Patent Application
20220098236
The present invention generally relates to a fusion peptide and, more particularly, to a fusion peptide for forming a virus-like particle.
Virus-like particle (VLP) with great biocompatibility and biosafety is considered as a potential delivery vehicle or a potential therapeutic vehicle. As an example, the therapeutic agent (such as chemotherapy drugs) can be enveloped in the virus-like particle (VLP) and then delivered to its target site.
In order to target the specific target site, it is known to chemical conjugated a biomolecule (such as an antibody) to the virus-like particle (VLP). The virus-like particle (VLP) carrying the antibody can thus target the specific target site (such as the target antigen that can specifically bind to the antibody or a cell includes the target antigen) via the interaction between the antibody and the target antigen. However, a crosslinker is needed for performing the chemical conjugation reaction, and the chemical conjugation reaction needs a longer time period, affecting the stability of the biomolecule (such as the antibody).
In light of this, it is necessary to provide a fusion peptide for forming the virus-like particle.
It is therefore an objective of the present invention to provide a fusion peptide for forming a virus-like particle, which is able to bind to a target biomolecule.
It is another objective of the present invention to provide a fusion peptide for forming a virus-like particle, which is able to bind to the target biomolecule without any crosslinker.
One embodiment of the present invention discloses a fusion peptide for forming virus-like particle. The fusion peptide can include a vehicle fragment and a capture fragment connected to the vehicle fragment. The vehicle fragment is used to form a virus-like particle. The capture fragment is used to form a capture peptide that is not enveloped by the virus-like particle and is able to specifically bind to a target biomolecule.
Accordingly, instead of enveloping in the virus-like particle formed by the vehicle fragment, the fusion peptide for forming virus-like particle according to the present invention has the capture peptide formed by the capture fragment that is exposed outside the virus-like particle. Therefore, the capture peptide can specifically bind to a target biomolecule to form the virus-like particle with the target biomolecule, and the target biomolecule can be delivered to a predetermined location by the virus-like particle. Alternatively, by the specific binding between the capture peptide and the target biomolecule, the targeting of the virus-like particle can also be improved.
Moreover, the virus-like particle carrying the target biomolecule is formed by a reversible specific binding (such as the interaction of hydrogen bond, van der Waals force, electrostatic force, hydrophobic interaction, etc.) between the capture peptide and the target biomolecule. Therefore, the process for conjugating the target biomolecule to the virus-like particle (VLP) using the crosslinker can be omitted. As such, the virus-like particle (VLP) carrying the target biomolecule can be manufactured at a decreased cost, and the virus-like particle (VLP) carrying the target biomolecule can be manufactured with a better yield.
In a preferred form shown, the fusion peptide can further include a linker fragment connected between the vehicle fragment and the capture fragment. As such, the folding of the vehicle fragment and the folding of the capture fragment will not affect each other; and thus, the biological activity of the capture peptide formed by the capture fragment can be remained.
In a preferred form shown, the vehicle fragment can has an amino acid sequence set forth as SEQ ID NO: 3, and the capture fragment can has an amino acid sequence set forth as SEQ ID NO: 4. Preferably, the fusion peptide can consist of an amino acid sequence set forth as SEQ ID NO: 6. As such, the capture peptide with an IgG Fc-binding domain that is able to bind to a Fc domain of an antibody (such as the Fc domain of an anti-RBD antibody); and thus, the fusion peptide can form the virus-like particle (VLP) carrying the antibody by the specific binding between the IgG Fc-binding domain and the Fc domain. The antibody carried by the virus-like particle (VLP) can specifically bind to a virus particle (such as such as specifically bind to the virus particle of SARS-CoV-2 via the receptor-binding domain of SARS-CoV-2). As a result, the specific binding between the virus particle and the receptor of the target cell (such as angiotensin-converting enzyme 2 (ACE2)) can be blocked, and the infection of the target cell by the virus particle can be prevented.
In a preferred form shown, the vehicle fragment can has an amino acid sequence set forth as SEQ ID NO: 3, and the capture fragment can has an amino acid sequence set forth as SEQ ID NO: 8. Preferably, the fusion peptide can consist of an amino acid sequence set forth as SEQ ID NO: 10. As such, the capture peptide with a z domain that is able to bind to a Fc domain of an antibody (such as the Fc domain of an anti-RBD antibody); and thus, the fusion peptide can form the virus-like particle (VLP) carrying the antibody by the specific binding between the z domain and the Fc domain. The antibody carried by the virus-like particle (VLP) can specifically bind to a virus particle (such as such as specifically bind to the virus particle of SARS-CoV-2 via the receptor-binding domain of SARS-CoV-2). As a result, the specific binding between the virus particle and the receptor of the target cell (such as angiotensin-converting enzyme 2 (ACE2)) can be blocked, and the infection of the target cell by the virus particle can be prevented.
The present invention will become more fully understood from the detailed description given hereinafter and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:
Herein, the term “fusion peptide” indicates a peptide manufactured by recombinant DNA technology. The peptide includes at least two amino acid fragments respectively encoded by corresponding genes that originally coded for separate portions, which can be appreciated by a person having ordinary skill in the art; and therefore, detail description is not given to avoid redundancy.
Specifically, the fusion peptide for forming a virus-like particle (VLP) according to the present invention can include a vehicle fragment and a capture fragment connected to the vehicle fragment. The vehicle fragment is used for forming the virus-like particle (VLP), while the capture fragment is used for forming a capture peptide that is not enveloped by and is exposed outside the virus-like particle (VLP). The capture peptide can specifically bind to a target biomolecule. As an example, the target biomolecule can be an antibody, an antigen, an enzyme, a substrate, an aptamer or a ligand.
The fusion peptide can be expressed by E. coli cells. As an example, an expression plasmid for expressing the fusion peptide can be constructed and transformed into the E. coli cells. The fusion peptide expressed by the E. coli cells can be purified, and the purified fusion peptide can therefore be obtained.
Specifically, the expression plasmid includes a first DNA fragment corresponding to the vehicle fragment and a second DNA fragment corresponding to the capture fragment. Moreover, the first and second DNA fragments preferably have the codon usage of E. coli, thus the E. coli cells can show preferable expression efficiency. In the first embodiment of the present invention, the first and second DNA fragments have nucleic acid sequences set forth as SEQ ID NOS: 1 and 2, respectively. In addition, the vehicle fragment and the capture fragment expressed by the E. coli cells have the amino acid sequences set forth as SEQ ID NOS: 3 and 4, respectively.
Furthermore, a linker fragment can be provided to connect between the vehicle fragment and the capture fragment. The sequence of the linker fragment can be appreciated by a person having ordinary skill in the art; and therefore, detail description is not given to avoid redundancy. In the first embodiment of the present invention, the expression plasmid comprises a nucleic acid sequence set forth as SEQ ID NO: 5, while the fusion peptide expressed by the E. coli cells consists of an amino acid sequence set forth as SEQ ID NO: 6.
The construction of the expression plasmid is the ordinary skill in the art, and therefore is not limited to the following statement. In the first embodiment of the present invention, the DNA fragment with the nucleic acid sequence set forth as SEQ ID NO: 5 is synthesized and digested by the restriction enzyme. The digested DNA fragment is then ligated to a pCDFDuet-1 vector by a ligase, and the expression plasmid for expressing the fusion peptide according to the first embodiment is obtained.
Subsequently, after the expression plasmid is transformed into the E. coli BL21(DE) cells, the E. coli BL21(DE) cells can express the fusion peptide by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The fusion peptide expressed by the E. coli BL21(DE) cells is obtained and is precipitated by ammonium sulfate ((NH4)2SO4) to obtain a crude sample. The crude sample is then resuspended in phosphate buffered saline (PBS) and is extracted by a mixture including n-butanol and chloroform in a volume ratio of 1:1. A upper aqueous layer solution is collected and is purified by sucrose gradient ultracentrifugation. After precipitating by a salt solution including polyethylene glycol 8000 (20% w:v PEG 8000-NaCl solution), the obtained precipitate is resuspended in phosphate buffered saline (PBS). The purified fusion peptide can be obtained after dialysis with phosphate buffered saline (PBS).
The purified fusion peptide has the capture peptide with an immunoglobulin G (IgG) fragment-crystllizable (Fc)-binding domain that is able to bind to a fragment-crystllizable (Fc) domain of an antibody; and thus, the fusion peptide can be used as a vehicle of the antibody for specific binding to an antigen of a virus particle. As a result, the specific binding between the virus particle and a receptor of a target cell can be blocked, and the infection of the target cell by the virus particle can be prevented. As an example, in order to prevent the target cell from infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the antibody can be an anti-RBD antibody that is able to specific bind to the receptor-binding domain (RBD domain) of SARS-CoV-2. By the specific binding between the anti-RBD antibody and the RBD domain of SARS-CoV-2, the specific binding between the RBD domain of SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2) of the target cell can be blocked.
Based on the same technical concept, in the second embodiment of the present invention, the first and second DNA fragments have nucleic acid sequences set forth as SEQ ID NOS: 1 and 7, respectively. In addition, the vehicle fragment and the capture fragment expressed by the E. coli cells have the amino acid sequences set forth as SEQ ID NOS: 3 and 8, respectively. The expression plasmid comprises a nucleic acid sequence set forth as SEQ ID NO: 9, while the fusion peptide expressed by the E. coli cells consists of an amino acid sequence set forth as SEQ ID NO: 10.
Moreover, after the construction of the expression plasmid for expressing the fusion peptide according to the second embodiment (the vector that used for the expression plasmid is pCDFDuet-1 vector), the purified fusion peptide can be obtained according to the same procedure. The purified fusion peptide has the capture peptide with a z domain that is able to bind to the Fc domain of the antibody; and thus, the fusion peptide can be used as the vehicle of the antibody for specific binding to the antigen of the virus particle. As a result, the specific binding between the virus particle and the receptor of the target cell can be blocked, and the infection of the target cell by the virus particle can be prevented.
To evaluate both the fusion peptides according to the first and second embodiments of the present invention form the virus-like particles (VLPs), and both the capture fragments formed by the capture peptides are not enveloped in the virus-like particles (VLPs) and are able to specifically bind to the target biomolecule, the following trials are carried out.
Trial (A).
In trial (A), the fusion peptide according to the first embodiment of the present invention (5 μL) is pipetted onto the Formvar-coated copper mesh grids for 5 minutes, followed by exposure to the aqueous uranyl acetate solution (8 μL) for 2 minutes as a negative stain. Excess stain is then removed, and the Formvar-coated copper mesh grids are left to dry in air overnight. The obtained sample is analyzed by transmission electron microscopy (TEM) (75 keV accelerating voltage). Moreover, the fusion peptide according to the second embodiment of the present invention is analyzed according to the same procedure.
Referring to
Trial (B).
In trial (B), the virus-like particle (VLP) formed by the fusion peptide according to the first embodiment of the present invention (group B1) and the virus-like particle (VLP) formed by the vehicle fragment (group B2) are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 15%). The proteins on PAGE are transferred to a poly(vinylidene) fluoride (PVDF) membrane. The poly(vinylidene) fluoride (PVDF) membrane is then blocked using a milk solution (5%). The poly(vinylidene) fluoride (PVDF) membrane is then performed antibody binding using a rabbit anti-mouse IgG primary antibody. After tris(hydroxymethyl)aminomethane (Tris) buffer (TBST) with polysorbate 20 (Tween 20) washing for three times, the poly(vinylidene) fluoride (PVDF) membrane is stained using a horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibody. Finally, after washing for three times, the poly(vinylidene) fluoride (PVDF) membrane is photographed after soaking in a Western luminal substrate.
Referring to
Trial (C).
In trial (C), the virus-like particle (VLP) formed by the vehicle fragment (group C1) and the virus-like particle (VLP) formed by the fusion peptide according to the second embodiment of the present invention (group C2) are analyzed according to the same procedure in trial (B).
Referring to
Trial (D).
In trial (D), the virus-like particle (VLP) formed by the fusion peptide according to the first embodiment of the present invention is immobilized on the wells of a 96-well plate. The primary antibody and/or the second antibody is added into the 96-well plate according to TABLE 1. After the color reaction, the absorbance at 450 nm is measured by a spectrometer.
Referring to
Trial (E).
In trial (E), the mixture including the virus-like particle (VLP) and the primary antibody as shown in TABLE 2 is analyzed by agarose gel electrophoresis.
Referring to
To evaluate both the virus-like particles (VLPs) formed by the fusion peptide according to the first and second embodiments of the present invention are able to carry the anti-RBD antibody, and to evaluate both the virus-like particles (VLPs) formed by the fusion peptide according to the first and second embodiments of the present invention that carries the anti-RBD antibody are able to specifically bind to SARS-CoV-2, blocking the specific binding between SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2), the following trials are carried out.
Trial (F).
In trial (F), the platinum nanoparticle (PtNP, 70 nm, purchased from Sigma) is co-incubated with the thiolated SARS-CoV-2 spike recombinant protein (LEADGENE® SARS-CoV-2 trimeric spike protein, His tag, HEK293, CAT: 63233) at 4° C. for 12 hours to obtain the SARS-CoV-2 mimicking signal source. The SARS-CoV-2 mimicking signal source includes receptor-binding domain of SARS-CoV-2, and thus is able to specifically bind to angiotensin-converting enzyme 2 (ACE2).
Trial (G).
In trial (G), the virus-like particle (VLP) formed by the fusion peptide according to the first embodiment of the present invention is co-incubated with the anti-RBD antibody (LEADGENE® Human Anti-SARS-CoV & CoV-2 Spike Antibody (IgG)) at 37° C. for 30 minutes. The capture peptide which is formed by the capture fragment of the fusion peptide and is not enveloped by the virus-like particle (VLP) can bind to the Fc domain of the anti-RBD antibody to form the virus-like particle (VLP) formed by the fusion peptide according to the first embodiment of the present invention carrying the anti-RBD antibody.
Moreover, the virus-like particle (VLP) formed by the fusion peptide according to the second embodiment of the present invention is co-incubated with the anti-RBD antibody (LEADGENE® Human Anti-SARS-CoV & CoV-2 Spike Antibody (IgG)) at 37° C. for 30 minutes. The capture peptide which is formed by the capture fragment of the fusion peptide and is not enveloped by the virus-like particle (VLP) can bind to the Fc domain of the anti-RBD antibody to form the virus-like particle (VLP) formed by the fusion peptide according to the second embodiment of the present invention carrying the anti-RBD antibody.
Trial (H).
In trial (H), angiotensin-converting enzyme 2 (ACE2) is diluted to a solution of 200 ng/mL in a carbonate-bicarbonate buffer (CBC buffer, 0.05 M Na2CO3 and 0.05 M NaHCO3, pH 9.6). The diluted angiotensin-converting enzyme 2 (ACE2) solution (100 μL) is added into each well of a 96-well plate. After incubation overnight at 4° C., the wells are washed with the tris(hydroxymethyl)aminomethane (Tris) buffer (TBST) with polysorbate 20 (Tween 20) for three times, then blocked with the blocking buffer (5% bovine serum albumin (BSA), dissolved in TBST) at room temperature for 1 hour. Each well is then washed with the tris(hydroxymethyl)aminomethane (Tris) buffer (TBST) with polysorbate 20 (Tween 20) for three time to obtain the well immobilized with angiotensin-converting enzyme 2 (ACE2).
The mixture (100 μL) as shown in TABLE 3 is added to each well, incubating at room temperature for 1 hour. Each well is washed with the tris(hydroxymethyl)aminomethane (Tris) buffer (TBST) with polysorbate 20 (Tween 20) for three time. Next, horseradish peroxidase (HRP)-labelled angiotensin-converting enzyme 2 (ACE2) antibody is used to stain each well. Finally, the commercial chromogenic solution (with 3,3′,5,′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), 100 μL) is added to each well, and the reaction is terminated by adding the aqueous hydrochloric acid (HCl) solution (1 M). The absorbance of each well at 450 nm is measured by the spectrometer (SpectraMax M2).
1with 2.5 ng/mL of receptor-binding domain of SARS-CoV-2, dissolved in phosphate buffeted saline (PBS);
2with 10 ng/mL of anti-RBD antibody, dissolved in phosphate buffeted saline (PBS);
3with 10 ng/mL of anti-RBD antibody, dissolved in phosphate buffeted saline (PBS).
Referring to
Trial (I).
Referring to
The first sprayer S1 and the second sprayer S2 are filled in the solutions shown in TABLE 4 and are turned on to form ultrasonic spray of respective solutions. After spraying for 60 minutes, the first sprayer S1 and the second sprayer S2 are turned off, and the ultrasonic spray of respective solutions is settled for 60 minutes. After washing with the tris(hydroxymethyl)aminomethane (Tris) buffer (TBST) with polysorbate 20 (Tween 20) for three time, the upper chip C1 and the lower chip C2 are stained by the horseradish peroxidase (HRP)-labelled angiotensin-converting enzyme 2 (ACE2) antibody. Finally, the commercial chromogenic solution (with 3,3′,5,′-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), 100 μL) is added to the upper chip C1 and the lower chip C2, and the reaction is terminated by adding the aqueous hydrochloric acid (HCl) solution (1 M). The absorbance of the upper chip C1 and the lower chip C2 at 450 nm is measured by the spectrometer (SpectraMax M2).
1with 10 ng/mL of anti-RBD antibody, dissolved in phosphate buffeted saline (PBS);
2with 2.5 ng/mL of SARS-CoV-2 receptor-binding domain, dissolved in phosphate buffeted saline (PBS).
Referring to
Trial (J).
Referring to
1with 10 ng/mL of anti-RBD antibody, dissolved in phosphate buffeted saline (PBS);
2with 2.5 ng/mL of receptor-binding domain of SARS-CoV-2, dissolved in phosphate buffeted saline (PBS).
Referring to
Accordingly, instead of enveloping in the virus-like particle formed by the vehicle fragment, the fusion peptide for forming virus-like particle according to the present invention has the capture peptide formed by the capture fragment that is exposed outside the virus-like particle. Therefore, the capture peptide can specifically bind to a target biomolecule to form the virus-like particle with the target biomolecule, and the target biomolecule can be delivered to a predetermined location by the virus-like particle. Alternatively, by the specific binding between the capture peptide and the target biomolecule, the targeting of the virus-like particle can also be improved.
Moreover, the virus-like particle carrying the target biomolecule is formed by a reversible specific binding (such as the interaction of hydrogen bond, van der Waals force, electrostatic force, hydrophobic interaction, etc.) between the capture peptide and the target biomolecule. Therefore, the process for conjugating the target biomolecule to the virus-like particle (VLP) using the crosslinker can be omitted. As such, the virus-like particle (VLP) carrying the target biomolecule can be manufactured at a decreased cost, and the virus-like particle (VLP) carrying the target biomolecule can be manufactured with a better yield.
Also, the capture peptide with an IgG Fc-binding domain that is able to bind to a Fc domain of an antibody (such as the Fc domain of an anti-RBD antibody); and thus, the fusion peptide can form the virus-like particle (VLP) carrying the antibody by the specific binding between the IgG Fc-binding domain and the Fc domain. The antibody carried by the virus-like particle (VLP) can specifically bind to a virus particle (such as such as specifically bind to the virus particle of SARS-CoV-2 via the receptor-binding domain of SARS-CoV-2). As a result, the specific binding between the virus particle and the receptor of the target cell (such as angiotensin-converting enzyme 2 (ACE2)) can be blocked, and the infection of the target cell by the virus particle can be prevented.
In addition, the capture peptide with a z domain that is able to bind to a Fc domain of an antibody (such as the Fc domain of an anti-RBD antibody); and thus, the fusion peptide can form the virus-like particle (VLP) carrying the antibody by the specific binding between the z domain and the Fc domain. The antibody carried by the virus-like particle (VLP) can specifically bind to a virus particle (such as such as specifically bind to the virus particle of SARS-CoV-2 via the receptor-binding domain of SARS-CoV-2). As a result, the specific binding between the virus particle and the receptor of the target cell (such as angiotensin-converting enzyme 2 (ACE2)) can be blocked, and the infection of the target cell by the virus particle can be prevented.
Although the invention has been described in detail with reference to its presently preferable embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.
The application claims the benefit of U.S. provisional application No. 63/083,872, filed on Sep. 26, 2020, and the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63083872 | Sep 2020 | US |
