Efficient transcription terminator

Abstract

The invention relates to the technical field of biology, in particular to a nucleic acid molecule, a carrier containing the nucleic acid molecule, a host cell, a pharmaceutical composition and application of the nucleic acid molecule and the carrier. The nucleic acid molecule is particularly suitable for gene therapy and can be applied to the scene of treatment of various diseases.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a nucleic acid molecule, a carrier thereof, a host cell thereof, a pharmaceutical composition thereof, and uses thereof. Background Technology

[0002] Currently, most gene therapy products on the market are viral vectors. In contrast, gene therapy using plasmids has the advantages of high safety and very low immunogenicity. However, plasmid gene therapy also has disadvantages such as low cell transfection rate and low protein expression level. Therefore, although a considerable number of projects have entered clinical trials, only Japan and Russia have approved or conditionally approved intramuscularly injected plasmid drugs within their borders. No plasmid gene therapy drugs have yet been approved in major countries around the world.

[0003] For plasmid gene therapy drugs, low transfection rates and poor stability make the expression level (yield) of the target protein a key factor affecting their efficacy. The transcription terminator sequence significantly influences the protein expression level of the plasmid. This study provides a novel transcription terminator sequence that effectively increases the expression levels of various proteins in plasmids used for gene therapy, thereby enhancing the drug-likeness of plasmid gene therapy projects and promoting their clinical application. Summary of the Invention

[0004] This application provides a modified transcription terminator that significantly increases the yield of the target protein expressed by plasmids containing it compared to the unmodified transcription terminator or transcription termins modified in different ways.

[0005] Therefore, in a first aspect, this application provides a nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO:3 or a complementary sequence thereof.

[0006] In some implementations, the nucleic acid molecule is a transcription terminator.

[0007] In some embodiments, the nucleic acid molecule has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, or at least 99.5% identity with SEQ ID NO: 3.

[0008] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit is present (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For instance, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match).

[0009] It should be understood that this application obtains modified transcription terminators that can significantly increase protein yield by modifying and screening the 30 bases at the beginning of existing transcription terminators. Therefore, under the guidance of this application, those skilled in the art are capable of modifying or selecting the remaining sequences in the transcription terminator sequence, i.e., sequences other than the 30 bases at the beginning of the transcription terminator provided in this application.

[0010] In some embodiments, the nucleic acid molecule further comprises a target nucleic acid molecule operatively linked to the nucleic acid molecule as described above.

[0011] In some embodiments, the target nucleic acid molecule contains a target nucleotide sequence encoding a target RNA or a target protein.

[0012] In some implementations, the target nucleic acid molecule further comprises an intron sequence.

[0013] In some embodiments, the target nucleotide sequence encoding the target RNA or target protein further includes an intron sequence.

[0014] In some embodiments, the target nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide.

[0015] In some embodiments, the signal peptide is a natural or non-natural signal peptide sequence of the target protein.

[0016] In some embodiments, the target nucleic acid molecule contains a nucleotide sequence encoding a hepatocyte growth factor (HGF) protein.

[0017] In some embodiments, the nucleotide sequence encoding the hepatocyte growth factor protein comprises the nucleotide sequence shown in SEQ ID NO: 8.

[0018] In some embodiments, the hepatocyte growth factor is human hepatocyte growth factor.

[0019] In some embodiments, the human hepatocyte growth factor comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0020] In some embodiments, the target nucleic acid molecule contains a nucleotide sequence encoding the parathyroid hormone (PTH) protein.

[0021] In some embodiments, the nucleotide sequence encoding parathyroid hormone protein comprises the nucleotide sequence shown in SEQ ID NO:11.

[0022] In some embodiments, the parathyroid hormone is human parathyroid hormone.

[0023] In some embodiments, the human parathyroid hormone comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0024] In some embodiments, the nucleic acid molecule further comprises a selective marker, a replicon, a promoter, or any combination thereof.

[0025] In some embodiments, the selective marker is selected from genes for antibiotic resistance or tolerance (e.g., kanamycin, streptomycin, chloramphenicol, hygromycin B).

[0026] On the other hand, this application provides a vector containing the nucleic acid molecules as described above.

[0027] In some embodiments, the vector comprises an element selected from the following: one or more expression control elements (e.g., promoters, enhancers, terminators), selection marker genes, multiple cloning sites, reporter genes, tags, or any combination thereof.

[0028] In some embodiments, the vector contains transcriptional control elements, such as promoters, enhancers, terminators, etc. In some embodiments, the promoter is selected from constitutive promoters (e.g., CMV promoters, SV40 promoters, EF-1α promoters), inducible promoters, tissue-specific promoters, or any combination thereof.

[0029] In some embodiments, the selective marker is selected from antibiotics (e.g., kanamycin, streptomycin, chloramphenicol, hygromycin B), resistance or tolerance genes (e.g., bar gene).

[0030] In this document, the term "multiple cloning site" refers to a short, contiguous region of DNA on a vector for inserting a target gene. Typically, a multiple cloning site contains recognition sequences for multiple different restriction endonucleases, and these sequences are unique and do not appear elsewhere on the vector. Common restriction endonuclease sites include EcoRI, BamHI, XhoI, HindIII, NotI, etc. Therefore, in some embodiments, the vector contains one or more restriction endonuclease sites.

[0031] In some embodiments, the reporter gene is selected from green fluorescent protein (GFP), luciferase, β-galactosidase (LacZ), or any combination thereof.

[0032] In some embodiments, the vector may contain a tag to facilitate the expression, detection, tracing, and / or purification of the target protein. Such epitope tags are well known to those skilled in the art, and examples include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select an appropriate epitope tag based on the desired purpose (e.g., purification, detection, or tracing). In some embodiments, the vector is a cloning vector or an expression vector.

[0033] A plasmid is a double-stranded DNA molecule capable of autonomous replication. In some embodiments, the plasmid is artificially constructed based on a natural plasmid. For example, compared to a natural plasmid, the plasmid typically carries one or more selective marker genes (such as antibiotic resistance genes) and a synthetically produced multiple cloning site sequence containing multiple restriction endonuclease recognition sites, with most non-essential sequences removed. In some embodiments, the plasmid also contains a target nucleic acid molecule. In some embodiments, the target nucleic acid molecule contains a target nucleotide sequence encoding a target RNA or a target protein.

[0034] In some embodiments, the vector is used to express (e.g., in a subject (e.g., a mammal, such as a human)) a target RNA or a target protein.

[0035] In some embodiments, the target protein is HGF protein.

[0036] In some embodiments, the target protein is human HGF protein.

[0037] In some embodiments, the target protein is the PTH protein.

[0038] In some embodiments, the target protein is human PTH protein.

[0039] In some embodiments, the vector comprises a nucleotide sequence as shown in SEQ ID NO: 10, 13 or 14.

[0040] Based on the verified content of this application, the transcription terminator of this application can significantly increase protein yield (e.g., increase it to about 2 to 3 times the original amount). Therefore, it is reasonable to expect that the transcription terminator of this application can significantly increase the protein yield of other plasmids, regardless of what target nucleic acid molecules they contain.

[0041] On the other hand, this application provides a host cell containing the nucleic acid molecules described above or the vector described above.

[0042] In some embodiments, the host cell is selected from prokaryotic cells or eukaryotic cells.

[0043] In some embodiments, the host cell is an Escherichia coli cell (e.g., an Escherichia coli DH5α cell).

[0044] In some implementations, the host cell is a human cell (e.g., 293T cell).

[0045] On the other hand, this application provides a pharmaceutical composition comprising the nucleic acid molecule as described above, the carrier as described above, or the host cell as described above.

[0046] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0047] In some embodiments, the pharmaceutical composition is administered by injection.

[0048] In some embodiments, the pharmaceutical composition is an injection or a lyophilized powder.

[0049] In another aspect, this application provides a method for expressing or generating a target RNA and / or a target protein, the method comprising using a nucleic acid molecule as described above, a vector as described above, a host cell as described above, or a pharmaceutical composition as described above.

[0050] In some embodiments, the method includes expressing the nucleic acid molecule or the vector in the host cell under conditions that allow the expression of the target RNA and / or the target protein; and optionally, recovering the target RNA and / or the target protein expressed in the host cell.

[0051] In some implementations, the target protein is hepatocyte growth factor.

[0052] In some embodiments, the hepatocyte growth factor is human hepatocyte growth factor.

[0053] In some embodiments, the human hepatocyte growth factor comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0054] In some embodiments, the nucleotide sequence encoding the human hepatocyte growth factor comprises the nucleotide sequence shown in SEQ ID NO:8.

[0055] In some embodiments, the target protein is parathyroid hormone.

[0056] In some embodiments, the parathyroid hormone is human parathyroid hormone.

[0057] In some embodiments, the human parathyroid hormone comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0058] In some embodiments, the nucleotide sequence encoding the parathyroid hormone comprises the nucleotide sequence shown in SEQ ID NO: 11.

[0059] On the other hand, this application provides the use of the nucleic acid molecules, vectors, host cells, or pharmaceutical compositions described above in increasing the yield of target RNA and / or target protein.

[0060] In some implementations, the target protein is hepatocyte growth factor.

[0061] In some embodiments, the hepatocyte growth factor is human hepatocyte growth factor.

[0062] In some embodiments, the human hepatocyte growth factor comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0063] In some embodiments, the nucleotide sequence encoding the human hepatocyte growth factor comprises the nucleotide sequence shown in SEQ ID NO:8.

[0064] In some embodiments, the target protein is parathyroid hormone.

[0065] In some embodiments, the parathyroid hormone is human parathyroid hormone.

[0066] In some embodiments, the human parathyroid hormone comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0067] In some embodiments, the nucleotide sequence encoding the parathyroid hormone comprises the nucleotide sequence shown in SEQ ID NO: 11.

[0068] On the other hand, this application provides the use of the nucleic acid molecule as described above, the vector as described above, or the host cell as described above in the preparation of a pharmaceutical product for treating a disease in which a subject may benefit from the expression of a target RNA and / or a target protein; wherein a nucleotide encoding the target RNA and / or the target protein is operatively linked to the nucleic acid molecule.

[0069] In some embodiments, the medicament is used to treat diseases in subjects that may benefit from the activity of natural HGF or PTH proteins.

[0070] In some embodiments, the disease is selected from ischemic diseases (e.g., coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower extremity arterial ischemia), metabolic syndrome, diabetes and its complications (e.g., diabetic peripheral neuropathy), restenosis (e.g., postoperative restenosis and post-perfusion restenosis), and nerve injury (e.g., neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (e.g., diabetic peripheral neuropathy)), osteoporosis, or hypoparathyroidism syndrome.

[0071] In some implementations, the subject is a mammal, such as a human.

[0072] In some implementations, the drug is administered by injection.

[0073] In some embodiments, the drug is an injection or a lyophilized powder.

[0074] Terminology Definition

[0075] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0076] As used in this article, the term "transcription terminator" is a functional unit of nucleic acid, also known as a terminator (terminator T). These terms have the same meaning and are used interchangeably. Specifically, a transcription terminator is a specific DNA sequence located downstream of the coding region of a gene that provides a signal to RNA polymerase to terminate transcription.

[0077] If a gene driven by a strong promoter on a plasmid (such as CMV or T7) does not have a valid terminator, RNA polymerase may "read through" the gene's endpoint and continue transcribing downstream DNA sequences. Therefore, in some implementations, transcription terminators can improve the accuracy of expressed target proteins.

[0078] As used herein, the terms "hepatocyte growth factor" or "HGF" or "HGF protein" refer to naturally occurring, biologically active hepatocyte growth factor (HGF), and they have the same meaning and are used interchangeably. As used herein, the terms "human hepatocyte growth factor" or "hHGF" or "hHGF protein" refer to naturally occurring, biologically active human hepatocyte growth factor, and they have the same meaning and are used interchangeably. The amino acid sequences of HGF protein or hHGF protein can be readily obtained from various public databases (e.g., GenBank). For example, the amino acid sequence of natural hHGF protein is available in GenBank accession number BAA14348.1.

[0079] As used herein, the terms “parathyroid hormone” or “PTH” or “PTH protein” refer to naturally occurring, biologically active parathyroid hormone (PTH), and they have the same meaning and are used interchangeably. As used herein, the terms “human parathyroid hormone” or “hPTH” or “hPTH protein” refer to naturally occurring, biologically active human parathyroid hormone, and they have the same meaning and are used interchangeably. The amino acid sequences of PTH protein or hPTH protein can be readily obtained from various public databases (e.g., GenBank). For example, the amino acid sequence of natural hPTH protein is available in GenBank accession number AAA60215.1.

[0080] Natural HGF and PTH proteins can exist in multiple versions, possessing substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), as well as substantially the same biological function, although minor differences in their amino acid sequences may still exist. Furthermore, HGF and PTH proteins have been shown to be highly conserved across different species, with homology exceeding 80% or 90%. Therefore, in this application, the natural HGF protein is not limited to the protein shown in SEQ ID NO: 9, and the natural PTH protein is not limited to the protein shown in SEQ ID NO: 12, but is intended to cover all known natural HGF and PTH proteins. In particular, this application verifies that the nucleic acid molecules provided in this application can significantly enhance the expression levels of human HGF and PTH proteins. In some embodiments, the N-terminus of the HGF or PTH protein further includes a signal peptide sequence.

[0081] The nucleotide sequences encoding HGF and PTH proteins can be either native or modified HGF and PTH protein-coding sequences, provided that the resulting HGF and PTH proteins retain or partially retain the activity of the native HGF and PTH proteins. In some embodiments, the nucleotide sequence encoding the HGF protein is modified (e.g., codon-optimized). In some embodiments, the nucleotide sequence encoding the HGF protein is shown in SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the PTH protein is shown in SEQ ID NO: 11.

[0082] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including, but not limited to, replicon sequences, promoter sequences, transcription terminator sequences, selection elements, and reporter genes. In some embodiments, the vector is a plasmid.

[0083] As used herein, the term "nucleotide" is intended to include ribonucleotides and deoxyribonucleotides. For example, adenine nucleotides are intended to include adenine ribonucleotides and adenine deoxyribonucleotides, and may be selected as needed. Similarly, guanine nucleotides are intended to include guanine ribonucleotides and guanine deoxyribonucleotides, and may be selected as needed. In some preferred embodiments, the nucleotide is a deoxyribonucleotide. In some preferred embodiments, the nucleotide is a ribonucleotide. In this document, the nucleotide may be modified (e.g., chemically modified) or unmodified.

[0084] As used herein, the term "nucleic acid" is intended to include ribonucleic acid, deoxyribonucleic acid, and combinations thereof. Therefore, in this application, the nucleic acid molecule encoding hepatocyte growth factor (HGF) and parathyroid hormone (PTH) can be RNA, DNA, or an RNA / DNA hybrid. In some preferred embodiments, the nucleic acid molecule is RNA. In some preferred embodiments, the nucleic acid molecule is DNA. In some preferred embodiments, the nucleic acid molecule is an RNA / DNA hybrid. In this application, the nucleic acid molecule can be modified (e.g., chemically modified) or unmodified.

[0085] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0086] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars and NaCl. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0087] As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this application, a beneficial or desired clinical outcome includes (but is not limited to) relief of symptoms, reduction of the extent of disease, stabilization (i.e., cessation of disease progression) of the disease state, delay or slowing of disease progression, improvement or relief of the disease state, and relief of symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to extended survival compared to expected survival (if no treatment was received).

[0088] Beneficial effects of the invention

[0089] This application provides a modified transcription terminator that significantly increases the yield of the target protein expressed by plasmids containing the modified terminator compared to the original terminator or other modified termins. For example, when applied to HGF or PTH protein expression plasmids for in vitro cell transfection, the modified transcription terminator of this application increases protein yield by at least three times. Furthermore, the effect of this modified transcription terminator is not limited to specific plasmids or target proteins, and has broad application prospects.

[0090] The embodiments of the present invention will now be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments.

[0091] Sequence information

[0092] Information on a portion of the sequence involved in this invention is provided below.

[0093] Among them, the underlined sequences in SEQ ID NO: 2~7 indicate that their nucleotide sequences at the corresponding positions shown in SEQ ID NO: 1 have been modified. Detailed Implementation

[0094] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, the experiments and methods described in the embodiments are generally carried out in accordance with conventional methods well known in the art and described in various references.

[0095] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0096] Example 1. Construction and preparation of recombinant plasmids

[0097] 1. Obtaining the target sequence

[0098] According to the sequence of the recombinant plasmid pYJC-HGF-MUT1 in Chinese patent application CN110577954A (SEQ ID NO: 7 in CN110577954A), the HGF intron sequence was removed to synthesize a plasmid, which is called the original plasmid. This plasmid contains the original transcription terminator sequence as shown in SEQ ID NO: 1, and the nucleotide sequence encoding HGF as shown in SEQ ID NO: 8, and expresses the HGF protein as shown in SEQ ID NO: 9.

[0099] 1.1 Obtaining the transcription terminator sequence

[0100] The original transcription terminator sequence (SEQ ID NO: 1) of the plasmid was obtained, and it was modified using conventional genetic engineering methods to obtain the modified transcription terminator sequences shown in SEQ ID NO: 2-7. The full text of Chinese patent application CN110577954A is incorporated herein by reference.

[0101] Specifically, partial bases in the first 6, 10, 20, 25, and 30 bases of the transcription terminator were modified to obtain the modified transcription terminator sequences shown in SEQ ID NO: 2-7. Wherein:

[0102] (1) SEQ ID NO: 2 is based on SEQ ID NO: 1, with the first base changed from G to A, the sixth base changed from G to A, and the tenth base changed from G to A;

[0103] (2) SEQ ID NO: 3 is based on SEQ ID NO: 1, with the first base changed from G to A, the sixth base changed from G to A, the tenth base changed from G to A, the twelfth base changed from G to A, and the twentieth base changed from G to A;

[0104] (3) SEQ ID NO: 4 is based on SEQ ID NO: 1, with the first base changed from G to A and the sixth base changed from G to A;

[0105] (4) SEQ ID NO: 5 is based on SEQ ID NO: 1, with the first base changed from G to T, the sixth base changed from G to T, the tenth base changed from G to T, the twelfth base changed from G to T, and the twentieth base changed from G to T;

[0106] (5) SEQ ID NO: 6 is based on SEQ ID NO: 1, with the first base changed from G to A, the sixth base changed from G to A, the tenth base changed from G to A, the twelfth base changed from G to A, the twentieth base changed from G to A, and the twentieth base changed from G to A;

[0107] (6) SEQ ID NO: 7 is based on SEQ ID NO: 1, with the first base changed from G to A, the sixth base changed from G to A, the tenth base changed from G to A, the twelfth base changed from G to A, the twentieth base changed from G to A, the twentieth base changed from G to A, the twentieth base changed from G to A, and the twentieth base changed from G to A.

[0108] 1.2 Construction of pYJC-HGF related plasmids:

[0109] In the original plasmids described above, the modified transcription terminator sequence was replaced with the modified transcription terminator sequence to obtain the corresponding modified plasmids pYJC-HGF containing the modified transcription terminator sequences shown in SEQ ID NO: 2-7. The nucleotide sequence of the pYJC-HGF plasmid containing the modified transcription terminator sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 10.

[0110] 1.3 Construction of pYJC-PTH related plasmids:

[0111] The parathyroid hormone (PTH) gene was synthesized according to SEQ ID NO: 11. The HGF target gene in the original plasmid was replaced with this sequence using conventional genetic engineering methods to obtain the plasmid pYJC-PTH containing the foreign gene PTH coding sequence. This plasmid contains the original transcription terminator sequence as shown in SEQ ID NO: 1.

[0112] Furthermore, the original transcription terminator sequence was replaced by the modified transcription terminator sequence SEQ ID NO: 3, and the recombinant plasmid pYJC-PTH was constructed to obtain the pYJC-PTH plasmid containing the modified transcription terminator sequence shown in SEQ ID NO: 3. The nucleotide sequence of the plasmid is shown in SEQ ID NO: 13.

[0113] 1.4 Construction of pVAX-HGF related plasmids:

[0114] The pVAX vector was synthesized according to the sequence of SEQ ID NO: 15, and the HGF target gene was synthesized according to the sequence of SEQ ID NO: 8. The target gene was constructed into the pVAX vector using conventional genetic engineering methods to obtain the plasmid pVAX-HGF containing the coding sequence of the exogenous gene HGF. This plasmid contains the original transcription terminator sequence as shown in SEQ ID NO: 1.

[0115] Furthermore, the original transcription terminator sequence was replaced by the modified transcription terminator sequence SEQ ID NO: 3, and constructed into the recombinant plasmid pVAX-HGF to obtain the pVAX-HGF plasmid containing the modified transcription terminator sequence shown in SEQ ID NO: 3. The nucleotide sequence of this plasmid is shown in SEQ ID NO: 14.

[0116] 2. Preparation of recombinant plasmids

[0117] 2.1 Plasmid Transformation

[0118] E. coli DH5α competent cells were prepared using the calcium chloride method. The plasmids described above were transformed into E. coli DH5α cells and plated on LB agar plates containing 50 μg / mL kanamycin (Kan). Positive colonies were selected and cultured on LB agar plates containing 50 μg / mL kanamycin. Strains with high in vitro expression levels of recombinant plasmids and correct restriction endonuclease identification were screened and stored below -70°C for large-scale culture.

[0119] 2.2 Shake flask culture and seed culture preparation

[0120] 2.2.1 Seed activation

[0121] (1) Take the bacterial strain stored at -70℃, revive it, streak it onto LB agar plates (containing 50 μg / mL Kan), and incubate it at 37℃ overnight to become activated seed.

[0122] (2) Pick the activated seeds from the above plates and inoculate them into a test tube containing 5 mL LB medium (containing 50 μg / mL Kan). Incubate overnight at 37°C and 230 rpm.

[0123] (3) Take the above overnight cultured seeds and transfer them at a ratio of 1:20 into a shake flask containing 100 mL LB medium (containing 50 μg / mL Kan). Incubate at 37°C and 230 rpm for about 10 hours.

[0124] (4) Take the above culture solution and transfer it at a ratio of 1:20 into a shake flask containing 1800mL LB medium (containing 50μg / mL Kan). Incubate overnight at 37℃ and 230 rpm to obtain the seed culture for the upper tank.

[0125] 2.2.2 Fermentation

[0126] (1) Culture medium and feed solution

[0127] a) M9CA medium (upper tank medium):

[0128] Na2HPO4: 90g, KH2PO4: 45g, NaCl: 22.5g, NH4Cl: 5g.

[0129] The above components were dissolved in 2L of distilled water, adjusted to pH 7.4, and placed in a 30L fermenter. Distilled water was added to bring the volume to 18L, and the mixture was sterilized at 121°C for 30 minutes. After cooling, the following two components were aseptically added: 180mL of 20% glucose solution (each 100mL contains 20mL of 1M MgSO4, 1mL of 1M CaCl2, and 100mg of vitamin B1) and 180mL of 20% casein acid solution. After aseptic filtration, both components can be stored at 4°C for one week.

[0130] b) Feeding solution

[0131] Glucose: 75g; Yeast extract: 30g; Tryptone: 30g; MgSO4·7H2O: 3g. After aseptic filtration, it can be stored at 4°C for 1 week.

[0132] (2) Control of fermentation parameters

[0133] a) Temperature: 37℃

[0134] b) Dissolved oxygen and rotation speed: When the dissolved oxygen in the culture medium drops below 30%, increase the rotation speed by 50 rpm each time until it reaches 600 rpm, then introduce pure oxygen and maintain the dissolved oxygen at around 30%.

[0135] c) pH: Automatically add 30% ammonia water to maintain the pH at around 7.0.

[0136] d) Glucose feeding: According to the fermentation process, feeding is divided into two stages, depending on the cell growth. Feed containing 100 g of glucose is fed after about 2 hours, and feed containing 200 g of glucose is fed after 3-5 hours.

[0137] (3) Fermentation in a 30-liter fermenter

[0138] Take 1.8 L of the above-mentioned seed culture and add it to the fermenter at a 1:10 inoculation rate. Then, aseptically add 540 mg of kanamycin. Incubate at 37°C, with an aeration rate of 1.5 L / min and 300 rpm. Automatically add 30% ammonia water to maintain the pH at 7.0. After 2 hours of incubation, start adding feed containing 100 g of glucose. After 4-5 hours, add feed containing 200 g of glucose. At OD... 600 When the bacterial count is above 60, centrifuge to collect the bacterial cells.

[0139] 2.2.3 Purification of recombinant plasmids

[0140] (1) Sterilization: The strain was lysed using alkaline lysis.

[0141] (2) Clarification: The supernatant is clarified by filter cartridge filtration.

[0142] (3) Pretreatment concentration: Use hollow fiber ultrafiltration with a molecular weight of 300kD to concentrate and reduce volume.

[0143] (4) Chromatography 1: Gel filtration chromatography was used to separate plasmid DNA and RNA under high salt conditions.

[0144] (5) Chromatography II: Affinity chromatography was used to separate supercoiled plasmid DNA and open circular plasmid DNA under high salt conditions.

[0145] (6) Chromatography 3: Ion exchange chromatography is used for purification and removal of endotoxins.

[0146] (7) Ultrafiltration concentration: 300kD hollow fiber is used for concentration, and the original solution is obtained by sterile filtration.

[0147] Example 2. Plasmid concentration and purity detection

[0148] The purified recombinant plasmid sample was diluted with water for injection to approximately 30 μg / mL. The sample was placed in a quartz cuvette, and the absorbance was measured at 260 nm using a UV spectrophotometer. Three measurements were taken, and the average value was used to calculate the plasmid concentration: plasmid concentration (μg / mL) = absorbance × 50 μg / mL × dilution factor. The concentrations of various plasmids were all within the range of 1.9–2.1 mg / mL. The results are shown in Table 1.

[0149] Table 1. Results of plasmid concentration determination

[0150]

[0151] HPLC analysis showed that the supercoiling ratio of all plasmids was greater than 95.0%.

[0152] Example 3. Protein yield detection

[0153] 3.1 Transfection of HEK293T cells with recombinant plasmid

[0154] (1) Cell preparation: Take HEK293T cells to be transfected, seed them in 24-well cell culture plates, 500 μl / well, and incubate overnight at 37°C and 5% CO2, so that the cell confluence rate is 90-95% on the day of transfection.

[0155] (2) Plasmid transfection: For each well of cells, dilute 2 μL of Lipofectamine 2000 with 50 μL of serum-free DMEM medium; dilute the plasmid with 50 μL of serum-free DMEM medium for each well of cells, and add the recombinant plasmid accordingly; after incubating at room temperature for 5 min, gently mix the diluted DNA and diluted Lipofectamine 2000. Incubate at room temperature for 20 min. The experimental group consisted of: culture medium + plasmid + transfection reagent; the negative control group consisted of: culture medium + transfection reagent; 100 μL of each of the experimental and control groups was added to the cells to be transfected. The cells were cultured at 37℃ and 5% CO2 for 48 h, and the transfection supernatant was collected.

[0156] 3.2 HGF expression level determination

[0157] Quantitative detection was performed using an HGF detection kit (R&D). The absorbance was measured at 450 nm using a microplate reader to calculate and analyze the HGF content. The results are shown in Table 2.

[0158] 3.3 PTH Expression Measurement

[0159] Quantitative detection was performed using a PTH detection kit (abcam). The absorbance was measured at 450 nm using a microplate reader to calculate and analyze the PTH content. The results are shown in Table 2.

[0160] Table 2. Results of protein expression level determination for various plasmids

[0161]

[0162] As shown in the table, the protein expression levels of plasmids containing the transcription terminator sequence shown in SEQ ID NO: 3 were significantly increased. Conversely, the protein expression levels (e.g., HGF protein) of plasmids containing other modified transcription terminators (e.g., the modified transcription terminators shown in SEQ ID NO: 2, 5, 6, and 7) were slightly decreased. Specifically, without altering the host bacteria or fermentation process, the protein expression levels of plasmids containing the transcription terminator sequence shown in SEQ ID NO: 3 increased approximately 2-3 times compared to other modified plasmids and the original plasmid. Furthermore, even when using different plasmid backbones and different target proteins, the modified transcription terminator sequence shown in SEQ ID NO: 3 significantly increased the expression levels of the target protein. Even for proteins with low native expression levels (e.g., PTH protein), the modified transcription terminator sequence shown in SEQ ID NO: 3 still significantly increased their expression levels. This indicates that the transcription terminator sequence shown in SEQ ID NO: 3 is applicable to a variety of different plasmids, and its ability to increase protein expression is independent of the specific exogenous target protein it is linked to. It can effectively increase the yield of the target protein in the expression of various different plasmids and target proteins.

[0163] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO: 3 or a complementary sequence thereof; Preferably, the nucleic acid molecule is a transcription terminator.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule further comprises a target nucleic acid molecule operably linked to the nucleic acid molecule of claim 1; Preferably, the target nucleic acid molecule contains a target nucleotide sequence encoding the target RNA or the target protein; Preferably, the target nucleic acid molecule contains a nucleotide sequence encoding a hepatocyte growth factor (HGF) protein; Preferably, the nucleotide sequence encoding the hepatocyte growth factor protein comprises the nucleotide sequence shown in SEQ ID NO: 8; Preferably, the hepatocyte growth factor is human hepatocyte growth factor; Preferably, the human hepatocyte growth factor comprises the amino acid sequence shown in SEQ ID NO: 9; Preferably, the target nucleic acid molecule contains a nucleotide sequence encoding the parathyroid hormone (PTH) protein; Preferably, the nucleotide sequence encoding parathyroid hormone protein comprises the nucleotide sequence shown in SEQ ID NO: 11; Preferably, the parathyroid hormone is human parathyroid hormone; Preferably, the human parathyroid hormone comprises the amino acid sequence shown in SEQ ID NO:

12.

3. The nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule further comprises a selective marker, a replicon, a promoter, or any combination thereof; Preferably, the selective marker is selected from genes for antibiotic resistance or tolerance (e.g., kanamycin, streptomycin, chloramphenicol, hygromycin B).

4. A vector comprising the nucleic acid molecule according to any one of claims 1-3; Preferably, the vector is a cloning vector or an expression vector; Preferably, the vector comprises an element selected from the following: one or more expression control elements (e.g., promoter, enhancer, terminator), selection marker gene, multiple cloning site, reporter gene, tag, or any combination thereof; Preferably, the promoter is selected from a constitutive promoter (e.g., CMV promoter, SV40 promoter, EF-1α promoter), an inducible promoter, a tissue-specific promoter, or any combination thereof; Preferably, the selective marker is selected from antibiotics (e.g., kanamycin, streptomycin, chloramphenicol, hygromycin B), resistance or tolerance genes (e.g., bar gene).

5. The vector of claim 4, wherein the vector is used to express (e.g., in a subject (e.g., a mammal, such as a human)) a target RNA or a target protein; Preferably, the target protein is HGF protein; Preferably, the target protein is human HGF protein; Preferably, the target protein is PTH protein; Preferably, the target protein is human PTH protein; Preferably, the vector comprises a nucleotide sequence as shown in SEQ ID NO: 10, 13 or 14.

6. A host cell comprising a nucleic acid molecule as described in any one of claims 1-3 or a vector as described in claim 4 or 5; Preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; Preferably, the host cell is an Escherichia coli cell (e.g., an Escherichia coli DH5α cell). Preferably, the host cell is a human cell (e.g., 293T cell).

7. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1-3, a carrier according to claim 4 or 5, or a host cell according to claim 6; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition is administered by injection; Preferably, the pharmaceutical composition is an injection solution or a lyophilized powder.

8. A method for expressing or producing a target RNA and / or a target protein, the method comprising using a nucleic acid molecule according to any one of claims 1-3, a vector according to claim 4 or 5, a host cell according to claim 6, or a pharmaceutical composition according to claim 7; Preferably, the method includes expressing the nucleic acid molecule or the vector in the host cell under conditions that allow the expression of the target RNA and / or the target protein; and optionally, recovering the target RNA and / or the target protein expressed in the host cell. Preferably, the target protein is hepatocyte growth factor; Preferably, the hepatocyte growth factor is human hepatocyte growth factor; Preferably, the human hepatocyte growth factor comprises the amino acid sequence shown in SEQ ID NO: 9; Preferably, the nucleotide sequence encoding the human hepatocyte growth factor comprises the nucleotide sequence shown in SEQ ID NO: 8; Preferably, the target protein is parathyroid hormone; Preferably, the parathyroid hormone is human parathyroid hormone; Preferably, the human parathyroid hormone comprises the amino acid sequence shown in SEQ ID NO: 12; Preferably, the nucleotide sequence encoding the parathyroid hormone comprises the nucleotide sequence shown in SEQ ID NO:

11.

9. Use of the nucleic acid molecule according to any one of claims 1-3, the vector according to claim 4 or 5, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in increasing the yield of target RNA and / or target protein; Preferably, the target protein is hepatocyte growth factor; Preferably, the hepatocyte growth factor is human hepatocyte growth factor; Preferably, the human hepatocyte growth factor comprises the amino acid sequence shown in SEQ ID NO: 9; Preferably, the nucleotide sequence encoding the human hepatocyte growth factor comprises the nucleotide sequence shown in SEQ ID NO: 8; Preferably, the target protein is parathyroid hormone; Preferably, the parathyroid hormone is human parathyroid hormone; Preferably, the human parathyroid hormone comprises the amino acid sequence shown in SEQ ID NO: 12; Preferably, the nucleotide sequence encoding the parathyroid hormone comprises the nucleotide sequence shown in SEQ ID NO:

11.

10. The use of the nucleic acid molecule according to any one of claims 1-3, the vector according to claim 4 or 5, or the host cell according to claim 6 in the preparation of a pharmaceutical product, wherein the pharmaceutical product is used to treat a disease in which a subject may benefit from the expression of a target RNA and / or a target protein; wherein, The nucleotides encoding the target RNA and / or target protein are operatively linked to the nucleic acid molecule; Preferably, the medicament is used to treat diseases in subjects that may benefit from the activity of natural HGF protein or PTH protein; Preferably, the disease is selected from ischemic diseases (e.g., coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower extremity arterial ischemia), metabolic syndrome, diabetes and its complications (e.g., diabetic peripheral neuropathy), restenosis (e.g., postoperative restenosis and post-perfusion restenosis), and nerve injury (e.g., neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (e.g., diabetic peripheral neuropathy)), osteoporosis, or hypoparathyroidism syndrome; Preferably, the subject is a mammal, such as a human; Preferably, the drug is administered by injection; Preferably, the drug is an injection solution or a lyophilized powder.

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