Polypeptide translated from circular RNA Circ-ace2 and application thereof

Abstract

The application provides a polypeptide translated by a circular RNA Circ-ACE2 and application thereof. The circular RNA molecule comprises at least one of the following sequences: 1) an RNA sequence shown in SEQ ID NO: 1; 2) an RNA sequence with at least 70% identity, preferably an RNA sequence with at least 80% identity, preferably an RNA sequence with at least 85% identity, preferably an RNA sequence with at least 90% identity, preferably an RNA sequence with at least 95% identity, more preferably an RNA sequence with at least 99% identity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, the present application relates to a polypeptide translated from a circular RNA Circ-ACE2 and its application, more particularly, the present application relates to an isolated circular RNA molecule, a polypeptide encoded by the isolated circular RNA molecule, a fusion protein, an isolated nucleic acid encoding the fusion protein, and the use of the polypeptide, the fusion protein, and the isolated nucleic acid encoding the fusion protein in the preparation of a medicine or a kit, and a method for inhibiting the infection of a new coronavirus into a cell. BACKGROUND

[0002] The placenta is not only an important temporary organ for material exchange between the fetus and the mother during pregnancy, but also can protect the fetus from infection by pathogenic microorganisms during fetal development; placental cells may play an important role in inhibiting the transmission of viruses from a pregnant mother to a fetus. Some research groups have reported that the vertical transmission of COVID-19 virus from mother to child is limited, and data shows that the proportion of newborns transmitting COVID-19 from the mother is less than 5%, which suggests that there is a unique antiviral substance in the placental tissue that can inhibit the transmission of the virus from a COVID-19 infected mother to the fetus.

[0003] Polypeptide drugs have been widely used in clinical practice, and a typical representative is insulin polypeptide drugs. The metabolic products of polypeptide drugs are amino acids, and amino acids are essential elements for the human body. Therefore, polypeptide drugs are similar to protein drugs, and thus have low toxicity and high safety. Compared with large protein and antibody drugs, polypeptide drugs have low immunogenicity, and are easy to synthesize artificially, suitable for injection and nasal aerosol administration, and have good drug properties.

[0004] In recent years, a large number of circular RNA molecules have been found to exist in various organisms, and circular RNA is widely present in eukaryotes and has important biological functions in individual development and the occurrence and development of diseases. There is no effective and side-effect-free treatment drug for the disease caused by the new coronavirus, so it is necessary to discover and develop ideal drugs against the new coronavirus from a new perspective. SUMMARY

[0005] The present application is based on the discovery and understanding of the inventors of the following facts and problems:

[0006] The novel coronavirus binds to ACE2 (angiotensin 2) to infect cells, and the inventors found that circularization and splicing of the ACE2 gene specifically occurs in human placental tissue to form a circular RNA Circ-ACE2 through deep analysis and mining of the circular RNA circAtlas comprehensive database and experiments, and the polypeptide encoded by the circular RNA and the recombinant polypeptide obtained by using the polypeptide have the ability to inhibit COVID-19 from infecting eukaryotic cells.

[0007] To this end, in a first aspect of the present application, the present application provides an isolated circular RNA molecule. According to an embodiment of the present application, the circular RNA molecule comprises at least one of the following sequences: 1) the RNA sequence shown in SEQ ID NO: 1; 2) an RNA sequence having at least 70% identity with 1), preferably an RNA sequence having at least 80% identity, preferably an RNA sequence having at least 85% identity, preferably an RNA sequence having at least 90% identity, preferably an RNA sequence having at least 95% identity, more preferably an RNA sequence having at least 99% identity.

[0008] According to an embodiment of the present application, the specific sequence of SEQ ID NO: 1 is:

[0009] CGCCCAACCCAAGUUCAAAGGCUGAUAAGAGAGAAAAUCUCAUGAGGAGGUU UUAGUCUAGGGAAAGUCAUUCAGUGGAUGUGAUCUUGGCUCACAGGGGACGAUGUCAAGCUCUUCCUGGCUCCUUCUCAGCCUUGUUGCUGUAACUGCUGCUCAGUCCA CCAUUGAGGAACAGGCCAAGACAUUUUUGGACAAGUUUAACCACGAAGCCGAAGACCUGUUCUAUCAAAGUUCACUUGCUUCUUGGAAUUAUAACACCAAUAUUACUGAA GAGAAUGUCCAAAACAUG.

[0010] The polypeptide translated from the circular RNA sequence according to an embodiment of the present application can significantly inhibit the ability of the novel coronavirus to infect cells.

[0011] According to an embodiment of the present application, the above-mentioned circular RNA molecule can further include at least one of the following additional technical features:

[0012] According to an embodiment of the present application, the circular RNA molecule is formed by circularization of the second exon of the mRNA sequence encoding angiotensin-converting enzyme 2 alone.

[0013] According to an embodiment of the present application, the circularization occurs in a placental tissue.

[0014] According to an embodiment of the present application, the circular RNA molecule is formed by connecting the initial nucleotide and the terminal nucleotide of the nucleotide sequence shown in 1) or 2), and the initial nucleotide is arranged as the first nucleotide of the circular RNA molecule.

[0015] According to an embodiment of the present application, the circular RNA coding region comprises the 104th nucleotide to the 45th nucleotide of the circular RNA molecule.

[0016] According to an embodiment of the present application, the nucleic acid sequence of the circular RNA coding region comprises at least one of the following sequences: 1) the RNA sequence shown in SEQ ID NO: 2; 2) an RNA sequence having at least 70% identity with 1), preferably an RNA sequence having at least 80% identity, preferably an RNA sequence having at least 85% identity, preferably an RNA sequence having at least 90% identity, preferably an RNA sequence having at least 95% identity, more preferably an RNA sequence having at least 99% identity.

[0017] According to an embodiment of the present application, the specific sequence of SEQ ID NO: 2 is:

[0018] AUGUCAAGCUCUUCCUGGCUCCUUCUCAGCCUUGUUGCUGUAACUGCUGCUC AGUCCACCAUUGAGGAACAGGCCAAGACAUUUUUGGACAAGUUUAACCACGAAGC CGAAGACCUGUUCUAUCAAAGUUCACUUGCUUCUUGGAAUUAUAACACCAAUAUUACUGAAGAGAAUGUCCAAAACAUGCGCCCAACCCAAGUUCAAAGGCUGAUAAGAGAGAAAAUCUCAUGA.

[0019] The polypeptide translated from the nucleic acid sequence of the circular RNA coding region according to an embodiment of the present application can significantly inhibit the ability of the novel coronavirus to infect cells.

[0020] In a second aspect, the present application provides a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide comprises at least one of the following sequences: 1) the amino acid sequence set forth in SEQ ID NO: 3; 2) an amino acid sequence having at least 70% identity, preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, and more preferably at least 99% identity to 1). According to an embodiment of the present application, the polypeptide can significantly inhibit the infection ability of the novel coronavirus to cells.

[0021] According to an embodiment of the present application, the specific sequence of SEQ ID NO: 3 is:

[0022] MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMRPTQVQRLIREKIS.

[0023] According to an embodiment of the present application, the polypeptide can further comprise at least one of the following additional technical features:

[0024] According to an embodiment of the present application, the polypeptide contains a signal peptide sequence.

[0025] According to an embodiment of the present application, the amino acid sequence of the signal peptide is MSSSSWLLLSLVAVTAA.

[0026] According to an embodiment of the present application, the polypeptide exists in placental tissue.

[0027] In a third aspect, the present application provides a fusion protein. According to an embodiment of the present application, the fusion protein comprises the polypeptide of the first or second aspect and an Fc, wherein the C-terminus of the polypeptide is connected to the N-terminus of the Fc. According to an embodiment of the present application, the fusion protein can significantly inhibit the infection ability of the novel coronavirus to cells.

[0028] According to an embodiment of the present application, the fusion protein can further comprise at least one of the following additional technical features:

[0029] According to an embodiment of the present application, the fusion protein comprises at least one of the following sequences: 1) the amino acid sequence shown in SEQ ID NO: 4; 2) an amino acid sequence having at least 70% identity with 1), preferably an amino acid sequence having at least 80% identity, preferably an amino acid sequence having at least 85% identity, preferably an amino acid sequence having at least 90% identity, preferably an amino acid sequence having at least 95% identity, more preferably an amino acid sequence having at least 99% identity.

[0030] According to an embodiment of the present application, the specific sequence of SEQ ID NO: 4 is:

[0031] MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEE NVQNMRPTQVQRLIREKISLVPRGSGGGGDPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0032] In a fourth aspect of the present application, an isolated nucleic acid is provided. According to an embodiment of the present application, the isolated nucleic acid encodes the fusion protein of the third aspect. The fusion protein translated from the isolated nucleic acid sequence according to an embodiment of the present application can significantly inhibit the ability of the novel coronavirus to infect cells.

[0033] According to an embodiment of the present application, the isolated nucleic acid can further comprise at least one of the following additional technical features:

[0034] According to an embodiment of the present application, the nucleic acid comprises at least one of the following sequences: 1) the nucleotide sequence shown in SEQ ID NO: 5; 2) a nucleotide sequence having at least 70% identity with 1), preferably a nucleotide sequence having at least 80% identity, preferably a nucleotide sequence having at least 85% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 95% identity, more preferably a nucleotide sequence having at least 99% identity.

[0035] According to the embodiment of the present application, the specific sequence of SEQ ID NO: 5 is:

[0036] ATGTCAAGCTCTTCCTGGCTCCTTCTCAGCCTTGTTGCTGTAACTGCTGCTCAGTCCACCATTGAGGAACAGGCCAAGACATTTTTGGACAAGTTTAACCACGAAGCCGAAGACCTGTTCTATCAAAGTTCACTTGCTTCTTGGAATTATAACACCAATATTACTGAAGAGA ATGTCCAAAACATGCGCCCAACCCAAGTTCAAAGGCTGATAAGAGAGAAAATCTCACTGGTGCCCAGAGGCTCCGGCGGCGGCGGCGATCCTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA.

[0037] In a fifth aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector comprises the RNA molecule of the first aspect or the isolated nucleic acid of the fourth aspect. The polypeptide translated from the expression vector according to an embodiment of the present application can significantly inhibit the infection ability of the novel coronavirus to the cell.

[0038] In a sixth aspect, the present application provides a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the RNA molecule of the first aspect, the isolated nucleic acid of the fourth aspect or the expression vector of the fifth aspect. The recombinant cell has a stronger ability to resist the infection of the novel coronavirus.

[0039] In a seventh aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect or the recombinant cell of the sixth aspect. According to an embodiment of the present application, the pharmaceutical composition can be used to resist the infection of the novel coronavirus to the cell.

[0040] According to an embodiment of the present application, the pharmaceutical composition can further comprise at least one of the following additional technical features:

[0041] According to an embodiment of the present application, the pharmaceutical composition is at least one of a solution, a powder, a microsphere or a microcapsule. Further, the pharmaceutical composition according to an embodiment of the present application is convenient for administration and suitable for maintaining the effect of the pharmaceutical composition.

[0042] According to an embodiment of the present application, the administration dose of the pharmaceutical composition is not particularly limited, and in practical applications, it can be flexibly selected according to the health status of the administration subject.

[0043] In an eighth aspect, the present application provides the use of the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect or the pharmaceutical composition of the seventh aspect in the preparation of a pharmaceutical or a kit for inhibiting the infection of the novel coronavirus to the cell. According to an embodiment of the present application, the pharmaceutical or the kit can significantly inhibit the infection ability of the novel coronavirus to the cell, and the pharmaceutical or the kit can be used for clinical diagnosis or scientific research.

[0044] In a ninth aspect, the present application provides a method for inhibiting SARS-CoV-2 from infecting a cell, comprising contacting SARS-CoV-2 or a cell to be infected with the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect. According to embodiments of the present application, the method can significantly inhibit SARS-CoV-2 from infecting a cell.

[0045] According to embodiments of the present application, the method can further comprise at least one of the following additional technical features:

[0046] According to embodiments of the present application, the contacting is in vivo or ex vivo. For example, the in vivo contacting can be achieved by at least one of direct microinjection, oral administration, intranasal administration, transdermal administration, intravenous injection, respiratory inhalation, rectal administration, i.e., introducing the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect into a subject by the above-mentioned ways, so as to contact the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect with a cell to be infected, such as a lung epithelial cell. For example, the ex vivo contacting means directly mixing and incubating or contacting the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect with SARS-CoV-2 or a cell to be infected in vitro.

[0047] According to specific embodiments of the present application, the contacting is achieved by: 1) performing a first mixing process on SARS-CoV-2 and the RNA molecule of the first aspect, the polypeptide of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid of the fourth aspect, the expression vector of the fifth aspect, the recombinant cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect; and 2) performing a second mixing process on the mixture obtained in step 1) and a cell to be infected, so as to inhibit SARS-CoV-2 from infecting a cell.

[0048] According to an embodiment of the present application, the final concentration of the polypeptide or fusion protein in the mixed system is 10 μg / mL or 160 μg / mL. According to an embodiment of the present application, the polypeptide can inhibit the ability of the new coronavirus pseudovirus to infect cells by 91.61% when the final concentration in the mixed system is 10 μg / mL, and by 95.50% when the final concentration in the mixed system is 160 μg / mL; the fusion protein (recombinant polypeptide) can inhibit the ability of the new coronavirus pseudovirus to infect cells by 52.11% when the final concentration in the mixed system is 10 μg / mL, and by 92.18% when the final concentration in the mixed system is 160 μg / mL, and the final concentration of the polypeptide or fusion protein in the mixed system according to the embodiment of the present application is suitable for maintaining the efficacy of the drug.

[0049] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0050] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the accompanying drawings, wherein:

[0051] Figure 1 is a distribution diagram of the expression amount of ACE2 mRNA molecules in each tissue of the human body according to an embodiment of the present application;

[0052] Figure 2 is a distribution diagram of the expression amount of circular RNA circ-ACE2 in each tissue of the human body according to an embodiment of the present application;

[0053] Figure 3 is a mode diagram of the formation of circular RNA Circ-ACE2 according to an embodiment of the present application, wherein the ACE2 RNA is spliced and circularized at the position of the second exon (Exon2) to form the circular RNA Circ-ACE2;

[0054] Figure 4 is a result diagram of the sequencing verification of the circular RNA Circ-ACE2 circular sequence of the PCR product of the RNA Circ-ACE2 according to an embodiment of the present application, wherein the PCR result diagram is from left to right in the direction of the 3' end to 5' of the PCR product sequence;

[0055] Figure 5 is an analysis diagram of the coding reading frame across the interface of the circular RNA Circ-ACE2 according to an embodiment of the present application, wherein the coding sequence of the circular RNA molecule is the nucleotide sequence across the circularization interface from the 104th nucleotide to the 45th nucleotide of the sequence of the circular RNA molecule, and the specific sequence is the underlined sequence in the diagram;

[0056] Figure 6 is a comparison analysis chart of the amino acid sequence of the CircACE2-76aa polypeptide and the protein encoded by ACE2 according to the embodiment of the present application, wherein, compared with the amino acid sequence of the ACE2 protein, the CircACE2-76aa contains the N-terminal 62 amino acids of ACE2 and the C-terminal carrying specific 14 amino acids (RPTQVQRLIREKIS);

[0057] Figure 7 is a mass spectrometry identification result chart of the polypeptide translated from the circular RNA Circ-ACE2 molecule according to the embodiment of the present application;

[0058] Figure 8 is an online program analysis chart of the amino acid sequence of the CircACE2-76aa according to the embodiment of the present application;

[0059] Figure 9 is a cell localization chart of the CircACE2-76aa under a fluorescence microscope according to the embodiment of the present application;

[0060] Figure 10 is a result chart of the difference fold of the expression amount of the EGFP gene carried by the pseudovirus in the experiment of detecting the pseudovirus infection of cells by the chemically synthesized polypeptide CircACE2-76aa according to the embodiment of the present application; and

[0061] Figure 11 is a result chart of the difference fold of the expression amount of the EGFP gene carried by the pseudovirus in the experiment of detecting the pseudovirus infection of cells by the recombinant protein CircACE2-76aa-FC according to the embodiment of the present application. DETAILED DESCRIPTION

[0062] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0063] TERMS EXPLANATION

[0064] In the present application, the terms “new coronavirus”, “coronavirus”, “COVID-19” all refer to the pathogen causing COVID-19.

[0065] Identity, the present invention, to compare two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical with the nucleotides in the corresponding positions of the second sequence] by [the total number of nucleotides in the first sequence], and then multiplying by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered a difference at a single nucleotide (position).

[0066] Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings, using known computer algorithms for sequence alignment, such as NCBI Blast v2.0.

[0067] Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0068] Identity, the present invention, to compare two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical with the nucleotides in the corresponding positions of the second sequence] by [the total number of nucleotides in the first sequence], and then multiplying by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered a difference at a single nucleotide (position).

[0069] Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings, using known computer algorithms for sequence alignment, such as NCBI Blast v2.0.

[0070] Generally, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence having the largest number of amino acid residues is taken as the "first" amino acid sequence, and the other amino acid sequence is taken as the "second" amino acid sequence.

[0071] Likewise, in determining the degree of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which the amino acid residue is replaced with an amino acid residue that has similar chemical structure and that has little or no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. from WO 04 / 037999, GB-A-2357768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and WO 01 / 09300. The type and / or combination of such substitutions can be selected and / or (preferably) based on the relevant teachings from WO 04 / 037999 as well as WO 98 / 49185 and other references cited therein.

[0072] The inventors of the present embodiment conducted in-depth analysis and mining on the comprehensive database of circular RNA circAtlas, and found that the ACE2 gene specifically formed a circular RNA molecule in the placental tissue, which was formed by circularization of the second exon of ACE2 RNA alone, and was named Circ-ACE2 by the inventors. Sequence analysis and experimental verification results of the circular RNA Circ-ACE2 suggest that CircACE2 can translate a polypeptide containing a secretion signal peptide, which is a completely new polypeptide molecule, and the inventors named it CircACE2-76aa. The polypeptide CircACE2-76aa is 76 amino acids in length. The present application obtains a recombinant polypeptide CircACE2-76aa-FC by chemically synthesizing the polypeptide CircACE2-76aa and expressing the FC fragment of human immunoglobulin G in eukaryotic cells. The anti-viral function of CircACE2-76aa is verified by the strategy of in vitro infection of human hACE2-293T cells with a new crown pseudovirus, and the results show that chemically synthesized CircACE2-76aa polypeptide and recombinant polypeptide CircACE2-76aa-FC can significantly inhibit the cell infection ability of the new crown pseudovirus. The CircACE2-76aa polypeptide and the recombinant polypeptide CircACE2-76aa-FC involved in the present application can become candidate molecules for inhibiting COVID-19 virus infection.

[0073] The following will be specifically introduced. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0074] Example 1 Identification and characterization of circular RNA Circ-ACE2 in isolated placental tissue

[0075] The human placental tissue in the present embodiment is from the discarded placenta after natural delivery of pregnant women in clinical obstetrics and gynecology.

[0076] 1. Identification of circular RNA Circ-ACE2 in isolated placental tissue

[0077] ACE2 mRNA is widely expressed in multiple tissues of humans, and the expression distribution of the gene in various tissues of the human body is shown in Figure 1 The inventors found that circular splicing of ACE2 gene specificity occurred in isolated placental tissue through deep analysis and mining of the circular RNA circAtlas comprehensive database, forming circular RNA Circ-ACE2, as shown in Figure 2

[0078] 2. Sequencing identification of circular RNA Circ-ACE2 in isolated placental tissue

[0079] PCR detection primers specific for detecting circular RNA Circ-ACE2 were designed, the upstream sequence of the primer was 5'GAAGCCGAAGACCTGTTCTA3', the downstream sequence was 5'TCTTATCAGCCTTTGAACTTGG 3', and the amplified fragment size was 114 bp. Total RNA of the isolated placental tissue sample was extracted by Trizol method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme Company) with random primers. The PCR amplification reaction system and conditions are as follows: the PCR is 50 μL total system, specifically 2x PCR MIX (Vazyme Company) 25 μL, upper and lower primers (10 mM) each 2 μL, cDNA template 1 μL, and sterilized water is added to 50 μL system. The reaction conditions are as follows: cDNA pre-denaturation at 95°C for 3 min, first amplification: denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 35 cycles of PCR reaction, and then 72°C for 3 min of extension, followed by 16°C storage. The PCR product was separated by 1.5% agarose gel, and the gel was purified for sanger DNA sequencing. Figure 3 As can be seen, the circular RNA Circ-ACE2 is a circular RNA molecule formed by circular splicing of the second separate exon of ACE2 mRNA. As shown in Figure 4 The inventors identified the accurate circular interface of circular RNA Circ-ACE2 by the method of PCR product Sanger DNA sequencing, and the sequence length of Circ-ACE2 RNA was 289 nt, and the specific nucleic acid sequence is shown in SEQ ID NO: 1.

[0080] ​3. Coding potential analysis of RNA Circ-ACE2

[0081] According to the nucleic acid sequence of RNA Circ-ACE2, the coding potential analysis was performed by using the ORF finder online program, and it was found that the circular RNA Circ-ACE2 contained a reading frame across the interface of the circular RNA, as shown in Figure 5 The coding region of the polypeptide translated from the circular RNA Circ-ACE2 was composed of 231 bases, and the base sequence was shown as SEQ ID NO: 2; the length of the polypeptide translated from the RNA Circ-ACE2 was 76 amino acids, which was named as CircACE2-76aa in the present application, and the amino acid sequence was shown as SEQ ID NO: 3. The amino acid sequences of CircACE2-76aa and the protein encoded by RNA ACE2 were compared and analyzed, and the results showed that CircACE2-76aa contained the N-terminal 62 amino acids of ACE2 and the C-terminal carrying specific 14 amino acids (RPTQVQRLIREKIS), as shown in Figure 6

[0082] Example 2 Identification of the polypeptide CircACE2-76aa translated from the circular RNA Circ-ACE2

[0083] 1. Design and construction of circular RNA Circ-ACE2 expression plasmid

[0084] According to the sequence information of the circular RNA Circ-ACE2, the target sequence was obtained by the method of whole gene chemical synthesis, and then the sequence was constructed into the circular RNA expression vector pCD5-ciR (Ji Sai Biological) by restriction endonuclease EcoRI and BamHI.

[0085] 2. Construction of red fluorescent protein fusion expression plasmid CircACE2-76aa-mcherry

[0086] ​According to the circular RNA expression vector CircRNA Mini Vector (Addgene ID: #60648) circular RNA expression design method, the CircACE2-76aa and red fluorescent protein mcherry fusion circular RNA expression vector CircACE2-76aa-mcherry is designed, the start codon ATG and the stop codon of the mcherry gene fragment itself are removed, and are inserted into the front end of the stop codon of CircACE2-76aa, and the circularization mediated sequences of the CircRNA Mini Vector carrier are added at both ends of the sequence, and the final designed expression framework is shown in SEQ ID NO: 7; after the design of the CircACE2-76aa-mcherry circular RNA fusion expression framework, the expression framework is obtained by the method of whole gene chemical synthesis, and then the expression framework is constructed into the pcDNA3.1(+) expression vector by using the EcoRI and BamHI restriction endonuclease sites.

[0087] 3. Transfection of CircACE2-76aa-mcherry into hACE2-293T cells

[0088] After transfecting the 293T cells with the circular RNA Circ-ACE2 expression plasmid, the circular RNA Circ-ACE2 expression product was subjected to SDS-PAGE protein electrophoresis and Coomassie blue staining, and the gel was cut for mass spectrometry identification. As shown in Figure 7 , the peptide segment translated by the circular RNA Circ-ACE2 across the circularization interface was identified by mass spectrometry; the peptide segment sequence obtained by mass spectrometry was QNMRPTAVQR, and the sequence showed the terminal specific amino acid sequence translated by the circular RNA circ-ACE2; amino acid sequence analysis of the CircACE2-76aa polypeptide found that the analysis used the signalP-4.1 online program, as shown in Figure 8 , the CircACE2-76aa did not contain a transmembrane amino acid sequence, and contained a typical secretion signal peptide sequence, the secretion signal peptide sequence of CircACE2-76aa was composed of 17 amino acids at the N terminus, and the sequence was MSSSSWLLLSLVAVTAA; through the fluorescence microscope, the localization of the CircACE2-76aa polypeptide was observed by using the fused mcherry red fluorescent protein, and it was found that the CircACE2-76aa polypeptide had the characteristics of extracellular secretion, and the results are shown in Figure 9 .

[0089] Example 3: Chemical synthesis of polypeptide CircACE2-76aa

[0090] The polypeptide was synthesized by Shanghai Polypeptide Biological Company. The polypeptide was synthesized by solid phase synthesis. The peptide chain was synthesized by Fmoc / PyBOP method. The Fmoc protecting group was removed by 30% hexahydropyridine DMF solution; the peptide chain was cut off from the resin by peptide cutting reagent (trifluoroacetic acid / crystalline phenol / water / ethanedithiol / methyl ethyl sulfide / = 81.5 / 5 / 5 / 5 / 2.5 / 1). Polypeptide purification and detection: C18 reverse phase column was used, the conditions were as follows: A phase was 95% water (methanol ratio), B phase was 95% methanol (methanol ratio), then 0.1% TFA was added respectively, the conventional conditions were as follows: the column was equilibrated with A phase for 15 minutes before sample loading, then sample loading was performed, gradient elution from A phase to B phase was 25 minutes. The detection wavelength was 220 nm, the flow rate was 1 mL / min, the column was equilibrated with A solution, after sample loading, gradient elution from A to B solution was 25 min, and the target peptide was collected; the synthesized polypeptide was identified by mass spectrometer.

[0091] Example 4 Construction of circACE2-76aa-FC polypeptide recombinant expression plasmid and eukaryotic cell expression and purification

[0092] 1. Construction of polypeptide recombinant expression plasmid

[0093] According to the coding nucleic acid sequence of circACE2-76aa and the human immunoglobulin IGg sequence, a recombinant expression framework was designed, and the nucleic acid sequence is shown as SEQ ID NO: 5; the expression framework of circACE2-76aa-FC was obtained by using the strategy of segmented PCR amplification and overlap PCR splicing amplification, and then the framework was linked to pcDNA3.1 (+) vector by EcoRI and BamHI endonuclease sites.

[0094] 2. Eukaryotic cell transfection of expression plasmid

[0095] The cells were cultured to a density of 2.5-3.0 million / mL, and 500 mL of cells were cultured in a 2L culture bottle under the conditions of temperature 37℃, rotation speed 120 rpm and 8% CO2; 0.5 mg of plasmid was transfected per 500 mL of cells, and PEI was 2.7 times the amount of plasmid. First, prepare 10 mL of fresh culture medium (take 500 mL as an example), first add 50 μg of plasmid, mix well, slowly add the corresponding amount of PEI, incubate for 5-10 min (can be observed that the culture medium changes from the original transparent state to slightly turbid), after incubation, add to the cells, and express the cells under the conditions of 33℃, 8% CO2 and 120 rpm, collect the cell supernatant after 6 days of expression, and end the expression.

[0096] 3. Purification of circACE2-76aa-FC recombinant polypeptide

[0097] On each tube of the collection tube, add 5 mL of neutralizing liquid (1 M tris-HCl pH 8.0), AKTA prue column (AKTA prue column: containing protein A (Cytiva Company, item number: 17040301)) is balanced with Binding buffer (20 mM phosphate), and the cell supernatant is 500 mL. Add to the purification column with a volume setting of 1500 mL. After column loading, wash the impurities with Binding buffer (20 mM phosphate) for 80 mL. Elution buffer (0.1 M citric acid, pH 3.0) is used for elution for 100 mL. The collection tube is 5 mL per tube. Run the gel for the collection tube at the peak position to determine the collection tube with protein. Concentrate the protein, and finally replace the protein in PBS.

[0098] Example 5 COVID-19 novel coronavirus pseudovirus infection cell experiment of CircACE2-76aa polypeptide and recombinant CircACE2-76aa-FC

[0099] The process of virus invading cells can be simulated by using commercial novel coronavirus S protein recombinant pseudovirus and stable overexpression human ACE2 full-length gene 293T cell line hACE2-293T (Guangzhou Pisheng Biotechnology Co., Ltd.). The chemically synthesized CircACE2-76aa polypeptide (SEQ ID NO: 6) and the recombinant polypeptide CircACE2-76aa-FC (SEQ ID NO: 4) obtained by eukaryotic expression in 293T were mixed and incubated with the new crown pseudovirus, and then infected hACE2-293T cells. The specific operation is as follows: hACE2-293T cells were plated in a 96-well plate at a density of 1 x 10 4 cells / well; 12 hours of cell adhesion, COVID-19 pseudovirus infection experiment; add 5 μL of pseudovirus (about 10,000 virus particles) and different concentrations of polypeptide to each well of cells. The pseudovirus and polypeptide were mixed in 100 μL of DMEM complete culture medium in vitro according to the proportion, and were placed at room temperature for 30 min. Before the pseudovirus infected the cells, 8 μg / mL of polybrene was added.

[0100] By the EGFP gene sequence carried in the new crown pseudo-virus, the genomic DNA of the infected cells is extracted, and QPCR fluorescence quantitative detection is performed. The effects of chemically synthesized CircACE2-76aa polypeptide and recombinant CircACE2-76aa-FC on inhibiting the new crown pseudo-virus COVID-19 from infecting cells are quantitatively detected, and the specific operation is as follows: after the pseudo-virus infects hACE2-293T cells for 15h; the cell culture medium is removed, 100μL of cell genomic DNA extraction lysis solution is directly added to each well, the cells are mixed and lysed in a 96-well plate, then transferred to a 1.5mL centrifuge tube, and then the genomic DNA is extracted. The extracted DNA is dissolved in sterile water, 1μL of genomic DNA is used for QPCR fluorescence quantitative detection, the integration of the EGFP gene carried by the pseudo-virus into the cell genome is detected, and the GAPDH gene is used as an internal reference to evaluate the ability of the new crown pseudo-virus to infect hACE2-293T cells.

[0101] QPCR detection: The reaction system is configured according to the instruction manual of the fluorescent quantitative reaction kit QPCR kit (Vazyme Company). The QPCR reaction system is 20μL, 10μL of 2x SYBR Green PCR Master Mix, 0.4μL of upstream and downstream primers (10μM) each, 1μL of template DNA, and finally sterile deionized water is added to make up to 20μL; the fluorescent quantitative PCR reaction conditions are as follows: cDNA pre-denaturation at 95℃ for 5min; first amplification: denaturation at 95℃ for 10s, annealing at 60℃ for 35s (collect fluorescence signal in this step); 40 cycles, then perform melting curve analysis: collect fluorescence signal at temperature 60℃-95℃. The sequence of the QPCR detection primer is: EGFP-upstream primer: 5'TTCAAGGAGGACGGCAACAT 3', EGFP-downstream primer: 5'TGGCGGATCTTGAAGTTCAC 3', and the primer amplification size is 119bp.

[0102] From Figure 10 It can be seen that the chemically synthesized polypeptide CircACE2-76aa can inhibit the ability of the new crown pseudo-virus to infect cells by 91.61% at a concentration of 10μg / mL, and can inhibit the ability of the new crown pseudo-virus to infect cells by 95.50% at a concentration of 160μg / mL. Figure 11 It is shown that the recombinant CircACE2-76aa-FC can inhibit the ability of the new crown pseudo-virus to infect cells by 52.11% at a concentration of 10μg / mL, and can inhibit the ability of the new crown pseudo-virus to infect cells by 92.18% at a concentration of 160μg / mL, and the specific results are shown in Figure 11 .

[0103] Table 1:

[0104]

[0105]

[0106]

[0107] Note: The underlined sequences are circular RNA mediated sequences

[0108] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0109] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application. SEQUENCE LISTING <110> Bioisland Laboratory, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences <120> Polypeptide translated from circular RNA Circ-ACE2 and application thereof <130> BI3210816A <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 289 <212> RNA <213> Artificial Sequence <220> <223> 1 <400> 1 cgcccaaccc aaguucaaag gcugauaaga gagaaaaucu caugaggagg uuuuagucua 60 gggaaaguca uucaguggau gugaucuugg cucacagggg acgaugucaa gcucuuccug 120 gcuccuucuc agccuuguug cuguaacugc ugcucagucc accauugagg aacaggccaa 180 gacauuuuug gacaaguuua accacgaagc cgaagaccug uucuaucaaa guucacuugc 240 uucuuggaau uauaacacca auauuacuga agagaauguc caaaacaug 289 <210> 2 <211> 231 <212> RNA <213> Artificial Sequence <220> <223> 2 <400> 2 augucaagcu cuuccuggcu ccuucucagc cuuguugcug uaacugcugc ucaguccacc 60 auugaggaac aggccaagac auuuuuggac aaguuuaacc acgaagccga agaccuguuc 120 uaucaaaguu cacuugcuuc uuggaauuau aacaccaaua uuacugaaga gaauguccaa 180 aacaugcgcc caacccaagu ucaaaggcug auaagagaga aaaucucaug a 231 <210> 3 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 3 <400> 3 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Arg Pro 50 55 60 Thr Gln Val Gln Arg Leu Ile Arg Glu Lys Ile Ser 65 70 75 <210> 4 <211> 320 <212> PRT <213> Artificial Sequence <220> <223> 4 <400> 4 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Arg Pro 50 55 60 Thr Gin Val Gin Arg Leu lie Arg Glu Lys lie Ser Leu Val Pro Arg 65 70 75 80 Gly Ser Gly Gly Gly Gly Asp Pro Glu Pro Lys Ser Cys Asp Lys Thr 85 90 95 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 100 105 110 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 115 120 125 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 130 135 140 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 145 150 155 160 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 165 170 175 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 180 185 190 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 195 200 205 Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 210 215 220 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 225 230 235 240 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 245 250 255 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 260 265 270 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 275 280 285 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 290 295 300 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 305 310 315 320 <210> 5 <211> 963 <212> DNA <213> Artificial Sequence <220> <223> 5 <400> 5 atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60 attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120 TATCAAAGTT CACTTGCTTC TTGGAATTAT AACACCAATA TTACTGAAGA GATGTCCAA 180 AACATGCGCC CAACCCAAGT TCAAAGGCTG ATAAGAGAGA AAATCTCACT GGTGCCCAGA 240 GGCTCCGGCG GCGGCGGCGA TCCTGAGCCC AAATCTTGTC ACAAAACTCA CACATGCCC 300 CCGTGCCCAG CACCTGAACT CCTGGGGGGA CCgtcagtct TCCTCTTCCC CCCAAAACC 360 AAGGACACCC Tcatgatctc CCggacccct GAGgtcacat GCgtggtggt GGACGTGAGC 420 CACGAAGACC CTGAGGTCAA GTTCACTGGT ACgtggacgg Cgtggaggtg CATAATGCC 480 AAGACAAAGC CGGggaggaG CAGTACAAC AGCACGTACC GTGTGgtcag CGTCCTCACC 540 GTCTTCTGTC CTGTCCTGTC CTGTCCTGTC CTGTCCTGTC CTGTCCTGTC CTGTCCTGTC 600 CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG 660 GTGTACACCC TGCCCCCATC CCgggatgag CTGACCAAGA ACCAGGTCAG CCTGACCTGC 720 CTGgtcaaag Gcttctatcc CAGcgacatc GCCGTggagt GGGAGAGcaa TGGGCAGCCG 780 GAGAACAACt ACAAGACCAC GCCTCCCgtg CTGGACTCCG ACggctcctt CTCTCTAC 840 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 900 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 960 tga 963 <210> 6 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> 6 <400> 6 Gln Ser Thr Ile Glu Glu Gin Ala Lys Thr Phe Leu Asp Lys Phe Asn 1 5 10 15 His Glu Ala Glu Asp Leu Phe Tyr Gin Ser Ser Leu Ala Ser Trp Asn 20 25 30 Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gin Asn Met Arg Pro Thr 35 40 45 Gln Val Gin Arg Leu Ile Arg Glu Lys Ile Ser 50 55 <210> 7 <211> 1132 <212> DNA <213> Artificial Sequence <220> <223> 7 <400> 7 agtgctgaga ttacaggcgt gagccaccac ccccggccca ctttttgtaa aggtacgtac 60 taatgacttt ttttttatac ttcagcgccc aacccaagtt caaaggctga taagagagaa 120 aatctcaaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt 180 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 240 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt 300 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc 360 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt 420 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 480 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat 540 gcagaagaag actatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc 600 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga 660 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa 720 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg 780 CGCCGAGGGCCGCCACTCCACC GGC GG CAT GGACGAGCTGTACAAGTGAGGAGGTTTAG 840 TCTAGGGAAAGTCATTCAGTGGATGTGATCTTGGCTCACAGGGGACGATGTCAAGCTCTT 900 CCTGGCTCCTTCTCAGCCTTTGT TGCTGTAACTGCTGCTCAGTCCACCATTGAGGAACAGG 960 CCAAGACATTTTTGGACAAGTTTAACCACGAAGCCGAAGACCTGTTCTATCA AAGTTCAC 1020 TTGCTTCTTGG AATTATAACACCAATATTA CTGAAGAGAATGTCCAAAACATGGTAAGAA 1080 GCAAGGAAGAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA 1132 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide <400> 8 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence upstream of PCR detection of circular RNA Circ-ACE2 <400> 9 GAAGCCGAAGACCTGTTCTA 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RNA Circ-ACE2 PCR detection primer downstream sequence <400> 10 tcttatcagc ctttgaactt gg 22 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CircACE2-76aa secretion signal peptide sequence <400> 11 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFP-upstream primer <400> 12 ttcaaggagg acggcaacat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EGFP-downstream primer <400> 13 tggcggatct tgaagttcac 20

Claims

1. An isolated circular RNA molecule, characterized in that, The circular RNA molecule is the RNA sequence shown in SEQ ID NO:

1. The circular RNA molecule is formed by the circularization of the second exon of the mRNA sequence encoding angiotensin-converting enzyme 2, and this circularization occurs in placental tissue. The circular RNA molecule is formed by linking the starting nucleotide and the terminal nucleotide of the nucleotide sequence shown in SEQ ID NO: 1, wherein the starting nucleotide is set as the first nucleotide of the circular RNA molecule, and the nucleic acid sequence of the coding region of the circular RNA includes the RNA sequence shown in SEQ ID NO:

2.

2. A polypeptide, characterized in that, The polypeptide has the amino acid sequence shown in SEQ ID NO:

3.

3. The polypeptide according to claim 2, characterized in that, The polypeptide contains a signal peptide sequence.

4. The polypeptide according to claim 3, characterized in that, The amino acid sequence of the signal peptide is MSSSSWLLLSLVAVTAA.

5. The polypeptide according to any one of claims 2 to 4, characterized in that, The polypeptide is present in placental tissue.

6. A fusion protein, characterized in that, The fusion protein comprises the amino acid sequence shown in SEQ ID NO:

4.

7. An isolated nucleic acid, characterized in that, Encodes the fusion protein of claim 6.

8. The nucleic acid according to claim 7, characterized in that, The nucleic acid is the nucleotide sequence shown in SEQ ID NO:

5.

9. The use of the RNA molecule of claim 1, the polypeptide of any one of claims 2 to 5, the fusion protein of claim 6, and the isolated nucleic acid of claim 7 or 8 in the preparation of a drug or kit for inhibiting SARS-CoV-2 infection of cells.

10. A method for inhibiting the invasion of cells by the novel coronavirus, characterized in that, The method is used for non-therapeutic purposes and includes: The novel coronavirus or cells to be infected are brought into contact with the RNA molecule of claim 1, the polypeptide of any one of claims 2 to 5, the fusion protein of claim 6, or the isolated nucleic acid of claim 7 or 8.

11. The method of claim 10, wherein the contact is performed in the following manner: 1) The novel coronavirus is first mixed with the RNA molecule of claim 1, the polypeptide of any one of claims 2 to 5, the fusion protein of claim 6, and the isolated nucleic acid of claim 7 or 8; 2) The mixture obtained in step 1) is mixed with the cells to be infected for a second mixing treatment in order to inhibit the infection of cells by the novel coronavirus.

12. The method according to claim 10, characterized in that, The final concentration of the polypeptide in the mixed system is 10 μg / mL or 160 μg / mL.

13. The method according to claim 10, characterized in that, The final concentration of the fusion protein in the mixing system was 160 μg / mL.

Images