Production of bioscalable nanorods
Through the BSFnano replication assembly box and auxiliary plasmid system, the length and yield of filamentous phage Ff nanorods were controlled, and the problems of low yield and contamination in the prior art were solved, and efficient and safe nanorod production was achieved.
Patent Information
- Application Number
- CN202380023054.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-10-17
- Filing Date
- 2023-02-20
- Publication Date
- 2025-01-07
AI Technical Summary
In the prior art, nanorods produced using filamentous phage Ff are difficult to achieve high yield and low pollution, and nanorods often contain antibiotic resistance genes, which affect medical and diagnostic applications.
Using a single plasmid or dual plasmid system, the assembly box and auxiliary plasmids are replicated through BSFnano to control the length and yield of nanorods, avoid the presence of antibiotic resistance genes, and regulate the expression of replica protein pII through inducible promoters to delay the nanorods to improve yield.
High yield bioscalable nanorod production is achieved, with adjustable nanorod lengths and free of antibiotic resistance genes, which improves the purity and safety of nanorods.
Smart Images
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Abstract
Claims
1. A nanorod production system (NPS) comprising a nucleic acid expression construct, the nucleic acid expression construct comprising The replication assembly cassette includes the filamentous phage (+)ori1, the packaging signal (PS) and the (+)ori2. at least one plasmid origin of replication that is not located within said replication cassette that allows said construct to replicate in bacteria, at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff bacteriophage replication protein, The expression construct expresses the Ff phage replication protein and produces a DNA sequence that is excised and replicated from the replication assembly box, and the DNA sequence forms a circular single-stranded DNA encapsulated in the nanorod.
2. The NPS according to claim 1, wherein: The at least one Ff bacteriophage replication protein is pII.
3. The NPS according to claim 1 or 2, wherein: The replication assembly cassette also includes a (-)ori located between the PS and the (+)ori2.
4. The NPS according to any one of claims 1 to 3, wherein: The nucleic acid expression construct is a plasmid.
5. The NPS according to any one of claims 1 to 4, wherein: The NPS lacks a second nucleic acid construct encoding one or more filamentous bacteriophage proteins.
6. The NPS according to any one of claims 1 to 5, wherein: The nucleic acid expression construct comprises a nucleic acid sequence encoding the Ff bacteriophage pI-pXI protein.
7. The NPS according to claim 6, wherein: Any or all of the nucleic acid sequences encoding each of the Ff bacteriophage pi-pXI proteins encode modified Ff bacteriophage proteins.
8. The NPS according to any one of claims 1 to 7, wherein: The nucleic acid construct comprises a nucleic acid sequence encoding a modified Ff bacteriophage protein comprising mutations that allow chemical or enzymatic conjugation of small molecules, synthetic or biological polymers to the protein, optionally wherein the modified Ff bacteriophage protein is pill and / or pVIII.
9. The NPS according to any one of claims 1 to 8, wherein: The nucleic acid construct includes a nucleic acid sequence encoding a modified Ff bacteriophage protein pVIII including an amber mutation.
10. The NPS according to any one of claims 1 to 9, wherein: The nucleic acid construct comprises a nucleic acid sequence encoding at least one of pill, pVI, pVII, pVIII and pIX fused to a nucleic acid sequence encoding a heterologous polypeptide.
11. The NPS according to any one of claims 1 to 10, wherein: The nucleic acid expression construct also includes a nucleic acid sequence encoding an auxotrophic marker.
12. The NPS according to any one of claims 1 to 11, wherein: The nucleic acid sequence between the (+)ori1 and the PS or between the PS and the (+)ori2 is a stuffer nucleic acid sequence encoding at least one Ff phage protein.
13. The NPS of claim 12, wherein: The stuffer nucleic acid sequence encodes pVII, pVIII and / or pIX.
14. The NPS according to claim 12 or 13, wherein: The stuffer nucleic acid sequence also encodes a prokaryotic or eukaryotic protein of interest.
15. The NPS according to any one of claims 1 to 14, wherein: The bacteria is Escherichia coli.
16. An isolated host cell comprising the NPS according to any one of claims 1 to 15.
17. A method for producing nanorods, comprising culturing an isolated host cell comprising the NPS according to any one of claims 1 to 15, and providing an inducer of an inducible promoter to the host cell during an optimal growth phase, thereby expressing the Ff bacteriophage replication protein in the cell, producing an excised and replicated DNA sequence, which forms a circular single-stranded DNA encapsulated in the nanorod.
18. The method according to claim 17, wherein: The optimal growth phase is determined by the optical density (OD600) of the host cells.
19. The method according to claim 17 or 18, wherein: The Ff bacteriophage replication protein is pII.
20. A nanorod with a length of 60-800 nm, which encapsulates a circular single-stranded DNA excised by pII cleavage of a replication assembly box comprising a filamentous phage (+)ori1, a packaging signal (PS) and (+)ori2, and a filler nucleic acid sequence between (+)ori1 and the PS or between the PS and (+)ori2, wherein the filler nucleic acid sequence encodes at least one Ff phage protein.
21. The nanorod according to claim 20, wherein The replication assembly cassette also includes a (-)ori between the PS and (+)ori2.
22. The nanorod according to claim 20 or 21, wherein The stuffer nucleic acid sequence encodes pVII, pVIII and / or pIX or encodes modified pVII, pVIII and / or pIX or a combination thereof.
23. The nanorod according to claim 22, wherein The nucleic acid sequence encoding the pVII, pVIII and / or pIX and / or the modified pVII, pVIII and / or pIX is fused to a nucleic acid sequence encoding a heterologous polypeptide.
24. The nanorod according to any one of claims 20 to 23, wherein The stuffer nucleic acid sequence also encodes a heterologous polypeptide that may or may not be fused to the Ff bacteriophage protein or modified Ff bacteriophage protein.
25. The nanorod of any one of claims 20 to 24, having a length of about 95-125 nm.
26. A population of nanorods encapsulating circular single-stranded DNA excised by pII cleavage of a replication assembly box comprising a filamentous bacteriophage (+)ori1, a packaging signal (PS) and (+)ori2, and a stuffer nucleic acid sequence between (+)ori1 and the PS or between the PS and (+)ori2, wherein the stuffer nucleic acid sequence encodes at least one Ff bacteriophage protein, wherein at least 70% of the nanorods in the population have a length of about 40 nm to about 800 nm.
27. The nanorod population according to claim 26, wherein The replication assembly cassette further includes a (-)ori between the packaging signal and the (+)ori2, wherein at least 70% of the nanorods in the population have a length of about 60 nm to about 800 nm.
28. The nanorod population according to claim 27, wherein At least 70% of the nanorods in the population have a length of about 60 nm to about 400 nm.
29. The nanorod population of claim 27, wherein: At least 70% of the nanorods in the population have a length of about 60 nm to about 300 nm.
30. The nanorod population of claim 27, wherein: At least 70% of the nanorods in the population have a length of about 95 nm to about 125 nm.
31. A nanorod having a length of about 35 to about 45 nm, encapsulating a circular single-stranded DNA excised by pII cleavage of a replication assembly cassette comprising a filamentous bacteriophage (+)ori1, a packaging signal (PS) and (+)ori2 and lacking (-)ori.
32. A nanorod population comprising a plurality of nanorods according to claim 31, wherein: At least 70% of the nanorods in the population have a length of about 38 nm to about 42 nm.
33. The nanorod population of claim 32, wherein: At least 70% of the nanorods in the population have a length of about 40 nm.
34. A nanorod production system (NPS), comprising i) A nucleic acid nanorod replication assembly construct comprising A replication assembly cassette comprising a filamentous bacteriophage (+)ori1, a packaging signal (PS) and (+)ori2, and at least one plasmid origin of replication that is not located within said replication cassette that allows said construct to replicate in bacteria, and ii) an auxiliary nucleic acid expression construct comprising at least one selection marker, and at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff bacteriophage replication protein, The auxiliary nucleic acid construct expresses the Ff phage replication protein and produces a DNA sequence that is excised and replicated from the replication assembly box, which forms a circular single-stranded DNA encapsulated in the nanorod.
35. The NPS of claim 34, wherein: The replication assembly cassette also includes a (-)ori between the PS and (+)ori2.
36. The NPS of claim 34 or 35, wherein: The Ff bacteriophage replication protein is pII.
37. An isolated host cell comprising the NPS according to any one of claims 34 to 36.
38. A method for producing nanorods, comprising Cultivating an isolated host cell comprising the NPS according to any one of claims 34 to 36; and The inducer of the inducible promoter is provided to the host cell during the optimal growth phase, thereby expressing the Ff phage replication protein pII, generating a DNA sequence that is excised and replicated from the replication assembly box, which forms a circular single-stranded DNA encapsulated in the nanorod.
39. A method for detecting a target molecule in a sample, comprising c) contacting a sample containing or suspected of containing the target molecule with the nanorod binding agent conjugate, and d) detecting the binding of the nanorod binding agent conjugate to the target molecule, wherein the nanorod binder conjugate comprises at least one modified Ff phage coat protein, wherein the nanorod in the nanorod binder conjugate is produced by the NPS according to any one of claims 1 to 15 or 34 to 36, or is a nanorod according to any one of claims 20 to 25 or 31, or is one of the nanorod populations according to any one of claims 26 to 30, 32 or 33, or is prepared by the method according to any one of claims 17 to 19 or 38.
40. The method of claim 39, wherein: The nanorod binder conjugate comprises at least one detection moiety covalently bound to the at least one modified Ff bacteriophage coat protein.
41. The method according to claim 39 or 40, wherein: The nanorod binder conjugate includes one detection moiety per about 7 copies of the Ff bacteriophage coat protein pVIII or modified pVIII included in the nanorod.
42. The method according to any one of claims 39 to 41, wherein: The nanorod binder conjugate comprises a plurality of detection moieties covalently bound to a plurality of the at least one modified Ff bacteriophage coat proteins.
43. A method according to any one of claims 39 to 42, wherein: The nanorod binder conjugate comprises at least three, four, five, six, seven, eight, nine or more detection moieties.
44. A method according to any one of claims 39 to 42, wherein: The nanorod binder conjugate includes at least two different types of modified Ff bacteriophage coat proteins.
45. A method according to any one of claims 39 to 44, wherein: The nanorod binder conjugate includes at least two different types of detection moieties.
46. A method according to any one of claims 39 to 45, wherein: The nanorod binder conjugate comprises at least three, four, five, six, seven, eight, nine or more different types of detection moieties.
47. A method according to any one of claims 39 to 46, wherein: The nanorod binder conjugate comprises a plurality of each of at least two different detection moieties, each detection moiety being covalently bound to a plurality of at least two different types of modified Ff bacteriophage coat proteins.
48. A method according to any one of claims 39 to 47, wherein: The nanorod binding agent conjugate is included in the population of nanorod binding agent conjugates.
49. The method of claim 48, wherein: At least some of the nanorod binder conjugates in the population comprise different detection moieties.
50. The method according to any one of claims 39 to 49, wherein: The target molecules are immobilized on the solid support by binding to capture molecules attached to the support.
51. The method of claim 50, wherein: The binding agent is selected from the group consisting of small molecules or polypeptides.
52. The method of claim 51, wherein: The polypeptide is selected from the group consisting of antibodies, antibody derived single chain variable domains (scFv), camelid single chain antibody domains VHH and other types of antibodies and analyte binding polypeptides.
53. A method according to any one of claims 39 to 52, wherein: Detection includes detection of chemical, spectroscopic, linear-dichroic, fluorescent, visual, chemiluminescent, paramagnetic, acoustic, electrical, surface plasmon resonance, isotopic, radioactive or other chemical or physical signals.
54. A method according to any one of claims 39 to 53, wherein: Detection includes fluorescent signal detection or visual detection by enzymatic reactions using chromogenic or chemiluminescent substrates.
55. The method according to any one of claims 39 to 54, which is a dot blot assay, a lateral flow assay (LFA) or an enzyme-linked immunosorbent assay (ELISA).