A linear DNA, a linear RNA and a plasmid expression vector for encoding methicillin-resistant Staphylococcus aureus endolysin

Abstract

The invention provides a linear DNA, a linear RNA and a plasmid expression vector thereof for encoding a methicillin-resistant Staphylococcus aureus endolysin, and provides a therapeutic scheme for the treatment and prevention of methicillin-resistant Staphylococcus aureus infection.

Description

[0001] This application is a divisional application of an invention patent application with an application date of October 11, 2023, application number 202311312869.5, and invention name “A method for encoding methicillin-resistant Staphylococcus aureus endolysin based on circular RNA and its application”. Technical Field

[0002] The present application belongs to the field of nucleotide technology application. Specifically, the present application relates to a method for preparing a circular RNA encoding methicillin-resistant Staphylococcus aureus endolysin. Background Art

[0003] Circular RNA is a type of single-stranded RNA characterized by a covalently closed topological structure, so there is no free end. Compared with linear RNA, circular RNA is not easily digested by exonucleases and exhibits higher stability, so it has many practical application prospects both in vivo and in vitro.

[0004] Staphylococcus aureus is a Gram-positive bacteria. As a common pathogen in humans and animals, it brings great trouble to clinical practice, especially postoperative infection. It also causes various diseases, such as sepsis, septicemia, pneumonia, meningitis, myelitis, etc. In order to prevent and treat diseases caused by Staphylococcus aureus, antibiotics are currently used. However, long-term use of antibiotics has led to the emergence of multiple drug-resistant Staphylococcus aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA). The emergence of this drug-resistant strain poses a major threat to human health.

[0005] In order to overcome the drug resistance of Staphylococcus aureus, bacteriophages and endolysins encoded by phage genes are used to kill drug-resistant strains. Endolysins are proteins produced in bacterial cells in the later stage of progeny phage proliferation after phage infects bacteria. The protein can lyse bacterial cells from the inside, release progeny and kill bacteria at the same time. Since endolysins kill bacteria differently from antibiotics and phage endolysins are highly specific to bacteria, no drug-resistant strains have been found so far.

[0006] At present, the general way to obtain endolysin is to lyse Escherichia coli and purify the endolysin protein in vitro after induction expression in Escherichia coli, and clinical applications also require special delivery methods. Since circular RNA (circular RNA, CircRNA) has certain advantages in terms of efficient and persistent expression of proteins in vivo, it has a considerable application prospect in the development of a new generation of biomedical therapies. Compared with the endolysin obtained by the traditional preparation method, the endolysin expressed intracellularly by circular RNA has the advantage that it can directly act on bacteria in the cell and the risk of producing immunogenicity is relatively smaller. The present invention uses circular RNA to encode the endolysin protein, so that it is directly expressed in the supernatant of mammalian cells and exerts a pharmacodynamic effect. Summary of the invention

[0007] The purpose of the present disclosure is to provide a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, a preparation method thereof and pharmaceutical use thereof.

[0008] On the one hand, the present invention provides a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin. On the other hand, the present invention provides a linear DNA for encoding a methicillin-resistant Staphylococcus aureus endolysin. On the other hand, the present invention provides a linear RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin. On the other hand, the present invention provides a plasmid expression vector for preparing the linear DNA encoding the methicillin-resistant Staphylococcus aureus endolysin or the linear RNA of the methicillin-resistant Staphylococcus aureus endolysin. On the other hand, the present invention provides a circular RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin. On the other hand, the present invention provides the use of the circular RNA in the preparation of a drug for treating or preventing methicillin-resistant Staphylococcus aureus infection. On the other hand, the present invention also provides an RNA sequence for expressing a methicillin-resistant Staphylococcus aureus endolysin.

[0009] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, comprising the following steps:

[0010] Step (a), designing and preparing a linear DNA template, wherein the linear DNA template comprises elements arranged in the following order from the 5' to the 3' end:

[0011] 5'-5' homology arm-3' Group I intron-3' exon (Exon2)-internal ribosome entry site (IRES)-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon (Exon1)-5' Group I intron-3' homology arm-3';

[0012] Step (b), purifying the linear DNA template prepared in step (a);

[0013] Step (c), transcribing the linear DNA template in step (b) into RNA in vitro, wherein the reaction system for in vitro transcription comprises: a linear DNA template, T7 polymerase, an RNase inhibitor, NTPs, an inorganic pyrophosphatase and a reaction buffer;

[0014] Step (d), adding DNase I to the system of step (c) and heating to remove DNA from the product obtained by in vitro transcription;

[0015] Step (e), heating the system of step (d) to circularize the linear RNA, and purifying the reaction product to obtain circular RNA.

[0016] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is selected from: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is preferably SEQ ID NO: 2 or SEQ ID NO: 3.

[0017] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the internal ribosome entry site (IRES) is a CVB3 virus IRES sequence as shown in SEQ ID NO: 12 (see Jennifer M Bailey et al., J Virol. 2007 Jan; 81(2): 650-668, Figure 1 ), wherein the signal peptide is the IGK signal peptide as shown in SEQ ID NO:13.

[0018] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the condition in step (d) is 50-60° C. for 8-60 min. The temperature can be specifically selected from 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., and 60° C. The time can be specifically selected from any value of 8-45 min, 8-30 min, and 8-15 min.

[0019] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the step (e) is carried out in Mg 2+In the presence of 1,2-dihydro-1,4 ...

[0020] In some embodiments, the present invention relates to a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the circularization in the step (e) is achieved by connecting the 3' exon (Exon2) and the 5' exon (Exon1) while removing the 5' homology arm, 3' Group I intron, 5' Group I intron, and 3' homology arm elements.

[0021] In some embodiments, the present invention relates to a linear DNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the linear DNA comprises elements arranged in the following order from the 5' to 3' end: 5'-5' homology arm-3' Group I intron-3' exon (Exon2)-internal ribosome entry site (IRES)-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon (Exon1)-5' Group I intron-3' homology arm-3'. Codon optimization was performed on the basis of the wild-type sequence SEQ ID NO: 1 of methicillin-resistant Staphylococcus aureus endolysin, and SEQ ID NO: 2 and SEQ ID NO: 3 were screened out, thereby significantly improving the expression amount of methicillin-resistant Staphylococcus aureus endolysin. The sequence encoding the methicillin-resistant Staphylococcus aureus endolysin in the present invention is selected from: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, preferably SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the present invention relates to a linear DNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the internal ribosome entry site (IRES) is a CVB3 virus IRES sequence as shown in SEQ ID NO:12, and wherein the signal peptide is an IGK signal peptide as shown in SEQ ID NO:13.

[0022] As used herein, elements of a vector are "operably linked" if they are located on the vector such that they can be transcribed to form a linear RNA, which can then be circularized to form a circular RNA using the methods provided herein.

[0023] In some embodiments, the present invention relates to a linear RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the linear RNA is prepared by in vitro transcription of a linear DNA provided by the present invention.

[0024] In some embodiments, the present invention relates to a plasmid expression vector, which is used to prepare the linear DNA encoding the methicillin-resistant Staphylococcus aureus endolysin provided by the present invention or the linear RNA encoding the methicillin-resistant Staphylococcus aureus endolysin provided by the present invention.

[0025] In some embodiments, the present invention relates to a circular RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, wherein the circular RNA is prepared by cyclizing the linear DNA provided by the present invention. In some embodiments, the circular RNA provided by the present invention has the following characteristics: Fig. 9 The structure shown.

[0026] In some embodiments, the present invention relates to use of the circular RNA encoding the methicillin-resistant Staphylococcus aureus endolysin provided by the present invention in the preparation of a medicament for treating or preventing methicillin-resistant Staphylococcus aureus infection.

[0027] In some embodiments, the present invention relates to an RNA sequence for expressing methicillin-resistant Staphylococcus aureus endolysin, wherein the RNA sequence comprises a sequence as shown in SEQ ID NO:4 or SEQ ID NO:5.

[0028] In some embodiments, the 5' homology arm sequence is selected from the sequence shown in SEQ ID NO:6. In some embodiments, the 3' Group I intron sequence is selected from the sequence shown in SEQ ID NO:7. In some embodiments, the 3' exon (Exon2) sequence is selected from the sequence shown in SEQ ID NO:8. In some embodiments, the 5' exon (Exon1) sequence is selected from the sequence shown in SEQ ID NO:9. In some embodiments, the 5' Group I intron sequence is selected from the sequence shown in SEQ ID NO:10. In some embodiments, the 3' homology arm sequence is selected from the sequence shown in SEQ ID NO:11. In some embodiments, the present invention relates to a circular RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, and the circular RNA is prepared by cyclizing a linear RNA provided by the present invention. In some embodiments, the RNA sequence is a circularized RNA sequence or an mRNA sequence.

[0029] Other Implementations

[0030] Embodiment 1, a method for preparing a circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that it comprises the following steps:

[0031] Step (a), designing and preparing a linear DNA template, wherein the linear DNA template comprises elements arranged in the following order from the 5' to the 3' end:

[0032] 5'-5' homology arm-3' Group I intron-3' exon-internal ribosome entry site-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon-5' Group I intron-3' homology arm-3';

[0033] Step (b), purifying the linear DNA template prepared in step (a);

[0034] Step (c), transcribing the linear DNA template in step (b) into RNA in vitro, wherein the reaction system for in vitro transcription comprises: a linear DNA template, T7 polymerase, an RNase inhibitor, NTPs, an inorganic pyrophosphatase and a reaction buffer;

[0035] Step (d), adding DNase I to the system of step (c) and heating to remove DNA from the product obtained by in vitro transcription;

[0036] Step (e), heating the system of step (d) to circularize the linear RNA, and purifying the reaction product to obtain circular RNA.

[0037] Embodiment 2: The preparation method according to embodiment 1 is characterized in that the sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is selected from: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0038] Embodiment 3: The preparation method according to Embodiment 1 or 2 is characterized in that the internal ribosome entry site is the CVB3 virus IRES sequence as shown in SEQ ID NO:12, and the signal peptide is the IGK signal peptide as shown in SEQ ID NO:13.

[0039] Embodiment 4: The preparation method according to Embodiment 1 or 2 is characterized in that the conditions in step (d) are treating at 50-60°C for 8-60 min.

[0040] Embodiment 5: The preparation method according to Embodiment 1 or 2, characterized in that step (e) is carried out in Mg 2+ In the presence of 1,4-dihydro-1,4-dihydro-2-nitropropene and 1,4-dihydro-1,4-dihydro-2-nitropropene, ...

[0041] Embodiment 6: The preparation method according to Embodiment 1 or 2 is characterized in that the cyclization in the step (e) is achieved by connecting the 3' exon and the 5' exon, and simultaneously removing the 5' homology arm, the 3' Group I intron, the 5' Group I intron, and the 3' homology arm element.

[0042] Embodiment 7, a linear DNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that the linear DNA comprises elements arranged in the following order from the 5' to the 3' end:

[0043] 5'-5' homology arm-3' Group I intron-3' exon)-internal ribosome entry site-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon-5' Group I intron-3' homology arm-3';

[0044] The sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is selected from: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

[0045] The internal ribosome entry site is the CVB3 virus IRES sequence shown in SEQ ID NO:12, and the signal peptide is the IGK signal peptide shown in SEQ ID NO:13.

[0046] Embodiment 8: A linear RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that the linear RNA is prepared by in vitro transcription of the linear DNA described in Embodiment 7.

[0047] Embodiment 9. A plasmid expression vector, characterized in that the plasmid expression vector is used to prepare the linear DNA encoding the methicillin-resistant Staphylococcus aureus endolysin according to Embodiment 7 or the linear RNA encoding the methicillin-resistant Staphylococcus aureus endolysin according to Embodiment 8.

[0048] Embodiment 10: A circular RNA for encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that the circular RNA is prepared by cyclizing the linear DNA described in Embodiment 7.

[0049] Embodiment 11. Use of the circular RNA according to Embodiment 10 in the preparation of a medicament for treating or preventing methicillin-resistant Staphylococcus aureus infection.

[0050] Embodiment 12: An RNA sequence for expressing methicillin-resistant Staphylococcus aureus endolysin, characterized in that the RNA sequence comprises the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5.

[0051] Embodiment 13: The RNA sequence for expressing methicillin-resistant Staphylococcus aureus endolysin according to Embodiment 12 is characterized in that the RNA sequence is a circularized RNA sequence or an mRNA sequence.

[0052] The prior art cited in this specification is hereby incorporated by reference in its entirety and for all purposes.

[0053] definition

[0054] Listed below are definitions of various terms used to describe nucleic acid combinations and compositions disclosed herein. These definitions apply to the terms used throughout this specification and claims, except where otherwise limited in specific cases, and these terms are used either individually or as part of a larger group.

[0055] As used herein, the terms "optional" and "optionally" include both the cases of being selected or not being selected. For example, "optionally modified" includes both the cases of being modified and not being modified.

[0056] As used herein, the terms "a," "an," and "the" are generally construed to cover both the singular and the plural.

[0057] The term "include" used herein means the phrase "including (but not limited to)", and can be used interchangeably therewith. The term "comprising" used herein means the phrase "including (but not limited to)", and can be used interchangeably therewith. The technical solutions using "include" or "comprising" in this patent can be further defined as "consisting of" or "composed of".

[0058] Throughout this specification, reference to "one embodiment," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "further embodiments," or combinations thereof, means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] As used herein, the terms "circRNA," "circular RNA," or "cyclic polyribonucleotide" or "circular RNA" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.

[0060] As used herein, a "homologous arm" is any of the following contiguous sequences, 1) predicted to form base pairs with at least about 75% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 100%) of another sequence in the RNA (such as another homology arm), 2) at least 7 nt and no more than 250 nt in length, located before and near or contained in the 3' intron fragment, and / or after and near or contained in the 5' intron fragment, and optionally, 4) predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with an unexpected sequence in the RNA (e.g., a non-homologous arm sequence). As used herein, the 5' and 3' homology arms can be synthetic sequences that are different from the internal homology regions, but have similar functions. The homology arms can be, for example, about 5-50 nucleotides in length, about 9-19 nucleotides in length, for example, about 5, about 10, about 20, about 30, about 40, or about 50 nucleotides in length.

[0061] As used herein, examples of Group I intron (also referred to as "Group I intron" or "Group I Intron") self-splicing sequences include, but are not limited to, intron-exon sequences derived from the self-splicing arrangement of the T4 bacteriophage gene td or the cyanobacterium Anabaena pre-tRNA-Leu gene.

[0062] As used herein, a 3' Group I intron fragment is a contiguous sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) homologous to the 3' proximal fragment of a native Group I intron, including the 3' splice site dinucleotide, and, optionally, an adjacent exonic sequence, that is at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 50 nucleotides in length). In certain embodiments, a 5' Group I intron fragment is a contiguous sequence that is at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 100%) homologous to a 5' proximal fragment of a native Group I intron, including a 5' splice site dinucleotide and, optionally, an adjacent exonic sequence, and is at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 50 nucleotides in length).

[0063] As used herein, "vector" refers to a segment of DNA that is synthesized (e.g., using PCR) or extracted from a virus, plasmid, or cell of a higher organism into which a foreign DNA segment can be or has been inserted for cloning and / or expression purposes. In some embodiments, the vector of the present invention is an E. coli cloning plasmid.

[0064] In some embodiments, the circular RNA of the present invention further comprises an internal ribosome entry site (IRES) sequence. In some embodiments, the internal ribosome entry site (IRES) sequence is a CVB3 virus, EV71 virus, EMCV virus, PV virus or CSFV virus IRES sequence, preferably a CVB3 virus IRES, more preferably a CVB3 virus IRES as shown in SEQ ID NO: 12.

[0065] Beneficial technical effects:

[0066] 1. The present invention provides a therapeutic scheme for the treatment and prevention of methicillin-resistant Staphylococcus aureus infection. The methicillin-resistant Staphylococcus aureus endolysin prepared by the method of the present application has a significant antibacterial effect on MRSA.

[0067] 2. The inventors of the present application performed codon optimization based on the wild-type sequence SEQ ID NO: 1 of methicillin-resistant Staphylococcus aureus endolysin, and screened out SEQ ID NO: 2 and SEQ ID NO: 3, thereby significantly improving the expression level of methicillin-resistant Staphylococcus aureus endolysin.

[0068] 3. Compared with the traditional preparation method of endolysin, the methicillin-resistant Staphylococcus aureus endolysin expressed by the circular RNA of the present invention can directly act on the bacteria in the cell to exert its pharmacological effect, while reducing the risk of immunogenicity.

[0069] 4. Compared with conventional mRNA expression technology, the technology of expressing proteins through circular RNA provided by the present invention is simpler, the circular RNA structure produced is simpler, and it is easier to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 An exemplary method for in vitro generation of circRNA based on rearranged Group I introns is shown: the linear RNA precursor 5' to 3' comprises: 5' homology arm, 3' Group I intron, 3' exon (Exon 2), internal ribosome entry site (IRES), signal peptide, insertion sequence, 5' exon (Exon 1), 5' Group I intron and 3' homology arm. The insertion sequence may comprise a nucleic acid sequence encoding an endolysin polypeptide. In Mg 2+ In the presence of 1,4-dihydro-1,4-dihydro-2-nitropropene and 1,4-dihydro-1,4-dihydro-2-nitropropene, ...

[0071] Figure 2 The results of amplifying the methicillin-resistant Staphylococcus aureus endolysin circEndoWT gene fragment, circEndo1 and circEndo2 gene fragments from the plasmid are shown: Lane 1: amplified fragment of circEndoWT; Lane 2: amplified fragment of circEndo1; Lane 3: amplified fragment of circEndo2; M: Trans 2k plusⅡDNA Maker.

[0072] Figure 3 PCR identification of the recombinant vector containing the methicillin-resistant Staphylococcus aureus endolysin gene: Lane 1: identification result of circEndoWT gene; Lane 2: identification result of circEndo1 gene; Lane 3: identification result of circEndo2 gene; M: Trans 2k plusⅡDNA Maker.

[0073] Figure 4The results of PmeⅠ endonuclease digestion: lane 1-1: plasmid circEndoWT; lane 1-2: linearized plasmid circEndoWT; lane 2-1: plasmid circEndo1; lane 2-2: linearized plasmid circEndo1; lane 3-1: plasmid circEndo2; lane 3-2: linearized plasmid circEndo2; M: Trans 2k plusⅡDNA Maker.

[0074] Figure 5 This is the electrophoresis result of in vitro preparation of circular RNA (CircRNA). After GTP cyclization, the Precursor in the RNA component decreases: M: ssRNA Ladder;

[0075] WT: IVT, DNaseⅠ, and GTP represent the results of in vitro transcription of the linearized plasmid circEndoWT, DNaseⅠ digestion of DNA, and circularization of linear RNA into circRNA, respectively;

[0076] 1: IVT, DNaseⅠ, and GTP represent the results of in vitro transcription of linearized plasmid circEndo1, DNaseⅠ digestion of DNA, and circularization of linear RNA into circRNA, respectively;

[0077] 2: IVT, DNaseⅠ, and GTP represent the results of in vitro transcription of linearized plasmid circEndo2, DNaseⅠ digestion of DNA, and circularization of linear RNA into circRNA, respectively.

[0078] Figure 6 To detect the expression of circRNA-encoded methicillin-resistant Staphylococcus aureus endolysin protein circEndo (34KD) in cell supernatant by Western Blot: M: BeyoColor TM Colored pre-stained protein molecular weight standard (6.5-270kD); Lane 1: protein expression of circular RNA circEndoWT after in vitro transfection of 293T cells; Lane 2: protein expression of circular RNA circEndo1 after in vitro transfection of 293T cells; Lane 3: protein expression of circular RNA circEndo2 after in vitro transfection of 293T cells; Non represents the negative control of untransfected 293T cells.

[0079] Figure 7 Grayscale analysis of Western Blot protein electrophoresis band quantification.

[0080] Figure 8Analysis of the anti-methicillin-resistant Staphylococcus aureus activity of endolysin proteins in the cell supernatant expressed by circular RNA: treatment group containing endolysin protein circEndoWT; treatment group containing endolysin protein circEndo1; treatment group containing endolysin protein circEndo2; vehicle is the solvent control group (DMEM high glucose medium + 10% fetal bovine serum, cell supernatant without any treatment). The OD values ​​of the treatment groups containing endolysin proteins expressed by circular RNA circEndo1 and circEndo2 with optimized sequences were much lower than those of the solvent control group, showing obvious anti-methicillin-resistant Staphylococcus aureus activity.

[0081] Fig. 9 Schematic diagram of circular RNA. DETAILED DESCRIPTION

[0082] The embodiments of the present disclosure will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific conditions are not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0083] The test of the present invention uses methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, deposit number: BNCC337371). After activation treatment, 40% glycerol is used for storage. The endolysin sequence was synthesized by Synthetic Nucleotide Service Company (Anshengda Life Sciences), and was named circEndoWT, circEndo1 and circEndo2, respectively, and the corresponding DNA sequences were SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

[0084] Example 1: Construction of circEndo expression vector

[0085] (1) Examples of in vitro generation of circRNA based on Group I catalytic introns include Figure 1As shown. Synthesize endolysin sequence fragments: According to the amino acid sequence of candidate endolysin PlySs2 derived from Streptococcus suis phagestrain 89 / 1591 (GenBank: AGF87539.1), add Mouse Ig Kappa (IGK) signal peptide sequence at the N-terminus, add 3×flag tag (SEQ ID NO: 14) at the C-terminus, optimize the amino acid sequence according to codon preference, and obtain two optimized sequences. Then synthesize the designed sequence with the original sequence of PlySs2 that has not been optimized (GenBank: KC348602.1, 287-1024). The gene sequence is shown in the sequence table. The sequence that has not been codon-optimized is SEQ ID NO: 1, and the two optimized sequences are SEQ ID NO: 2 and SEQID NO: 3, respectively;

[0086] (2) Design a pair of primers: P1: 5'-GTTGAATACAGCAAAACTAGTG CCACC-3' (SEQ ID NO: 15); P2: 5'-CGGTAACGCATAATAGCC GTTTTG-3' (SEQ ID NO: 16); Use the above primers to amplify the synthetic fragment;

[0087] (3) Vector digestion: Use restriction endonucleases SpeⅠ and AgeⅠ to double digest the empty vector for vector recombination;

[0088] The above products were subjected to 1% agarose gel electrophoresis to identify the fragment size, and the gel blocks of the target bands were recovered. The amplified fragments and the restriction vectors were recovered using Zymoclean Gel DNA Recovery Kits (ZYMO, USA);

[0089] (4) Vector recombination: The amplified fragment and the restriction-cut vector were recombined according to the instruction manual of Gibson Assembly Master Mix (NEB, USA), and then transformed into Escherichia coli T1 chemically competent cells and plated on LB plates containing kanamycin resistance (50 μg / mL). Single clones were picked the next day and amplified and identified using 2× Rapid Tap Master Mix (Vazyme, Nanjing, China). Primers P1 and P2 were used, and positive clones were selected for sequencing identification. The plasmids with correct sequencing were named P(circEndoWT), P(circEndo1), and P(circEndo2);

[0090] like Figure 2As shown in the figure, after PCR amplification, the target gene bands circEndoWT, circEndo1 and circEndo2 appeared at 952 bp, and the sizes were in line with expectations, proving that the fragments were amplified correctly. Figure 3 As shown, after PCR amplification of the monoclonal colonies, the target gene bands circEndoWT, circEndo1 and circEndo2 appeared at 952 bp, and the sizes were consistent with expectations, proving that the vector was constructed correctly.

[0091] Example 2: Preparation and purification of linearized plasmid

[0092] (1) The positive clones with correct sequencing were inoculated into LB liquid culture medium containing kanamycin resistance (50 μg / mL), cultured at 37°C overnight, and the plasmids were extracted according to the instructions of EndoFree Mini Plasmid Kit II (TIANGEN, Beijing, China).

[0093] (2) The plasmid was digested with restriction endonuclease PmeⅠ and treated at 37℃ for 3h to linearize it. Then, DNA Clean& The linearized plasmid was purified according to the ZYMO-25 (ZYMO, USA) operating instructions, and the purified product was identified by 1% agarose gel electrophoresis to determine whether the plasmid was successfully linearized.

[0094] like Figure 4 After digestion with restriction endonuclease PmeⅠ, a linearized vector band appeared at 4.8 kb, and the size was consistent with the expectation, indicating that the recombinant vector was successfully linearized.

[0095] Example 3: In vitro transcription (IVT) and RNA circularization

[0096] (1) Perform in vitro transcription on the linearized plasmid. Perform in vitro transcription according to the following system in Table 1:

[0097] Table 1

[0098] name Reaction system (μL) 10× reaction buffer 6.0 100mM ATP 3.0 100mM CTP 3.0 100mM UTP 3.0 100mM GTP 6.0 T7 polymerase (200 U / μL) 0.99 RNase inhibitor (40U / μL) 1.65 Ppase (0.1U / μL) 0.24 Linear DNA (1.2 μg) 36.12 total 60μL

[0099] The RNA product was obtained by reacting at 37°C for 3 h;

[0100] (2) Then, according to the system in Table 2 below, DNase I was added to remove DNA from the in vitro transcription product and the reaction was carried out at 37°C for 15-30 minutes.

[0101] Table 2

[0102] name Reaction system (μL) RNA Mix 60 DNase I 3 total 63μL

[0103] (3) In vitro RNA cyclization and purification. To further obtain the circularized circRNA, the RNA product was treated at 55°C for 15 min and in Mg 2+ In the presence of GTP, two transesterification reactions occurred. The RNA circularization product was purified according to the instructions of RNACleanup Kit (NEB, USA);

[0104] (4) Identify the RNA from the in vitro transcription, DNase I, and cyclization steps by 2% agarose gel electrophoresis. Figure 5 The main products after in vitro transcription (IVT) shown include uncircularized precursor RNA and circularized CircRNA. After cyclization by GTP, the band of the precursor RNA becomes weaker, indicating that part of the uncircularized precursor RNA has become CircRNA.

[0105] Example 4: Detection of expression in 293T cells

[0106] (1) Prepare cells. 293T cells were cultured in advance using DMEM high-glucose medium + 1% double antibody + 10% fetal bovine serum. When the cell confluence reached more than 90%, trypsin was used to treat the cells into a cell suspension and counted in a 12-well plate at 3.5 × 10 cells per well. 5 Cells were plated;

[0107] (2) Lipofectamine circRNA transfection of 293T cells. The next day, when the cell confluence reached 60%-70%, the medium in the 12-well plate was removed and DMEM high-glucose medium + 10% fetal bovine serum was added. TM MessengerMAX TM Cell transfection was performed according to the instructions of Invitrogen (Invitrogen, USA) and cultured for 48 h in a 5% CO2, 37°C cell culture incubator.

[0108] (3) Detection of cell supernatant protein expression

[0109] a. Collect the cell supernatant. Transfer the cell supernatant into a 1.5 mL centrifuge tube, centrifuge at 1500 rpm, 4°C for 5 min, and carefully transfer the supernatant to a new centrifuge tube. Take 40 μL of the supernatant sample, add 10 μl of 5×Dual color protein loading buffer, and treat at 100°C for 15 min to denature the protein;

[0110] b. Western blot protein expression detection. Polyacrylamide gel was prepared according to the instructions of 12.5% ​​ExpressCast PAGE Color Gel Rapid Kit (NCM Biotech, Suzhou, China). The separation gel concentration was 12.5%, the sample volume was 20 μL, and the electrophoresis conditions of the concentrated gel were 80V, 30min; the separation gel electrophoresis conditions were 120V, 60min. Electrotransfer: using polyvinylidene fluoride membrane (PVDF membrane), the semi-dry transfer method was used at 25V for 10min. Blocking: using 5% skim milk (prepared by TBST) for 1h. Primary antibody incubation: using 5% skim milk, the primary antibody DYKDDDDK tag Monoclonal antibody (Proteintech, Wuhan, China) was diluted 1:2000 and incubated at 4℃ overnight. Wash with TBST three times, 10min each time. Secondary antibody incubation: Use 5% skim milk to dilute the secondary antibody HRP-labeled Goat Anti-Mouse IgG (H+L) at 1:1000 and incubate at room temperature for 1 hour. Wash with TBST 3 times, 10 minutes each time. Development: Prepare developer A solution: B solution at a ratio of 1:1, use ChemiDocMP Imaging System (BIO-RAD) to develop the PVDF membrane, and Western blot to detect protein expression. Figure 6 As shown in Figure 3, the three groups of samples, circEndoWT, circEndo1 and circEndo2, all showed bands at 34KD, proving that the target protein was successfully expressed in the cell supernatant and the protein size was in line with expectations. No protein expression was detected in the negative control group. The grayscale analysis of the developed images was then performed using ImageJ, and the results are shown in Table 3. Figure 7 As shown, the protein band encoded by circular RNA circEndoWT was significantly weaker than that of the optimized groups circEndo1 and circEndo2, and the protein expression level of the optimized endolysin circular RNA sequence was significantly increased, by 5 times and 3.8 times, respectively.

[0111] Table 3 Grayscale statistics of Western Blot protein electrophoresis bands

[0112] name Response protein expression circEndoWT 1 circEndo1 5.02 circEndo2 3.83

[0113] Example 5: Antimicrobial activity detection

[0114] (1) Resuscitate and activate MRSA freeze-dried powder according to the operating instructions provided by NBCC.

[0115] a. Prepare a sterile shaking tube containing 5 mL of NB medium and two NA plates;

[0116] b. Open the safety cabinet, burn the top with an alcohol lamp, then quickly drip sterile water to break it, and then break it with tweezers;

[0117] c. Pipette 0.5 mL of liquid culture medium into the freeze-drying tube, and after fully dissolving, return it to the liquid test tube and mix well;

[0118] d. Pipette 0.2 mL of bacterial suspension into the plate and spread evenly. Repeat twice to obtain two plates. Add the remaining bacterial suspension into a sterile shaking tube containing 5 mL of NB medium.

[0119] e. Place all the liquid test tubes and plates at 37°C and culture them overnight under aerobic conditions until the strain grows;

[0120] f. Finally, the activated bacteria were made into 40% glycerol bacteria and stored at -80°C. The bacteria were inoculated into NB medium at a ratio of 1:1000 and cultured at 37°C until the OD value reached 0.1-0.08.

[0121] (2) Antibacterial activity detection

[0122] a. Dilute MRSA with OD value of 0.1-0.08 100 times using NB medium;

[0123] b. Add 50 μL NB medium into the 96-well cell culture plate;

[0124] c. Add 50 μL of cell supernatant sample to the treatment group, add untreated cell supernatant to the solvent control group, and the negative control contains only NB culture medium;

[0125] d. In addition to the negative control, add 50 μL of the prepared MRSA bacterial solution to the 96-well plate, incubate at 37°C for 16-20 hours, and measure the OD600 value using an ELISA reader. When processing the data, subtract the OD value of the negative control group from the OD values ​​of the treatment group and the solvent control group. The results are shown in Figure 8 As shown, each data group has two replicates. The growth of MRSA in the solvent control group vehicle was not inhibited, and the OD value reached about 0.15 after overnight. The endolysin protein circEndoWT in the treatment group had no inhibitory difference compared with the solvent control group vehicle due to the low expression of the protein encoded by circular RNA. In the treatment group containing endolysin proteins encoded by circular RNA circEndo1 and circRNA2, the OD value was only 0.07-0.09, which reached a very significant level compared with the solvent control group vehicle, and significantly inhibited the growth of MRSA.

[0126] The results showed that the expression level of the endolysin protein encoded by the circular RNA sequences circEndo1 and circEndo2 optimized by the present invention increased by 3.8-5 times in cells, and the inhibitory effect on methicillin-resistant Staphylococcus aureus of MRSA was significantly higher than that of circEndoWT, and the antibacterial effect was positively correlated with protein expression.

Claims

1. A linear DNA encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that: The linear DNA comprises elements arranged in the following order from the 5' to the 3' end: 5'-5' homology arm-3' Group I intron-3' exon-internal ribosome entry site (IRES)-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon-5' Group I intron-3' homology arm-3'; The sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is selected from: SEQ ID NO: 2 or SEQ ID NO: 3; The internal ribosome entry site (IRES) is the CVB3 virus IRES sequence shown in SEQ ID NO: 12, and the signal peptide is the IGK signal peptide shown in SEQ ID NO:

13.

2. A linear RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that The linear RNA is prepared by in vitro transcription of the linear DNA according to claim 1.

3. A plasmid expression vector, characterized in that: The plasmid expression vector is used to prepare linear DNA encoding methicillin-resistant Staphylococcus aureus endolysin or linear RNA encoding methicillin-resistant Staphylococcus aureus endolysin; The linear DNA encoding the methicillin-resistant Staphylococcus aureus endolysin is characterized in that the linear DNA comprises elements arranged in the following order from the 5' end to the 3' end: 5'-5' homology arm-3' Group I intron-3' exon-internal ribosome entry site (IRES)-signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus endolysin-5' exon-5' Group I intron-3' homology arm-3'; The sequence encoding the methicillin-resistant Staphylococcus aureus endolysin is selected from: SEQ ID NO: 2 or SEQ ID NO: 3; wherein the internal ribosome entry site (IRES) is the CVB3 virus IRES sequence shown in SEQ ID NO: 12, and wherein the signal peptide is the IGK signal peptide shown in SEQ ID NO: 13; The linear RNA of the methicillin-resistant Staphylococcus aureus endolysin is prepared by in vitro transcription of the linear DNA encoding the methicillin-resistant Staphylococcus aureus endolysin.

4. A circular RNA encoding a methicillin-resistant Staphylococcus aureus endolysin, characterized in that The circular RNA is prepared by cyclizing the linear DNA according to claim 1.

5. Use of the circular RNA according to claim 4 in the preparation of a medicament for treating or preventing methicillin-resistant Staphylococcus aureus infection.

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