CN113975380B

CN113975380B - Application of SUCLG2 in preparing medicine for preventing or treating Japanese encephalitis virus infection

Info

Publication number: CN113975380B
Application number: CN202111054804.6A
Authority: CN
Inventors: 朱勇喆; 吴昌贵; 邱屹洲; 戚中田; 赵平; 任浩; 陈梦晨
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2023-01-17
Anticipated expiration: 2041-09-09
Also published as: CN113975380A

Abstract

The invention relates to the technical field of biomedicine, in particular to a new target for resisting Japanese Encephalitis Virus (JEV) infection: succinate coenzyme A ligase GDP forms beta subunit (SUCLG 2), and application thereof in preparing medicine for preventing or treating Japanese encephalitis virus infection. The invention takes human neuroblastoma SH-SY5Y as a target cell, and analyzes the influence of dihydroartemisinin on gene expression of a JEV infected target cell after the action by adopting a transcriptome sequencing technology, and the invention discovers that SUCLG2 plays an important role in JEV infected human neuroblastoma SHSY5Y, the expression of SUCLG2 is up-regulated, and the JEV infection can be obviously inhibited. The invention provides a new target and a treatment scheme for clinically preventing and treating the incapacity of the nervous system caused by the Japanese encephalitis virus infection.

Description

Application of SUCLG2 in preparing medicine for preventing or treating Japanese encephalitis virus infection

Technical Field

The invention relates to the technical field of biomedicine, in particular to a new target for resisting Japanese Encephalitis Virus (JEV) infection: succinate coenzyme A ligase GDP forms beta subunit (SUCLG 2), and application thereof in preparing medicine for preventing or treating Japanese encephalitis virus infection.

Background

Japanese Encephalitis Virus (JEV) is a Flaviviridae (Flaviviridae) Flavivirus, which is an enveloped single positive strand RNA virus [ Gubler, D., G.Kuno, and L.Markoff. Flaviviviruses, p.2007.1153-1252.In D.M.Knipe and P.M.Howley (ed.), field virology,5th ed., vol.1.Lippincott-Williams & Wilkins, philadelphia, PA ]. JEV is a pathogen of epidemic encephalitis B, is mainly transmitted by taking mosquitoes as a medium, can cause serious acute nervous system symptoms such as encephalitis or meningitis and the like after infection, and has great threat to human health. Encephalitis B is predominantly prevalent in east Asia, southeast Asia, and parts of the continent, with vaccine prophylaxis and mosquito eradication currently the major control measures, however over the past decade the global cases of encephalitis B counted by the world health organization averaged 69,000 cases/year, over 10,000 deaths [ Turtle L, solomon T.Japanese encephalitis-the prospects for new diseases Nature Reviews Neurology,2018,14 (5): 298-313 ], actual cases of infection and death exceed these numbers due to imperfect diagnostic and reporting mechanisms, which can range from 5 to 17.5 million cases/year and JEV-induced neurological disease results in a large number of infected persons with lifelong disabilities. Therefore, the research on the pathogenic mechanism and the prevention means of JEV and the search of a new antiviral strategy are the leading subjects to be solved urgently.

Although JEV can infect cells of different tissue types, its primary threat is also invasion of the central nervous system, causing encephalitis. JEV infection is neurotropic and is prone to cause serious Central Nervous System (CNS) diseases and complications thereof, which are also the main causes of severe and even death of patients with japanese encephalitis. After the JEV is amplified in vivo, the JEV can break through blood brain barrier to infect the central nervous system through blood circulation. Studies have shown that JEV infection can directly or indirectly trigger apoptosis of nerve cells. JEV invades nerve cells to cause inflammatory reaction, activate a series of signal channels related to cell inflammation, and trigger host cells to produce a large amount of inflammatory factors, thereby promoting disease process [ Denizot M, new JW, gasque P.Encephalitis product to engineering viruses: CNS input immunity and potential therapeutic targets.J. Infect.2012.65 (1): 1-16 ]. Currently, there is relatively little research on the mechanisms of JEV infection of the nervous system. Previously, we found that JEV invades neuroblastoma cells via the crypt-mediated endocytosis pathway, unlike the mechanism of virus invasion into non-neural cells [ Zhu YZ, xu QQ, wu DG, et al. Japanese encephalitis virus enterers rate neuroblastoma cells via pH-dependent, dynamin and caveola-mediated endocytosis path.J Virol.2012,86 (24): 13407-22 ]. Studies by Kalia et al also found that the pathway of JEV invasion into mouse neuroblastoma cells (Neuro 2 a) differs from the pathway of invasion into other host cell types, and does not depend on the clathrin pathway [ Kalia M, khasa R, sharma M, nain M, vrti S.Japan encephalititis virus in nuclear cells through a clathrin-independent endogenous metabolism. J Virol,2013,87 (1): 148-62 ]. These studies suggest that the mechanism of JEV invasion into nerve cells is different from its invasion into other host cell types, and the specificity of JEV invasion into nerve cells may be related to the neurotropic and pathogenic properties of JEV.

At present, although there are some reports on Japanese encephalitis virus replication cycle antiviral drugs, vaccine development, RNA and the like, until now, no specific drug or effective method for treating Japanese encephalitis exists. The anti-JEV drugs currently studied mainly include the following: nucleic acid targeted therapeutics, nucleic acid analogs, cytokines and flavonoids, and the like. Researchers at home and abroad can be attributed to several main directions in the research of anti-encephalitis medicines: firstly, the encephalitis B is treated by using the existing antiviral drugs; secondly, designing and synthesizing derivatives of the existing antiviral drugs according to the molecular biology characteristics of the JEV; and thirdly, searching a new target spot and further screening a new anti-JEV medicament. Dihydroartemisinin (DHA) is a clinically safe and effective antimalarial drug. In addition, researches show that DHA also has the functions of antivirus, anti-inflammation and the like. The artemisinin compound has inhibitory effect on herpesviruses such as cytomegalovirus, hepatitis B virus, hepatitis C virus, zika virus, etc. DHA can regulate viral infection and viral replication by regulating inflammatory signaling pathways as well as oxidative stress pathways. The influence of DHA on JEV infection before and after the DHA acts on human neuroblastoma SH-SY5Y is analyzed, and the result shows that the DHA acts on the JEV infection to be obviously inhibited. And further comparing the change of gene transcription level in the SH-SY5Y cell infected by the JEV before and after the action of the DHA by adopting a genome sequencing method, and screening a group of key host cell molecules participating in the JEV infected nerve cell from the gene transcription level, wherein the action of the molecules in the JEV infection is not reported.

Succinate-CoA Ligase GDP forms a Beta Subunit (Succinate-CoA ligand GDP-Forming Beta. Subunit, SUCLG 2) which is mainly distributed in mitochondria, provides the nucleotide specificity of the enzyme and is combined with a substrate succinic acid to play an important role in the tricarboxylic acid cycle. SUCLG2 is involved in the progression of various diseases in humans, and experiments have shown that SUCLG2 has been shown to play an important role in the clearance of cerebrospinal amyloid-1-42 in functional microglia and in slowing down Alzheimer's disease [ SUCLG2 identified as a bouth a determiner of CSF beta (1-42) levels and an initiator of cognitive degrees in Alzheimer's disease [ J ]. Human Molecular Genetics,2014,23 (24): 6644-6658 ]. In addition, SUCLG2 has been found to play an important role in the development of autoimmune diseases, and the absence of the mitochondrial protein SUCLG2 in T Cells leads to the arrest of tricarboxylic acid (TCA) cycle, leading to the accumulation of excess acetyl-CoA and to the acetylation of the microtubule system, ultimately promoting the migratory behavior of T Cells, which undermines the immune tolerance of the body [ Wu B, qiu J, ZHao T V, et al. SuCLG2 was also found to be involved in the virus infection process, and SUCLG2 was found to inhibit the replication of HIV-1 in HeLa cells after down-regulation [ Zhou H, min X, qian H, et al, genome-scale RNAi screens for host factors required for HIV replication. [ J ]. Cell host & microbe,2008,4 (5): 495-504 ].

At present, no report about the effect of SUCLG2 in JEV infection exists, and the deep study on the molecule can not only improve the understanding of JEV infection and pathogenic mechanism, but also provide a new thought and target point for preventing and treating JEV infection.

Disclosure of Invention

The invention aims to provide a novel target for resisting Japanese Encephalitis Virus (JEV) infection, namely succinate coenzyme A ligase GDP forming beta subunit (SUCLG 2).

The invention also aims to provide a new application of succinate coenzyme A ligase GDP to form beta subunit (SUCLG 2), in particular to an application in preparing medicaments for preventing or treating Japanese encephalitis virus infection.

The third purpose of the invention is to provide a reagent for promoting or up-regulating the expression level of beta subunit (SUCLG 2) formed by succinate coenzyme A ligase GDP and application thereof.

In order to achieve the purpose, the main technical scheme of the invention is as follows:

according to the invention, human neuroblastoma SH-SY5Y cells are used as target cells to infect JEV, and the change of transcription level of the target cells before and after the action of dihydroartemisinin (20 mu M) is compared, so that a host factor capable of effectively inhibiting JEV from infecting human nerve cells is searched, and the function of a nervous system is protected. And screening a group of host molecules which are obviously reduced after the dihydroartemisinin acts and are obviously increased in JEV infection through gene expression amount change. The invention discovers that SUCLG2 plays an important role in JEV infection SH-SY5Y, the expression of SUCLG2 is up-regulated, and the JEV infection can be obviously inhibited.

In the first aspect of the invention, a novel target for resisting Japanese Encephalitis Virus (JEV) infection, namely a beta subunit formed by a succinate coenzyme A ligase GDP (SUCLG 2), is provided.

In a second aspect of the invention, the application of SUCLG2 in preparing a medicament for preventing or treating Japanese encephalitis virus infection is provided.

Further, the invention also provides an application of SUCLG2 in preparing a medicament for preventing or treating Japanese encephalitis.

Furthermore, the medicament for preventing or treating Japanese encephalitis virus infection takes SUCLG2 protein as an active ingredient.

In a third aspect of the invention, an application of an agent for promoting or up-regulating SUCLG2 expression level in preparing a medicament for preventing or treating Japanese encephalitis virus infection is provided.

Furthermore, the medicament for preventing or treating Japanese encephalitis virus infection takes an agent for promoting or up-regulating SUCLG2 expression as an active ingredient.

Further, the application refers to that the SUCLG2 gene is used as an intervention target for preventing or treating Japanese encephalitis virus infection.

Furthermore, the medicine is a medicine for preventing or treating Japanese encephalitis virus infection by promoting or up-regulating SUCLG2 expression level.

Further, the agent for promoting or up-regulating SUCLG2 expression level refers to a recombinant vector (such as a plasmid) comprising SUCLG2 gene.

In one embodiment of the present invention, the agent for promoting or up-regulating the expression level of SUCLG2 is a pEGFP-N1 expression plasmid containing the SUCLG2 gene (NM-001177599.2).

In the fourth aspect of the present invention, a medicament for preventing or treating japanese encephalitis virus infection is provided, wherein the medicament comprises SUCLG2 protein or an agent for promoting/up-regulating SUCLG2 expression level.

The invention has the advantages that:

1. according to the invention, human neuroblastoma SH-SY5Y is taken as a target cell, and the influence of dihydroartemisinin on gene expression of a JEV infected target cell after action is analyzed by adopting a transcriptome sequencing technology, so that a host factor capable of effectively inhibiting the JEV from infecting the human nerve cell is searched, the function of a nervous system is protected, and encephalitis caused by virus infection of the central nervous system is prevented. Experiments show that SUCLG2 plays an important role in JEV infected human neuroblastoma SHSY5Y, the expression of SUCLG2 is up-regulated, and the JEV infection can be obviously inhibited.

2. The invention screens a new host cell molecule SUCLG2 which can inhibit the Japanese encephalitis virus from infecting SH-SY5Y cells. After the SUCLG2 gene is up-regulated, the normal physiological function of cells is not influenced, but the infection of the SH-SY5Y cells by encephalitis B virus is obviously inhibited.

3. The invention provides a new target and a treatment scheme for clinically preventing and treating the incapacity of the nervous system caused by the Japanese encephalitis virus infection.

Drawings

FIG. 1 shows the 20 most significant changes in the transcription levels of target cell molecules (rank from high to low according to fold-difference) before and after dihydroartemisinin acted on JEV-infected SH-SY5Y cells. The ordinate axis of the graph shows the up-regulation multiple of host cell molecule expression after the action of dihydroartemisinin, and the abscissa axis shows the molecule name.

Fig. 2 is a graph of the effect of transfection of SUCLG2 eukaryotic expression plasmid on expression efficiency and cell viability, wherein a is a graph of detection of mRNA level of SUCLG2 gene (×, p <0.01, statistically significantly different from CTRL), B is a graph of detection of SUCLG2 protein expression by Western Blot, and C is a graph of the effect of SUCLG2 gene up-regulation on cell viability;

CTRL: SH-SY5Y cell group without transfection of any plasmid vector (empty cell group);

VT-CTRL: SH-SY5Y cell group (negative control group) transfected with empty plasmid vector;

SUCLG2: SH-SY5Y cell group of eukaryotic plasmid vector for expressing SUCLG2 gene.

Fig. 3 is a graph of JEV viral load detected by fluorescence quantitative PCR with the effect on JEV infection after SUCLG2 upregulation (p <0.001, statistically significantly different from CTRL).

VT-CTRL: SH-SY5Y cell group (negative control group) of the transfection empty plasmid vector;

SUCLG2: SH-SY5Y cell group of eukaryotic plasmid vector for expressing SUCLG2 gene by transfection.

Fig. 4 is an immunofluorescence assay for detecting the effect of SUCLG2 upregulation on JEV infection, wherein a is an immunofluorescence assay for the effect of SUCLG2 upregulation on viral infectivity, fluorescence is shown for cells positive for JEV envelope protein, and B is a statistical plot of the inhibition of viral infection after upregulation of the molecule (p <0.001, statistically significantly different compared to CTRL);

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), either according to the usual conditions or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Example 1:

1. experimental methods

1. Transcriptome sequencing analysis of the change of SH-SY5Y cell gene expression level before and after the dihydroartemisinin treatment.

1.1 SH-SY5Y cells (purchased from ATCC, accession number: HTB-11) were plated 12-16 hours in advance and cultured in 24-well cell culture plates so that the cell density at transfection was 80% -90%. Inoculating SK-N-SH cells in a 96-or 24-well plate, 37 ℃, 5% ₂ Culturing for 12-16h under the condition to make the cell density be 80% -90%. The experiment was set up with 3 replicate wells each, with drug treatment groups and general infection groups, and the experiment was replicated 3 times independently. After adding dihydroartemisinin (20 μ M) into each well, incubating at 37 deg.C for 1h, and adding equal amount of whole culture solution into general infected group. Subsequently, a JEV virus (infectious agent MOI = 1) solution containing drugs is added into the cells, the cells are placed in an incubator at 37 ℃ to be infected for 2h, then the virus solution is discarded, the cells are rinsed for 3 times by using pre-warmed PBS, a fresh culture medium containing dihydroartemisinin drugs is added for continuous culture, and an equal amount of solvent is added into a general infected group. After 48h of culture, collecting cells of each group, and respectively extracting nucleic acid components of a control group, a JEV infected group and a dihydroartemisinin intervention group (3 independent samples in each group) by using a nucleic acid extraction kit.

1.2 after extracting total RNA of a sample and digesting DNA by using DNase, enriching mRNA by magnetic beads with Oligo (dT); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a six-base random primer by using the broken mRNA as a template, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail, connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by Agilent 2100Bioanalyzer quality testing and then analyzed by sequencing using an Illumina HiSeqTM2500 sequencer. And performing large-scale data analysis based on the sequencing result, respectively comparing the gene expression profiles of the control group and the JEV infected group, and the gene expression profiles of the JEV infected group and the dihydroartemisinin treatment group, and screening the genes with significant difference through statistical cluster analysis.

Identification of SuCLG2 overexpression Effect

2.1 plasmid transfection

Transfection procedures were performed according to Lipofectamine 3000 instructions

1) SH-SY5Y cells (purchased from ATCC, accession number: HTB-11) were plated on 24-well cell culture plates to achieve a cell density of 80% -90% at transfection.

2) Adding 2 mu.L Lipofectamine 3000 into 50 mu.L opti-MEM, mixing, and incubating at room temperature for 5 min; mu.L of SUCLG2 expression plasmid at a concentration of 200 ng/. Mu.L was mixed with 50. Mu.L of opti-MEM. After incubation, diluted Lipofectamine 3000 transfection reagent was added to the diluted RNA and gently pipetted and mixed. After incubation at room temperature for 20min, the cells were added to SH-SY5Y cells and supplemented with 400. Mu.L of opti-MEM.

3) Fresh medium containing the double antibody was replaced 6-8 hours after transfection.

2.2 real-time fluorescent quantitative PCR (RT-PCR) detection of mRNA levels of SUCLG2 molecules

1) TRIzol extracts total RNA of cells of a control group and an interference group, and comprises the following specific steps:

after 48 hours of transfection, the culture supernatant was removed, 1ml of TRIzol was added to the cells, and the cells were lysed by well mixing for 3 to 5 minutes at room temperature. Add 1/5 volume of chloroform and mix vigorously by hand for 15 seconds. Centrifugation was carried out at 4 ℃ for 15 minutes at 12,000 rpm. The upper aqueous phase was taken and transferred to a new EP tube, an equal volume of isopropanol was added, mixed well and precipitated at room temperature for 10 minutes. Centrifugation was carried out at 4 ℃ for 10 minutes at 12,000 rpm. The supernatant was discarded and 1ml of pre-cooled 75% ethanol was added. Centrifuge at 12,000 ℃ for 5 minutes. And fully discarding the supernatant, airing the RNA precipitate at room temperature, and adding DEPC (diethyl phthalate) treatment water to dissolve the precipitate to obtain the total RNA.

2) The method comprises the following steps of (1) acquiring cDNA of cells of a control group and an interference group by using a takara reverse transcription kit:

the following reaction system was added to the PCR tube,

the mixture was gently mixed and mixed, reacted at 37 ℃ for 15 minutes, and then heated at 85 ℃ for 5 seconds to inactivate the reverse transcriptase.

3) Fluorescent quantitative RT-PCR detection

The reaction was carried out using the SYBR Premix Ex Taq kit from takara in the following reaction system,

SYBR Premix Ex Taq 10μL

two-step amplification was performed using a Rotor Gene 3000A instrument, with pre-denaturation at 95 ℃ for 2min, 40 PCR cycles at 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds.

3. Cytotoxicity test

The CCK-8 method is adopted to detect the influence of the over-expression of SUCLG2 molecules on cell proliferation, and comprises the following specific steps: cells in the logarithmic growth phase were collected and seeded in 96-well plates at a density of 3000 cells per well. After the cells adhere to the wall overnight, each siRNA is transfected, and the cell proliferation condition is detected after 48 hours of culture. Discarding the original culture medium, adding 110 μ L of fresh culture medium containing 10 μ L of CCK-8 into each well, culturing for 3h, and detecting the absorbance value of each well at 450nm wavelength by using a multifunctional microplate reader. The experiment was independently repeated 3 times and the mean was calculated.

JEV Virus infection of SH-SY5Y cells

4.1 JEV Virus infection experiment of SH-SY5Y cells

JEV infection experiments were performed 48 hours after SH-SY5Y cells overexpress SUCLG2 molecules. The culture supernatant was aspirated, rinsed with pre-warmed PBS 2 times, inoculated with JEV at a virus amount of MOI =1, incubated at 37 ℃ for 2h, and then virus solution was discarded, rinsed with pre-warmed PBS 3 times, and added with fresh medium to continue culturing.

4.2 immunofluorescence staining detection of JEV antigen expression

SH-SY5Y cells are continuously cultured for 48h after being infected with viruses, and the expression of virus antigens is detected by adopting an immunofluorescence method, which comprises the following specific steps:

1) Cell fixation: the culture medium in the 96-well plate was removed, cells were washed 2 times with PBS, 100. Mu.l of pre-chilled methanol was added to each well, fixed at-20 ℃ for 20min, and cells were washed 3 times with pre-chilled PBS.

2) And (3) membrane penetration: the fixed cells were incubated at room temperature for 15min by adding 0.1% Triton X-100 in 100. Mu.l per well, and washed 3 times with pre-cooled PBS.

3) And (3) sealing: add 100. Mu.l 3% BSA per well and incubate for 1h at room temperature.

4) Primary anti-incubation: 100 μ l of JEV-specific murine mab (1 diluted 500) was added to each well, incubated at room temperature for 1h, and washed 3 times with pre-cooled PBS.

5) And (3) secondary antibody incubation: 100 μ l of AF 488 fluorescence-labeled anti-mouse IgG (1 diluted 1000) was added to each well, incubated at room temperature for 1h in the dark, and washed 2 times in the dark with pre-cooled PBS.

6) Marking cell nucleus: cell nucleus fluorescent dye DAPI (1, 5000, PBS diluted) was added to each well, incubated at room temperature for 15min in the dark, and washed 3 times with pre-cooled PBS in the dark.

7) And detecting and calculating the clone number of the green fluorescence positive cells under a fluorescence microscope.

4.3 Western blot

(1) And extracting the total protein of SH-SY5Y cells of the control group and the SUCLG2 molecule overexpression group respectively by using protein lysate.

(2) After the protein quantification, 30. Mu.g of the protein was added to 12.5% polyacrylamide gels for electrophoresis, and the corresponding bands were cut off and transferred to PVDF membranes using an electrotransfer.

(3) Non-specific sites of the protein were blocked with 5% skim milk, followed by SUCLG2 antibody overnight at 4 deg.C, washed three times with TBST buffer, and the primary antibody washed away.

(4) The cells were then incubated with HRP-labeled secondary antibody for 2 hours at room temperature, followed by three washes with TBST buffer.

(5) And finally, developing by using a developing solution and photographing for analysis.

4.4RT-PCR detection of the amount of JEV Virus in cells

Culturing for 48h after SH-SY5Y cells are infected with virus, extracting total RNA of cells of a control group and an interference group by TRIzol, carrying out reverse transcription to obtain cDNA, and detecting the amount of JEV virus by RT-PCR. The specific steps are as shown in 2.2.

2. Results of the experiment

JEV infection SH-SY5Y cell key host molecule screening

SH-SY5Y cells are used as an infection model, the transcriptome of each group of samples is deeply sequenced by a second generation sequencing platform after the action of dihydroartemisinin, large-scale data analysis is carried out based on the sequencing result, and the gene expression profile difference of a JEV infected group and a dihydroartemisinin intervention group is compared. The results are shown in figure 1, and the dihydroartemisinin induced expression changes of multiple host cell molecules compared with the JEV-only infected group, and figure 1 lists the 20 host molecules with the most significant downregulation after dihydroartemisinin action. The beta subunit (SUCLG 2) formed by the succinate coenzyme A ligase GDP is most obviously up-regulated, and the difference multiple is 6.0 times (figure 1), which suggests that SUCLG2 may be a key molecule participating in JEV infection of SH-SY5Y cells.

SuCLG2 overexpression plasmid efficiency detection and cytotoxicity detection

Human SUCLG2 gene transcript 1 (NM-001177599.2) was selected for SUCLG2 target gene sequence, and the gene sequence was cloned into pEGFP-N1 expression plasmid vector (Clontech), amplified by E.coli in vitro, and used for cell transfection.

The expression plasmid of SUCLG2 gene is transfected into SH-SY5Y cell by in vitro transfection method, the expression efficiency of gene is detected by RT-PCR method after 48h, the protein expression efficiency is detected by western blot method, and the influence on SH-SY5Y cell toxicity after transfection is detected by CCK 8.

The results are shown in fig. 2.A, compared with the group transfected with empty vector, the group transfected with SUCLG2 overexpression plasmid can significantly improve the expression level of SUCLG2 gene, and the gene expression level is improved by 4.3 times (P < 0.01).

The immunoblotting method detects the influence of SUCLG2 on protein expression after up-regulation, the result is consistent with the detection result of gene expression level, the expression level of SUCLG2 protein is obviously up-regulated, and is improved by 2.8 times compared with a control group (figure 2. B).

Cytotoxicity experiments show that after the SUCLG2 gene is over-expressed, obvious cytotoxicity (P is more than 0.05) is not generated, and the normal physiological function of cells is not influenced (figure 2. C), so that the SUCLG2 gene can be used for subsequent experiments.

Effect of SUCLG2 on JEV Virus infection after Up-Regulation

After transfection of SUCLG2 molecule expression plasmids, the effect on viral infectivity was observed. The results show that the inhibition rate of the SUCLG2 gene expression level reaches 79.5% of that of the control group when the SUCLG2 gene expression level is increased to 6.0 times of that of the control group (figure 3), and the results suggest that the SUCLG2 molecule is up-regulated to have a remarkable inhibition effect on JEV infection, and indicate that the SUCLG2 molecule plays an important role in JEV infection SH-SY 5Y.

Further, after the SUCLG2 molecule expression plasmid is transfected, the expression of host cells SUCLG2 is up-regulated, and JEV virus with the same dose is infected, the influence of the up-regulated SUCLG2 molecule on JEV infection is detected by an immunofluorescence method 48h after the infection, and the result shows that compared with a control group, after the SUCLG2 gene is up-regulated by transfecting the SUCLG2 molecule expression plasmid, the number of cells positive to the JEV envelope protein antigen is obviously reduced, and the fact that the up-regulation of the SUCLG2 gene can reduce the SH-SY5Y cell infection caused by JEV is further confirmed (figure 4. A). Through calculation of virus amount, the inhibition rate of the SUCLG2 gene on the virus after up-regulation reaches 81.7% compared with a control group, and the infection of SH-SY5Y cells by JEV is obviously inhibited (figure 4. B).

The experimental results prove that: a new host cell molecule SUCLG2 capable of inhibiting JEV from infecting SH-SY5Y cells is screened by the invention. After the SUCLG2 gene is up-regulated, the normal physiological function of cells is not influenced, but the infection of the SH-SY5Y cells by JEV is obviously inhibited. Therefore, the invention provides a new target and a treatment scheme for clinically preventing and treating the nervous system injury caused by JEV infection.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. Application of a reagent for promoting or up-regulating SUCLG2 expression level in preparing a medicament for treating Japanese encephalitis virus infection.

2. The use of the agent for promoting or up-regulating SUCLG2 expression in the preparation of a medicament for treating Japanese encephalitis virus infection according to claim 1, wherein the agent for promoting or up-regulating SUCLG2 expression refers to a recombinant vector containing SUCLG2 gene.