Application of AW112010 ORF protein in preparation of medicine for preventing and treating bacterial infection

Abstract

The invention relates to an application of AW112010ORF protein in preparation of a medicine for preventing and treating bacterial infection. According to the application disclosed by the invention, an AW112010ORF protein overexpression mouse model is constructed, and the influence of AW112010ORF protein overexpression on inflammatory diseases caused by bacterial infection is researched. Immunofluorescence experimental research proves that the medicine can mediate elimination of bacteria by macrophages, animal experiments prove that the medicine remarkably improves the survival rate of infected mice, and meanwhile, AW112010ORF protein can remarkably inhibit expression and activity of AKT protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of AW112010 ORF protein in preparing drugs for preventing and treating bacterial infections. 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. Most of them are non-coding RNA, but it has been recently reported that 1% can encode proteins and play an important physiological function. For example, the small peptides encoded by lncRNA MIR22HG, LincRNA00961, and LincRNA HOXB-AS3 mRNA regulate influenza virus infection, stem cell proliferation, muscle cell regeneration, and tumor growth, respectively. It has been proven that lncRNA genes play an important role in regulating biological processes such as cell proliferation, differentiation, and apoptosis, and therefore can regulate the pathological process of trauma, sepsis, and inflammatory diseases. Its functional regulation of macrophages mainly includes: regulating LPS-induced inflammatory responses (such as NEAT1) and mediating the killing of intracellular tuberculosis bacteria (such as HOTAIR and MEG3). There are only four literature reports on AW112010, of which three reported that it is related to infection response: (1) Inflammatory response: Aw112010 expression is upregulated when CD4+T cells (especially Th1 and TH17) are activated, and it directly binds to the histone demethylase KDM5A to regulate the H3K4 methylation modification of IL10. (2) Viral infection: La Crosse virus (LACV) infects microglia and astrocytes, and AW112010 mRNA is significantly upregulated. (3) Bacterial infection: A 2018 Nature article used RiboTag RNA-seq sequencing and translation group sequencing analysis to identify Aw112010, and reported for the first time that the full sequence of Aw112010 is 789 nt, and the sequence contains 246 nt of ORF that does not start with ATG, encoding 82 amino acids. The Aw112010Stop knock-in mice were prepared using CRISPR-Cas9 technology, which made their ribosomes unable to effectively translate proteins, making them susceptible to Salmonella typhi, accompanied by blocked IL-12p40 and IL-6 production. However, 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. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention proposes an application of AW112010 ORF protein in the preparation of a drug for preventing and treating bacterial infection, which can mediate the clearance of bacteria by macrophages and improve the survival rate of mice with inflammatory diseases caused by bacteria.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] Application of AW112010 ORF protein in the preparation of drugs for preventing and treating bacterial infections.

[0006] Furthermore, the AW112010 ORF protein is highly expressed during bacterial infection.

[0007] Furthermore, the AW112010 ORF protein can mediate macrophages to clear bacteria.

[0008] Furthermore, the drug can inhibit the expression and activity of AKT protein.

[0009] Furthermore, the amino acid sequence of the AW112010 ORF protein is shown in SEQ ID NO:1.

[0010] Furthermore, the gene sequence of AW112010 encoding the AW112010 ORF protein is shown in SEQ ID NO:2.

[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] Compared with the prior art, the present invention has at least the following advantages:

[0015] 1) The expression of AW112010 ORF protein in peritoneal macrophages was highest 6 hours after mice were infected with E. coli. When the body was infected, AW112010 ORF protein was mainly expressed in the cytoplasm of peritoneal macrophages in a resting state (0 hours), and migrated to the nucleus of peritoneal macrophages 3 hours and 6 hours after infection with E. coli and MRSA, and gradually decreased in the nucleus of peritoneal macrophages 12 and 24 hours after infection, while expression in the cytoplasm increased. And with different infection times, AW112010 ORF protein and HMGB1 molecules related to mediating inflammation were co-localized in the nuclei of peritoneal macrophages. The experiments of this application have proved that overexpression of AW112010 ORF protein can improve the survival rate of MRSA-infected mice.

[0016] 2) The application of the AW112010 ORF protein in the preparation of drugs for preventing and treating bacterial infections. The AW112010 ORF protein can significantly inhibit the expression and activity of AKT protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation of the present invention, the following will briefly introduce the drawings required for use in the specific implementation. In all the drawings, each element or part is not necessarily drawn according to the actual scale.

[0018] Figure 1 This is the principle for constructing the AW112010 ORF protein overexpression mouse model provided by the present invention.

[0019] Figure 2 Strategies for overexpression mouse identification.

[0020] Figure 3 PCR identification results of gene-edited mice.

[0021] Figure 4 For the survival study of AW112010 ORF mice.

[0022] Figure 5 QPCR was used to detect the expression of LncRNA AW112010 ORF mRNA at different times after wild-type mice were infected with Escherichia coli.

[0023] Figure 6 Western blotting was used to detect the expression of AW112010 ORF protein in peritoneal macrophages at different time points after Escherichia coli infection.

[0024] Figure 7 Flow cytometry analysis of the frequency of splenic macrophages in wild-type and AW112010 ORF-overexpressing mice infected with Escherichia coli for 12 hours.

[0025] Figure 8 The frequencies of spleen B cells and granulocytes were studied by flow cytometry analysis after wild-type and AW112010 ORF-overexpressing mice were infected with Escherichia coli for 12 hours.

[0026] Fig. 9 Flow cytometric analysis of DC cell frequencies in spleens of wild-type and AW112010 ORF-overexpressing mice infected with E. coli for 12 hours.

[0027] Fig.10 The frequency of NK cells in the spleen of wild-type and AW112010 ORF-overexpressing mice was studied by flow cytometry after infection with Escherichia coli for 12 hours.

[0028] Fig.11 Flow cytometry analysis of splenic CD4 in wild-type and AW112010 ORF-overexpressing mice infected with E. coli for 12 hours + T, CD8 + T cell frequency studies.

[0029] Fig.12 Immunofluorescence was used to detect the co-localization of ORF protein and HMGB1.

[0030] Fig.13 Immunofluorescence was used to detect the co-localization of ORF protein with molecules related to bacterial phagocytosis and clearance.

[0031] Fig.14 Western blotting was used to detect the expression of AKT in peritoneal macrophages of wild-type mice and AW112010 ORF-overexpressing mice infected with Escherichia coli for 6 hours. DETAILED DESCRIPTION

[0032] The lncRNA AW112010 gene sequence (SEQ ID NO: 2) is as follows: CTG is the start codon of the protein-coding ORF; TAG is the stop codon.

[0033] CAACTAATTGTGTAAACTTCTCTCAGACTCGGGGTTTTTCAGTCCACTCCTGTTTAGCAAATAGTGCCCCAAAGCAATTCAAAGGGAAAGTGAAACTGACATTCTCAAACTTTATATAAAAAACCAAAGAGAAGATCAGCAGGAGTCTAGCAGACAAC CTGAGC T GC AAG ATG TCT CCC ATC CCT CTG ATA TTT ATC TTT GGTGGT GTG CTC ATC ATC TGC CTG ATG CAA CAA TAC CTG GCG TAT AAG TCT TCT AAG A ACGTC GTT AAA GTC TTC TGC CAT CAA GCC AAT GAT GTA CAT ATA TA C CAG ACC CAG GTCGTC ATG ACA AAC ACA CTG GAA ACC TCA AGT GG A AAA AGC CAC CCA CTG GGT CGT TCAGGA GAG ATC CAG TCT TTA AAG AAG CAA AAC TAG AGACCAAAATAAGTCCTCTTCCTTCTAGATGTGCATCATCTGCTTCTTCCTTCCCTAGAAGACTGAGGGAGCTATCTTTCACAGAGTGTATTCAGTGGTATACGTGGAGCTCAGAAGTAGACACTTGCCTTGCCTCTATTAGGACTTGATTGAACCCAAATACCACAAAATTCTTTCTCTC TGCTGAGAAAATGGAAATTTAAGATGGGAACCAAGCACTGTGTGGTCAACATCCATGAGCATGGTTTAATATACATACCATGTGCTTGTTTTTCTGCTTGTGAACTTGTGAGATAGAAAAACTCCCAAGTCATCCACTTTTACTTGTTATCATTCAGTCACATAATAAATTTCGATTGTGCA

[0034] AW112010 ORF encodes 82 amino acids, and its protein sequence (SEQ ID NO: 1) is as follows:

[0035] LSCKMSPIPLIFIFGGVLIICLMQQYLAYKSSKNVVKVFCHQANDVHIYQTQVVMTNTLETSSGKSHPLGRSGEIQSLKKQN

[0036] Embodiment 1

[0037] 1.1 Gene preparation information

[0038]

[0039] 1.2 Design Strategy Diagram

[0040] This model uses CRISPR / Cas9 technology to insert the CAG-mAW112010 (ORF)-tag gene fragment into the H11 site of mouse chromosome 11. The schematic diagram is shown below. Figure 1 shown.

[0041] 1.3 Vector construction

[0042] According to the design plan, gRNA was designed, constructed and transcribed in vitro, and a homologous recombination vector (Donor vector) was constructed. The correctness of the vector sequence was verified by sequencing.

[0043] 1.4 Microinjection

[0044] Cas9, gRNA and Donor vector samples were microinjected into fertilized eggs of mice with C57BL / 6JGpt background; the fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth.

[0045] 1.5 Identification of F0 generation mice

[0046] The F0 generation pups born to the recipient mice were numbered after their tails and toes were clipped at 5-7 days old, and their genomic DNA was extracted for PCR and sequencing to confirm their genotypes.

[0047] 1.6 Breeding of positive F0 generation mice

[0048] After the positive F0 generation mice reach sexual maturity, they are mated with wild-type background mice; the F1 generation mice born are numbered after their tails and toes are cut off at 5-7 days old, and their genomic DNA is extracted for PCR and sequencing identification to confirm their genotype.

[0049] 1.7 Experimental Results

[0050] 1.8 gRNA sequence information

[0051]

[0052] 1.9 Information on F0 positive mice

[0053] ID gender color genotype Algebra 159 male black KI positive F0

[0054] Example 2: Results of Identification of F1 Generation Mice

[0055] 2.1 Schematic diagram of F1 generation mouse identification results Figure 2 shown.

[0056] 2.2 Overexpression mouse identification strategy

[0057] Wild type: ①②PCR reaction did not obtain the Target band; ③PCR reaction can obtain a single WT band

[0058] Heterozygote: ①②PCR reaction can obtain Target band; ③PCR reaction can obtain a single WT band

[0059] Homozygous: ①②PCR reaction can obtain Target band; ③PCR reaction does not obtain a single WT band

[0060] 2.3 PCR primer information

[0061]

[0062] Sequencing protocol

[0063]

[0064] 2.4 F1 mouse identification results

[0065] According to the PCR strategy diagram, PCR and sequencing confirmed that mice 247#, 248#, 249#, 250#, 254#, 258#, and 259# were positive F1 mice. Figure 3 shown.

[0066] 2.5 PCR reaction conditions

[0067] serial number PCR reaction components Volume (μl) 1 2XTaq Master Mix 12.5 2 <![CDATA[ddH2O]]> 9.5 3 Primer A (10 pmol / μl) 1 4 Primer B (10 pmol / μl) 1 5 Template (≈100 ng / μl) 1

[0068] PCR procedure

[0069]

[0070] 2.6 F1 positive mouse information

[0071]

[0072]

[0073] 2.7 Conclusion

[0074] Mice #247, #248, #249, #250, #254, #258, and #259 were identified as positive by PCR and sequencing.

[0075] Example 3 Functional study of AW112010 ORF protein overexpression mice

[0076] 3.1 Survival study of mice overexpressing AW112010 ORF protein

[0077] The study found that AW112010 ORF overexpression mice survived significantly better than wild-type mice after infection with a lethal dose of MRSA (methicillin-resistant Staphylococcus aureus), indicating that ORF overexpression can significantly improve the survival rate of MRSA-infected mice (see Figure 4 ).

[0078] 3.2 Study on the expression of AW112010 ORF protein in cells and organs

[0079] Escherichia coli was used to infect C57BL / 6J wild-type mice at different times (0, 6, and 12 hours), and the expression of AW112010 ORF protein mRNA in peripheral blood mononuclear cells (PBMC), peritoneal macrophages (PM), lymph nodes (LN), thymus, heart, lung, liver, and spleen was detected. Figure 5 The results showed that ORF mRNA expression was highest in peritoneal macrophages after infection, reaching its highest level 6 hours after infection and decreasing to a certain extent 12 hours after infection, but still higher than that in peritoneal macrophages of uninfected mice. The AW112010 ORF protein was highest in peritoneal macrophages 6 hours after mice were infected with E. coli (see Figure 6 ).

[0080] 3.3 Effects of AW112010 ORF protein overexpression on the differentiation of spleen cell subsets during E. coli infection

[0081] Wild-type mice and mice overexpressing AW112010 ORF protein at about 8 weeks of age were infected with E. coli for 12 hours, and the frequency of each cell subset in the spleen was analyzed. Studies have shown that 12 hours after E. coli infection, the frequency of each cell subset in macrophages of mice overexpressing AW112010 ORF protein (see Figure 7 ), granulocytes (see Figure 8 ) decreased (p≤0.05); while the number of B cells increased (see Figure 8 )(p≤0.05); DC(see Fig. 9 ), NK (see Fig.10 ), CD4+T, CD8+T (see Fig.11 ) The cell frequency did not change significantly (p≥0.05).

[0082] 3.4 Study on the expression of ORF protein in cells

[0083] During infection, LncRNA AW112010 ORF mRNA was mainly expressed in peritoneal macrophages, and immunofluorescence was further used to clarify the localization of its protein in peritoneal macrophages at different time points of infection. Fig.12 The results of immunofluorescence showed that at different time points of E. coli and MRSA infection (0, 3, 6, 12, and 24 hours of infection), ORF protein was mainly expressed in the cytoplasm in the resting state (0 hours), migrated to the nucleus 3 and 6 hours after E. coli and MRSA infection, and gradually decreased in nuclear expression and increased in cytoplasm 12 and 24 hours after infection. In addition, HMGB1 molecules related to mediating inflammation co-localized in the nuclei of peritoneal macrophages at different times of infection.

[0084] 3.5 Study on the co-expression of ORF protein and molecules related to bacterial phagocytosis and clearance in macrophages

[0085] It is planned to further clarify the expression of AW112010 ORF in peritoneal macrophages at different time points during infection, including the expression of apoptotic molecules caspase1, early endosome molecules (EEA1), and molecules Catalase (catalase) that degrade bacteria in an acidic environment after macrophages engulf bacteria, and late peroxisome molecules (Drp1). Fig.13 The results of immunofluorescence show that at different time points of E. coli infection (0, 3, 6, 12, and 24 hours of infection), the ORF protein co-localized with the early endosome EEA1 at 3 hours and 6 hours after E. coli infection, and strongly co-localized with the apoptotic molecule caspase1 at 12 hours after infection; and there was a certain co-localization with Drp1 (shown in white), indicating that the ORF-encoded protein mediates the clearance of bacteria by macrophages.

[0086] 3.6 ORF protein inhibits AKT expression and phosphorylation activity

[0087] To further study the function of AW112010 and its ORF-encoded protein, wild-type mice (WT), LncRNAAW112010 KO (AW- / -) mice, and LncRNA AW112010 ORF-overexpressing (ORF-overexpressing) mice were infected with E. coli for 6 hours, and peritoneal macrophages were isolated to detect the expression of signaling pathway molecules ( Fig.14), the study found that compared with wild-type mice and LncRNA AW112010 KO mice, the total protein and activity (pAKT) of AKT in KO mice were higher than those in peritoneal macrophages of wild-type mice; while the expression of LncRNA AW112010 ORF-overexpressing mice was lower than that in peritoneal macrophages of wild-type mice. This indicates that the protein encoded by AW112010 ORF can significantly inhibit the expression and activity of AKT protein.

[0088] 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 protein in the preparation of drugs for preventing and treating bacterial infections.

2. The use according to claim 1, characterized in that: The AW112010ORF protein is highly expressed during bacterial infection.

3. The use according to claim 1, characterized in that: The AW112010ORF protein can mediate macrophages to clear bacteria.

4. The use according to claim 1, characterized in that: The drug can inhibit the expression and activity of AKT protein.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of the AW112010ORF protein is shown in SEQ ID NO:

1.

6. The use according to any one of claims 1 to 4, characterized in that: The gene sequence of AW112010 encoding the AW112010 ORF protein is shown in SEQ ID NO:2.

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