An interfering polypeptide targeting novel coronavirus ORF7b and its application

Abstract

The present invention relates to the field of biotechnology, and specifically to an interfering polypeptide against the novel coronavirus ORF7b and its application. The present invention targets a polypeptide sequence obtained by modifying, replacing, adding or deleting one or more amino acids in a partial amino acid sequence of the ORF7b sequence, and connects an HIV TAT transmembrane peptide to the C-terminus of the interfering polypeptide. The HIV TAT transmembrane peptide is used to improve the transmembrane effect of the ORF7b interfering polypeptide. The peptide segment carrying the transmembrane peptide interferes with the formation of ORF7b polymers, inhibits the formation of polymers, and improves the host's anti-infection ability. The present invention has broad application prospects and important scientific and social value in the diagnosis and treatment of ORF7b infection, and has the potential for clinical treatment of the novel coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to an interfering polypeptide targeting novel coronavirus ORF7b and applications thereof. Background Art

[0002] The new coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) is a single-stranded RNA virus. The complete genome sequence shows that it is 79% similar to the SARS-CoV genome, and most of the proteins are similar. They all have 4 main structural proteins: Spike Protein (S protein), Envelop Protein (E protein), Membrane Protein (M protein), Nucleoprotein (N protein), and 16 non-structural proteins (nsp1-16), as well as some small auxiliary proteins, such as ORF3a, 3b, 6, 7a, 7b, 8a, 8b, 9b. Studies have shown that these small auxiliary proteins play an important role in the process of virus invasion of host cells.

[0003] SARS-CoV-2 Spike protein (S protein) is the most important surface protein of coronavirus, which is closely related to the virus's infectious ability and pathogenesis. S protein is a large trimeric transmembrane protein that can be divided into two functional units: S1 and S2 protein subunits. S1 contains the receptor binding domain (RBD) part, which is responsible for recognizing and binding to cell receptors.

[0004] Studies have found that the human-to-human transmission of coronaviruses is achieved through the interaction between the capsid surface spike protein (S protein) and the host cell surface receptor. Therefore, all existing inventions are polypeptides targeting the novel coronavirus Spike (S) protein, which are used to inhibit the membrane fusion mediated by the novel coronavirus S protein and the cell receptor ACE2, thereby inhibiting the novel coronavirus from entering cells and preventing or slowing down the virus infection.

[0005] ORF7b is an auxiliary protein of coronavirus, which can inhibit the formation of polymers, thereby relieving the inhibitory effect of ORF7b on the natural immunity of host cells. Therefore, the development of more effective therapeutic drugs for the new coronavirus based on ORF7b polypeptide has important scientific significance and broad application prospects for the clinical treatment of the new coronavirus and the research and development of diagnostic reagents. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned technical defects, the present invention provides a group of interfering polypeptides against SARS-CoV-2 ORF7b and their applications in the preparation of SARS-CoV-2 vaccines, SARS-CoV-2 infection treatment drugs, and SARS-CoV-2 detection reagents or kits.

[0007] The present invention targets a polypeptide sequence obtained by modifying, replacing, adding or deleting one or more amino acids in a partial amino acid sequence shown in the ORF7b sequence, and connects an HIV TAT penetrating peptide to the C-terminus of the interfering polypeptide, and utilizes the HIV TAT penetrating peptide to improve the transmembrane effect of the ORF7b interfering polypeptide. The peptide segment carrying the penetrating peptide interferes with the formation of ORF7b polymers and inhibits the formation of polymers, thereby relieving the inhibitory effect of ORF7b on the natural immunity of host cells, improving the host's anti-infection ability, and reducing the virus infection rate, thereby improving the antiviral effect.

[0008] On the one hand, the present invention provides an interfering polypeptide against novel coronavirus ORF7b, wherein the amino acid sequence of the interfering polypeptide is selected from the following:

[0009] (1) the amino acid sequence shown in SEQ ID NO.1; or an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function;

[0010] (2) the amino acid sequence shown in SEQ ID NO.2; or an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO.2 and having the same function;

[0011] (3) the amino acid sequence as shown in SEQ ID NO.3; or an amino acid sequence having 90% or more homology with the amino acid sequence as shown in SEQ ID NO.3 and having the same function;

[0012] (4) the amino acid sequence shown in SEQ ID NO.4; or an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO.4 and having the same function;

[0013] (5) The amino acid sequence shown in SEQ ID NO.5; or an amino acid sequence that has 90% or more homology with the amino acid sequence shown in SEQ ID NO.5 and has the same function.

[0014] On the other hand, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the interfering polypeptide in the above scheme.

[0015] In another aspect, the present invention provides a nucleic acid derivative, which is formed by connecting, transforming, or modifying the nucleic acid molecules in the above scheme;

[0016] In some specific embodiments, the substance linked to the nucleic acid molecule includes one or more of a fluorescent label, an isotope label, a therapeutic substance, an enzyme label, and a biotin label;

[0017] In some specific embodiments, the modified substance of the nucleic acid molecule includes a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid molecule or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleotide sequence of the nucleic acid molecule.

[0018] The present invention also provides the use of interfering polypeptides, nucleic acid molecules and nucleic acid derivatives in the preparation of new coronavirus vaccines.

[0019] The present invention also provides the use of interfering polypeptides, nucleic acid molecules and nucleic acid derivatives in the preparation of drugs for treating novel coronavirus vaccine infection.

[0020] The present invention also provides the use of interfering polypeptides, nucleic acid molecules and nucleic acid derivatives in the preparation of new coronavirus vaccine detection reagents or kits.

[0021] Beneficial effects: Compared with the prior art, the present invention obtains a polypeptide sequence by modifying, replacing, adding or deleting one or more amino acids in a partial amino acid sequence of the ORF7b sequence, and connects the HIVTAT transmembrane peptide to the C-terminus of the interfering polypeptide. The HIVTAT transmembrane peptide is used to improve the transmembrane effect of the ORF7b interfering polypeptide. The peptide segment carrying the transmembrane peptide interferes with the formation of ORF7b polymers, inhibits the formation of polymers, and improves the host's anti-infection ability. It has broad application prospects and important scientific and social value in the diagnosis and treatment of ORF7b infection, and has the potential for clinical treatment of new coronary pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the three-dimensional structure diagram of ORF7b tetramer;

[0023] Figure 2 This is a diagram of the co-localization of interference peptides in cells;

[0024] Figure 3 This is a diagram showing the effect of interfering peptides on the biological activity of IFNβ;

[0025] Figure 4 This is a Western blot detection image of the interfering peptide;

[0026] Figure 5 This is a diagram for identifying the expression of polypeptide proteins that are interfered with by VSV virus infection. DETAILED DESCRIPTION

[0027] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0028] Example 1 Obtaining Interfering Polypeptides

[0029] The amino acid sequence of ORF7b is shown as: SEQ ID NO.6:

[0030] MESUOFVICFUAFLLFLVLIMLIIFWFSIELODHNETCHA; The full-length sequence of ORF7b shows that the middle part is a tetrameric transmembrane domain, and the Outside and Inside parts at both ends are exposed to the outside. Therefore, the polypeptide targeting the amino acid sequence of ORF7b is mainly composed of Outside OIP (red area) and Inside IIP (blue area) (e.g. Figure 1 shown).

[0031] Outside OIP and Inside IIP were modified, substituted, added or deleted with one or more amino acids, and a Biotin tag was added to the N-terminus. The obtained peptide sequences are as follows:

[0032] SEQ ID NO.7: Biotin-MIELSLIDF

[0033] SEQ ID NO.8: Biotin-ELSLIDF

[0034] SEQ ID NO.9: Biotin-ELSLIDFYL

[0035] SEQ ID NO.10: Biotin-ELQDHNETCH

[0036] SEQ ID NO.11: Biotin-SLELQDHNET

[0037] HIVTAT transmembrane peptide PPRRRQRRKKRG was connected to the C-terminus of the interfering peptide, and the final interfering peptide sequence was:

[0038] SEQ ID NO.1: Biotin-MIELSLIDFPPRRRQRRKKRG

[0039] SEQ ID NO.2: Biotin-ELSLIDFPPRRRQRRKKRG

[0040] SEQ ID NO.3: Biotin-ELSLIDFYLPPRRRQRRKKRG

[0041] SEQ ID NO.4: Biotin-ELQDHNETCHPPRRRQRRKKRG

[0042] SEQ ID NO.5: Biotin-SLELQDHNETPPRRRQRRKKRG

[0043] Example 2 Analysis of specific binding of interfering polypeptides to cells

[0044] Place an 18mm round glass slide in a 12-well plate, add 1 ml of 1% gelatin to each well, and coat the glass slide in a 37°C incubator for 1 hour. Take 293T cells growing in the logarithmic phase, digest with trypsin and divide the cells into 12-well plates, with 100,000 cells per well. After culturing with 10% FBS DMEM medium for 24 hours, the medium was replaced with 1% FBS DMEM culture medium, and the ORF7b-FLAG plasmid was transfected into the cells using liposome transfection technology. After 6 hours of transfection, the medium was replaced with 5% FBS DMEM culture medium. 18 hours after transfection, 10μg / mL of poly(I:C) was added to the cell supernatant to stimulate for 6 hours. At the same time, 50uMol / mL of interfering peptide was added for 6 hours, and 3 controls were set for each interfering peptide. 24 hours after transfection, the supernatant was discarded, washed twice with PBS, and fixed with 4% paraformaldehyde for 10 minutes. 0.1% Triton-X100 was used to permeabilize the membrane for 10 minutes, and 5% BSA was added to block the membrane for 1 hour at room temperature. A 1:100 dilution of biotin antibody was added, and the membrane was incubated for 1 hour at room temperature. The membrane was washed three times with 0.1% Triton-X100 in TBST, each for 10 minutes. A 1:200 dilution of FITC-labeled donkey anti-rabbit secondary antibody was added. The membrane was incubated for 1 hour at room temperature, and the membrane was washed three times with 0.1% Triton-X100 in TBST, each for 10 minutes. The membrane was observed and photographed under a laser confocal microscope at a wavelength of 561. The results are shown in Figure 2. Figure 2 shown. Figure 2 It can be seen that high concentrations of ORF7b interfering polypeptides can enter the cells and act on the location of the ORF7b polymer.

[0045] Example 3 Interfering polypeptide relieves immunosuppression effect

[0046] HEK293T cells and ORF7b-GFP cells were seeded into 12-well plates. After culturing in 10% FBS DMEM medium for 24 hours, the reporter plasmids of IFN-β-pGL3 and pRL-SV40 and the MAVS plasmid were co-transfected using liposome transfection technology. 18 hours after transfection, the cells were treated with 10μg / mL poly(I:C) and 50uMol / mL of interfering peptides for 6 hours, with 3 replicates for each peptide. 24 hours after transfection, the cells were lysed for 15 minutes using 1× lysis buffer (Promega, E1910), and the firefly and Renilla luciferase activities were measured using a SpectraMax i3x microplate reader (Molecular Devices, Afghanistan) according to the reagent instructions. Using Renilla luciferase activity as a reference, each experimental group was repeated 3 times. The results are shown in the figure. Figure 3 As shown in A.

[0047] Similarly, HEK293T cells and ORF7b-GFP cells were trypsinized and divided into 12-well plates. After 24 hours of culture, the MAVS plasmid was transfected into the cells. After 18 hours, the cells were treated with 10μg / mL poly(I:C) and 50uMol / mL of interfering peptide for 6 hours. After 24 hours, the supernatant was discarded, the cells were washed twice with PBS, and the total RNA of the cells was extracted with TRIGene reagent. The RNA was reverse transcribed into cDNA using QPCR reverse transcription reagent (vazyme, R323-01). Finally, the mRNA level of IFN-β in the cells was detected using fluorescent quantitative PCR reagent (vazyme, Q712-02). The results are shown in Figure 3 shown. Figure 3 It was shown that the interfering polypeptides SEQ IP I, SEQ IP II and SEQ IP V enhanced the IFNβ promoter activity and the transcriptional expression of IFNβ;

[0048] Digest the ORF7b-GFP cells with trypsin to a 12-well plate, culture them in 10% FBS DMEM medium for 24 hours, and add 50uMol / mL interfering peptide for treatment for 6 hours. Digest the cells with trypsin to an EP tube and wash twice with PBS. Lyse with 100uL of cell lysate for 30 minutes, shaking every 10 minutes. Add 1× non-denaturing loading buffer. Prepare a non-denaturing gel and perform protein immunoblotting analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, transfer the protein bands to a polyvinylidene fluoride (PVDF) membrane to separate the protein extracts. After blocking with 5% skim milk, wash the PVDF membrane 5 times with TBST. Add a 1:2000 diluted GFP antibody and incubate overnight at 4°C. After washing five times with TBST, add a 1:2000 diluted goat anti-mouse secondary antibody and incubate at 4°C for 3 hours. Wash 5 times with TBST, incubate with a chemiluminescent reagent and collect the signal in an imaging laboratory. The results are as follows. Figure 4 shown. Figure 4 It was shown in the results that the interfering peptide IPV can prevent the production of ORF7b multimers;

[0049] HEK293T cells were seeded in 12-well plates. 293T cells were cultured in 10% FBS DMEM medium for 24 hours, and ORF7b FLAG or PCAGGS plasmid and pMD2G plasmid were co-transfected into the cells. After 24 hours of transfection, the cells were washed twice with PBS and infected with VSV-GFP virus for 2 hours. After 2 hours of infection, the cells were washed twice with PBS and cultured with 2% FBS DMEM virus maintenance medium for 12 hours. After 12 hours, the cells were photographed using a fluorescence microscope. Each experimental group was repeated three times. The results are shown in Figure 2. Figure 5 shown. Figure 5 It was shown that the interfering peptide IPV could relieve the inhibitory effect of ORF7b on IFNβ and reduce the infection rate of VSV virus.

[0050] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the scope of protection of the present invention.

Claims

1. An interfering peptide targeting novel coronavirus ORF7b, Features: The amino acid sequence of the interfering polypeptide is selected from the following: (1) SEQ ID NO.1: Biotin-MIELSLIDFPPRRRQRRKKRG; (2) SEQ ID NO.2: Biotin-ELSLIDFPPRRRQRRKKRG; (3) SEQ ID NO. 5: Biotin-SLELQDHNETPPRRRQRRKKRG.

2. Use of the interfering polypeptide described in claim 1 in the preparation of a new coronavirus vaccine.

3. Use of the interfering polypeptide described in claim 1 in the preparation of drugs for the treatment of novel coronavirus vaccine infection.

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