A target antigen artificial lipid droplet surface display system, and a preparation method and application thereof

Abstract

The application provides a target antigen artificial lipid droplet surface display system and a preparation method and application thereof. The first aspect of the application provides a target antigen artificial lipid droplet surface display system, which comprises an artificial lipid droplet and an amphiphilic target antigen molecule; the artificial lipid droplet is assembled into a particle with a hydrophilic surface and a hydrophobic inner core in a hydrophilic medium; the amphiphilic target antigen molecule is a target antigen molecule modified by a hydrophobic group; and the target antigen molecule is displayed on the hydrophilic surface of the artificial lipid droplet. The target antigen artificial lipid droplet surface display system is used to replace streptavidin magnetic beads, which helps to reduce the non-specific binding with the target antigen molecule in the antibody screening process, and improves the specificity and screening efficiency of the target antibody; and the display system provided by the application greatly simulates the spatial structure of a virus, does not contain genetic material, and is safer and more effective.

Description

Technical Field

[0001] This invention relates to a target antigen artificial lipid droplet surface display system, its preparation method and application, and relates to the field of biotechnology. Background Technology

[0002] Antibodies are immunoglobulins produced by B cells and plasma cells in the human and animal body, forming the body's primary immune defense against pathogens and foreign invasions. They are used in drug development, disease diagnosis, and treatment, providing precise and efficient solutions for medical research and clinical applications. With the increasing demand for antibody drugs, antibody screening technologies are developing rapidly. Currently, commonly used technologies include hybridoma technology, antibody library screening, nanobody technology, and transgenic mouse antibody screening. Antibody library screening further includes phage display technology, yeast surface display technology, ribosome display technology, and mRNA display technology.

[0003] Ribosome display technology is a method that displays antibodies on the surface of ribosomes in vitro, and then selects and screens these antibodies to obtain those with high specificity and affinity. Because it does not require transformation, it can be used with libraries containing extremely large amounts (10^6 ribosomes). 14 The antibody gene library is used for screening; the target antigen molecules that can be screened are very broad, including toxic proteins, DNA, RNA, etc.; it has high screening efficiency and accuracy. The process mainly involves incubating the "mRNA-ribosome-antibody" complex obtained by in vitro transcription and translation with streptavidin magnetic beads linked to specific antigens, and then obtaining the target antibody by magnetic separation and washing.

[0004] While ribosome display technology offers numerous advantages, it also has certain limitations. Due to the strong adsorption properties of streptavidin magnetic beads and the immobilized streptavidin on their surface, non-specific binding can occur between the antibody displayed on the ribosome surface and the target antigen bound to the bead surface. This non-specific binding can lead to false positives, severely impacting the accuracy of antibody screening. It causes some antibodies that do not interact with the target protein to be incorrectly selected, thus affecting screening efficiency and antibody specificity. Summary of the Invention

[0005] This invention provides a target antigen artificial lipid droplet surface display system and its preparation method, which is used to improve the specificity of antibody-antigen binding process and improve antibody screening efficiency.

[0006] The present invention also provides the application of the above-described target antigen artificial lipid droplet surface display system in antibody display technology.

[0007] The first aspect of the present invention provides a target antigen artificial lipid droplet surface display system, comprising artificial lipid droplets and amphiphilic target antigen molecules;

[0008] The artificial lipid droplets are assembled into particles with a hydrophilic surface and a hydrophobic core in a hydrophilic medium;

[0009] The amphiphilic target antigen molecule is a target antigen molecule modified with a hydrophobic group;

[0010] The target antigen molecules are displayed on the hydrophilic surface of the artificial lipid droplets.

[0011] In the solution of the present invention, such as Figure 1 As shown, artificial lipid droplets 1 are formed by surfactants in a hydrophilic medium. Since surfactants have hydrophilic and hydrophobic ends, they can form particles with cell membrane-like structures in a hydrophilic medium. The hydrophilic end is located on the particle surface to form a hydrophilic surface, and the hydrophobic end is located inside the particle to form a hydrophobic core. Amphiphilic target antigen molecule 2 is formed by hydrophobically modifying the target antigen molecule. The hydrophilic target antigen molecule 21 is displayed on the hydrophilic surface of artificial lipid droplets 1 to facilitate binding with antibodies during antibody screening. The hydrophobic end is located inside artificial lipid droplets 1, so that the target antigen molecule 21 is tightly displayed on the surface of artificial lipid droplets 1.

[0012] This invention uses an artificial lipid droplet surface display system for target antigens to replace streptavidin magnetic beads. Because artificial lipid droplets possess properties such as high fluidity, a hydrophobic core (making them less prone to adhering to other proteins), and electroneutrality, non-specific binding during the screening process can be reduced, improving the specificity and screening efficiency of the target antibody. Furthermore, the display system provided by this invention largely simulates the spatial structure of viruses but does not contain genetic material, making it safer and more effective. In addition, the antigen molecules displayed by the system provided by this invention can be recovered, making it more economical and environmentally friendly. Moreover, it can display different types of antigens, enabling high-throughput screening to obtain high-affinity antibodies. Furthermore, those skilled in the art can precisely control the structural and functional parameters of the artificial lipid droplets by controlling the size, quantity, and type of antigens displayed on the surface, thus possessing high controllability.

[0013] In the present invention, the target antigen molecule can be a polypeptide, protein, polysaccharide, lipid, nucleic acid, or other substance. Further, the target antigen molecule is a polypeptide or protein.

[0014] In the present invention, the hydrophilic medium can be pure water.

[0015] In one specific embodiment, an amphiphilic target antigen molecule is obtained by modifying the target antigen molecule with poly(N-isopropylacrylamide) activated with mercaptothiazoline or N-hydroxysuccinimide (NHS). Specifically, poly(N-isopropylacrylamide) is activated with mercaptothiazoline or N-hydroxysuccinimide, and the activated poly(N-isopropylacrylamide) is mixed with the target antigen molecule and reacted to obtain the target antigen molecule.

[0016] Furthermore, the molar ratio of activated poly(N-isopropylacrylamide) to target antigen molecules was 2:1, the pH of the reaction system was controlled at 8.0, and the reaction was carried out at 4°C for 12 hours before lysine was added to terminate the reaction.

[0017] Furthermore, the number average molecular weight of poly(N-isopropylacrylamide) is 8000–15000 g / mol. By controlling the molecular weight of the polymer, it is helpful to balance the hydrophilicity and hydrophobicity of the antigen-polymer, so that the antigen molecules can be better displayed on the surface of the artificial lipid droplets.

[0018] In another specific embodiment, the target antigen molecule is modified by isopentenylation to obtain an amphiphilic target antigen molecule; specifically, the target antigen molecule can be modified using farnesyltransferase (FTase) or geraniyltransferase (GGTase) to form a hydrophobic group at the C-terminus of the target antigen molecule.

[0019] In one specific embodiment, the artificial lipid droplets are assembled from glyceryl tributyrate in a hydrophilic medium; the density of glyceryl tributyrate is greater than that of water, which facilitates the settling of the assembled artificial lipid droplets to the bottom of the aqueous medium, helps to enrich the artificial lipid droplets, and prevents the artificial lipid droplets from suspending at the air-water interface, thus inactivating the antigen molecules.

[0020] In one specific embodiment, in order to facilitate the recycling of the magnetic enrichment and display system, the artificial lipid droplets are assembled in a hydrophilic medium and have magnetic nanoparticles 3 inside. The magnetic nanoparticles 3 are highly hydrophobic and can be dispersed inside the hydrophobic interior of the artificial lipid droplets formed by glyceryl tributyrate.

[0021] Furthermore, the magnetic nanoparticles can be Fe3O4 nanoparticles. The mass of the magnetic nanoparticles is 1%-2% of the mass of glycerol tributyrate.

[0022] A second aspect of the present invention provides a method for preparing the artificial lipid droplet surface display system for the target antigen as described above, comprising the following steps:

[0023] Hydrophobic groups were modified on the target antigen molecules to obtain amphiphilic target antigen molecules;

[0024] A surfactant is mixed with an amphiphilic target antigen molecule and dispersed in a hydrophilic medium to form the target antigen artificial lipid droplet surface display system.

[0025] In one specific embodiment, the target antigen molecule is modified with hydrophobic groups to obtain an amphiphilic target antigen molecule, specifically including the following steps:

[0026] Poly(N-isopropylacrylamide) was activated using mercaptothiazoline or N-hydroxysuccinimide. The activated poly(N-isopropylacrylamide) was then mixed with the target antigen molecule and reacted. After the reaction was completed, lysine was added to terminate the reaction, resulting in an amphiphilic target antigen molecule.

[0027] Furthermore, the molar ratio of activated poly(N-isopropylacrylamide) to target antigen molecules was 2:1, the pH of the reaction system was controlled at 8.0, and the reaction was carried out at 4°C for 12 hours before lysine was added to terminate the reaction.

[0028] Next, glyceryl tributyrate and amphiphilic target antigen molecules are dispersed in a hydrophilic medium to form the target antigen artificial lipid droplet surface display system.

[0029] In addition, magnetic nanoparticles can be added.

[0030] A third aspect of the present invention provides the application of the above-described target antigen artificial lipid droplet surface display system or the target antigen artificial lipid droplet surface display system prepared by any of the above-described preparation methods in antibody screening technology.

[0031] A fourth aspect of the present invention provides a method for screening target antibodies using antibody display technology, comprising the following steps:

[0032] An antibody library was constructed, and a complex for displaying the antibody was prepared.

[0033] The complex is brought into contact with any of the target antigen artificial lipid droplet surface display systems described above or the target antigen artificial lipid droplet surface display systems prepared by any of the preparation methods described above, and target complexes that bind to the target antigen molecules are screened out.

[0034] The genetic information of the target antibody is obtained based on the target complex.

[0035] In the solution provided by this invention, the target antibody can be an ScFv antibody.

[0036] In one specific implementation, taking ribosome display technology as an example, an antibody gene library is first constructed. Based on the antibody formation mechanism in vivo, mutations are introduced into highly mutated regions of the antibody gene library. Further, the highly mutated regions include CDR3.

[0037] In the scheme provided by the present invention, the antibody gene may further include a TonB group located at the 3' end of the antibody gene to improve the binding stability of the complex formed during mRNA translation.

[0038] In the solution provided by this invention, stem-loop structures are also connected to the 5' and 3' ends of the antibody gene, and a promoter and ribosome binding site RBS are also connected to the 5' end.

[0039] The prepared antibody gene library was transcribed into mRNA in vitro, and then translated into an in vitro expression system rich in ribosomal cell extract to form an "mRNA-ribosome-antibody" complex. The complex was incubated with the target antigen artificial lipid droplet surface display system provided in the first aspect of this invention, allowing the target antigen displayed on the artificial lipid droplet surface to bind to the polypeptide in the complex. After the non-specific antibody binding was removed, the mRNA in the complex was released. The antibody gene was then amplified and restored using RT-PCR and Taq polymerase.

[0040] The binding specificity and affinity of the selected antibody genes to the target antigen can then be detected using ELISA or RBD assays.

[0041] It is understood that the target antigen artificial lipid droplet surface display system provided by the present invention can also be used for mRNA display or DNA display.

[0042] The antibody screening method provided by this invention helps reduce non-specific binding; the screening efficiency is faster and more efficient; reusable artificial cells are more economical and environmentally friendly; the artificial cell surface display system can display different types of antigens; and high-throughput screening yields high-affinity antibodies. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the target antigen artificial lipid droplet surface display system provided in Embodiment 1 of the present invention;

[0044] Figure 2 A bright-field microscope image of the artificial lipid droplet surface display system for target antigens provided in Embodiment 1 of the present invention;

[0045] Figure 3 This is a fluorescence microscope image of the artificial lipid droplet surface display system for target antigens provided in Embodiment 1 of the present invention, with a scale bar of 50 micrometers;

[0046] Figure 4 This is a schematic diagram of the construction method of the ScFv antibody gene library. In the diagram, a represents the amplification of the ScFv gene, b represents the amplification of the TonB spacer sequence and the addition of the ribosome binding site RBS before the ScFv gene, and c represents the addition of the T7 promoter before the ribosome binding site RBS and the introduction of mutations in the two CDR3 regions of the ScFv gene.

[0047] Figure 5 Agarose gel electrophoresis image of mRNA generated from the ScFv antibody gene library through in vitro transcription, M is the RNA molecular weight marker;

[0048] Figure 6 The image shows an agarose gel electrophoresis image of DNA from the restored ScFv antibody gene library after screening, where M is the DNA molecular weight marker.

[0049] Figure 7 To screen for antibodies that can bind to RBD for ELISA;

[0050] Figure 8 To determine antibody affinity using SPR.

[0051] Explanation of reference numerals in the attached figures:

[0052] 1-Artificial lipid droplets;

[0053] 2-Amphiphilic target antigen molecules;

[0054] 21-Target antigen molecule;

[0055] 3-Magnetic nanoparticles. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0057] The primers used in the embodiments of this invention are shown in Table 1:

[0058] Table 1 Nucleic acid sequences of primers used in this invention

[0059]

[0060] Example 1: Screening of target antibodies using ribosome display technology

[0061] 1. Rational design and construction of antibody gene libraries for screening

[0062] like Figure 4 As shown, in step a), the heavy chain variable region VH and light chain variable region VL of the ScFv antibody are amplified using primers ScFv-F and ScFv-R. The heavy chain variable region VH and light chain variable region VL each include the highly mutated region CDR3, and are linked by a linker to remove the stop codon located at the 3' end.

[0063] Step b) PCR was used to add a ribosome binding site RBS to the 5' end of the ScFv antibody, and a TonB group and stem-loop structure to the 3' end. The complete sequence was then amplified using SDA-ScFv-F and tonB-ScFv-R.

[0064] In step c), building upon step b), a T7 promoter and stem-loop structure were added to the 5' end, and the complete sequence was amplified using T7B-F and tonB-ScFv-R. Then, by mimicking the amino acid distribution and length of the highly mutated region CDR3 of naturally occurring antibodies, mutations were introduced into the highly mutated region (CDR3) of ScFv, with amino acid lengths of 9, 12, 15, and 18 amino acids. The types and ratios of amino acids in the highly mutated region CDR3 are shown in Table 2, and an antibody gene library was constructed.

[0065] The constructed antibody gene is approximately 1617-1653 bp in length. The PCR reaction components used are shown in Table 3.

[0066] Table 2. Amino acid types and proportions in the highly mutant region CDR3.

[0067]

[0068] Table 3 PCR components

[0069]

[0070] Reaction conditions: 98℃ pre-denaturation for 30 seconds, 30 cycles: 98℃ denaturation for 10 seconds, 72℃ annealing for 20 seconds, 72℃ extension for 30 seconds, and final extension at 72℃ for 2 minutes. The amplified products were identified by 1% agarose gel electrophoresis, and the target fragment was recovered from the gel.

[0071] 2. Preparation of the target antigen display system

[0072] Taking the SARS-CoV-2 RBD protein as an example, the SARS-CoV-2 RBD protein was reacted with thiol-thiazoline-activated poly(N-isopropylacrylamide). Reaction conditions: protein concentration 2 mg / mL; polymer concentration 1 mg / mL; reaction pH = 8.0; reaction temperature 4°C; reaction time 12 h. The reaction was terminated by adding a 60 mM lysine solution. A two-phase solution was formed by mixing 6 μL of glycerol tributyrate (1% w / w Fe3O4 nanoparticles) and 200 μL of polymer-modified antigen solution. The mixture was manually shaken for 30 seconds to obtain an antigen-polymer-stable artificial lipid droplet emulsion. Then, 1 mL of buffer was added for later use. Bright-field micrographs and fluorescence images of the artificial lipid droplets are shown below. Figure 2-3 As shown, according to Figure 3 As shown by fluorescence, the surface of the artificial lipid droplets exhibits the SARS-CoV-2 RBD protein.

[0073] 3. Ultra-efficient screening of specific antibodies

[0074] The prepared antibody gene library (1 μg) was transcribed into mRNA in vitro. The agarose gel electrophoresis image of the transcribed mRNA is shown below. Figure 5As shown. In a ribosome-rich cell-derived in vitro expression system, 10 μg of mRNA was translated to form an "mRNA-ribosome-antibody" complex, which was then incubated with the antigen display system constructed in step 2 at 4°C for 1 h. The complex was washed six times with WBT solution (50 mM HEPES-KOH, 150 mM NaCl, 50 mM MgOAc, 0.2% BSA, pH 7.4) to remove non-specific antibody binding. The mRNA was released using EDTA solution (50 mM HEPES-KOH, 150 mM NaCl, 20 mM EDTA, 50 μg / mL yeast RNA, 0.1% BSA, pH 7.4). The antibody gene was reconstructed by RT-PCR and Taq polymerase amplification (primers: SDA-ScFv-F and tonB-R). The agarose gel electrophoresis image of the reconstructed antibody gene is shown below. Figure 6 As shown in Table 4.

[0075] Table 4 PCR components

[0076]

[0077] PCR reaction conditions: 94℃ pre-denaturation for 5 minutes, 30 cycles: 94℃ denaturation for 30 seconds, 66℃ annealing for 30 seconds, 72℃ extension for 1 minute, and final extension at 72℃ for 10 minutes. The amplified products were identified by 1% agarose gel electrophoresis, and the target fragment was recovered from the gel.

[0078] 4. Identification of specific antibodies

[0079] Antibodies specifically binding to the SARS-CoV-2 RBD protein, selected using primers (ScFv-transfer-F and ScFv-transfer-R), were amplified using high-fidelity Q5 DNA polymerase. The polymerase was then transferred to an expression vector via Gibson DNA assembly. The antibody gene linked to the vector was transformed into *E. coli* TG1 cells by electroporation. Single clones were selected and cultured overnight at 37°C in DYT liquid medium containing ampicillin (Amp). The bacterial culture was then transferred to fresh medium and cultured at 37°C for 2.5 h. A final concentration of 1 mM IPTG was added, and expression was induced overnight at 26°C. The bacterial lysate was screened by ELISA to obtain antibodies specifically binding to RBD. Figure 7 The antibody's genetic information was obtained through sequencing. The PCR components and procedures used were the same as in step 1.

[0080] 5. Purification and Binding Affinity Analysis of RBD Antibodies

[0081] The positive clone C7 identified in step 4 was inoculated into 50 mL of DYT liquid medium containing Amp and cultured overnight at 37°C. The next day, the culture was transferred back to 1 L of DYT liquid medium containing Amp and cultured at 37°C until OD. 600 Approximately 0.6-0.8, add a final concentration of 1 mM IPTG, and induce expression overnight at 18°C. Collect bacterial cells by centrifugation, resuspend the cells in PBS, disrupt the bacterial culture using an ultrasonic homogenizer, and centrifuge at low temperature. The supernatant is then purified by Nitrogen chromatography. 2+ Protein separation and purification were performed using a column followed by linear gradient elution. Elution peaks were collected and analyzed by SDS-PAGE electrophoresis, and elution peaks showing a single band for the target protein were combined. Based on the purity of the purified protein, further purification can be achieved through ion-exchange chromatography and gel filtration to improve the purity of the target antibody.

[0082] Biotinylated RBD protein was coated onto a streptavidin (SA) sensor chip. The antibody was serially diluted five times (80 nM, 40 nM, 20 nM, 10 nM, 5 nM) and flowed through the chip at a flow rate of 30 μl / min. The binding kinetic constant was determined by surface plasmon resonance (SPR) spectroscopy, and K was calculated based on the five curves using Biacore analysis software. D It is 2.61 ± 0.46 × 10 -9 M( Figure 8 ).

[0083] 6. Comparison of artificial lipid droplets and streptomycin magnetic beads in display

[0084] The maximum library capacity and single-round screening efficiency of artificial lipid droplet and streptomycin magnetic bead display technologies were compared. The comparison results are shown in Table 5. It can be seen that, compared with streptomycin magnetic bead display technology, the artificial lipid droplet display provided by this invention helps to improve the specificity of antigen-antibody binding, thereby improving antibody screening efficiency.

[0085] Table 5 Comparison of artificial lipid droplets and streptomycin magnetic beads

[0086] Streptomycin magnetic beads <![CDATA[10 14 ]]> <![CDATA[10 4 ]]> Artificial lipid droplets <![CDATA[10 14 ]]> <![CDATA[10 11 ]]>

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A system for displaying target antigens on the surface of artificial lipid droplets, characterized in that, Including artificial lipid droplets and amphiphilic target antigen molecules; The artificial lipid droplets are assembled in a hydrophilic medium into particles with a hydrophilic surface and a hydrophobic core; the artificial lipid droplets are obtained by assembling glyceryl tributate in a hydrophilic medium, and the particles are encapsulated with magnetic nanoparticles. The amphiphilic target antigen molecule is a target antigen molecule modified with a hydrophobic group; the amphiphilic target antigen molecule is obtained by modifying the target antigen molecule with poly(N-isopropylacrylamide) activated with mercaptothiazoline or N-hydroxysuccinimide. The target antigen molecules are displayed on the hydrophilic surface of the artificial lipid droplets.

2. The method for preparing the target antigen artificial lipid droplet surface display system according to claim 1, characterized in that, Includes the following steps: Hydrophobic groups were modified on the target antigen molecules to obtain amphiphilic target antigen molecules; A surfactant is mixed with an amphiphilic target antigen molecule and dispersed in a hydrophilic medium to form the target antigen artificial lipid droplet surface display system.

3. The preparation method according to claim 2, characterized in that, Modifying the target antigen molecule with hydrophobic groups to obtain an amphiphilic target antigen molecule involves the following steps: Poly(N-isopropylacrylamide) was activated using mercaptothiazoline or N-hydroxysuccinimide. The activated poly(N-isopropylacrylamide) was then mixed with the target antigen molecule and reacted. After the reaction was completed, lysine was added to terminate the reaction, resulting in an amphiphilic target antigen molecule.

4. The preparation method according to claim 2, characterized in that, The surfactant is mixed with amphiphilic target antigen molecules and dispersed in a hydrophilic medium to form the target antigen artificial lipid droplet surface display system, specifically including the following steps: Glyceryl tributyrate and amphiphilic target antigen molecules are dispersed in a hydrophilic medium to form the target antigen artificial lipid droplet surface display system.

5. The application of the target antigen artificial lipid droplet surface display system according to claim 1 or the target antigen artificial lipid droplet surface display system prepared by the preparation method according to any one of claims 2-4 in antibody screening technology.

6. A method for screening target antibodies using antibody display technology, characterized in that, Includes the following steps: An antibody gene library was constructed, and a complex for displaying the antibody was prepared. The complex is brought into contact with the target antigen artificial lipid droplet surface display system according to claim 1 or the target antigen artificial lipid droplet surface display system prepared by the preparation method according to any one of claims 2-4, and the target complex that binds to the target antigen molecule is screened out. The genetic information of the target antibody is obtained based on the target complex.

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