Application of AW112010 ORF coding protein in preparation of antibacterial drugs

Abstract

According to the present invention, the LncRNA AW112010KO + AW112010ORF overexpression cell strain is prepared and is named as the KF cell strain so as to strip the influence of LncRNA AW112010 in cells, and the experiment results prove that the AW112010ORF coding protein can be used for preparing antibacterial drugs, the drugs up-regulate the upstream molecules Hnmpu and Tbk1 generated by IFN beta, and the downstream signal channel molecule Art2 of IFN beta, such that the effective scheme is provided for the treatment of bacterial infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of AW112010 ORF-encoded protein in the preparation of antibacterial drugs. Background Art

[0002] lncRNA (long non-coding RNA) refers to RNA molecules with a transcript length of more than 200nt, which is 3-100 times that of mRNA. Among them, humans have about 58,050 total genes, of which lncRNA has 13,870; mice have about 38,926 total genes, of which lncRNA has 4,074. In the cell nucleus, lncRNA mainly regulates the transcription of target genes through chromatin modification and transcriptional regulation; in the cytoplasm, it mainly regulates the degradation, stability and translation modification of target mRNA through post-transcriptional regulation. However, the functions of most lncRNA AW112010 genes in humans and mice have yet to be revealed. The vast majority of lncRNAs are non-coding RNAs, but it has recently been reported that 1% can encode proteins and play important physiological functions. At present, studies have shown that lncRNAs and their encoded proteins may play related functions or completely different functions; most of the existing literature on the functions of lncRNAs and their encoded proteins is a confounding factor of lncRNAs and their encoded proteins.

[0003] The full sequence of LncRNA AW112010 is 789 nt, and the sequence contains 246 nt of ORF that does not start with ATG, encoding 82 amino acids. It has been reported that when La Crosse Virus (LACV) infects microglia and astrocytes, LncRNA AW112010 mRNA is significantly upregulated; when CD4+Th1 and CD4+TH17 cell subsets are activated, LncRNA AW112010 expression is upregulated, and it directly binds to histone demethylase KDM5A to regulate the H3K4 methylation modification of IL10; when mouse ribosomes cannot effectively translate Aw112010 protein, they are susceptible to Salmonella typhi infection, and the production of IL-12p40 and IL-6 is reduced. Although this study revealed that ORF-encoded proteins can positively regulate the expression of IL-12p40 and IL-6 to increase the body's ability to resist infection, this study did not rule out the role of Aw112010 as a lncRNA in the pathological process, and its research conclusions still come from the mixed effects of the two. When studying lncRNA, it is necessary to pay attention to whether its sequence contains ORF. If so, its function needs to be studied separately to fully reveal the physiological regulatory functions of lncRNA and its encoded protein that cooperate or antagonize each other. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes an application of a protein encoded by AW112010 ORF in the preparation of antibacterial drugs.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] Application of AW112010 ORF-encoded protein in the preparation of antibacterial drugs.

[0007] Furthermore, the drug upregulates the secretion of IFNβ.

[0008] Furthermore, the amino acid sequence of the protein encoded by the AW112010 ORF is shown in SEQ ID NO:1.

[0009] Furthermore, the gene sequence encoding the AW112010 ORF-encoded protein is shown in SEQ ID NO:2.

[0010] Furthermore, the sequence of the cell line constructed by the protein encoded by the AW112010 ORF and the knockout of the LncRNA AW112010 gene is shown in SEQ ID NO:3.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0012] The pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0013] The administration route of the pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, topical skin administration or systemic transdermal administration.

[0014] Beneficial effects of the present invention: The present invention prepares a KF cell line (LncRNA AW112010 KO+AW112010ORF overexpression), and the KF cell line removes the influence of LncRNA AW112010 in the cell. Experiments have confirmed that the protein encoded by AW112010 ORF can be used to prepare antibacterial drugs. The drugs upregulate the upstream molecules Hnmpu and Tbk1 generated by IFNβ, as well as the downstream signaling pathway molecule Art2 of IFNβ, providing an effective solution for treating bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described in detail below in conjunction with the accompanying drawings, the purpose of which is to better understand the content of the present invention rather than to limit the protection scope of the present invention.

[0016] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0017] Figure 1 The LncRNA AW112010 gene information provided by the present invention.

[0018] Figure 2 This is the gene targeting position of LncRNA AW112010 cell line.

[0019] Figure 3 Construction of ORF overexpression vector.

[0020] Figure 4 The transfection efficiency of cells was detected by immunofluorescence after cells were transfected with ORF-overexpressing lentivirus.

[0021] Figure 5 QPCR was used to verify the expression of LncRNA AW112010 and AW112010 ORF mRNA in each cell line.

[0022] Figure 6 LncRNA AW112010 is expressed in tissues, organs and peritoneal macrophages of super mice.

[0023] Figure 7 QPCR was used to detect the expression changes of AW112010 and ORF in various tissues and cells after E.coli infection.

[0024] Figure 8 Immunofluorescence (A, B) and immunoblot analysis (C) of the expression changes of LncRNA AW112010 and ORF in cells infected with E. coli at different times.

[0025] Fig. 9 QPCR was used to analyze the expression changes of AW112010 and ORF mRNA in each cell line after LPS stimulation for 12 hours.

[0026] Fig.10 To find ORF-regulated specific expression genes for transcriptomics research.

[0027] Fig.11 It is the ORF up-regulation signaling pathway.

[0028] Fig.12 The expression changes of Ifnb1 and its regulated transcription factors irf3 / irf7 in four cell lines.

[0029] Fig.13 The expression of some genes regulated by the IFN-I signaling pathway changed in the four cell lines.

[0030] Fig.14 In the CLP model, the expression levels of IFNβ, IRF3, and ISG15 in peritoneal macrophages were rapidly upregulated at 1 h.

[0031] Fig.15 Mice were infected with Escherichia coli, and peritoneal macrophages were isolated and QPCR was used to detect the expression levels of lncRNA Aw112010 and Ifnβ1 mRNA.

[0032] Fig.16 The amount of endocytosed and extracellular residual bacteria was detected for each cell line infected with E. coli.

[0033] Fig.17 Research strategies for obtaining proteins that interact with ORF proteins.

[0034] Fig.18 Western blotting was used to detect the upstream and downstream signaling molecules that regulate IFNβ production in each cell line.

[0035] Fig.19 ELISA was used to detect the amount of IFNβ secreted by each cell line at different times after infection with E. coli. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0037] In this study, we designed to completely knock out LncRNA AW112010 in the mouse RAW264.7 cell line, and then overexpress the sequence of the protein encoded by AW112010 ORF in this cell line (adding transcription start codon and stop codon), and named it KF cell line (LncRNA AW112010 KO+AW112010 ORF overexpression), in order to remove the effect of LncRNA AW112010 in the cells and study the function of the protein encoded by AW112010 ORF alone.

[0038] Example 1 LncRNA AW112010 gene and protein encoded by ORF

[0039] 1.1LncRNA AW112010 in vitro transcription factor

[0040] 1.1.1LncRNA AW112010 Close Update (SEQ ID NO:2):

[0041] CAACTA ATTGTG TAAACT TCTCTC AGACTC GGGGTT TTTCAG TCCACT CCTGTT

[0042] TAGCAA ATAGTG CCCCAA AGCAAT TCAAAG GGAAAG TGAAAC TGACAT

[0043] TCTCAA ACTTTA TATAAA AAACCA AAGAGA AGATCA GCAGGA GTCTAG

[0044] CAGACA ACCTGA GCTGCA AGATGT CTCCCA TCCCTC TGATAT TTATCT TTGGTG

[0045] GTGTGC TCATCA TCTGCC TGATGC AACAAT ACCTGG CGTATA AGTCTT CTAAGA

[0046] ACGTCG TTAAAG TCTTCT GCCATC AAGCCA ATGATG TACATA TATACC AGACCC

[0047] AGGTCG TCATGA CAAACA CACTGG AAACCT CAAGTG GAAAAA GCCACC

[0048] CACTGG GTCGTT CAGGAG AGATCC AGTCTT TAAAGA AGCAAA ACTAGA

[0049] FATHERS TCCTCT TCCTTC TAGATG TGCATC ATCTGC TTCTTC CTTCCC

[0050] TAGAAG ACTGAG GGAGCT ATCTTT CACAGA GTGTAT TCAGTG GTATAC GTGGAG

[0051] CTCAGA AGTAGA CACTTG CCTTGC CTCTAT TAGGAC TTGATT GAACCC AAATAC

[0052] CACAAA ATTCTT TCTCTC TGCTGA GAAAAT GGAAAT TTAAGA TGGGAA CCAAGC

[0053] ACTGTG TGGTCA ACATCC ATGAGC ATGGTT TAATAT ACATAC CATGTG CTTGTT

[0054] TTTCTG CTTGTG AACTTG TGAGAT AGAAAA ACTCCC AAGTCA TCCACT TTTACT

[0055] TGTTAT CATTCA GTCACA TAATAA ATTTCG ATTGTG CACTTG AAAAAA AAAAAA

[0056] AAAA

[0057] 1.1.2 Gene transcript information

[0058] See Figure 1 , is the LncRNA AW112010 gene information.

[0059] 1.1.2 Gene targeting location

[0060] See Figure 2 , which is the gene targeting position of LncRNA AW112010 cell line.

[0061] 1.1.3 Construction of knockout cell lines

[0062] Using CRISPR / Cas9 technology, the mouse AW112010 gene was knocked out in mouse mononuclear macrophages (RAW264.7).

[0063] CRISPR / Cas9 Information:

[0064] gRNA-A1:TCCCAATCTGACCAGTCAGCTGG(SEQ ID NO:4)

[0065] gRNA-A2:CTCATGCTGTGGCGTGTAAGGGG (SEQ ID NO:5)

[0066] Subsequently, PCR method was used to verify the LncRNA AW112010 gene cell line obtained.

[0067] 1.2 Protein encoded by AW112010 ORF

[0068] 1.2.1 AW112010 ORF encodes 82 amino acids, and its protein sequence is: (SEQ ID NO: 1)

[0069] LSCKMSPIPLIFIFGGVLIICLMQQYLAYKSSKNVVKVFCHQANDVHIYQTQVVMTNTLETSSGKSHPLGRSGEIQSLKKQN.

[0070] 1.2.2 Preparation of cell line sequence by overexpression of AW112010 ORF

[0071] AW112010_ORF / GGSGx3 / FLAG(309bp): (SEQ ID NO:3)

[0072] CTGAGC TGCAAG ATGTCT CCCATC CCTCTG ATATTT ATCTTT GGTGGT GTGCTC ATCATCTGCCTG ATGCAA CAATAC CTGGCG TATAAG TCTTCT AAGAAC GTCGTT AAAGTC TTCTGC CATCAAGCCAAT GATGTA CATATA TACCAG ACCCAG GTCGTC ATGACA AACACA CTGGAA ACCTCA AGTGGAAAAAGC CACCCA CTGGGT CGTTCA GGAGAG ATCCAG TCTTTA AAGAAG CAAAAC GGTGGT TCTGGTGGTGGT TCTGGT GGTGGT TCTGGT GACTAC AAAGAC GATGAC GACAAG TGA

[0073] The AW112010 ORF lentiviral overexpression vector (LV-EF1a>AW112010_ORF / GGSGx3 / FLAG-CMV>mCherry / T2A / Puro) was designed using the broad-spectrum EF1a promoter (Human eukaryotic translation elongation factor 1α1 promoter). For information on ORF overexpression vectors, see Figure 3 .

[0074] 1.2.3 Obtaining overexpression cell lines

[0075] The above-mentioned overexpression lentivirus was used to transduce mouse mononuclear macrophage leukemia cells (RAW264.7), and the corresponding antibiotics were used for selection to obtain the AW112010 ORF overexpression stable cell line. Figure 4 The transfection efficiency of cells was detected by immunofluorescence after cells were transfected with ORF-overexpressing lentivirus.

[0076] 1.3.1 Construction of LncRNA AW112010 gene knockout (KO) + AW112010 ORF-encoded protein overexpression (ORF) cell line (KF cell line for short)

[0077] Using CRISPR / Cas9 technology, the mouse AW112010 gene was knocked out in mouse mononuclear macrophages (RAW264.7) to obtain the above-mentioned 1.1.3 cell line. Then, using lentiviral transduction technology, the AW112010_ORF / FLAG gene was overexpressed in the above-mentioned 1.1.3 mouse mononuclear macrophages (RAW264.7), and the AW112010_ORF / GGSGx3 / FLAG overexpression stable transfectant was obtained by screening with Hygro and Puro antibiotics.

[0078] 1.3.2 QPCR detection 2.3.1 KF cell line acquisition

[0079] QPCR detection was performed on the AW112010_ORF / GGSGx3 / FLAG overexpression stable cell after LncRNA AW112010 gene knockout (KO), confirming that it was a KF cell line.

[0080] Example 2 QPCR verification of the above cell lines

[0081] Conventional QPCR method was used to verify each cell line obtained. The QPCR primer sequences are as follows:

[0082] LncRNA AW112010-F:AAAAACCAAAGAGAAGAT(SEQ ID NO:6)

[0083] LncRNA AW112010-R:ATACCACTGAATACACTCTG(SEQ ID NO:7)

[0084] AW112010-ORF-F:TGTCTCCCATCCCTCTGA(SEQ ID NO:8)

[0085] AW112010-ORF-R:GGCTTTTTCCACTTGAGGT(SEQ ID NO:9)

[0086] mGAPDH-F:ACATCATCCCTGCATCCACT(SEQ ID NO:10)

[0087] mGAPDH-R:CACATTGGGGGTAGGAACAC (SEQ ID NO:11)

[0088] See Figure 5 The QPCR results show that each cell line was successfully verified.

[0089] Example 3 LncRNA AW112010 plays an important role in super mice

[0090] In previous studies, mice were continuously challenged with multiple lethal doses of Staphylococcus aureus, Escherichia coli, LPS, and methicillin-resistant Staphylococcus aureus (MRSA). Those that survived were named "super mice", that is, mice that can survive attacks by different bacteria / toxins. Figure 6 ), and found that the expression of LncRNA AW112010 in the abdominal MΦ, thymus, lung, liver, and skeletal muscle tissues of the "super mice" was significantly higher than that in the non-induced infection group, and was highest in the abdominal MΦ, indicating that this molecule, especially AW112010 in the abdominal MΦ, may be involved in the host's immune response against bacterial infection.

[0091] Example 4 Study on the expression of LncRNA AW112010 and AW112010 ORF mRNA in various tissues and organs

[0092] 4.1 Expression of LncRNA AW112010 and AW112010 ORF mRNA in peritoneal MΦ and tissues

[0093] After E. coli infection in C57BL / 6 mice for 6 and 12 hours, the expression levels of Aw112010 and its ORF mRNA in the main tissues, organs and cells of the mice were detected. Figure 7A QPCR study found that the expression level of ORF mRNA in PBMC (peripheral blood mononuclear cells), PM (peritoneal macrophages), lymph nodes, thymus, heart, lung, liver and spleen of wild-type mice was significantly higher than that of LncRNA Aw112010 by at least 1000 times; after 6 hours (Figure B) and 12 hours (Figure C) of E. coli infection, the expression level of ORF mRNA was the highest in peritoneal macrophages; D shows the expression of ORF mRNA and LncRNA Aw112010 in peritoneal macrophages at different times after infection (0, 6, and 12 hours), indicating that the expression of both was upregulated in various tissues and organs after infection, but the expression level of ORF mRNA was significantly higher than that of Aw112010 by more than 1000 times, and the expression level was the highest in peritoneal macrophages, and the changes of the two in the body's response after infection were inconsistent. This indicates that some LncRNA molecules may have different expression profiles from their protein-coding ORFs, and thus may play different functions in the subsequent disease.

[0094] 4.2 Clarifying the localization of LncRNA AW112010 and its AW112010ORF-encoded protein in cells

[0095] Immunofluorescence (AW112010ORF encoding protein, green) and RNA fish (LncRNA AW112010, red) were used to detect the expression of both in cells. Figure 8 As shown in A, the protein encoded by AW112010 ORF is mainly expressed in the nucleus, and the expression reaches a peak 6 hours after E. coli infection; LncRNA AW112010 is mainly expressed in the cytoplasm in the resting state, and enters the nucleus 6 hours after E. coli infection. LncRNA AW112010 and its AW112010 ORF-encoded protein have little co-localization, suggesting that they may play different functions. Figure 8 B shows that the fluorescence intensity of AW112010 ORF is higher than that of LncRNA AW112010 at different times of E. coli infection; Figure 8 Immunoblotting in C shows that the ORF protein reaches peak expression 6 hours after infection.

[0096] The cytoplasm and nucleus of each gene-edited cell line were further separated, among which the control cell lines were mCherry and RAW; ORF overexpression cell line: ORF; LncRNA AW112010 full sequence overexpression cell line: LncRNA; LncRNA AW112010 full sequence knockout cell line: KO; LncRNA AW112010 full sequence knockout + ORF overexpression cell line: KF. The cytoplasm and nucleus were separated, and the expression difference of LncRNA AW112010 and AW112010 ORF mRNA in the cytoplasm and nucleus was detected by QPCR. Fig. 9 The results showed that when the cells were in a resting state and activated by LPS stimulation for 12 hours, LncRNA AW112010 was mainly expressed in the cytoplasm, and AW112010 ORF mRNA was mainly expressed in the nucleus.

[0097] Example 5 Functional study of lncRNA Aw112010 and ORF-encoded protein

[0098] mCherry (control cell line), Aw112010 ORF high expression, Aw112010 LncRNA full sequence high expression, Aw112010 LncRNA full sequence knockout (KO), LncRNA AW112010 full sequence knockout + ORF overexpression cell line (KF) were used. Each cell line was stimulated with LPS for 6 hours, and then sent to Wuhan BGI for transcriptome sequencing with their respective control cells (without any treatment), and genes that responded to LPS stimulation were screened (gene expression levels were compared with the control strain after LPS stimulation, and those that were 2 times up and down were genes that responded after LPS stimulation). Bioinformatics analysis found that ORF upregulated NOS2 (iNOS) (suggesting that it can promote the secretion of bactericidal pathogen mediators), Dusp5, Apol9a, Tnc and other genes. Figure 10-13 Transcriptome sequencing analysis of each gene-edited cell line after LPS stimulation for 6 hours. Fig.10 The heat map lists the differentially regulated genes in each cell line. Fig.11 The main signaling pathways of ORF upregulation were shown. Further analysis revealed that ORF positively regulated ifnβ1 mRNA and its transcription factor irf7 ( Fig.12 ) and regulates IFNβ downstream signaling pathway molecules ( Fig.13 ).

[0099] Wild-type mice were used to establish a CLP model. One hour after the model was established, QPCR showed that the expression levels of IFNβ, IRF3, and ISG15 mRNA in peritoneal macrophages were rapidly upregulated (see Fig.14 ).

[0100] Mice were infected with Escherichia coli, peritoneal macrophages were extracted, and the expression of lncRNA Aw112010 and Ifnβ1 at different time points after infection was analyzed by QPCR (see Fig.15 ), and found that its mRNA expression level remained high after infection.

[0101] Example 6 Study on the antibacterial ability of various cell lines

[0102] The effects of lncRNA Aw112010 and Aw112010 ORF gene-edited cell lines on bacterial killing ability were studied using mCherry (control cell line), Aw112010 ORF high expression, Aw112010 LncRNA full sequence high expression, Aw112010 LncRNA full sequence knockout (KO), and LncRNA AW112010 full sequence knockout + ORF overexpression cell line (KF). The cells were resuspended in serum-free 1640 medium at 2*10^5 cells / well and 4*10^5 cells / well and added to 24-well plates. Four wells were plated for each order of magnitude of cells to allow them to grow adherently. 4×10^6 cfu of Escherichia coli were added to each well, equivalent to a cell-to-bacteria ratio of (1:10) and (1:20), respectively. Incubate at 37 degrees Celsius for 1 hour, aspirate 350ul of each cell supernatant (for subsequent analysis of the amount of bacteria in the supernatant), and directly plate the cells; wash the cells 3 times with 1ml of PBS containing antibiotics, 3 minutes each time; add 80 microliters of RIPA to lyse the cells for 10 minutes, label the name of each cell line, aspirate the cell suspension, and plate the plate to calculate the amount of bacteria. Fig.16 The results show that when MOI 10 (cell number: bacterial number = 1:10) was used in Figure A, the amount of bacteria phagocytosed by the KF cell line was the highest (statistically significant compared with KO); when MOI 20 (cell number: bacterial number = 1:20) was used in Figure B, the amount of bacteria phagocytosed by the KF cell line was also the highest. Figure C shows that when MOI 10 was used, the number of bacteria in the supernatant of KO cells was more than that in the supernatant of KF cells, indicating that the number of bacteria phagocytosed by KO cells in the supernatant was less; Figure D shows the situation when MOI 20 was used. This indicates that knockout of LncRNA AW112010 affects the phagocytic function of cells; while the KF cell line shows that high expression of ORF can restore the bactericidal effect of cells, indicating that it is mainly ORF rather than lncRNA Aw112010 that exerts the bactericidal effect.

[0103] Example 7 Study on the mechanism of ORF-encoded protein mediating macrophages

[0104] The RAW264.7 ORF overexpression gene editing cell line (AW112010-ORF-FLAG) was used, LPS was stimulated for 6 hours, Flag and IgG antibodies were immunoprecipitated (IP), gel recovery was performed after electrophoresis, and interacting proteins were identified by mass spectrometry. The design scheme and steps are shown below Fig.17 A, 17B, analysis revealed 59 proteins that bound to the ORF-encoded protein, among which Hdac1 was histone deacetylase 1 ( Fig.17 C).

[0105] Example 8 Study on the mechanism of ORF regulating IFNβ production

[0106] The cell lines LncRNA Aw112010 full sequence knockout (LncRNA-AW112010-knock out), LncRNA Aw112010 full sequence overexpression (LncRNA-Aw112010-overexpression), LncRNA AW112010 full sequence knockout + ORF overexpression cell line (LncRNA-AW112010-knock out / ORF overexpression), Aw112010 ORF overexpression (ORF overexpression), RAW264.7-MCherry (control cell line) were infected or not infected with Escherichia coli (E. coli), and the cytoplasm and nucleus were isolated to detect the upstream molecules and downstream effector molecules regulated by IFNβ production ( Fig.18 ), ORF protein (i.e., KF cell line) up-regulated the upstream molecules Hnmpu and Tbk1 (activating IRF3 and IRF7) that promote IFNβ production in the nucleus, as well as the total amount and activation level (p-IRF3 and p-IRF7) of the transcription factors IRF3 and IRF7 that regulate IFNβ. However, the molecules TET3 (DNA demethylase) and Hdac1, AKT, and ATF2 (transcriptional inhibitors of IFNβ1 and downstream type I IFN signals) that inhibit the transcriptional activity of IFNβ did not change significantly. Art2 (i.e., ADP-ribosyl transferase 2, type I and type II IFN can activate and stably up-regulate the expression of ART2 enzyme), a downstream pathway molecule of IFNβ, was up-regulated in KF cell line. This indicates that ORF protein directly binds to Hdac1 and activates the upstream molecules that regulate IFNβ production indirectly.

[0107] Example 9 Study on whether each cell line regulates IFNβ secretion

[0108] The above cell lines were infected with E. coli (0.5 MOI), and the cell supernatants were collected 1 hour and 6 hours after infection. Fig.19), the IFNβ secretion in the cell culture supernatant was detected by VeriKineTM Mouse IFN Beta ELISA Kit (PBL ASSAY SCIENCE, 424001-1), and the results were as follows: IFNβ secretion was very low at 1 hour, and there was no difference among the cell lines; IFNβ secretion was highest in the ORF cell line at 6 hours after infection, indicating that the ORF cell line regulates the secretion of IFNβ by cells during infection.

[0109] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments. On the basis of the present invention, some modifications or improvements can be made thereto, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Application of AW112010ORF-encoded protein in the preparation of antibacterial drugs.

2. The application according to claim 1, characterized in that: The drug upregulates the secretion of IFNβ.

3. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the protein encoded by the AW112010ORF is shown in SEQ ID NO:

1.

4. The use according to claim 1 or 2, characterized in that: The gene sequence encoding the AW112010ORF-encoded protein is shown in SEQ ID NO:

2.

5. The use according to claim 1 or 2, characterized in that: The sequence of the cell line constructed by the protein encoded by the AW112010 ORF and the knockout of the LncRNA AW112010 gene is shown in SEQ ID NO:3.

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