CN117866985A - RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin - Google Patents
RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin Download PDFInfo
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- CN117866985A CN117866985A CN202410048481.7A CN202410048481A CN117866985A CN 117866985 A CN117866985 A CN 117866985A CN 202410048481 A CN202410048481 A CN 202410048481A CN 117866985 A CN117866985 A CN 117866985A
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- staphylococcus aureus
- methicillin
- endolysin
- resistant staphylococcus
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Abstract
The invention provides an RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin, which is a cyclized RNA sequence or mRNA sequence, wherein the expressed methicillin-resistant staphylococcus aureus endolysin can directly act on bacteria in cells, and meanwhile, the risk of immunogenicity is reduced, so that a treatment scheme is provided for the treatment and prevention of methicillin-resistant staphylococcus aureus infection.
Description
The application is a divisional application of an invention patent application of which the application date is 2023, 10 and 11, the application number is 202311312869.5 and the invention name is 'an methicillin-resistant staphylococcus aureus endolysin based on circular RNA coding'.
Technical Field
The application belongs to the field of nucleotide technology application, and in particular relates to a preparation method of a circular RNA for encoding methicillin-resistant staphylococcus aureus endolysin.
Background
Circular RNAs are a class of single-stranded RNAs characterized by a covalently closed topology, and therefore have no free ends. Compared with linear RNA, circular RNA is not easy to digest by exonuclease, and shows higher stability, so that the circular RNA has a plurality of practical application prospects in vivo and in vitro.
Staphylococcus aureus (Staphylococcus aureus) belongs to one of gram-positive bacteria, is a pathogen commonly existing between humans and animals, brings great clinical trouble, particularly postoperative infection, and can cause various diseases such as septicemia, sepsis, pneumonia, meningitis, myelitis and the like. In order to prevent and treat diseases caused by staphylococcus aureus, antibiotics are mainly used at present, but the use of long-term antibiotics leads to the generation of various drug-resistant staphylococcus aureus strains, such as Methicillin-resistant staphylococcus aureus (MRSA), and the occurrence of the drug-resistant strains presents a great threat to human health.
In order to overcome the drug resistance of staphylococcus aureus, phages and endolysins encoded by phage genes are used to kill drug resistant strains. Endolysin is a protein produced in bacterial cells after infection of bacteria by phage, which can lyse bacterial cells from the inside, releasing the progeny while killing the bacteria. Since endolysins have a different mechanism to kill bacteria than antibiotics and phage endolysins are highly specific for bacteria, no drug resistant strain is currently found.
At present, the mode of obtaining endolysin is generally to purify endolysin protein in vitro by cracking escherichia coli after induced expression in escherichia coli, and a special delivery mode is also required for clinical application. The circular RNA has certain advantages in the aspects of high-efficiency and durable expression of proteins in vivo, so that the circular RNA has quite expected application prospect in the development of new-generation biological medicine therapies. Endolysins expressed in cells mediated by circular RNAs have the advantage over endolysins obtained by traditional preparation methods that they can act directly on bacteria in cells and that the risk of developing immunogenicity is relatively smaller. The invention uses the circular RNA to code the endolysin protein, so that the endolysin protein can be directly expressed in the supernatant of the mammalian cells and exert the drug effect.
Disclosure of Invention
The purpose of the present disclosure is to provide a cyclic RNA encoding methicillin-resistant staphylococcus aureus endolysin, a preparation method and pharmaceutical uses thereof.
In one aspect, the invention provides a method for preparing a circular RNA for encoding methicillin-resistant staphylococcus aureus endolysin. In another aspect, the invention provides a linear DNA encoding methicillin-resistant staphylococcus aureus endolysin. In another aspect, the invention provides a linear RNA for encoding methicillin-resistant staphylococcus aureus endolysin. In another aspect, the invention provides a plasmid expression vector for preparing the linear DNA encoding methicillin-resistant staphylococcus aureus endolysin or the linear RNA of methicillin-resistant staphylococcus aureus endolysin. In another aspect, the invention provides a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin. In another aspect, the invention provides the use of the circular RNA in the manufacture of a medicament for the treatment or prevention of methicillin-resistant staphylococcus aureus infection. In another aspect, the invention also provides an RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin.
In some embodiments, the invention relates to a method for preparing a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, comprising the steps of:
step (a) of designing and preparing a linear DNA template comprising elements arranged in the following order from 5 'to 3' end:
5'-5' homology arm-3 'group I intron-3' Exon (Exon 2) -Internal Ribosome Entry Site (IRES) -signal peptide-sequence encoding methicillin-resistant staphylococcus aureus endolysin-5 'Exon (Exon 1) -5' group I intron-3 'homology arm-3';
a step (b) of purifying the linear DNA template prepared in the step (a);
step (c) of in vitro transcribing the linear DNA template of step (b) into RNA, the reaction system of in vitro transcription comprising: linear DNA templates, T7 polymerase, RNase inhibitors, NTPs, inorganic pyrophosphatase, and reaction buffers;
step (d), adding DNase I into the system of the step (c) and heating to remove DNA in the product obtained by in vitro transcription;
step (e) heating the system of step (d) to circularize the linear RNA, and purifying the reaction to obtain circular RNA.
In some embodiments, the invention relates to a method for the preparation of a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, wherein the sequence encoding methicillin-resistant staphylococcus aureus endolysin is selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. In some embodiments, the sequence encoding methicillin-resistant staphylococcus aureus endolysin is preferably SEQ ID No. 2 or SEQ ID No. 3.
In some embodiments, the present invention relates to a method for the preparation of a circular RNA for encoding methicillin-resistant staphylococcus aureus endolysin, wherein the Internal Ribosome Entry Site (IRES) is the CVB3 virus IRES sequence as shown in SEQ ID NO:12 (see Jennifer M Bailey et al, J Virol.2007Jan;81 (2): 650-668, FIG. 1), wherein the signal peptide is an IGK signal peptide as shown in SEQ ID NO: 13.
In some embodiments, the invention relates to a method for the preparation of a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, wherein the conditions in step (d) are treatment at 50-60 ℃ for 8-60min. The temperature may be specifically selected from 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃,60 ℃. The time can be specifically selected from any value of the time period of 8-45min, 8-30min and 8-15 min.
In some embodiments, the present invention relates to a method for the preparation of a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, wherein step (e) is in Mg 2+ And conducting transesterification twice in the presence of GTP such that the 5 'homology arm-3' Group I intron at the 5 'end and the 5' Group I intron-3 'homology arm at the 3' end are linked to form a loop, and further cleaving the homology arm and Group I intron and ligating the-3 'Exon (Exon 2) and the 5' Exon (Exon 1) to form the circular RNA of the present invention. See Natalia Akopyanz et al Nucleic Acids Research,1992,20 (19): 5137-5142 and US11203767B2, column 31, paragraph 1.
In some embodiments, the invention relates to a method for the preparation of a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, wherein said circularization in step (e) is achieved by ligating the 3 'Exon (Exon 2) and the 5' Exon (Exon 1) while removing the 5 'homology arm, the 3' group I intron, the 5'group I intron, the 3' homology arm element.
In some embodiments, the invention relates to a linear DNA for encoding methicillin-resistant staphylococcus aureus endolysin comprising elements arranged in the following order from 5 'to 3' end: 5'-5' homology arm-3 'group I intron-3' Exon (Exon 2) -Internal Ribosome Entry Site (IRES) -Signal peptide-sequence encoding methicillin-resistant Staphylococcus aureus-5 'Exon (Exon 1) -5' group I intron-3 'homology arm-3'. Codon optimization is carried out on the basis of a wild type sequence SEQ ID NO. 1 of the methicillin-resistant staphylococcus aureus endolysin, and SEQ ID NO. 2 and SEQ ID NO. 3 are screened out, so that the expression quantity of the methicillin-resistant staphylococcus aureus endolysin is obviously improved. The sequence for encoding methicillin-resistant staphylococcus aureus endolysin in the invention is selected from the group consisting of: 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 encoding methicillin-resistant staphylococcus aureus endolysin, wherein the Internal Ribosome Entry Site (IRES) is a CVB3 virus IRES sequence as set forth in SEQ ID NO. 12, wherein the signal peptide is an IGK signal peptide as set forth in SEQ ID NO. 13.
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 linear RNAs, which can then be circularized using the methods provided herein to form circular RNAs.
In some embodiments, the invention relates to a linear RNA encoding methicillin-resistant staphylococcus aureus endolysin, said linear RNA being prepared by in vitro transcription of a linear DNA provided by the invention.
In some embodiments, the invention relates to a plasmid expression vector that is used to prepare the linear DNA encoding methicillin-resistant staphylococcus aureus endolysin provided by the invention or the linear RNA encoding methicillin-resistant staphylococcus aureus endolysin provided by the invention.
In some embodiments, the invention relates to a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin prepared by circularizing the linear DNA provided herein. In some embodiments, the present invention provides circular RNAs having the structure shown in figure 9.
In some embodiments, the invention relates to the use of a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin provided by the invention in the manufacture of a medicament for the treatment or prevention of methicillin-resistant staphylococcus aureus infection.
In some embodiments, the invention relates to an RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin comprising a sequence as set forth in SEQ ID NO. 4 or SEQ ID NO. 5.
In some embodiments, the 5' homology arm sequence is selected from the sequences set forth in SEQ ID NO. 6. In some embodiments, the 3' group I intron sequence is selected from the sequences shown as SEQ ID NO. 7. In some embodiments, the 3' Exon (Exon 2) sequence is selected from the sequences set forth in SEQ ID NO. 8. In some embodiments, the 5' Exon (Exon 1) sequence is selected from the sequences set forth in SEQ ID NO. 9. In some embodiments, the 5' group I intron sequence is selected from the sequences shown as SEQ ID NO. 10. In some embodiments, the 3' homology arm sequence is selected from the sequences set forth in SEQ ID NO. 11. In some embodiments, the invention relates to a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, said circular RNA being prepared by cyclizing a linear RNA provided herein. In some embodiments, the RNA sequence is a circularized RNA sequence or an mRNA sequence.
Other embodiments
Embodiment 1, a method for preparing a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, comprising the steps of:
step (a) of designing and preparing a linear DNA template comprising elements arranged in the following order from 5 'to 3' end:
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';
a step (b) of purifying the linear DNA template prepared in the step (a);
step (c) of in vitro transcribing the linear DNA template of step (b) into RNA, the reaction system of in vitro transcription comprising: linear DNA templates, T7 polymerase, RNase inhibitors, NTPs, inorganic pyrophosphatase, and reaction buffers;
step (d), adding DNase I into the system of the step (c) and heating to remove DNA in the product obtained by in vitro transcription;
step (e) heating the system of step (d) to circularize the linear RNA, and purifying the reaction to obtain circular RNA.
Embodiment 2, the method of preparation according to embodiment 1, characterized in that the sequence encoding methicillin-resistant staphylococcus aureus endolysin is selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
Embodiment 3, the method of preparation according to embodiment 1 or 2, wherein the internal ribosome entry site is the CVB3 virus IRES sequence as set forth in SEQ ID NO. 12, and wherein the signal peptide is an IGK signal peptide as set forth in SEQ ID NO. 13.
Embodiment 4, the method of preparation according to embodiment 1 or 2, characterized in that the conditions in step (d) are treatment at 50-60 ℃ for 8-60min.
Embodiment 5, the production method according to embodiment 1 or 2, characterized in that the step (e) is performed in Mg 2+ And performing transesterification twice in the presence of GTP to form circular RNA.
Embodiment 6, the method of preparation according to embodiment 1 or 2, wherein the cyclizing in step (e) is achieved by ligating the 3 'exon and the 5' exon while removing the 5 'homology arm, 3' group I intron, 5'group I intron, 3' homology arm element.
Embodiment 7, a linear DNA for encoding methicillin-resistant staphylococcus aureus endolysin, comprising elements arranged from 5 'to 3' end in the following order:
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';
the sequence for encoding methicillin-resistant staphylococcus aureus endolysin is selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3;
wherein 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.
Embodiment 8, a linear RNA for encoding methicillin-resistant staphylococcus aureus endolysin, characterized in that said linear RNA is prepared by in vitro transcription of a linear DNA according to embodiment 7.
Embodiment 9, a plasmid expression vector for use in preparing the linear DNA encoding methicillin-resistant staphylococcus aureus endolysin according to embodiment 7 or the linear RNA encoding methicillin-resistant staphylococcus aureus endolysin according to embodiment 8.
Embodiment 10, a circular RNA encoding methicillin-resistant staphylococcus aureus endolysin, wherein said circular RNA is prepared by circularization of the linear DNA according to embodiment 7.
Use of the circular RNA of embodiment 11, according to embodiment 10, in the manufacture of a medicament for treating or preventing methicillin-resistant staphylococcus aureus infection.
Embodiment 12, an RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin, characterized in that the RNA sequence comprises the sequence as shown in SEQ ID No. 4 or SEQ ID No. 5.
Embodiment 13, the RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to embodiment 12, wherein the RNA sequence is a circularized RNA sequence or an mRNA sequence.
The prior art cited in the specification herein is incorporated by reference in its entirety for all purposes.
Definition of the definition
The definitions of various terms used to describe the nucleic acid combinations and compositions disclosed herein are set forth below. These definitions apply to terms as used throughout this specification and claims, unless otherwise limited, as such terms are used either individually or as part of a larger group.
As used herein, the term "optional" includes both the selection and non-selection of the two cases. For example, "optionally modified" includes both modified and unmodified cases.
The terms "a" and "an" as used herein are generally construed to cover both the singular and the plural.
The term "comprising" as used herein means, and is used interchangeably with, the phrase "including, but not limited to. The term "comprising" as used herein means, and is used interchangeably with, the phrase "including, but not limited to. The use of "including" or "comprising" in this patent may be further defined as "comprising" or "consisting of.
Throughout this specification, references to "one embodiment," "some embodiments," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "another embodiment," or "a further embodiment" or combinations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
As used herein, the terms "circRNA", "circular RNA" or "circular polyribonucleotides" or "circular RNA" are used interchangeably and refer to polyribonucleotides that form a circular structure through covalent bonds.
As used herein, a "homology arm" is any one of 1) predicts base pairing of at least about 75% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 100%) with another sequence in RNA (e.g., another homology arm), 2) is at least 7nt and no more than 250nt in length, is located before and near or contained in a 3 'intron fragment, and/or is located after and near or contained in a 5' intron fragment, and optionally, 4) predicts base pairing of less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) with an unexpected sequence in RNA (e.g., a non-homology arm sequence). As used herein, the 5 'and 3' homology arms may be synthetic sequences, different from the internal homology regions, but functionally similar. The homology arms may 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.
As used herein, examples of Group I introns (which may also be referred to as "Group I introns" or "Group I Intron") self-splicing sequences include, but are not limited to, intron-exon sequences derived from the T4 phage gene td or the self-splicing arrangement of the blue algae anabaena pre-tRNA-Leu gene.
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 a 3' proximal fragment of a native group I intron, including a 3' splice site dinucleotide, and optionally, adjacent exon sequences that are 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, a contiguous exon 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).
As used herein, "vector" refers to a piece of DNA that is synthetic (e.g., using PCR), or that is extracted from cells of a virus, plasmid, or higher organism into which a foreign DNA fragment may be inserted or has been inserted for cloning and/or expression purposes. In some embodiments, the vector of the invention is an E.coli cloning plasmid.
In some embodiments, the circular RNAs of the invention further comprise 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 having the sequence set forth in SEQ ID NO: 12.
The beneficial technical effects are as follows:
1. the invention provides a treatment scheme for the treatment and prevention of methicillin-resistant staphylococcus aureus infection, and the methicillin-resistant staphylococcus aureus endolysin prepared by the method has a remarkable antibacterial effect on MRSA.
2. The inventor of the application optimizes codons based on a wild type sequence SEQ ID NO. 1 of the methicillin-resistant staphylococcus aureus endolysin, and screens out SEQ ID NO. 2 and SEQ ID NO. 3, thereby remarkably improving the expression quantity of the methicillin-resistant staphylococcus aureus endolysin.
3. Compared with the traditional preparation method of endolysin, the methicillin-resistant staphylococcus aureus endolysin expressed by the circular RNA can directly act on bacteria in cells to exert the drug effect, and meanwhile, the risk of immunogenicity is reduced.
4. Compared with the conventional mRNA expression technology, the technology for expressing the protein through the circular RNA is simpler and more convenient, and the prepared circular RNA has simpler structure and is easier to realize industrial production.
Drawings
FIG. 1 illustrates an exemplary method for in vitro generation of circRNA based on rearranged Group I introns: the linear RNA precursors 5 'to 3' comprise: a 5 'homology arm, a 3' group I intron, a 3 'Exon (Exon 2), an Internal Ribosome Entry Site (IRES), a signal peptide, an insertion sequence, a 5' Exon (Exon 1), a 5'group I intron and a 3' homology arm. The insertion sequence may comprise a nucleic acid sequence encoding an endolysin polypeptide. At Mg 2+ And performing transesterification twice in the presence of GTP to form circular RNA.
FIG. 2 shows the results of amplification of the methicillin-resistant Staphylococcus aureus endoWT gene fragment, the circEndo1 and the circEndo2 gene fragments from the plasmids: lane 1: amplified fragments of circEndoWT; lane 2: amplified fragments of circEndo1; lane 3: amplified fragments of circEndo2; m Trans 2k plus II DNA marker.
FIG. 3 is a PCR identification of recombinant vectors containing methicillin-resistant Staphylococcus aureus endolysin genes: lane 1: identification result of the circendoWT gene; lane 2: identification results of the circEndo1 gene; lane 3: identification results of the circEndo2 gene; m Trans 2k plus II DNA marker.
FIG. 4 shows the cleavage results of the pmeI endonuclease: lanes 1-1: plasmid circendoWT; lanes 1-2: linearization of plasmid circEndoWT; lane 2-1: plasmid circEndo1; lane 2-2: linearization of plasmid circEndo1; lane 3-1: plasmid circEndo2; lane 3-2: linearization of plasmid circEndo2; m Trans 2k plus II DNA marker.
FIG. 5 is a graph showing the results of electrophoresis for in vitro preparation of circular RNA (circRNA). After GTP cyclization, the Precursor in the RNA fraction decreases: m is ssRNA Ladder;
WT IVT, DNase I, GTP represents the in vitro transcription of linearized plasmid circEndoWT, respectively, DNase I enzymatically cleaves DNA and the result of cyclization of linear RNA into circRNA;
IVT, DNase I, GTP represents the in vitro transcription of linearization plasmid circEndo1, DNase I enzymatic hydrolysis of DNA and cyclization of linear RNA into circRNA, respectively;
IVT, DNase I, GTP indicates the result of in vitro transcription of the linearized plasmid circEndo2, respectively, after enzymatic DNA cleavage of DNase I and cyclization of the linear RNA into circRNA.
FIG. 6 shows the detection of the expression of the methicillin-resistant Staphylococcus aureus endolysin protein circEndo (34 KD) encoded by a circular RNA in the cell supernatant by Western Blot: m. BeyoColor TM Color pre-dye protein molecular weight standard (6.5-270 kD); lane 1: protein expression of circular RNA circEndoWT after transfection of 293T cells in vitro; lane 2: protein expression of circular RNA circEndo1 after transfection of 293T cells in vitro; lane 3: protein expression of circular RNA circEndo2 after transfection of 293T cells in vitro; non represents a negative control for untransfected 293T cells.
FIG. 7 shows a grayscale analysis of Western Blot protein electrophoresis band quantification.
FIG. 8 analysis of endolysin protein anti-methicillin-resistant Staphylococcus aureus activity in cell supernatants of circular RNA expression: a treatment group containing endolysin protein circEndoWT; a treatment group containing endolysin protein circEndo1; a treatment group containing endolysin protein circEndo2; vehicle is the solvent control group (DMEM high sugar medium+10% foetal calf serum, cell supernatant without any treatment). The OD values of the treated groups of the cyclic RNA circEndo1 and the endo2 expressed endolysin proteins containing the optimized sequences are far lower than those of a solvent control group, and the treated groups show obvious activity of resisting methicillin-resistant staphylococcus aureus.
FIG. 9 is a schematic diagram of circular RNAs.
Detailed Description
Embodiments of the present disclosure will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus, accession number: BNCC 337371) was used in the test of the present invention. After the activation treatment, 40% glycerol was used for storage. The endolysin sequences were synthesized by the synthetic nucleotide service company (An Sheng for life sciences) and were designated circEndoWT, circEndo and circEndo2, respectively, and their corresponding DNA sequences were SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, respectively.
Example 1: construction of the expression vector of circEndo
(1) An example of in vitro generation of circRNA based on Group I catalytic intron (catalytic intron) is shown in FIG. 1. Synthesis of endolysin sequence fragments: according to the amino acid sequence (GenBank: AGF 87539.1) of the candidate endolysin PlySs2 derived from Streptococcus suis phage Streptococcus suis phage strain/1591, a Mouse Ig Kappa (IGK) signal peptide sequence is added at the N-terminal, a 3 Xflag tag (SEQ ID NO: 14) is added at the C-terminal, and the amino acid sequence is optimized for codons according to codon preference, resulting in two optimized sequences. The designed sequence was then synthesized with the original sequence of the non-optimized PlySs2 (GenBank: KC348602.1,287-1024). The gene sequence is shown in a sequence table, the sequence which is not subjected to codon optimization is SEQ ID NO. 1, and the two optimized sequences are SEQ ID NO. 2 and SEQ ID NO. 3 respectively;
(2) Designing a pair of primers: p1:5'-GTTGAATACAGCAAAACTAGTGCCACC-3' (SEQ ID NO: 15); p2:5'-CGGTAACGCATAATAGCCGTTTTG-3' (SEQ ID NO: 16); amplifying the synthesized fragments by using the primers;
(3) And (3) carrier enzyme cutting: double-enzyme digestion is carried out on empty vectors by using restriction enzymes SpeI and AgeI for vector recombination;
subjecting the above product to 1% agarose gel electrophoresis to identify fragment size, recovering gel mass of target band, and recovering amplified fragment and enzyme-cleaved vector by Zymoclean Gel DNA Recovery Kits (ZYMO, U.S.A.);
(4) Vector recombination: vector recombination of amplified fragments and cut vectors according to Gibson Assembly Master Mix (NEB, U.S.) followed by transformation of E.coli T1 chemocompetent cells, plating onto LB plates containing kanamycin resistance (50. Mu.g/mL), picking up the monoclonal the next day, and amplification assay using 2X Rapid Tap Master Mix (Vazyme, nanjing, china), primers using P1 and P2, sequencing of positive clones, sequencing of correct plasmids designated P (circEndoWT), P (circEndo 1) and P (circEndo 2);
as shown in FIG. 2, after PCR amplification, the target gene bands circEndoWT, circEndo and circEndo2 appeared at 952bp, and the sizes were expected to confirm that the fragment amplification was correct. As shown in FIG. 3, after PCR amplification of the monoclonal colonies, the target gene bands circEndoWT, circEndo1 and circEndo2 appeared at 952bp, and the sizes were expected to confirm that the vector construction was correct.
Example 2: preparation and purification of linearized plasmids
(1) Positive clones sequenced correctly were inoculated into LB liquid medium containing kanamycin resistance (50. Mu.g/mL), cultured overnight at 37℃and plasmids were extracted according to the protocol of EndoFree Mini Plasmid Kit II (TIANGEN, beijing, china).
(2) Plasmid was digested with restriction enzyme pmeI, treated at 37℃for 3 hours to linearize it, and then purified according to DNA Clean&-25 (ZYMO, usa) protocol for purification of linearized plasmids and 1% agarose gel electrophoresis of the purified products to determine if the plasmids were successfully linearized.
After cleavage by the restriction enzyme pmeI shown in FIG. 4, a linearized vector band appears at 4.8kb, which is sized to match the expected size, indicating that the recombinant vector was successfully linearized.
Example 3: in Vitro Transcription (IVT) and RNA cyclization
(1) The linearized plasmid was transcribed in vitro. In vitro transcription was performed according to the following table 1 system:
TABLE 1
| Name of the name | Reaction system (mu L) |
| 10 Xreaction buffer | 6.0 |
| 100mM ATP | 3.0 |
| 100mM CTP | 3.0 |
| 100mM UTP | 3.0 |
| 100mM GTP | 6.0 |
| T7 polymerase (200U/. Mu.L) | 0.99 |
| RNase inhibitor (40U/. Mu.L) | 1.65 |
| Ppase(0.1U/μL) | 0.24 |
| Linear DNA (1.2 μg) | 36.12 |
| Totals to | 60μL |
Reacting for 3h at 37 ℃ to obtain an RNA product;
(2) DNase I was then added to the system of Table 2 below to remove DNA from the in vitro transcribed product, and reacted at 37℃for 15-30min.
TABLE 2
(3) And (5) RNA in vitro cyclization and purification. To further obtain circularized circRNA, the RNA product was treated at 55℃for 15min at Mg 2+ And in the presence of GTP, two transesterification reactions occur. After the reaction is finished, according to the following stepsRNA Clenup Kit (NEB, USA) protocol was used to purify the RNA circularized product;
(4) RNA from in vitro transcription, DNase I and cyclization steps was identified and subjected to 2% agarose gel electrophoresis. The major products after In Vitro Transcription (IVT) as shown in FIG. 5 include uncyclized precursor RNA and circularized circRNA, and the band of the precursor RNA becomes weaker after cyclization by GTP, indicating that part of the uncyclized precursor RNA has become circRNA.
Example 4: detection of expression in 293T cells
(1) The cells were prepared. Culturing 293T cells in advance, culturing with DMEM high sugar culture medium (1% diabody) and 10% foetal calf serum until cell confluence reaches above 90%, treating with pancreatin to obtain cell suspension, and counting in 12-well plate at 3.5X10 per well 5 Cell plating is carried out on individual cells;
(2) Liposome circRNA transfected 293T cells. The next day when the cell confluence reaches 60% -70%, removing the culture medium in the 12-hole plate, adding DMEM high-sugar culture medium and 10% fetal bovine serum, and according to Lipofectamine TM MessengerMAX TM Cell transfection was performed (Invitrogen, U.S.) protocol. 5% CO 2 The cells were cultured in a 37℃incubator for 48 hours.
(3) Detection of cell supernatant protein expression
a. Cell supernatants were collected. The cell supernatant was transferred to a 1.5mL centrifuge tube, centrifuged at 1500rpm at 4℃for 5min, and the supernatant carefully transferred to a new centrifuge tube. Taking 40. Mu.L of supernatant, adding 10. Mu.L of 5 XDual color protein loading buffer (color protein loading buffer), and treating at 100deg.C for 15min to denature proteins;
b. and (5) detecting the expression of the Western blot protein. Preparing polyacrylamide gel according to 12.5%ExpressCast PAGE color gel rapid kit (NCM Biotech, suzhou, china), separating gel concentration of 12.5%, loading 20 μl, and concentrated gel electrophoresis under 80V for 30min; the electrophoresis condition of the separating gel is 120V for 60min. Electric conversion: using a polyvinylidene fluoride film (PVDF film), the semi-dry transfer method was carried out at 25V for 10min. Closing: the block was performed with 5% skim milk (formulated by TBST) for 1h. Incubation resistance: a primary antibody DYKDDDDK tag Monoclonal antibody (Proteintech, wuhan, china) was diluted 1:2000 with 5% skim milk and incubated overnight at 4 ℃. Wash 3 times with TBST for 10min each. Secondary antibody incubation: secondary antibody HRP-labeled Goat Anti-Mouse IgG (H+L) was diluted 1:1000 with 5% skim milk and incubated for 1H at ambient temperature. Wash 3 times with TBST for 10min each. Developing: developing solution A and solution B are prepared according to a ratio of 1:1, a PVDF membrane is developed by using ChemiDoc MP Imaging System (BIO-RAD), the expression condition of protein detected by Western blot is shown as shown in figure 6, three groups of samples circEndoWT, circEndo and circEndo2 are respectively provided with a band at 34KD, the successful expression of target protein in cell supernatant is proved, the protein size accords with the expectation, and the protein expression is not detected in a negative control group. And then carrying out gray level analysis on the developed image by using imageJ, wherein the result is shown in the graph 7 of Table 3, the protein band coded by the cyclic RNA circEndoWT is obviously weaker than that of the optimized groups circEndo1 and circEndo2, and the protein expression quantity of the optimized endolysin cyclic RNA sequence is obviously improved by 5 times and 3.8 times respectively.
TABLE 3 Gray statistics of Western Blot protein electrophoresis bands
| Name of the name | Reactive protein expression |
| circEndoWT | 1 |
| circEndo1 | 5.02 |
| circEndo2 | 3.83 |
Example 5: antibacterial Activity detection
(1) Resuscitates and activates MRSA lyophilized powder according to the operation instruction provided by NBCC.
a. 1 sterile shake tube containing 5mL NB medium and 2 NA plates were prepared;
b. opening the safety cabinet, burning the top by using an alcohol lamp, then rapidly dripping sterile water to break the safety cabinet, and then breaking the safety cabinet by using tweezers;
c. sucking 0.5mL of liquid culture medium, pumping into a freeze-drying tube, fully dissolving, pumping back into the liquid test tube, and uniformly mixing;
d. sucking 0.2mL of bacterial suspension, pouring the bacterial suspension into a flat plate, uniformly coating, repeating the steps twice to obtain two flat plates, and adding the residual bacterial suspension into a sterile shaking tube containing 5mL of NB culture medium;
e. all the liquid test tubes and the flat plates are placed at 37 ℃ and cultivated overnight under aerobic cultivation conditions, and strains grow out;
f. finally, the activated bacteria are prepared into 40% glycerol bacteria and stored at-80 ℃. Bacteria were inoculated in NB medium at a ratio of 1:1000 and incubated at 37℃until OD reached 0.1-0.08.
(2) Antibacterial Activity detection
a. Diluting MRSA with OD value of 0.1-0.08 by 100 times by using NB culture medium;
b. 50. Mu.L of NB medium was added to a 96-well cell culture plate;
c. adding 50 mu L of cell supernatant into the treatment group, and adding cell supernatant which is not subjected to any treatment into the solvent control group, wherein the negative control is only NB medium;
d. in addition to the negative control, 50. Mu.L of the prepared MRSA bacteria solution was added to the 96-well plate, and incubated at 37℃for 16-20 hours, and OD600 was measured using a microplate reader. OD values of the negative control group were subtracted from OD values of the treatment group and the solvent control group when the data were processed. As a result, as shown in FIG. 8, the MRSA growth was not inhibited in each data group, the OD value reached about 0.15 after overnight, and the endolysin protein circEndoWT in the treated group showed no inhibition difference compared with the solvent control group, because of the lower expression level of the cyclic RNA-encoded protein, whereas the OD value was only 0.07-0.09 in the treated group containing the cyclic RNA circEndo1 and the cyclic RNA 2-encoded endolysin protein, which reached an extremely significant level compared with the solvent control group, and significantly inhibited the MRSA growth.
The result shows that the expression level of the coded endolysin protein in cells is improved by 3.8-5 times, the methicillin-resistant staphylococcus aureus effect of MRSA is obviously higher than that of the circendoWT, and the antibacterial effect is positively related to the protein expression.
Claims (10)
1. An RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin, wherein the RNA sequence is the sequence shown in SEQ ID No. 4.
2. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 1, wherein the RNA sequence is a circularized RNA sequence.
3. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 2, wherein the circularization is achieved by ligating the 3 'exon and the 5' exon, while removing the 5 'homology arm, the 3' group I intron, the 5'group I intron, the 3' homology arm element.
4. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 1, wherein the RNA sequence is an mRNA sequence.
5. An RNA sequence for expressing methicillin-resistant staphylococcus aureus endolysin, wherein the RNA sequence is the sequence shown in SEQ ID No. 5.
6. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 1, wherein the RNA sequence is a circularized RNA sequence.
7. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 6, wherein the circularization is achieved by ligating the 3 'exon and the 5' exon, while removing the 5 'homology arm, the 3' group I intron, the 5'group I intron, the 3' homology arm element.
8. The RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 1, wherein the RNA sequence is an mRNA sequence.
9. Use of a circularized RNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to any one of claims 2-3 or 6-7 in the manufacture of a medicament for the treatment or prevention of methicillin-resistant staphylococcus aureus infection.
10. Use of an mRNA sequence for expression of methicillin-resistant staphylococcus aureus endolysin according to claim 4 or 8 in the manufacture of a medicament for the treatment or prevention of methicillin-resistant staphylococcus aureus infection.
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