Plasmid for expression of KSP37

Abstract

This invention relates to a plasmid for expression of Ksp37 and more particularly, but not exclusively, to a plasmid for expression of Ksp37 in eukaryotic cells, including human cells. The plasmid includes a pNL4-3.luc plasmid backbone from which all HIV-1-associated DNA and an ampicillin resistance gene have been removed and a Ksp37 gene inserted between a PaeR7I-XhoI and a EagI-NotI restriction enzyme recognition site.

Description

[0001] PLASMID FOR EXPRESSION OF KSP37

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a plasmid for expression of Ksp37 and more particularly, but not exclusively, to a plasmid for expression of Ksp37 in eukaryotic cells, including human cells.

[0004] BACKGROUND TO THE INVENTION

[0005] A plasmid is a small, circular double-stranded DNA molecule commonly found in bacteria and eukaryotes. These plasmids exist as separate entities from the organism's chromosomal DNA and have the ability to replicate independently. Typically containing a limited number of genes, plasmids can be transferred from one cell to another, facilitating the exchange of genetic material.

[0006] The transfer of plasmids among bacterial cells often leads to the acquisition of advantageous genetic traits, such as resistance to antibiotics or other drugs. Plasmid sizes can vary significantly, ranging from a few thousand to hundreds of thousands of base pairs. Due to their capacity for intercellular transfer, plasmids play a crucial role in scientific research. For instance, researchers utilize recombinant plasmids, into which specific genes are inserted, to study gene properties in cells that naturally do not express them. Through a process called transfection, these recombinant plasmids can be introduced into eukaryotic cells, including human cells, to express desired genes for research, therapeutic, or immunization purposes. It's noteworthy that many plasmid DNAs are employed in animal vaccines, and their use in human immunization is increasingly prevalent.

[0007] Additionally, plasmids can be transferred to bacterial cells via transformation to express target genes. Notably, the transformation process is commonly employed in laboratories to "grow" plasmids, generating larger quantities of the same recombinant DNA.

[0008] The protein known as Ksp37, also known as Killer-specific secretory protein of 37 kDa or Fibroblast Growth Factor-Binding Protein 2 (FGF-BP2), is commonly expressed by various immune cells, including natural killer (NK), CD8+ T, CD4+ T, and CDT cells. It is believed to play a significant role in cytotoxic T lymphocyte (CTL) mediated immunity [1]-

[0009] Specifically, Ksp37 stimulates the cytotoxic effects of cells from the cellular arm of the immune system against diseased cells, including virus-infected cells and cancer cells. This protein is secreted and consists of 223 amino acids, with a molecular weight of 37 kDa. The Ksp37 sequence is stored under the accession number AB021123 for BLAST identification. This protein exhibits a 99% similarity to the Human Fibroblast Growth Factor-Binding Protein (FGFBP2). As such, FGFBP2 is also known as Killerspecific secretory protein of 37 kDa (Ksp37). The FGFBP2 gene is conserved across multiple species, including chimpanzee, rhesus monkey, chicken, zebrafish, and frog. Orthologs of the human FGFBP2 gene have been identified in at least 137 organisms.

[0010] Numerous studies have demonstrated that the human protein Ksp37 is expressed by cells involved in the cellular arm of the immune system. More precisely, this gene is expressed Th1-type CD4+ T cells, effector CD8+ T cells, y<5 T cells, and CD16+ NK cells. In many instances, Ksp37 is co-expressed with perforin, another protein responsible for mediating the response of cytotoxic T lymphocytes against various cancers and infectious diseases.

[0011] Several studies have indicated a potential association between Ksp37 expression and positive outcomes in different diseases. These include ovarian cancer [2], high-grade gliomas [3], and tuberculosis [4], Additionally, research has shown that Ksp37 is more highly expressed in long-term non-progressors (LTNPs) of HIV than in non-LTNP individuals, suggesting its role in HIV control [5],

[0012] Given these findings, there arises a necessity to develop and manufacture a plasmid for Ksp37 gene expression in human cells. This plasmid would serve as a valuable tool for further investigating the correlation between Ksp37 expression and protection against diseases.

[0013] Moreover, it presents an opportunity to explore the potential therapeutic and immunization applications of this protein against various human diseases. OBJECT OF THE INVENTION

[0014] It is an object of this invention to provide a plasmid for expression of Ksp37 which, at least partially, will help alleviate some of the above-mentioned difficulties in Ksp37- related research.

[0015] SUMMARY OF THE INVENTION

[0016] In accordance with the invention, there is provided a plasmid for gene expression that carries the Ksp37 gene for expression in eukaryotic cells, including human cells.

[0017] The plasmid is generated from a pNL4-3.luc plasmid backbone, which is a proviral clone plasmid containing HIV-1 DNA of approximately 15,500 base pairs.

[0018] All HIV-1-associated DNA, together with the ampicillin resistance gene, are removed from the pNL4-3 plasmid backbone, reducing its size by more than half.

[0019] The modified pNL4-3 plasmid retains the following restriction enzyme recognition sites: Zral, Aatll, FspAI, PfIMI, EcoRI, Sall, Nhel, Bmtl, BsaBI, Eagl-Notl, PaeR7l-Xhol, NgoMIV, Nael, and Btgl-Ncol. The Ksp37 gene is inserted between the PaeR7l-Xhol and Eagl-Notl restriction sites.

[0020] The modified plasmid further comprises an origin of replication that enables autonomous plasmid replication within host cells. The origin of replication is positioned between the Btgl-Ncol and Zral / Aatl I restriction enzyme recognition sites.

[0021] The promoter sequence used in the modified pNL4-3 plasmid is derived from the ampicillin resistance gene (AmpR) promoter. This AmpR promoter is positioned between the Btgl-Ncol and Zral / Aatll restriction enzyme recognition sites and is configured to initiate transcription of the Ksp37 gene.

[0022] The modified plasmid is initially constructed through chemical synthesis. Thereafter, it is propagated in large quantities by transformation into bacterial strains such as Escherichia coli DH5a or XL1-Blue. The plasmid is subsequently extracted and purified from the bacterial cultures using a commercially available plasmid isolation kit.

[0023] The invention further relates to the therapeutic use of the plasmid. Specifically, the plasmid may be used for delaying, preventing, reversing, and / or treating various conditions, including autoimmune diseases, biological aging, cancer, type 2 diabetes, malaria, neurological or degenerative diseases, and viral diseases. The autoimmune diseases include, but are not limited to, Ankylosing Spondylitis, Myasthenia Gravis, Multiple Sclerosis, Optic Neuritis, Polyarteritis Nodosa, Polymyalgia Rheumatica, Reiter Syndrome (Reactive Arthritis), Rheumatoid Arthritis, Type 1 Diabetes (Late Onset), Vasculitis Syndrome, and Giant Cell Arteritis. The biological aging-related conditions include, but are not limited to, general cellular aging, chronic inflammatory disorders, neurodegenerative conditions linked to aging, and mitochondrial dysfunction. The neurological or degenerative conditions include, but are not limited to, Alzheimer’s Disease, general Dementia, Motor Neuron Disease, Vascular Dementia, stroke-related neurological damage, traumatic brain injuries, and spinal cord injuries. The viral diseases include, but are not limited to, retroviruses such as HIV / AIDS and paramyxoviruses such as measles.

[0024] The invention further includes methods of treatment involving the administration of the plasmid to a person in need thereof for the treatment of such conditions.

[0025] In addition, the invention provides a method of expressing Ksp37 in a eukaryotic host cell, comprising transfecting the host cell with the plasmid and culturing the cell under conditions that permit expression of the Ksp37 polypeptide. The eukaryotic host cell may be a human cell, such as a HEK293 or HeLa cell.

[0026] These and other features of the invention are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] One embodiment of the invention is described below, by way of example only, with reference to the drawings in which figure 1 shows a diagram of the plasmid for Ksp37 gene expression.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood in the art. It must be noted that the singular form includes the plural unless the context clearly indicates otherwise.

[0030] In this embodiment, figure 1 shows a graphic representation of the plasmid for Ksp37 gene expression.

[0031] The Ksp37 plasmid is generated from a pNL4-3 plasmid backbone.

[0032] All HIV-1 associated DNA are first removed from the pNL4-3 plasmid. In addition, the antibiotic (ampicillin) resistant gene is also removed from the pNL4-3 plasmid. The modified pNL4-3 plasmid is left with, inter alia, the following restriction enzyme recognition sites: Zral, Aatll, FspAI, PfLMI, EcoRI, SaLI, Nhel, Bmtl, BasBI, Eagl-Notl,

[0033] PaeR7l-Xhol, NgoMIV, Nael, and Btgl-Ncol.

[0034] The Ksp37 gene is inserted between the PaeR71-Xhol and Eagl-Notl restriction enzyme recognition sites of the modified pNL4-3 plasmid.

[0035] The origin of DNA replication (Ori) is positioned between the Btgl-Ncol and Zral / Aatll restriction enzyme recognition sites.

[0036] Furthermore, the AmpR promoter used to promote the plasmid for gene expression, i.e. used for the initiation of single RNA expression once the plasmid is inside the cell, is also positioned between the Btgl-Ncol and Zral / Aatll restriction enzyme recognition sites.

[0037] This plasmid significantly advances the study of Ksp37, enabling detailed exploration of its immunological properties and potential therapeutic uses.

[0038] REFERENCES

[0039] [1], Ogawa, M., Tsukamoto, H., Sato, M., et al. (2001). A Novel Serum Protein That Is

[0040] Selectively Produced by Cytotoxic Lymphocytes. The Journal of Immunology, 166(10), 6404-6412.

[0041] [2], Elgaaen, B. V., Olstad, O. K., Haug, K. B. F., et al. (2010). POLD2 and KSP37 (FGFBP2)

[0042] Correlate Strongly with Histology, Stage and Outcome in Ovarian Carcinomas. PLoS ONE, 5(11), e13837.

[0043] [3], Yamanaka, R., Arao, T., Yajima, N., et al. (2006). Identification of expressed genes characterizing long-term survival in malignant glioma patients. Oncogene, 25(44), 5994- 6002.

[0044] [4], Kita, Y, Shibagaki, N., Shida, H., et al. (2013). Novel therapeutic vaccines [(HSP65 +

[0045] IL-12) DNA-, granulysin- and Ksp37-vaccine] against tuberculosis and synergistic effects in the combination with chemotherapy. Human Vaccines & Immunotherapeutics, 9(3), 526-533.

[0046] [5], Internal data, Viro-Gen (Pty) Ltd. Comparative analysis of Ksp37 expression in longterm non-progressors and non-LTNP individuals.

Claims

CLAIMS1. A plasmid for expression of Ksp37 in a eukaryotic cell, comprising: a. a pNL4-3.luc plasmid backbone from which all HIV-1-associated DNA and an ampicillin resistance gene have been removed and b. a Ksp37 gene inserted between a PaeR7l-Xhol and a Eagl-Notl restriction enzyme recognition site.

2. The plasmid as claimed in claim 1 in which an origin of replication is positioned between a Btgl-Ncol and a Zral / Aatl I restriction enzyme recognition site.

3. The plasmid as claimed in claim 2 in which a promotor is positioned between the Btgl-Ncol and the Zral / Aatl I restriction enzyme recognition site.

4. The plasmid as claimed in claim 3 in which the promotor is derived from the ampicillin resistance gene (AmpR) promoter.

5. The plasmid as claimed in any of the preceding claims in which the plasmid includes a Zral, Aatll, FspAI, PfLMI, EcoRI, SaLI, Nhel, Bmtl, BasBI, Eagl-Notl, PaeR7l-Xhol, NgoMIV, Nael, and the Btgl-Ncol restriction enzyme recognition site.

6. The plasmid as claimed in any of the preceding claims in which the eukaryotic cell is a human cell.

7. A method of producing a plasmid for expression of Ksp37 in eukaryotic cells, the method comprising: chemically synthesising a pNL4-3.luc plasmid backbone in which HIV- 1 -associated sequences and an ampicillin resistance gene have been removed, inserting a nucleic acid sequence encoding Ksp37 between a PaeR7l- Xhol and a Eagl-Notl restriction enzyme recognition site of the modified backbone, inserting an origin of replication between a Btgl-Ncol and a Zral / Aatll restriction enzyme recognition site of the modified backbone, and inserting a promoter sequence derived from an ampicillin resistance gene (AmpR) promoter between the Btgl-Ncol and the Zral / Aatll restriction enzyme recognition site, the promoter being configured to initiate transcription of the Ksp37 gene.

8. The method as claimed in claim 7 which includes the step of transforming the plasmid into a bacterial strain selected from Escherichia coli DH5a and XL1-Blue.

9. The method as claimed in any of claims 7 - 8 which includes extracting and purifying the plasmid from the bacterial strain using a commercially available plasmid isolation kit.

10. The method as claimed in any of claims 7 - 9 in which the plasmid is a proviral clone plasmid containing HIV-1 DNA of approximately 15,500 base pairs prior to modification.

11. The method as claimed in any one of claims 7 - 10 in which the plasmid includes one or more restriction enzyme recognition sites selected from Zral, Aatll, FspAI, PfIMI, EcoRI, Sall, Nhel, Bmtl, BsaBI, Eagl, Notl, PaeR7l, Xhol, NgoMIV, Nael, and Btgl.

12. The method as claimed in any of claims 7 - 11 in which the eukaryotic cell is a human cell.

13. Use of a plasmid as claimed in any one of claims 1 to 4 for the expression of Ksp37 in a eukaryotic cell.

14. A eukaryotic cell comprising the plasmid as claimed in any one of claims 1 to 4.

15. A human cell comprising the plasmid as claimed in any one of claims 1 to 4.

16. Use of the plasmid as claimed in any of claims 1 to 4 in the manufacture of a medicament for delaying, preventing, reversing and / or treating a person suffering from a condition.

17. The use of claim 16 wherein the conditions include autoimmune diseases, biological aging, cancer, type 2 diabetes, malaria, and neurological or degenerative diseases.

18. The plasmid of any of claims 1 - 4 for use in delaying, preventing, reversing, and / or treating a condition in a person.

19. The plasmid of any of claims 1 - 4 for use in the treatment of a condition.

20. The plasmid of claim 19 in which the condition is selected from selected from autoimmune diseases, biological aging, cancer, type 2 diabetes, malaria, and neurological or degenerative diseases.

21. A method for treating a condition in a person in need thereof comprising, administering to said person the plasmid as claimed in any of claims 1 - 4.

22. The method as claimed in claim 21 in which the disease is selected from the group including autoimmune diseases, biological aging, cancer, type 2 diabetes, malaria, and neurological or degenerative diseases.

23. A method of expressing Ksp37 in a eukaryotic host cell, comprising: a. transfecting the host cell with the plasmid as claimed in any one of claims 1 to 4; and b. culturing the host cell under conditions that permit expression of the Ksp37 polypeptide.

24. The method as claimed in claim 23 in which the eukaryotic host cell is a human cell selected from the group consisting of HEK293 and HeLa cells.

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