of SEQ ID No. 8 cloned into an adenovirus vector for expression and purification, with a purity of 1 ⁇ 10 11 PFU/ml, and Beijing Biotech Co., Ltd. is entrusted to carry out virus amplification.
  • Plasmids pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, p4cis-vRNA-Rluc, pCAGGS-T7, pCAGGS-Tim1 are all described in "Hoenen T,et al. Modeling The Lifecycle Of Ebola Virus Level Under 2 Biosafety With Virus-like Particles Containing Tetracistronic Minigenomes.J Vis Exp, 2014", the public can obtain it from the applicant, and it can only be used to repeat the experiment of the present invention, not for other purposes.
  • High-fidelity DNA polymerase KOD FX Neo, cDNA Reverse Mix, SYBR Green Mix are products of Toyobo (TOYOBO); dual luciferase detection kits are products of Promega; transfection reagent Lipofectamine 3000 is a product of Thermo; protease inhibitors Cocktail is a product of Roche; DMEM medium, RPMI1640 medium, MEM medium, NEAA and FBS are all GIBCO products; Forskolin (FSK) product number S2449, H89 product number S1582 are all products of Selleck; 666-15 (product number HY-101120 ) Is a product of MCE. KG-501 (product number S8409) is a product of Selleck.
  • HRP-labeled anti-Flag antibody FLP-HRP
  • HRP-labeled anti-Myc antibody Myc-HRP
  • anti-AKIP1 antibody is a product of Sigma
  • anti-CREB and anti-pCREB-133 antibodies are Abcam products
  • anti-Flag agarose beads and anti-Myc agarose beads are products of Sigma.
  • the initial voltage is set to 80V, after bromophenol blue migrates to the separation gel, adjust the voltage to 120V, continue electrophoresis until bromophenol blue migrates to the bottom of the gel to stop electrophoresis; use methanol for PVDF membrane Activate for 30 seconds, and then soak the filter paper in 1 ⁇ transfer buffer (Tris-HCl 24mM, glycine 5mM, 20% methanol) for 20 minutes; after the electrophoresis, place it in the halfway in the order of filter paper-glue-membrane-filter paper from top to bottom.
  • 1 ⁇ transfer buffer Tris-HCl 24mM, glycine 5mM, 20% methanol
  • the results are shown in Figure 1.
  • the Myc-AKIP1 band can be detected by immunoprecipitation using Flag agarose beads, while the Myc-AKIP1 band can not be detected by the immunoprecipitation and IgG immunoprecipitation of the control Flag carrier.
  • the results indicate that Ebola virus protein VP35 interacts with AKIP1.
  • FSK and H89 are soluble in DMSO.
  • the experiment also set up a control with the same amount of DMSO instead of FSK and H89.
  • the western blot results showed: VP35 promotes the phosphorylation of CREB serine 133 (pCREB-S133), and the presence of FSK promotes the phosphorylation of CREB, while H89 inhibits the phosphorylation of CREB.
  • the three repeats of TGACGTCA in the core region of the CRE promoter were cloned between the multiple cloning sites Xho I and Bgl II of the pGL3-Basic plasmid (Promega; Catalog No. E1751) to obtain the recombinant vector pGL-CRE-Luc.
  • E1910) Middle component Determine the luminescence value 2 (that is, the luminescence value of Renilla luciferase), and finally record the ratio of 1/2 to be the relative luciferase activity.
  • the detection of Ebola virus trVLP is performed by the Renilla luciferase reporter gene system.
  • the operation method is the same as that of the dual luciferase reporter system. The result is to record the luminescence value of Renilla luciferase.
  • Example 5 Using the Ebola virus minimal genome system to detect the replication of Ebola virus-like particles trVLP
  • the present invention uses the Ebola virus minimal genome system, which can express Ebola virus samples under the conditions of biosafety secondary laboratory. Particles (trVLP), through the expression of luciferase to simulate the Ebola virus life cycle (Hoenen T, et al. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes. JVis, 2014 ).
  • the experimental procedure is briefly as follows: on the first day, the virus-producing cells HEK293 cells (or HepG2 cells) (p0 for short) were seeded and cultured in a 6-well plate; on the second day, the plasmids pCAGGS-NP (125ng), pCAGGS-VP35 ( 125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA-Rluc (250ng) and pCAGGS-T7 (250ng) were transfected to p0; on day 3, the p0 supernatant was replaced with 5% FBS Medium; on the 4th day, the virus-targeted cells (p1 for short) were inoculated into a 6-well plate; on the 5th day, the plasmids pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS -L (
  • AKIP1siRNA To construct AKIP1siRNA, add 5 ⁇ l AKIP1siRNA and 3.75 ⁇ l TransIT-X2 reagent (Mirus, catalog number MIR 6000) to 125 ⁇ l opti-MEM serum-free medium, mix well, and place at room temperature for 20 minutes. AKIP1siRNA was transfected into HepG2 cells for 6 hours, washed with 1 ⁇ PBS buffer, replaced with fresh medium, and transfected with p0 cell related plasmids (i.e.
  • AKIP1siRNA is formed by annealing two single strands as follows:
  • AKIP1siRNA was transfected into HepG2 cells and cultured for 36-48h, the cells were harvested for qPCR to detect the transcription level of AKIP1 in the cells.
  • the detection primers are:
  • h-AKIP1-F 5’-AAggCTggCTCTAgAAgTgC-3’;
  • h-AKIP1-R 5'-CTgTTTCTCTAggTggggCg-3'.
  • AKIP1 KO1 and AKIP1 KO2 obtained through CRISPR-Cas9 technology, the sequences targeting AKIP1 are the following two:
  • Target sequence 1 5'-CATGCCCTGGAGCGTCCCAA-3' (SEQ ID No. 3);
  • Target sequence 2 5'-CATGTCTATCGTTATCACAG-3' (SEQ ID No. 4).
  • AKIP1 KO1 is the HepG2 endogenous AKIP1 knockout cell line obtained against target sequence 1
  • AKIP1 KO2 is the HepG2 endogenous AKIP1 knockout cell line obtained against target sequence 2.
  • AKIP1 KO1 and AKIP1 KO2 have been verified and verified that the endogenous AKIP1 of both has been successfully knocked out.
  • the HepG2 endogenous AKIP1 knockout cell lines AKIP1 KO1 and AKIP1 KO2 were transfected with minimal genome system-related plasmids, and the detection method was the same as above. The difference between this step and the above is that the HepG2 AKIP1 knockout cell lines AKIP1 KO1 and AKIP1 KO2 are used. The plasmids are transfected, and the operation and detection are the same as the above.
  • HepG2 cells were transfected with minimal genomic system-related plasmids. After transfection, 1 ⁇ M CREB inhibitor 666-15 was added for 48h or 25 ⁇ M CREB inhibitor KG-501 was added for 20min, then the cells were collected, lysed and RLU was detected. The detection method is the same as The above is the same.
  • the HepG2 endogenous AKIP1 knockout cell lines AKIP1 KO1 and AKIP1 KO2 were transfected with minimal genome system-related plasmids, and it was found that the AKIP1 knockout cell line can significantly inhibit the proliferation of Ebola virus-like particles trVLP in the cells ( Figure 6 C and D).
  • the addition of CREB inhibitors 666-15 and KG-501 to the minimal genome system will significantly inhibit the proliferation of Ebola virus-like particles trVLP ( Figure 7 A to C).
  • the research of the present invention found that the Ebola virus structural protein VP35 interacts with AKIP1 and activates PKA to promote its nuclear entry. And can promote the phosphorylation and transcriptional activity of the downstream transcription factor cAMP-response element binding protein (CREB) of PKA. At the same time, it was proved that the PKA inhibitor H89, knock down or knock out the AKIP1 gene can significantly inhibit the proliferation of Ebola virus in cells.
  • CREB cAMP-response element binding protein
  • the present invention clarifies that VP35-AKIP1 is combined to promote the phosphorylation of the PKA substrate CREB, and it is found that VP35 promotes Ebola virus replication through AKIP1. Therefore, the use of PKA, AKIP1 and CREB as targets provides a new basis for clinical use in the prevention and treatment of Ebola virus, and it will have broad application prospects in the treatment of Ebola virus disease.

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Abstract

一种可用于埃博拉病毒病治疗的靶点。提供了PKA、AKIP1和/或CREB作为靶点在制备具有如下任一功能的产品中的应用：治疗埃博拉病毒感染、治疗埃博拉病毒病、抑制埃博拉病毒复制、抑制埃博拉病毒在细胞中增殖。证明了PKA抑制剂H89、敲低或敲除AKIP1基因、CREB抑制剂666-15和KG-501能够显著抑制埃博拉病毒在细胞中的增殖。探索了PKA、AKIP1和CREB作为靶点在埃博拉病毒治疗中的潜力，为其在临床上用于防治埃博拉病毒提供了新的依据。

Description

一种可用于埃博拉病毒病治疗的靶点 技术领域
本发明涉及生物医药领域，具体涉及一种可用于埃博拉病毒病治疗的靶点。
背景技术
埃博拉病毒病又称埃博拉出血热，该病的病原体为埃博拉病毒。埃博拉病毒属于丝状病毒，是一种有包膜的单股负链RNA病毒，能够引起人类和非人灵长类急性出血性传染病，死亡率约在50％-90％。2013-2016年的埃博拉疫情在全球造成28000例感染，11000例死亡。2018年，埃博拉疫情再次在西非爆发，目前已报道1206例感染，764例死亡，死亡率约60％(截至2019年4月10日)。我国与西非国家合作往来日益密切，为保障出入境相关人员健康和安全，降低埃博拉病例输入风险，储备针对埃博拉病毒的防治药物非常必要。
埃博拉病毒基因组顺序为3’端非编码区-NP-VP35-VP40-GP-VP30-VP24-L-5’端非编码区，可以编码核蛋白NP、病毒粒子蛋白VP35、基质蛋白VP40、糖蛋白GP、VP30、VP24、RNA依赖的RNA聚合酶L共7个结构蛋白。埃博拉病毒尽管最初靶向巨噬细胞与树突状细胞，但最终它能够感染除淋巴细胞以外所有类型的细胞。VP35作为一种RNA聚合酶辅因子，其通过多种方式参与病毒复制和免疫逃避。最近研究结果VP35可以抑制I型干扰素的产生、破坏RNA沉默效应，在病毒复制中具有重要的作用。
A激酶相互作用蛋白1(AKIP1)是最初在乳腺癌和前列腺癌细胞系发现的蛋白质。AKIP1由五个外显子编码的210个氨基酸组成，分子量为23kDa。AKIP1是一种调控PKA信号通路的蛋白，作为衔接子或结构性胞内蛋白发挥作用，通过与人蛋白激酶A(PKA)催化亚基氨基端相互作用促进PKA催化亚基入核。PKA又称cAMP依赖性蛋白激酶A，作为cAMP信号的效应蛋白，接受cAMP刺激后被激活。PKA激活可导致一系列底物磷酸化，以CREB为例，作为一种转录因子，CREB磷酸化可促使诸如Bcl-2、CyclinA&D、IL-2、IL-6、等转录，进而调节细胞生长、代谢、免疫等一系列细胞活动。
发明公开
本发明的目的是提供一种可用于埃博拉病毒病治疗的靶点。
第一方面，本发明要求保护PKA、AKIP1和/或CREB作为靶点在如下任一中的应用：
(A1)制备用于治疗埃博拉病毒感染的产品，或治疗埃博拉病毒感染；
(A2)制备用于治疗埃博拉病毒病的产品，或治疗埃博拉病毒病；
(A3)制备用于抑制埃博拉病毒复制的产品，或抑制埃博拉病毒复制；
(A4)制备用于抑制埃博拉病毒在细胞中增殖的产品，或抑制埃博拉病毒在细胞中增殖。
第二方面，本发明要求保护PKA、AKIP1和/或CREB作为靶点在筛选埃博拉病 毒病防治的候选药物中的应用。
第三方面，本发明要求保护能够抑制PKA表达的物质、能够抑制AKIP1表达的物质和/或能够抑制CREB表达的物质在如下任一中的应用：
(A1)制备用于治疗埃博拉病毒感染的产品，或治疗埃博拉病毒感染；
(A2)制备用于治疗埃博拉病毒病的产品，或治疗埃博拉病毒病；
(A3)制备用于抑制埃博拉病毒复制的产品，或抑制埃博拉病毒复制；
(A4)制备用于抑制埃博拉病毒在细胞中增殖的产品，或抑制埃博拉病毒在细胞中增殖。
其中，所述能够抑制PKA表达的物质可为任何能够抑制PKA表达的物质。
进一步地，所述能够抑制PKA表达的物质可为PKA抑制剂。
在本发明的具体实施方式中，所述能够抑制PKA表达的物质具体为PKA抑制剂——H89。
H89全名为H89 2HCl，是一种有效的PKA抑制剂，化学式为C 20H 20BrN 3O 2S.2HCl，分子量为519.28，结构式如式I所示。
Figure PCTCN2019105871-appb-000001
其中，所述能够抑制AKIP1表达的物质可为任何能够抑制AKIP1表达的物质。
进一步地，所述能够抑制AKIP1表达的物质可为敲除AKIP1表达的物质或敲低AKIP1表达的物质。
更进一步地，所述敲低AKIP1表达的物质可为AKIP1siRNA。
在本发明的具体实施方式中，所述AKIP1siRNA具体为由SEQ ID No.1和SEQ ID No.2所示的两条单链退火形成的siRNA。
更进一步地，所述敲除AKIP1表达的物质可为用于敲除AKIP1表达的基因编辑工具。
在本发明的具体实施方式中，所述基因编辑工具为CRISPR/Cas9核酸酶，其特异性切割的靶序列具体为SEQ ID No.3或SEQ ID No.4。
其中，所述能够抑制CREB表达的物质可为任何能够抑制CREB表达的物质。
进一步地，所述能够抑制CREB表达的物质可为CREB抑制剂。
在本发明的具体实施方式中，所述能够抑制CREB表达的物质具体为CREB抑制剂——666-15或者KG-501。
666-15是有效选择性的CREB抑制剂，IC 50值为81nM。结构式如式II所示。
Figure PCTCN2019105871-appb-000002
KG-501是CREB抑制剂，破坏依赖于CREB的转录(Ki＝10μM)和CREB：CBP的相互作用(Ki＝50μM)。它还能破坏磷酸化CREB(Ser-133)与KIX的结合，Ki约为90μM。结构式如式III所示。
Figure PCTCN2019105871-appb-000003
在上述各方面中，所述PKA均可具体为SEQ ID No.5所示蛋白质。所述AKIP1均可具体为SEQ ID No.6所示蛋白质。所述CREB均可具体为SEQ ID No.7所示蛋白质。
第四方面，本发明要求保护如下任一方法：
方法A：一种治疗埃博拉病毒感染的方法，是以PKA、AKIP1和/或CREB作为靶点治疗埃博拉病毒感染；
方法B：一种治疗埃博拉病毒病的方法，是以PKA、AKIP1和/或CREB作为靶点治疗埃博拉病毒病；
方法C：一种抑制埃博拉病毒复制的方法，是以PKA、AKIP1和/或CREB作为靶点抑制埃博拉病毒复制；
方法D：一种抑制埃博拉病毒在细胞中增殖的方法，是以PKA、AKIP1和/或CREB作为靶点抑制埃博拉病毒在细胞中增殖；
方法E：一种筛选埃博拉病毒病防治的候选药物的方法，是以PKA、AKIP1和/或CREB作为靶点筛选埃博拉病毒病防治的候选药物。
进一步地，在所述方法A、所述方法B、所述方法C和所述方法D中，包括如下步骤：给宿主或宿主细胞施用能够抑制PKA表达的物质、能够抑制AKIP1表达的物质和/或能够抑制CREB表达的物质。
其中，所述能够抑制PKA表达的物质、所述能够抑制AKIP1表达的物质，以及所述能够抑制CREB表达的物质的进一步具体限定可见前文。
附图说明
图1为免疫沉淀检及免疫印迹测VP35与AKIP1的相互作用。图中lysate指的是全细胞裂解液，IP之前的细胞产物。
图2为免疫荧光染色检测VP35促进PKA的细胞核定位。
图3为免疫印迹检测VP35促进CREB的磷酸化。
图4为双荧光素酶报告系统检测VP35促进CREB的转录水平。
图5为H89显著抑制埃博拉病毒样粒子trVLP的增殖。A和B分别为p1和p2细胞中加入PKA抑制剂H89检测病毒增殖。
图6为敲低或敲除AKIP1会显著抑制埃博拉病毒样粒子trVLP的增殖。A为使用AKIP1 siRNA敲低AKIP1效果验证；B为使用AKIP1 siRNA敲低AKIP1后埃博拉病毒在细胞中增殖情况鉴定；C和D分别为向HepG2内源性AKIP1敲除细胞系AKIP1KO1与AKIP1 KO2转染最小基因组系统相关质粒检测病毒在p1和p0细胞中的增殖情况鉴定。
图7为666-15和KG-501显著抑制埃博拉病毒样粒子trVLP的增殖。A和B分别为p0和p1细胞中加入CREB抑制剂666-15检测病毒增殖。C为p1细胞中加入CREB抑制剂KG-501检测病毒增殖。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
以下实施例中的数据定量实验标准差使用SEM，数据间显著性差异采用t-检验(*表示p≤0.05，**表示p≤0.01，**表示p≤0.001)
下述实施例中所涉及的PKA蛋白的氨基酸序列如SEQ ID No.5所示；AKIP1蛋白的氨基酸序列如SEQ ID No.6所示；CREB蛋白的氨基酸序列如SEQ ID No.7所示。
1、细胞株质粒与病毒
HEK293细胞、HeLa细胞、HepG2细胞均为本实验室保存。质粒pcDNA3.0-Flag-VP35、pEGFP-VP35、pCMV-Myc-AKIP1、pGL3-CRE-Luc均为本研究构建pcDNA3.0-Flag-VP35、pEGFP-VP35、pCMV-Myc-AKIP1与pGL3-CRE-Luc均为本研究构建。其中VP35的核苷酸序列如SEQ ID No.8所示，将序列连同Flag 标签(DYKDDDDK)通过BamH I与EcoR I酶切位点克隆到pcDNA3.0载体中，进而得到重组载体pcDNA3.0-Flag-VP35。将VP35序列(SEQ ID No.8)通过EcoRI与BamH I酶切位点克隆到pEGFP-C1载体中，进而得到重组载体pEGFP-VP35。AKIP1的核苷酸序列如SEQ ID No.9所示，将序列通过酶切位点Xho I与Not I克隆到pCMV-Myc载体中，进而得到重组载体pCMV-Myc-AKIP1。腺病毒Ad-VP35为SEQ ID No.8序列克隆到腺病毒载体中进行表达纯化，纯度为1×10 11PFU/ml，委托北京百奥川生物科技有限责任公司进行病毒扩增。
质粒pCAGGS-NP、pCAGGS-VP35、pCAGGS-VP30、pCAGGS-L、p4cis-vRNA-Rluc、pCAGGS-T7、pCAGGS-Tim1均记载于“Hoenen T,et al.Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2Conditions With Virus-like Particles Containing Tetracistronic Minigenomes.J Vis Exp,2014”一文中，公众可从申请人处获得，仅可用于重复本发明实验使用，不得他用。
2、分子生物学试剂与抗体
高保真DNA聚合酶KOD FX Neo、cDNA反转Mix、SYBR Green Mix为东洋纺公司(TOYOBO)产品；双荧光素酶检测试剂盒为Promega公司产品；转染试剂Lipofectamine3000为Thermo公司产品；蛋白酶抑制剂Cocktail为罗氏公司产品；DMEM培养基、RPMI1640培养基、MEM培养基、NEAA与FBS均为GIBCO产品；Forskolin(FSK)货号S2449，H89货号S1582均为Selleck公司产品；666-15(货号HY-101120)为MCE公司产品。KG-501(货号S8409)为Selleck公司产品。
HRP标记的anti-Flag抗体(Flag-HRP)、HRP标记的anti-Myc抗体(Myc-HRP)为Sigma公司产品；anti-AKIP1抗体为Sigma公司产品；anti-CREB、anti-pCREB-133抗体为Abcam公司产品；anti-Flag琼脂糖珠、anti-Myc琼脂糖珠为Sigma公司产品。
实施例1、免疫沉淀与免疫印迹检测VP35与AKIPI的相互作用
以在HEK293细胞中共转染pCMV-Myc-AKIP1和pcDNA3.0-Flag-VP35为例，使用Thermo公司Lipofectamine 3000，按照说明书进行质粒转染：以质粒：P3000＝1:2的比例将1μg pCMV-Myc-AKIP1和1μg pcDNA3.0-Flag-VP35及4μl P3000加入50μl opti-MEM稀释；按质粒：Lipo3000＝1:3即将6μl Lipofectamine3000加入到50μl opti-MEM稀释，将稀释的opti-MEM、稀释的Lipofectamine3000相对应的加入稀释的质粒中混匀，室温放置5-10min，将混合液均匀滴加在细胞培养基中。转染后36-48h后，将转染后的细胞用PBS重悬细胞，清洗2次后，4℃、1000g/min离心3min收集细胞；加入150μl细胞裂解液(150mM NaCl，50mM Tris-HCl pH 8.0，含EDTA蛋白酶抑制剂1片/50ml，1％NP40，如果是磷酸化实验需按照每10ml再加入磷酸酶抑制剂1片)冰上裂解15min后，4℃12000rpm离心10min；将上清转移至1.5ml EP 管中，加入15μl anti-Flag琼脂糖珠，4℃旋转孵育2h进行免疫共沉淀，4℃、1000g离心3min，使用800μl不含蛋白酶抑制剂的细胞裂解液洗涤细胞3次；加入适量1×SDS上样缓冲液，沸水浴5min，4℃16000g/min离心5min，进行SDS-PAGE电泳及免疫沉淀。
取10μl样品进行SDS-PAGE电泳，电压初始设定为80V，待溴酚蓝迁移至分离胶后将电压调整至120V，继续电泳到溴酚蓝迁移至胶的底部停止电泳；将PVDF膜用甲醇激活30s，然后和滤纸在1×转膜缓冲液中(Tris-HCl 24mM，甘氨酸5mM，20％甲醇)浸泡20min；电泳结束后按照从上到下滤纸-胶-膜-滤纸的顺序放置于半干转膜仪上，18V转膜2h；将转膜完成后的PVDF膜室温封闭1h；1×TBST洗涤3次，每次5min(简称洗涤)；加入入Myc-HRP抗体和Flag-HRP抗体，室温孵育1h；洗涤后ECL显影。
结果如图1所示：通过Flag琼脂糖珠进行免疫沉淀，通过免疫印迹实验可检测到Myc-AKIP1条带，而对照Flag载体的免疫沉淀和IgG免疫沉淀则检测不到Myc-AKIP1条带。结果说明埃博拉病毒蛋白VP35与AKIP1存在相互作用。
实施例2、免疫荧光检测PKA的核定位
在HeLa细胞中转染pEGFP-VP35质粒，培养36-48h后将转染后的细胞用PBS洗涤3次，每次5min，吸尽残存液体后，加入4％多聚甲醛37℃固定30min；PBS洗涤后加入0.3％Triton X-100(用1×PBS配制)穿孔15min；PBS洗涤后加入2ml封闭液(含5％山羊血清的1×PBS)37℃封闭30min；PBS洗涤后加入孵育抗PRKACA(PKA催化亚基)抗体(BD Biosciences；货号：610980)(封闭液1:50稀释获得)，4℃孵育过夜；1×PBST洗涤细胞3次，每次10min(简称PBST洗涤)；向细胞上加入TRITC标记的山羊抗小鼠(二抗)(中杉金桥，货号为ZF-0313)(封闭液1:50稀释获得)，室温孵育1h(避光操作)；PBST洗涤后加入10μl含有DAPI(1μg/ml)的封片液，避光静置15min后在激光共聚焦显微镜(Carl Zeiss LSM800)下观察。
结果如图2所示，免疫荧光结果显示VP35存在时，会促进PRKACA的细胞核定位。
实施例3、免疫印迹检测VP35对CREB的磷酸化
用表达VP35的腺病毒Ad-VP35(MOI＝10)及对照Ad-GFP(MOI＝10)感染HepG2细胞；感染40小时后，加入PKA激活剂FSK(25μM)或PKA的抑制剂H89(10μM)处理3小时；细胞裂解后孵育CREB抗体(即anti-CREB抗体)及CREB第133位磷酸化抗体(即anti-pCREB-133抗体)进行免疫印迹。
其中，FSK和H89是溶于DMSO。实验同时设置了加入等量DMSO替代FSK和H89的对照。
结果如图3所示。用VP35的腺病毒Ad-VP35(MOI＝10)及对照Ad-GFP(MOI＝10) 感染HepG2细胞，加入PKA激活剂FSK(25μM)或PKA的抑制剂H89处理三小时，免疫印迹结果显示：VP35会促进CREB第133位丝氨酸(pCREB-S133)的磷酸化，且FSK存在会促进CREB的磷酸化，而H89会抑制CREB的磷酸化。
实施例4、荧光素酶活性检测
将CRE启动子核心区TGACGTCA三个重复序列克隆到pGL3-Basic质粒(Promega；货号E1751)的多克隆位点Xho I与Bgl II之间，得到重组载体pGL-CRE-Luc。
随后，将不同含量的pcDNA3.0-Flag-VP35质粒(0μg、0.2μg、0.4μg与0.8μg)连同表达萤火虫荧光素酶质粒pGL3-CRE-Luc及表达海参荧光素酶的质粒pRL-TK(Promega公司；货号E2241)，按照50:1(质量比)共同转染到HepG2细胞中；转染36h后弃去培养上清(FSK处理组则在转染33h后，加入25μM的FSK处理3h)，1×PBS洗涤2次后，每孔加入100μl 1×PLB裂解液(Promega公司，Dual-Luciferase Report Assay System货号E1910中组分)。在摇床上室温裂解15min，16000g/min离心5min获得细胞裂解上清；在100μl室温平衡的Luciferase Assay ReagentⅡ(Promega公司，Dual-Luciferase Report Assay System货号E1910中组分)中加入20μl细胞裂解上清，轻轻混匀后放入TD-20/20型荧光光度计测定发光值1(即萤火虫萤光素酶的发光值)，随后再加入100μl Stop&Glo Reagent(Promega公司，Dual-Luciferase Report Assay System货号E1910中组分)测定发光值2(即海肾萤光素酶的发光值)，最后记录1/2的比值即为荧光素酶相对活性。埃博拉病毒trVLP检测是通过海肾荧光素酶报告基因系统进行检测，操作方式与双萤光素酶报告系统一致，结果记录海肾萤光素酶的发光值。
结果如图4所示，VP35以剂量依赖的方式增加CREB的转录活性，而FSK处理会进一步增加CREB的转录活性。
实施例5、利用埃博拉病毒最小基因组系统检测埃博拉病毒样颗粒trVLP的复制
上述结果表明VP35通过与AKIP1相互作用，会影响PKA-CREB信号通路。为了探究AKIP1-PKA-CREB通路在埃博拉病毒的增殖中是否发挥重要的作用，本发明使用了埃博拉病毒最小基因组系统，可在生物安全二级实验室条件下表达埃博拉病毒样颗粒(trVLP)，通过荧光素酶的表达来模拟埃博拉病毒生命周期(Hoenen T,et al.Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2Conditions With Virus-like Particles Containing Tetracistronic Minigenomes.J Vis Exp,2014)。
实验操作流程简要如下：第1天，将病毒生产细胞HEK293细胞(或HepG2细胞)(简称p0)接种在6孔板中培养；第2天，将质粒pCAGGS-NP(125ng)、 pCAGGS-VP35(125ng)、pCAGGS-VP30(75ng)、pCAGGS-L(1000ng)、p4cis-vRNA-Rluc(250ng)与pCAGGS-T7(250ng)转染到p0；第3天将p0上清更换为5％FBS的培养基；第4天将病毒靶向细胞(简称p1)接种到6孔板中；第5天，将质粒pCAGGS-NP(125ng)、pCAGGS-VP35(125ng)、pCAGGS-VP30(75ng)、pCAGGS-L(1000ng)与pCAGGS-Tim1(250ng)转染到p1；第6天将p1细胞上清更换为p0的细胞上清；第7天将p1上清更换为5％FBS的培养基继续培养72h收取p1，如果需要继续传代病毒获得p2，则方法与p1获取流程一致。
1、在最小基因组系统中加入PKA抑制剂H89(在细胞收集前12h加入10μM H89处理)，将p1及p2用250μl PLB裂解液(Promega公司，货号E194A)裂解15min，离心后取40μl细胞上清与40μl Renilla Glo Reagent(Promega公司：Renilla-Glo Luciferase Assay System货号E2710中底物与缓冲液按照1:100混合)等比例混匀后室温放置10min后测海肾荧光素酶发光值RLU以评价病毒复制。p2的测量方法与p1相同。
2、构建AKIP1siRNA，在125μl opti-MEM无血清培养基中依次加入5μl AKIP1siRNA及3.75μl TransIT-X2试剂(Mirus，货号MIR 6000)，混合均匀，室温放置20min。AKIP1siRNA转染HepG2细胞6h后，1×PBS缓冲液洗涤后，更换新鲜培养基，并转染p0细胞相关质粒(即pCAGGS-NP 125ng、pCAGGS-VP35125ng、pCAGGS-VP30 75ng、pCAGGS-L 1000ng、p4cis-vRNA-Rluc 250ng与pCAGGS-T7 250ng)，转染24h后更换5％FBS培养基，72h后收集细胞根据上述方法测RLU值。
其中，AKIP1siRNA由如下两条单链退火形成：
5’-GCAGUUGAUUCUGGACAAATT-3’(SEQ ID No.1)；
5’-UUUGUCCAGAAUCAACUGCTT-3’(SEQ ID No.2)。
另外，在将AKIP1siRNA转染HepG2细胞，培养36-48h后，收细胞进行qPCR检测细胞中AKIP1的转录水平。检测引物为：
h-AKIP1-F：5’-AAggCTggCTCTAgAAgTgC-3’；
h-AKIP1-R：5’-CTgTTTCTCTAggTggggCg-3’。
3、构建HepG2内源性AKIP1敲除细胞系AKIP1 KO1与AKIP1 KO2，通过CRISPR-Cas9技术获得，靶向AKIP1的序列为如下两个：
靶序列1：5’-CATGCCCTGGAGCGTCCCAA-3’(SEQ ID No.3)；
靶序列2：5’-CATGTCTATCGTTATCACAG-3’(SEQ ID No.4)。
AKIP1 KO1为针对靶序列1所得的HepG2内源性AKIP1敲除细胞系，AKIP1 KO2为针对靶序列2所得的HepG2内源性AKIP1敲除细胞系。AKIP1 KO1和AKIP1 KO2经验证验证两者的内源性AKIP1已经被成功敲除。
向HepG2内源性AKIP1敲除细胞系AKIP1 KO1与AKIP1 KO2中转染最小基因组系统相关质粒，检测方法同上。此步与上述不同点在于用的是HepG2 AKIP1 敲除细胞系AKIP1 KO1与AKIP1 KO2，转染质粒，操作及检测与上述一致。
4、在最小基因组系统中加入CREB抑制剂666-15及KG-501检测对埃博拉病毒样颗粒增殖的影响。
HepG2细胞转染最小基因组系统相关质粒，转染后加入1μM的CREB抑制剂666-15处理48h或加入25μM的CREB抑制剂KG-501处理20min，随后收集细胞，裂解后并检测RLU，检测方法与上述一致。
结果显示：使用埃博拉病毒最小基因组感染细胞模型，在最小基因组系统中加入PKA抑制剂H89会显著抑制埃博拉病毒样颗粒trVLP的增殖(图5中A与B)。在最小基因组系统中加入AKIP1 siRNA敲低HepG2细胞中内源性AKIP1(图6中A)，结果发现埃博拉病毒复制受到抑制(图6中B)。向HepG2内源性AKIP1敲除细胞系AKIP1 KO1与AKIP1 KO2转染最小基因组系统相关质粒，发现AKIP1敲除细胞系可显著抑制埃博拉病毒样颗粒trVLP在细胞中的增殖(图6中C和D)。在最小基因组系统中加入CREB抑制剂666-15和KG-501会显著抑制埃博拉病毒样颗粒trVLP的增殖(图7中A至C)。
综合以上结果：上述埃博拉最小基因组系统结果显示AKIP1-PKA-CREB通路在埃博拉病毒增殖过程中发挥重要的作用。这提示AKIP1-PKA-CREB信号通路可作为埃博拉病毒的靶点，利用PKA抑制剂H89、干扰AKIP1及使用CREB抑制剂666-15或KG-501可作为埃博拉病毒防控的候选药物用于埃博拉病毒感染病例的治疗。本发明对埃博拉病毒病的治疗发面具有重大的应用价值。
工业应用
本发明研究发现埃博拉病毒结构蛋白VP35与AKIP1存在相互作用并激活PKA，促进其入核。且可促进PKA下游录因子cAMP-应答元件结合蛋白(CREB)的磷酸化以及转录活性。同时证明了PKA抑制剂H89、敲低或敲除AKIP1基因能够显著抑制埃博拉病毒在细胞中的增殖。
本发明通过对埃博拉病毒蛋白VP35与AKIP1相互作用及相关分子机制的研究，阐明了VP35-AKIP1结合后促进PKA底物CREB磷酸化，同时发现VP35通过AKIP1促进埃博拉病毒的复制。因此，以PKA、AKIP1及CREB作为靶点在临床上用于防治埃博拉病毒提供了新的依据，其在埃博拉病毒病的治疗领域将有广阔的应用前景。

Claims (35)

  1. PKA、AKIP1和/或CREB作为靶点在如下任一中的应用：
    (A1)制备用于治疗埃博拉病毒感染的产品，或治疗埃博拉病毒感染；
    (A2)制备用于治疗埃博拉病毒病的产品，或治疗埃博拉病毒病；
    (A3)制备用于抑制埃博拉病毒复制的产品，或抑制埃博拉病毒复制；
    (A4)制备用于抑制埃博拉病毒在细胞中增殖的产品，或抑制埃博拉病毒在细胞中增殖。
  2. PKA、AKIP1和/或CREB作为靶点在筛选埃博拉病毒病防治的候选药物中的应用。
  3. 能够抑制PKA表达的物质、能够抑制AKIP1表达的物质和/或能够抑制CREB表达的物质在如下任一中的应用：
    (A1)制备用于治疗埃博拉病毒感染的产品，或治疗埃博拉病毒感染；
    (A2)制备用于治疗埃博拉病毒病的产品，或治疗埃博拉病毒病；
    (A3)制备用于抑制埃博拉病毒复制的产品，或抑制埃博拉病毒复制；
    (A4)制备用于抑制埃博拉病毒在细胞中增殖的产品，或抑制埃博拉病毒在细胞中增殖。
  4. 根据权利要求3所述的应用，其特征在于：所述能够抑制PKA表达的物质为PKA抑制剂。
  5. 根据权利要求3所述的应用，其特征在于：所述能够抑制AKIP1表达的物质为敲除AKIP1表达的物质或敲低AKIP1表达的物质。
  6. 根据权利要求3所述的应用，其特征在于：所述能够抑制CREB表达的物质为CREB抑制剂。
  7. 根据权利要求4所述的应用，其特征在于：所述PKA抑制剂为H89。
  8. 根据权利要求7所述的应用，其特征在于：所述H89的结构式如式I所示；
    Figure PCTCN2019105871-appb-100001
  9. 根据权利要求5所述的应用，其特征在于：所述敲低AKIP1表达的物质为AKIP1 siRNA。
  10. 根据权利要求9所述的应用，其特征在于：所述AKIP1 siRNA为由SEQ  ID No.1和SEQ ID No.2所示的两条单链退火形成的siRNA。
  11. 根据权利要求5所述的应用，其特征在于：所述敲除AKIP1表达的物质为用于敲除AKIP1表达的基因编辑工具。
  12. 根据权利要求11所述的应用，其特征在于：所述基因编辑工具为CRISPR/Cas9核酸酶，其特异性切割的靶序列为SEQ ID No.3或SEQ ID No.4。
  13. 根据权利要求6所述的应用，其特征在于：所述CREB抑制剂为666-15或者KG-501。
  14. 根据权利要求13所述的应用，其特征在于：所述666-15的结构式如式II所示；
    Figure PCTCN2019105871-appb-100002
  15. 根据权利要求13所述的应用，其特征在于：所述KG-501的结构式如式III所示；
    Figure PCTCN2019105871-appb-100003
  16. 根据权利要求1-15中任一所述的应用，其特征在于：所述PKA为SEQ ID No.5所示蛋白质。
  17. 根据权利要求1-16中任一所述的应用，其特征在于：所述AKIP1为SEQ ID No.6所示蛋白质。
  18. 根据权利要求1-17中任一所述的应用，其特征在于：所述CREB为SEQ ID No.7所示蛋白质。
  19. 如下任一方法：
    方法A：一种治疗埃博拉病毒感染的方法，是以PKA、AKIP1和/或CREB作为靶点治疗埃博拉病毒感染；
    方法B：一种治疗埃博拉病毒病的方法，是以PKA、AKIP1和/或CREB作为靶点治疗埃博拉病毒病；
    方法C：一种抑制埃博拉病毒复制的方法，是以PKA、AKIP1和/或CREB作为靶点抑制埃博拉病毒复制；
    方法D：一种抑制埃博拉病毒在细胞中增殖的方法，是以PKA、AKIP1和/或CREB作为靶点抑制埃博拉病毒在细胞中增殖；
    方法E：一种筛选埃博拉病毒病防治的候选药物的方法，是以PKA、AKIP1和/或CREB作为靶点筛选埃博拉病毒病防治的候选药物。
  20. 根据权利要求19所述的方法，其特征在于：所述方法A、所述方法B、所述方法C和所述方法D中，包括如下步骤：给宿主或宿主细胞施用能够抑制PKA表达的物质、能够抑制AKIP1表达的物质和/或能够抑制CREB表达的物质。
  21. 根据权利要求20所述的方法，其特征在于：所述能够抑制PKA表达的物质为PKA抑制剂。
  22. 根据权利要求20所述的方法，其特征在于：所述能够抑制AKIP1表达的物质为敲除AKIP1表达的物质或敲低AKIP1表达的物质。
  23. 根据权利要求20所述的方法，其特征在于：所述能够抑制CREB表达的物质为CREB抑制剂。
  24. 根据权利要求21所述的方法，其特征在于：所述PKA抑制剂为H89。
  25. 根据权利要求24所述的方法，其特征在于：所述H89的结构式如式I所示；
    Figure PCTCN2019105871-appb-100004
  26. 根据权利要求22所述的方法，其特征在于：所述敲低AKIP1表达的物质为AKIP1 siRNA。
  27. 根据权利要求26所述的方法，其特征在于：所述AKIP1 siRNA为由SEQ ID No.1和SEQ ID No.2所示的两条单链退火形成的siRNA。
  28. 根据权利要求22所述的方法，其特征在于：所述敲除AKIP1表达的物质为用于敲除AKIP1表达的基因编辑工具。
  29. 根据权利要求28所述的方法，其特征在于：所述基因编辑工具为CRISPR/Cas9核酸酶，其特异性切割的靶序列为SEQ ID No.3或SEQ ID No.4。
  30. 根据权利要求23所述的方法，其特征在于：所述CREB抑制剂为666-15或者KG-501。
  31. 根据权利要求30所述的方法，其特征在于：所述666-15的结构式如式II所示；
    Figure PCTCN2019105871-appb-100005
  32. 根据权利要求30所述的方法，其特征在于：所述KG-501的结构式如式III所示；
    Figure PCTCN2019105871-appb-100006
  33. 根据权利要求19-32中任一所述的方法，其特征在于：所述PKA为SEQ ID No.5所示蛋白质。
  34. 根据权利要求19-33中任一所述的方法，其特征在于：所述AKIP1为SEQ ID No.6所示蛋白质。
  35. 根据权利要求19-36中任一所述的方法，其特征在于：所述CREB为SEQ ID No.7所示蛋白质。
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