Synthetic nano library for displaying and screening ribosome as well as construction and application of synthetic nano library

Abstract

The invention belongs to the technical field of antibody library construction, and particularly relates to a synthetic nano library for ribosome display screening and construction and application thereof. Aiming at the problems that the existing nano antibody acquisition depends on animal immunity, the diversity and screening efficiency of a synthetic library are insufficient and the like, the invention discloses a synthetic nano antibody library suitable for ribosome display and a construction method of the synthetic nano antibody library, and the synthetic nano antibody library suitable for ribosome display is constructed by combining a trinucleotide primer synthesis and Type IIS enzyme digestion connection technology through a consensus skeleton design and a CDR region differentiation randomization strategy. The library stability and the effective diversity are improved. The library does not need to immunize animals, is short in period and low in cost, is compatible with toxic antigens, can be screened through combination of ribosome and bacteriophage, can efficiently obtain nano-mole-level affinity nano-antibodies, can also assist structure research of a cryoelectron microscope, has the advantages of being complete in library, efficient in screening, low in sequence redundancy and the like, and provides powerful support for research and development of biological medicines.

Description

Technical Field

[0001] This invention belongs to the field of antibody library construction technology, specifically relating to a synthetic nanolibrary for ribosome display screening and its construction and application. Background Technology

[0002] Single-domain antibodies (sdAbs) are variable region fragments containing only heavy-chain-only antibodies (HCAbs) unique to camelids and some cartilaginous fish (such as sharks). With a molecular weight of approximately 14 kDa, only about one-tenth that of conventional IgG antibodies, they are also known as "nanobodies." Due to their small molecular weight and compact structure, nanobodies have demonstrated unique advantages in various biomedical and biotechnology applications. Specifically, nanobodies possess the following characteristics: (1) High tissue permeability and excellent pharmacokinetic properties: The single-domain structure makes it easy to penetrate dense tissues, and some nanobodies can even cross the blood-brain barrier; (2) Excellent physicochemical stability: Nanobodies have strong tolerance to thermal denaturation and reducing environments, and can be used for intracellular function blocking or protein activity regulation; (3) Easy to express and scale up: Nanobodies can be efficiently synthesized in microbial expression systems such as Escherichia coli and yeast, and the production cost is relatively low; (4) Strong potential for engineering modification: Nanobodies have simple structures and no light chain constraints, making them suitable for various protein engineering modifications, including affinity maturation, fusion protein construction, and multivalent antibody design.

[0003] With the FDA's approval of Caplacizumab, the first nanobody drug, for the treatment of acquired thrombotic thrombocytopenic purpura, the feasibility of nanobodies as therapeutic biological agents has been further validated. In recent years, numerous nanobody drugs or diagnostic agents targeting tumors, autoimmune diseases, and infectious diseases have entered different stages of clinical research, propelling nanobodies to become an important direction in antibody engineering and biomedical research and development. Traditionally, nanobodies are obtained primarily through immunization procedures on camel species such as alpacas and camels, or shark species. This method typically requires multiple rounds of cyclical immunization to induce a response in the animal's immune system to exogenous antigens and to induce somatic hypermutation and affinity maturation of B cells. Subsequently, relevant immune cells are isolated from the peripheral blood or lymphoid tissue of the immunized animals, total RNA is extracted and reverse transcribed to obtain cDNA, and then the variable region encoding a single-domain antibody (VHH) fragment is amplified using specific primers. The resulting amplified product is cloned into a phage display vector and transformed into host cells to construct a phage display library. Finally, using various screening strategies such as solid-phase screening and competitive screening, nanobody clones that can specifically bind to target antigens were screened from the library. Although this immune-dependent method has been widely used, it still has many limitations: (1) This method depends on the animal immunization cycle, which is not only time-consuming and costly, but also significantly limits the immunization induction efficiency when targeting highly toxic antigens, membrane protein antigens, low-stability antigens, or conformation-dependent antigens; (2) The applicability of the antibody library obtained from animal immunization is limited by the animal's physiological conditions, making it difficult to conduct experiments in special screening environments, such as extreme pH values, reducing environments, high ligand occupancy states, or insufficient exposure of the native conformation of membrane proteins; (3) The capacity of immunogenic antibody libraries is usually small, and they can only screen for target antigens that have already been immunized, and cannot cover other antigen types. If it is necessary to screen nanobodies for new antigens, it is necessary to re-implant animal immunization and construct new antibody libraries, which involves a large workload.

[0004] To overcome the aforementioned limitations, nanobody screening strategies based on in vitro display platforms have been developed in recent years. Typical in vitro display methods include phage display, yeast display, ribosome display, and mRNA display. Among these, phage display is one of the most convenient methods, achieving 10-1 times higher efficiency due to its high bacterial transformation efficiency. 9 Library diversity is limited; while yeast display offers advantages in eukaryotic expression and efficient sorting, its library diversity is only around 10% due to limitations in cell size (approximately 5 μm) and transformation scale. 8 Large-scale. Ribosome display and mRNA display do not require cell transformation and can construct highly diverse libraries in cell-free systems, with theoretical library sizes reaching 10^10^6. 12 -10 14By maintaining the ribosome-nascent peptide chain-mRNA ternary complex or forming a protein-mRNA covalent fusion, gene-phenotype coupling can be achieved, thereby enabling in vitro screening of highly diverse antibody libraries. mRNA display libraries are not constrained by the number of ribosomes and can have a larger library size, but require chemical modification of the mRNA, making the process relatively complex.

[0005] The effectiveness of in vitro display platforms highly depends on the design quality of synthetic libraries. The construction of synthetic antibody libraries typically involves assembling the backbone sequence of natural antibodies, or a backbone sequence optimized through sequence analysis, with artificially designed diverse complementarity-determining region (CDR) sequences. Theoretically, the chemical space of antibodies has infinite possibilities; the number of amino acid sequence combinations for just three CDR regions is extremely large, theoretically sufficient to screen for target binding proteins. However, in reality, even on the most diverse screening platforms (such as mRNA display, where library diversity can reach 10^6), the actual performance is significantly affected. 13 -10 14 The achievable library diversity is also far lower than the theoretical value of complete randomization (on a scale of [number]). For example, only 15 completely randomized amino acid sites can generate approximately 3.3 × 10 [number] libraries. 19 The theoretical diversity of these documents far exceeds the actual capacity of various display platforms. Therefore, semi-randomization strategies are crucial for constructing a library that combines operability with high effective diversity.

[0006] Several nanobody synthesis libraries have been reported to date, most of which rely on phage display technology for construction, and the diversity of these libraries is typically only 10%. 8 -10 9 Scale. Internationally, nanobody libraries employing ribosome display technology are relatively scarce. Only three types of synthetic libraries (classified as Concave, Loop, and Convex types based on CDR3 shape / length) developed by Seeger's laboratory and collaborators, along with the CeVICA platform library, have been reported, exhibiting library diversity reaching 10. 12 The scale is enormous, and there are no publicly reported cases of ribosome-displayed nanobody libraries in China. Existing synthetic nanobody libraries still have significant shortcomings: First, the number of randomized sites in the CDR region is limited, constraining library diversity; second, some libraries employ a completely randomized strategy without restrictive design of amino acid composition, easily generating a large number of unfoldable or low-stability sequences, thus affecting actual screening efficiency; third, the theoretical diversity of some libraries far exceeds the physical capacity of the in vitro display platform, resulting in a limited number of sequences that can actually be covered, reducing the screening success rate.

[0007] Based on the above situation, there is an urgent need to develop a synthetic nanobody library and its construction method that is optimized by randomization strategy and compatible with ribosome display platform, so as to improve the effective diversity and screening hit rate of the library and achieve efficient acquisition of nanobody molecules that specifically bind to target proteins. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, this invention provides a synthetic nanobody library suitable for ribosome display and its construction method. Through consensus-based backbone design, CDR region differential randomization strategy, combined with trinucleotide primer synthesis and Type IIS enzyme digestion and ligation technology, the stability and effective diversity of the library are improved. This library does not require animal immunization, has a short cycle time, low cost, is compatible with toxic antigens, and can yield nanomolar affinity nanobodies after screening. It can also assist in cryo-electron microscopy structural studies, exhibiting high screening efficiency and low sequence redundancy.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for constructing a synthetic nanobody library, the method comprising the following steps: S1. The sequence encoding the nanobody shown in SEQ ID NO.8 was seamlessly cloned into the ribosome display vector pRDV5 to obtain the basic vector pRDV5-Nb shown in SEQ ID NO.10. Then, using the obtained vector as a template, different functional modules of the nanobody coding region were amplified using high-fidelity DNA polymerase to obtain fragment 1 shown in SEQ ID No.11, fragment 2 shown in SEQ ID No.12, and fragment 3 shown in SEQ ID No.13. S2. A single-stranded DNA containing randomized sequences of CDR1, CDR2 and CDR3 is synthesized using a trinucleotide primer synthesis method. The single-stranded DNA contains sequences that overlap with adjacent frame region fragments at both ends. The single-stranded DNA includes rCDR1 shown in SEQ ID NO.14, rCDR2 shown in SEQ ID NO.15 and rCDR3 shown in SEQ ID NO.16. The amino acid sequence of CDR1 and its adjacent region is GaTbcdefMg, where the bolded letters are randomized sites. The randomization design is as follows: a: F(80), R(20); b: F(80), I(20); c: S(57), R(15), Y(10), G(9), N(6), W(3); d: S(35), R(20), E(11), Y(10), Q(9), L / G(6), W(3); e: Y(30), R(20), E(15), Q(10), S / H / G / N / A(5); f: A(43), R(24), E(20), Y(10), W(3); g: A / E / G(16); R / H(15), Y(10), N / M / T / W(3), where the numbers in parentheses represent the proportion of each amino acid. The amino acid sequence of CDR2 and its adjacent region is AIXSXGhBTZ, where the bolded letters are randomized sites. The randomization design is as follows: X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); h: S(40), R(20), E(20), Y(20); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3), where the numbers in parentheses indicate the proportion of each amino acid. The amino acid sequence of CDR3 and its adjacent region is ZVBi XGXBYZ, where the bold letters are randomized sites. The randomization design is as follows: Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3); X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); i: I(50), K(18), Y / D(15), G(2), where the numbers in parentheses represent the proportion of each amino acid. S3. Assemble fragment one from S1 with rCDR1 from S2 to obtain functional module F-CDR1 containing CDR1 as shown in SEQ ID No. 28. Then assemble fragment two from S1 with rCDR2 from S2 to obtain functional module F-CDR2 containing CDR2 as shown in SEQ ID No. 29. Then assemble fragment three from S1 with rCDR3 from S2 to obtain functional module F-CDR3 containing CDR3 as shown in SEQ ID No. 30. Then, ligate F-CDR1 and F-CDR2 to form an intermediate product containing the CDR1-CDR2 region using a Type IIS restriction endonuclease-mediated directional digestion and ligation method. The obtained intermediate product is amplified and then ligated with F-CDR3 to finally obtain a complete DNA fragment containing the T7 promoter, ribosome binding site, nanobody open reading frame, spacer region, and TolA ribosome stationary sequence, as shown in SEQ ID NO. 31. S4. The DNA product obtained in S3 is digested with BspQI to remove non-target or reverse complementary fragments, and the target fragment is purified to obtain the desired synthetic nanobody library.

[0010] This invention has the following advantages: (1) Consensus-based backbone sequence design enhances overall library stability: The backbone sequence of the library in this invention neither uses a single naturally derived nanobody backbone nor directly uses a developed nanobody framework as a library template, unlike existing technologies. Instead, it constructs a consensus framework by statistically analyzing a large number of nanobody sequences with resolved structures and selecting the amino acids with the highest frequency at each framework site. Compared to libraries constructed based on a single backbone sequence in existing technologies, this design better aligns with the evolutionary optimization patterns of natural nanobodies, thereby improving antibody folding stability and expression efficiency. (2) The CDR region randomization strategy is more rational, improving the effective diversity of the library: To match the theoretical diversity of the library with the processing capacity of the ribosome display system, this invention implements differentiated design for the three CDR regions: CDR1 adopts restricted randomization, and the restricted amino acid set is screened by combining structural analysis and natural sequence statistical results; based on structural alignment analysis, the 7th amino acid of CDR2 is limited to R, E, S, Y to reduce the diversity of the CDR2 region; CDR3 is a key region for antigen recognition. Compared with the Concave library of Seeger Laboratory, the CDR3 of this invention has one additional amino acid residue, and together with the adjacent region, a total of 7 randomization sites are set, giving this region higher sequence diversity. This design controls the overall library capacity to adapt to the physical upper limit of the ribosome display system while strengthening the diversity of the CDR3 region that determines the binding characteristics, effectively increasing the proportion of potential high-quality binding nanobodies in the library. (3) In the library construction process, this invention uses overlapping extension PCR combined with Type IIS restriction endonuclease-mediated directional enzyme digestion-ligation method to achieve modular assembly of CDR region, backbone region and other functional elements, instead of using an assembly strategy that relies entirely on PCR amplification. This method can effectively reduce sequence redundancy and sequence bias caused by biased amplification during the amplification process by reducing repeated amplification steps, thereby improving the sequence uniformity and actual coverage of the library. (4) The nanobody library constructed by this invention covers the key amino acids recognized by Fab8D3-2, can form a complex with Fab8D3-2, can be used for legobody assembly, increase the apparent molecular weight of target protein, and help the cryo-electron microscopy structure study of small molecular weight proteins.

[0011] Preferably, the nanobody in S1 has the sequence shown in SEQ ID NO.7, and it is capable of forming a stable complex with MBP-PrAc and Fab8D3-2.

[0012] More preferably, the nanobody is obtained by site randomization design of the consensus nanobody framework sequence shown in SEQ ID NO.6.

[0013] Preferably, the high-fidelity DNA polymerase in S1 includes Phusion high-fidelity DNA polymerase (Thermo Fisher) or PrimeSTAR Max DNA polymerase (Takara).

[0014] Preferably, before assembly in S3, fragments one, two, and three in S1 need to be separated and purified by agarose gel electrophoresis.

[0015] Preferably, the assembly described in S3 is performed using a multi-primer overlap extension PCR method.

[0016] Preferably, the Type IIS restriction endonuclease in S3 includes BsaI or BbsI.

[0017] The second aspect of the present invention also provides a synthetic nanobody library prepared by the construction method described in the first aspect, wherein the sequence of the synthetic nanobody library is shown in SEQ ID NO.32.

[0018] The third aspect of this invention also provides the application of the synthetic nanobody library described in the second aspect in screening specific nanobody molecules, wherein the screening method is a combination of ribosome display and phage display.

[0019] The fourth aspect of the present invention also provides the application of the synthetic nanobody library described in the second aspect in the construction of an in vitro nanobody high-throughput screening platform, the platform being used for the discovery of target antigen-specific nanobodies.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention addresses the problems of existing nanobody acquisition relying on animal immunization, insufficient diversity of synthetic libraries, and low screening efficiency. It discloses a synthetic nanobody library suitable for ribosome display and its construction method. The specific steps include: selecting a constant frame region sequence as the backbone; determining the randomization sites and amino acid composition of the CDR region and adjacent regions based on the statistical characteristics and structural analysis results of the nanobody's natural sequence; preparing CDR1, CDR2, and CDR3 randomized fragments using a trinucleotide primer synthesis method; and then assembling these fragments with the frame region fragments and functional fragments required for in vitro transcription and translation via overlap extension PCR to obtain a complete synthetic nanobody library. This invention improves library stability and effective diversity through a consensus-based backbone design, a differentiated randomization strategy for the CDR region, combined with trinucleotide primer synthesis and Type IIS enzyme digestion and ligation technology. This library does not require animal immunization, has a short cycle time, low cost, is compatible with toxic antigens, and can be screened by ribosomes and bacteriophages to efficiently obtain nanomolar affinity nanobodies. It can also assist in cryo-electron microscopy structural studies, possessing advantages such as complete library, high screening efficiency, and low sequence redundancy, providing strong support for biomedical research and development.

[0021] Specifically, the present invention has the following advantages: (1) This invention provides a synthetic nanobody library and its construction method; in terms of library design, a consensus backbone is selected as the library backbone, and a differentiated randomization strategy is adopted for the three CDR regions in combination with the structural analysis and the statistical results of natural nanobody sequences to improve the effective diversity of the library; in terms of library construction, the amino acid ratio and distribution are precisely controlled by the trinucleotide primer synthesis method, and the enzyme digestion-ligation method mediated by Type IIS restriction endonuclease is combined to reduce the sequence redundancy and bias caused by excessive PCR.

[0022] (2) The synthetic nanobody library constructed in this invention is compatible with the ribosome display system, has a large library capacity, and can also be used for high-specificity secondary screening through phage display, making the screening process more efficient. The obtained nanobody clones all have nanomolar affinity and good specificity.

[0023] (3) The synthetic nanobody library constructed in this invention can be used to build an in vitro nanobody discovery platform. It does not require animal immunization, has a short cycle and low cost, and is applicable to antigens such as toxic proteins that cannot be prepared into antibodies through immunization.

[0024] (4) The present invention can screen fluorescent proteins to obtain nanomolar level specific nanobodies.

[0025] (5) The nanobodies obtained by screening the synthetic nanobodies constructed in this invention can form complexes with known Fab8D3-2 and MBP-PrAc, effectively increasing the apparent molecular weight of the antigen, which is beneficial for assisting the cryo-electron microscopy structure study of the target protein. Attached Figure Description

[0026] Figure 1The physicochemical properties of the nanobody framework were tested as follows: a. Purification test results of the template nanobody: SDS-PAGE was used to analyze different components of the sample during the purification process. The supernatant was the supernatant from periplasmic extraction during the purification process; the flow-through buffer was the supernatant after binding with Ni-NTA affinity medium; and the elution buffer was the protein eluted after washing with Ni-NTA. b. Surface melting temperature determination of the template nanobody: The sample was heated at different temperatures for 20 minutes each. After removing the aggregated protein by high-speed centrifugation, the supernatant was taken for fluorescence molecular sieve (FSEC) analysis. The peak intensity ratios of different temperatures were plotted, and the curves were fitted using the sigmoidal function. The FSEC buffer contained 150 mM NaCl, 50 mM Tris-HCl, and pH 8.0. The column used for analysis was a Sepax Zenix C SEC-300. c. Non-specific binding test of the template nanobody: Biotin-labeled antigen was immobilized on a streptavidin-conjugated sensor and subjected to 200... nM nanobodies were incubated, and changes in interference signals were detected. The left figure shows the binding kinetics of the nanobody framework protein with different antigens, and the right figure shows the kinetics of the control antibody (nanobody targeting TGP) with its target protein (TGP) and other test antigens. d. Binding test of template nanobody with MBP-PrAc and Fab8D3-2. The nanobody, Fab8D3-2, and MBP-PrAc were mixed in a molar ratio of 3:1.1:1, and then MBP-PrAc was bound to amylose beads. The protein components in the eluent were then analyzed by SDS-PAGE.

[0027] Figure 2 For the analysis of the antigen-binding structure of nanobodies: a. Structure of the complex of a natural nanobodily with its antigen (PDBID: 7PHP); b. Structure of the complex of a thermostable fluorescent protein (TGP) with the Sb44 nanobodily derived from the Concave library (PDBID: 6LZ2); c. Structure of the complex of the receptor-binding domain of the novel coronavirus Spike protein with the nanobodily derived from the Concave library (PDB ID: 7F5G); d. Structure of the complex of TGP and Sb44 (PDB ID: 6LZ2).

[0028] Figure 3 A schematic diagram for constructing a synthetic nanobody library.

[0029] Figure 4The results are as follows: a. Nucleic acid gel analysis of PCR amplification of fragments one, two, and three; b. Nucleic acid gel analysis of overlapping PCR amplification of F-CDR1, F-CDR2, and F-CDR3; c. Nucleic acid gel analysis of the ligation products of F-CDR1 and F-CDR2, where CDR1 and CDR2 represent F-CDR1 and F-CDR2, respectively; d. Nucleic acid gel analysis of the ligation products of F-CDR1 / F-CDR2 fragments and CDR3 fragments, where CDR1&2 represent the ligated fragments of F-CDR1 and F-CDR2; CDR3 represents the F-CDR3 fragment; and CDR1&2&3 represent the ligated fragments of F-CDR1 / F-CDR2 and F-CDR3.

[0030] Figure 5 For the evaluation of mRNA library integrity: a. Agarose gel electrophoresis analysis results of mRNA library; b. Standard curve of 5′ primer-based quantitative PCR; c. Standard curve of 5′ primer-based quantitative PCR; d. Quantitative results of mRNA library after inversion into cDNA using quantitative PCR.

[0031] Figure 6 The results show the screening results of the synthetic nanobody library; a, b. Coomassie brilliant blue staining and fluorescent gel results of biotin labeling efficiency of thermostable green fluorescent protein (TGP) (a) and (b), where black M represents commercial protein markers, and the molecular weight of each protein band is marked on the left side of Figure a; green M represents laboratory-made fluorescent markers, and the actual molecular weight of each protein band is marked on the left side of Figure b; T is the abbreviation for TGP, representing thermostable green fluorescent protein; Str. is the abbreviation for streptavidin; T-Str. represents the complex formed by biotin-labeled TGP and streptavidin; c. Quantitative real-time PCR results of ribosome display and phage display during the screening process; d. Detection of the binding of synthetic nanobodies to TGP using the FSEC method, where the black dashed line represents the peak position of TGP; e, f. Binding kinetic curves of TGP and corresponding synthetic nanobodies (e) and fitted kinetic constants (f). During detection, biotin-labeled TGP was immobilized on a streptavidin-coupled sensor and subjected to 50 μL of ... nM synthesized nanobodies were incubated, and changes in interference signals were detected. The data were fitted using Data Analysis 10.0 software in a 1:1 binding mode. Detailed Implementation

[0032] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all commercially available. It should be noted that all DNA sequences are shown in 5' to 3' order. All amino acid sequences are shown from the N-terminus to the C-terminus.

[0034] Nanobodies have shown broad application potential in drug development, structural biology and molecular recognition due to their advantages such as small molecular weight, high stability and ease of engineering. However, existing technologies still have many limitations in obtaining high-quality nanobodies.

[0035] The traditional approach to discovering nanobodies mainly involves screening phage display libraries constructed after animal immunization. This method is not only time-consuming and costly, but also subject to the physiological state of the animals, making it unsuitable for screening highly toxic antigens, membrane protein antigens, and antibody screening scenarios that require non-physiological conditions. On the other hand, although existing synthetic nanobodies libraries have been applied to in vitro display platforms, they have the following significant shortcomings: (1) The number of random sites in the complementarity-determining region (CDR) is limited, resulting in insufficient effective diversity of the library; (2) The amino acid composition design lacks fine-grained control, and complete randomization easily generates a large number of sequences that cannot be correctly folded or do not have binding activity, thereby reducing screening efficiency; (3) The theoretical size of the library far exceeds the actual carrying capacity of the display system, resulting in low actual library coverage and difficulty in improving the screening hit rate.

[0036] In summary, there is an urgent need for a novel synthetic nanobody library that improves upon CDR randomization strategies, amino acid distribution optimization, and library adaptability, thereby enhancing the screening efficiency of in vitro display platforms and obtaining high-affinity nanobodies.

[0037] Therefore, this invention provides a synthetic nanobody library suitable for ribosome display systems and a method for constructing the same, to achieve the following technical effects: (1) Rational randomization design was implemented for complementarity-determining regions CDR1, CDR2 and CDR3 to significantly improve the effective diversity of the library while ensuring the structural stability of the nanobody; (2) Restricted amino acid sets were introduced at key sites to avoid a large number of non-functional sequences caused by complete randomization, thus effectively improving the quality of screening results; (3) The number and distribution of randomization sites were optimized to make the theoretical diversity of the library match the physical capacity of the ribosome display system, thereby improving the actual coverage of the library; (4) Finally, nanobody molecules that can form stable binding with the target protein and have affinity at the nanomolar level were obtained, greatly improving the application effectiveness of the in vitro screening platform.

[0038] The method for constructing the nanobody library specifically includes the following steps: (1) Determination of the framework region sequence: The framework region sequence is determined by sequence alignment of nanobodies in existing structural biology studies and consensus sequence analysis. (2) CDR randomization sequence design: Analyze the existing structural characteristics of concave sybody-antigen complexes, determine the randomization sites of CDR1, CDR2, and CDR3 and their adjacent positions, and rationally design the amino acid composition: 1) Seven randomization sites were designed for CDR1 and its adjacent locations, and a restricted randomization method was designed based on existing statistical and structural analysis of natural nanobody sequences; 2) Five randomization sites were designed for CDR2 and its adjacent positions. Complete randomization was used at multiple sites (except for cysteine ​​and proline), and the 7th amino acid of CDR2 was restricted to R, E, S, Y based on structural analysis. 3) A total of 7 randomization sites were designed for CDR3 and its adjacent positions. Complete randomization was adopted at multiple sites (except for cysteine ​​and proline), and the amino acid composition of some sites was restricted based on structural analysis. 4) The amino acid ratio and distribution at each random site are controlled by the trinucleotide primer synthesis method.

[0039] (3) Synthesis and amplification of randomized fragments: randomized fragments of CDR1, CDR2 and CDR3 were prepared by oligonucleotides synthesized using trinucleotide primers. A nanobody encoding a fixed sequence was used as a template to amplify fragments containing CDR and its neighboring elements.

[0040] (4) Library assembly: The fragments were digested and ligated using TypeIIS restriction endonuclease to obtain a synthetic nanobody library DNA fragment containing all functional elements, including the T7 promoter, ribosome binding site, nanobody open reading frame, spacer region and TolA.

[0041] (5) Obtaining mRNA library by in vitro transcription: The DNA library is transcribed into an mRNA library in vitro using T7 RNA polymerase, and the RNA fragment required for ribosome display is obtained after purification.

[0042] The nanobody encoding sequence consists of frame region 1 (FW1) -- CDR1 -- frame region 2 (FW2) -- CDR2 -- frame region 3 (FW3) -- CDR3 -- frame region 4 (FW4) -- tag, from the amino terminus to the carboxyl terminus.

[0043] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1: Selection and property testing of the framework region of the nanobody synthesis library 386 nanobody sequences with previously studied structures were retrieved from the Protein Structure Database (PDB) (https: / / www.rcsb.org / ). These sequences were systematically analyzed using sequence alignment and consensus analysis to determine the most frequently occurring amino acid residues in each frame region, thus obtaining a consensus nanobody frame sequence. This nanobody frame sequence consists of four frame regions: frame region 1 (FW1)-CDR1-frame region 2 (FW2)-CDR2-frame region 3 (FW3)-CDR3-frame region 4 (FW4). The sequences of FW1-4 are shown in SEQ ID NO. 1-4, respectively. Meanwhile, to ensure compatibility with the legobody components developed in (X. Wu, & TA Rapoport, Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies), Proc. Natl. Acad. Sci. USA 2021, 118 (41) e2115001118, https: / / doi.org / 10.1073 / pnas.2115001118.), a tag sequence was introduced at the carboxyl terminus of the nanobody library to form a complex with Fab8D3. The tag sequence is shown in SEQ ID NO. 5. Finally, the obtained nanobody framework region sequence is shown in SEQ ID NO. 6.

[0045] FW1 (SEQ ID NO.1): QVQLVESGGGLVQAGGSLRLSCAASG; FW2 (SEQ ID NO.2): MGWFRQAPGKEREWVAAIS; FW3 (SEQ ID NO.3): TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV; FW4 (SEQ ID NO.4): YAGQGTQVTVS; Tag (SEQ ID NO.5) SLEHHHHHHSA; Synthetic nanobody framework sequence (SEQ ID NO.6): QVQLVESGGGLVQAGGSLRLSCAASG-----MGWFRQAPGKEREWVAAIS -----TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAV-----YAGQGTQVTVS SLEHHHHHHSA (The dashed part is the CDR, and the underlined part is the tag sequence).

[0046] Based on the obtained consensus framework sequence and randomization design at each point, a nanobody sequence with the highest theoretical probability was obtained. The obtained nanobody sequence is shown in SEQ ID NO.7. Codon optimization was performed on the coding sequence of this nanobody to make it more suitable for the *E. coli* expression system, and the obtained nanobody coding sequence is shown in SEQ ID NO.8. Subsequently, the coding sequence was cloned into the expression vector pSb-init using a seamless ligation method for subsequent protein expression and physicochemical property analysis. The sequence of the obtained recombinant expression vector is shown in SEQ ID NO.9.

[0047] Nanobody sequence (SEQ ID NO.7): MGSQVQLVESGGGLVQAGGSLRLSCAASGFTFSSYAMYWFRQAPGKEREWVAAISSGTGSTYYADSVKGRFTISSRDNAKNTVYLQMNSLKPEDTAVYYCYVYVSGSSYTGQGTQVTVS SLEHHHHHHSA (The underlined part is the tag sequence).

[0048] Nanobody encoding sequence (SEQ ID NO.8): ATGGGTAGTCAAGTACAGTTAGTAGAAAGTGGTGGTGGTTTAGTTCAAGCGGGAGGTTCATTAAGGTTATCATGCGCAGCGTCAGGGTTTACTTTTTCAAGTTATGCAATGTATTGGTTTCGTCAAGCTCCAGGGAAGGAGCGCGAATGGGTTGCAGCGATTAGTTCGGGCACCGGTTCCACGTATTACGCTGATAGCGTGAAAGGCCGTTTTACCATCTCTCGTGACAACGCCAAAAATACTGTCTATCTGCAGATGAACTCATTAAAACCAGAAGATACAGCTGTTTACTACTGTTATGTTTATGTTTCAGGATCAAGTTATACAGGCCAGGGTACTCAAGTCACAGTAAGTAGCCTGGAGCATCATCATCATCATCATAGCGCA。

[0049] Recombinant expression vector sequence (SEQ ID NO.9):

[0050] The obtained recombinant expression vector was transformed into *Escherichia coli* strain MC1061, and single clones were obtained by screening on LB agar plates containing chloramphenicol. The single clones were then inoculated into TB medium containing chloramphenicol and cultured overnight at 37 °C with shaking. Subsequently, they were transferred to fresh TB / chloramphenicol medium at a ratio of 1:100 (v / v) and cultured until the bacterial culture reached OD500. 600 The value was 0.4–0.6. The culture temperature was then lowered to 22 °C, and cultured for another 1.5 h. Then, 0.02% (w / v) arabinose was added to induce protein expression. The cells were collected after 17 h of induction culture.

[0051] Periplasmic proteins were extracted using an osmotic method. The bacterial cell pellet was resuspended in a hypertonic buffer (0.5 M sucrose, 0.5 mM EDTA, and 0.2 M Tris-HCl, pH 8.0) and incubated at 4 °C for 30 minutes. Then, it was rapidly added to ice water for hypotonic lysis for 1 hour. After centrifugation, the supernatant containing nanobodies was collected and incubated with Ni-NTA affinity medium (Qiagen, Cat. 30430) in an imidazole-containing binding buffer. After washing to remove non-specific binding proteins, the supernatant was eluted with a high-concentration imidazole buffer (300 mM imidazole, 150 mM NaCl, and 20 mM HEPES, pH 7.5) to obtain the purified nanobodies. The purification yield was approximately 5 mg / L, and the protein purity was higher than 90%. Figure 1 a).

[0052] The thermal stability of the purified nanobodies was analyzed. Protein samples were heated at different temperatures, and then the aggregated proteins were removed by high-speed centrifugation. The supernatant was then analyzed by fluorescent molecular sieve (FSEC). The single-peak signal intensity of the protein after different temperature treatments was plotted against temperature, and an sigmoid curve was used for fitting to calculate the melting temperature (T0) of the nanobodies. m ) is 75℃ ( Figure 1 b).

[0053] The non-specific binding of the obtained nanobodies to soluble and transmembrane proteins was further investigated using biolayer interference (BLI) technology. Specifically, biotinylated labeled membrane proteins (PlsY, preparation procedure see reference: Li, Z., Tang, Y., Wu, Y. et al. Structural insights into the committedstep of bacterial phospholipid biosynthesis. Nat Commun 8, 1691 (2017). https: / / doi.org / 10.1038 / s41467-017-01821-9) or soluble proteins (thermally stable green fluorescent protein TGP, preparation procedure see reference: Cai H, Yao H, Li T, et al. An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification[J]. Communications biology, 2020, 3(1): 753) were immobilized onto streptavidin-conjugated sensors (ForteBio, Cat 18-5019), and after baseline equilibration in PBS-T buffer, they were incubated with 200 nM nanobodies, and the binding signal was recorded. The results showed that the control nanobody targeting TGP could specifically bind to the antigen TGP ( Figure 1 c) This confirms that the BLI detection system can effectively characterize antigen-antibody specific binding; while the nanobodies described in this invention did not show non-specific binding to the above-mentioned test proteins. Figure 1 c).

[0054] Furthermore, since the obtained nanobody framework contains the Fab8D3-2 recognition site reported in the literature (X. Wu, & TA Rapoport, Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies), Proc. Natl. Acad. Sci. USA 2021, 118 (41) e2115001118, https: / / doi.org / 10.1073 / pnas.2115001118.), a pull-down experiment was conducted to verify whether the nanobody framework could form a complex with MBP-PrAc and Fab8D3-2 (see the literature for specific procedures). Specifically, the purified nanobody, Fab8D3-2, and MBP-PrAc were mixed in a molar ratio of 3:1.1:1 and incubated on ice for 1 hour to promote complex formation. Amylose affinity packing material (NEB, Cat. E8021S) that specifically binds to MBP-PrAc was then added to the mixture, and the mixture was incubated at 4 °C for 1 hour. After incubation, the packing material was collected and thoroughly washed with buffer (1 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.0) to remove unbound proteins. The bound proteins were then eluted with elution buffer containing 20 mM maltose (20 mM maltose, 150 mM NaCl, 20 mM Tris pH 8.0). The elution products were then analyzed by SDS-PAGE to detect the individual protein components. Simultaneously, the nanobody, MBP-PrAc, and Fab8D3-2 were mixed in a 1:1:1 molar ratio and analyzed by SDS-PAGE as a control to determine the relative proportions of the protein components in the elution products. Figure 1 d). Experimental results show that the designed nanobody framework can form a stable complex with MBP-PrAc and Fab8D3-2.

[0055] Example 2: Analysis of existing nanobody structures and sequences to determine the amino acid length of the CDR region and randomization strategy. The Concave sybody library reported by the Seeger team (Zimmermann, I. et al., eLife,2018, 7:e34317, doi:10.7554 / eLife.34317; Zimmermann, I. et al., Nat Protoc,2020, 15:1707-1741, doi:10.1038 / s41596-020-0304-x) is characterized by a short CDR3 region, typically consisting of 6 amino acid residues. It also features a concave antigen-binding site formed by introducing a stable hydrophobic core into the framework region, making it suitable for recognizing antigenic epitopes with convex structures and providing a mature foundation for screening convex antigenic epitopes.

[0056] Based on this, analysis of the structures of multiple concave sybody-antigen complexes showed that ( Figure 2 The antigen-binding mechanism of these synthetic nanobodies differs significantly from that of natural nanobodies: structural analysis of the complexes between natural nanobodies and their antigens shows that antigen recognition is highly dependent on the CDR3 region (…). Figure 2 a); and structural analysis of the complex of the thermostable fluorescent protein (TGP) and the nanobody Sb44 derived from the Concave library showed that the antigen-binding interface of the concave sybody was significantly biased towards the CDR1 and CDR2 regions ( Figure 2 (b) This characteristic is directly related to the short length and limited randomization of the original Concave library's CDR3, as the library only randomized a few core residues and adjacent residues of CDR3; in addition, structural analysis confirmed the complex structure of the novel coronavirus Spike protein receptor-binding domain and the nanobody derived from the Concave library ( Figure 2 c) shows that the first half of the CDR1 region of some natural nanobodies participates in antigen binding; while the structure of the TGP / Sb44 complex ( Figure 2 d) indicates that the first half of the CDR1 region of the concave sybody usually does not participate in antigen binding, a feature consistent with the original library's design intention of immobilizing CDR1 and its adjacent residues to maintain the concave conformation.

[0057] Based on the above structural feature analysis results, while retaining the overall conformational advantages of the concave binding site, the original library design was improved to construct a novel synthetic nanobody library, making the antibody-antigen binding surface more biased towards the CDR3 region. The optimization schemes for its framework region and CDR region include the following aspects: (1) CDR1 randomization method: The number of randomization sites in the CDR1 region and its adjacent framework region was increased to 7. Simultaneously, based on natural nanobody sequence analysis, the degree of randomization was limited to reduce CDR1 diversity. The optimized CDR1 amino acid sequence is GaTbcdefMg (where bold letters indicate randomization sites). The randomization design is as follows: a: F(80), R(20); b: F(80), I(20); c: S(57), R(15), Y(10), G(9), N(6), W(3); d: S(35), R(20), E(11), Y(10), Q(9), L / G(6), W(3); e: Y(30), R(20), E(15), Q(10), S / H / G / N / A(5); f: A(43), R(24), E(20), Y(10), W(3); g: A / E / G(16); R / H(15), Y(10), N / M / T / W(3). The numbers in parentheses represent the proportion of each amino acid.

[0058] (2) CDR2 randomization method: Based on the resolved structural analysis of the nanobody-antigen complex, the 7th position of CDR2, which was originally completely randomized (except for cysteine ​​and proline), was changed to restricted randomization to reduce the overall diversity of the CDR2 region and improve library effectiveness. Given that this site is structurally oriented towards the antigen interface, the randomized amino acids were limited to one small and flexible residue (serine) and three larger residues (tyrosine, arginine, and glutamic acid) to control diversity while achieving a diverse antigen-binding surface shape. The optimized amino acid sequence of CDR2 is AIXSXGhBTZ (where the bolded letters represent randomized sites). The randomization design is as follows: X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); h: S(40), R(20), E(20), Y(20); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3). The numbers in parentheses represent the proportion of each amino acid.

[0059] (3) Randomization method of CDR3 and its neighboring regions: While maintaining the overall conformation of the concave binding site, the length of the CDR3 region was extended by one amino acid residue, and this residue and its adjacent regions were set as seven randomization sites to increase the conformational flexibility and sequence diversity of the CDR3 region. The optimized amino acid sequence of CDR3 and its adjacent regions is ZVBi XGXBYZ (where the bold letters are randomization sites). The randomization design is as follows: Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3); X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); i: I(50), K(18), Y / D(15), G(2). The numbers in parentheses represent the proportion of each amino acid.

[0060] Example 3: Assembling complete nanobody library DNA fragments The nanobody encoding sequence (SEQ ID NO.8) from Example 1 was seamlessly cloned into the ribosome display vector pRDV5 to obtain the base vector pRDV5-Nb. The obtained vector sequence is shown in SEQ ID NO.10. Then, using the obtained vector as a template, different functional modules of the nanobody encoding region were amplified using a high-fidelity DNA polymerase (e.g., Phusion or PrimeSTAR Max). Figure 3 The following fragment was obtained: (1) Fragment 1: A DNA fragment containing the T7 promoter, ribosome binding site (RBS) and coding framework 1 (FW1), the sequence of which is shown in SEQ ID No. 11; (2) Fragment 2: A DNA fragment containing the coding framework 3 (FW3) region, the sequence of which is shown in SEQ ID No. 12; (3) Fragment 3: A DNA fragment containing the coding framework region Framework 4 (FW4) and the tag, as well as the ribosome stationary sequence TolA and the BspQI restriction endonuclease recognition site, the sequence of which is shown in SEQ ID No. 13.

[0061] pRDV5-Nb vector sequence (SEQ ID NO.10):

[0062] Fragment 1 (SEQ ID NO.11): (The underlined section represents the T-starter sequence, the wavy underline represents the RBS sequence, and the uppercase letters represent the FW1 encoded sequence).

[0063] Fragment 2 (SEQ ID NO.12): TACGCTGATAGCGTGAAAGGCCGTTTTACCATCTCTCGTGACAACGCCAAAAATACTGTCTATCTGCA GATGAACTCATTAAAACCAGAAGATACAGCTGT GAGACCATGCAT (where the underlined part is the sequence encoding FW3).

[0064] Fragment 3 (SEQ ID NO.13): (The underlined part is the sequence encoding FW4, the bold part is the sequence encoding the tag, the gray background is the TolA sequence, and the wavy underline is the BspQI recognition and digestion sequence).

[0065] The amplified fragments were separated by agarose gel electrophoresis. Figure 4 a) After purification, it is used in subsequent modular assembly steps.

[0066] Meanwhile, according to the pre-designed randomization scheme, single-stranded DNA (rCDR1, rCDR2, rCDR3) containing randomized CDR1, CDR2, and CDR3 sequences was custom synthesized using the Trimer primer synthesis service at Nanjing GenScript Corporation. The single-stranded DNA also contained sequences that overlapped with adjacent frame region fragments at both ends.

[0067] The sequences of rCDR1, rCDR2, and rCDR3 are shown in SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively.

[0068] rCDR1 (SEQ ID NO.14): G TTA TCA TGC GCA GCG TCA GGG MiX1 ACT MiX2 MiX3 MiX4 MiX5 MiX6 ATGMiX7 T GG TTT CGT CAA GCT CCA GG (The underlined part is the sequence that overlaps with the adjacent frame region segment, and MiX is the trinucleotide randomization site).

[0069] rCDR2 (SEQ ID NO.15): GA AGA CCT TGG GTT GC A GCG ATT MiX8 TCG MiX8 GGC MiX9 MiX10 ACC MiX11 TAC GCT GAT AGC GTG AAA GG(The underlined part is the sequence that overlaps with the adjacent frame region segment, and MiX is the trinucleotide randomization site).

[0070] rCDR3 (SEQ ID NO.16): CGA AGA CCT GCT GTT TAC TAC TG T MiX11 GTT MiX10 MiX12 MiX8 GGA MiX8MiX10 TAT MiX11 GGC CAG GGT ACT CAA GTC AC (The underlined part is the sequence that overlaps with the adjacent frame region segment, and MiX is the trinucleotide randomization site).

[0071] The trinucleotide ratios used for randomization sites in rCDR1 / 2 / 3 are shown in Table 1.

[0072] Table 1. Trinucleotide ratio in randomized sites Subsequently, the assembly of each module was completed using a multi-primer overlap extension PCR method. Figure 3 , Figure 4b). Specifically, using pRDV5-Nb as a template, fragment one was amplified using primers 5'-Flank-F (CGAAATTAATACGACTCACTATAGGGAGAC; SEQ ID NO.17) and FW1-R (CCCTGACGCTGCGCATGATAAC; SEQ ID NO.18); fragment two was amplified using primers FW3-F (TACGCTGATAGCGTGAAAGG; SEQ ID NO.19) and FW3-R (ATGCATGGTCTCACAGCTGTATCTTCTGGTTTTAATGAG; SEQ ID NO.20); and fragment three was amplified using primers FW4-F (GGCCAGGGTACTCAAGTCAC; SEQ ID NO.21) and TolA-R1 (CAGCAGCTATAGCTCTTCAAAACCGCACACCAGTAAGGTGTGCGGTTTCAGTTGCCGCTTTCTTTCTTG; SEQ ID NO.22). Based on this, overlap extension PCR amplification was performed using primers 5'-Flank-F, FW2-R (ATGCATGGTCTCACCCATTCGCGCTCCTTCCCTGGAGCTTGACGAAACC; SEQ ID NO.23) and Link1-R (ATGCATGGTCTCACCCATTCG; SEQ ID NO.24). Fragment one was assembled with rCDR1 and introduced into FW2 to obtain a functional module containing CDR1 (F-CDR1), the sequence of which is shown in SEQ ID NO.28. Overlap extension PCR amplification was performed using primers Link1-F (GAATGGGTGAGACCATGCATGAAGACCTTGGGTTGC; SEQ ID NO.25) and FW3-R. Fragment two was assembled with rCDR2 to obtain a functional module containing CDR2 (F-CDR2), the sequence of which is shown in SEQ ID NO.29. Finally, primers Link2-F (GTGAGACCATGCATTATATCGAAGACCTGCTGTTTACTACTG; SEQ ID NO.24) were used. Fragment NO.26) and TolA-R2 (CAGCAGCTATAGCTCTTCAAAACC; SEQ ID NO.27) were subjected to overlap extension PCR amplification, and fragment 3 was assembled with rCDR3 to obtain a functional module containing CDR3 (F-CDR3), the sequence of which is shown in SEQ ID NO.30.

[0073] F-CDR1 (SEQ ID NO.28): (The downslope line represents the sequence of fragment one, the bold letters represent the sequence of fragment one overlapping with rCDR1, and the downslope wavy line represents the rCDR1 sequence; the gray background represents the introduced FW2 coding sequence; the trinucleotide ratio in the randomized site MiX is shown in Table 1).

[0074] F-CDR2 (SEQ ID NO.29): (The downslope line represents the rCDR2 sequence, the bold letters represent the sequence where fragment two overlaps with rCDR2, and the downslope wavy line represents the fragment two sequence; the gray background represents the introduced FW2 coding sequence; the trinucleotide ratio in the randomized site MiX is shown in Table 1).

[0075] F-CDR3 (SEQ ID NO.30): (The downslope line represents the rCDR3 sequence, the bold letters represent the sequence where fragment three overlaps with rCDR3, and the downslope wavy line represents the fragment three sequence; the trinucleotide ratio in the randomized site MiX is shown in Table 1).

[0076] F-CDR1 and F-CDR2 are ligated via a type IIS restriction endonuclease-mediated directional digestion and ligation method (e.g., BsaI or BbsI) to form an intermediate product containing the CDR1-CDR2 region. Figure 3 , Figure 4 c); After amplification, the intermediate product was digested with BsaI and then ligated with BbsI-digested F-CDR3 to obtain a complete DNA fragment containing the T7 promoter, ribosome binding site, nanobody coding region, spacer region, and TolA ribosome stationary sequence. Figure 3 , Figure 4 d), the specific sequence is shown in SEQ ID NO.31.

[0077] F-CDR3 + F-CDR2 + F-CDR3 (SEQ ID NO.31): (The underlined parts are sequences from F-CDR1; the bolded parts are sequences from F-CDR2; the wavy parts are sequences from F-CDR3; the trinucleotide ratios in the randomized site MiX are shown in Table 1).

[0078] Finally, the obtained DNA product was treated with BspQI restriction endonuclease to remove non-target or reverse complementary fragments. Electrophoresis was performed on a 3% agarose gel to check the BspQI digestion efficiency. After digestion, BspQI was inactivated by heating at 80°C for 5 minutes, and the DNA was purified using a commercial kit (QIAGEN, Cat. 28106) to obtain a synthetic nanobody DNA library suitable for ribosome display screening. The specific sequence is shown in SEQ ID NO. 32.

[0079] Synthetic nanobody library (SEQ ID NO.32): cgaaattaatacgactcactataggggacgacaacggtttccctctagaaataattttgtttaactttaagaaggagatatatccatgggtagtCAAGTACAGTTAGTAGAAAGTGGTGGTGGTTTAGTTCAAGCGGGAGGTTCATTAAGGTTATCATGCGCAGCGTCAGGG MiX1 ACT MiX2 MiX3 MiX4 MiX5 MiX6 ATG MiX7 TGGTTTCGTCAAGCTCCAGGGAAGGAGCGCGAATGG GTT GCA GCG ATT MiX8 TCG MiX8 GGC MiX9MiX10 ACC MiX11 TAC GCT GAT AGC GTG AAAGGCCGTTTTACCATCTCTCGTGACAACGCCAAAAATACTGTCTATCTGCAGATGAACTCATTAAAACCAGAAGATACAGCT GTT TAC TAC TGT MiX11 GTT MiX10MiX12 MiX8 GGA MiX8 MiX10 TAT MiX11 GGC CAG GGT ACT CAAGTCACAGTAAGTAGCCTGGAGCATCATCATCATCATCATAGCGCAaagctttatatggcctcgggggccgaattcggatctggtggccagaagcaagctgaagaggcggcagcgaaagcggcggcagatgctaaagcgaaggccgaagcagatgctaaagctgcggaagaagcagcgaagaaagcggctgcagacgcaaagaaaaagcagaagcagaagcagaagcagaagcagaagccgccaaagccgcagcagccgaagcgcagaaaaaagccgaggcagccgctgcggcactgaagaagaaagcggaagcgcagccgcagcgcagaaaaaagccgaggcagccgctgcggcactgaagaagaaagcggaagcggcagccgcagcactgaaACCGCACACCTTACTGGTGTGCGG (The trinucleotide ratios in the randomized site MiX are shown in Table 1; lowercase letters are sequences on the pRDV5 vector, and uppercase letters are sequences inserted during library construction).

[0080] Example 4: Generate an mRNA library through in vitro transcription and verify the integrity of the library. The synthesized nanobody DNA library (SEQ ID NO.19) was transcribed in vitro using a commercially available in vitro transcription kit (Promega Cat. P1300) to obtain the corresponding mRNA library.

[0081] To assess the integrity and stability of the obtained mRNA library, specific primers qPCR_RD_5'_for (GGGAGACGACAACGGTTTCCC; SEQ ID NO.33) and qPCR_RD_5'_rev (CCCTGACGCTGCGCATGATAAC; SEQ ID NO.34), and qPCR_RD_tolA_3'_for (GCCGAATTCGGATCTGGTGGC; SEQ ID NO.35) and qPCR_RD_tolA_3'_rev (CTGCTTCTTCCGCAGCTTTAGC; SEQ ID NO.36) were designed at the 5' and 3' ends of the mRNA library, respectively. The absolute number of mRNA molecules in the library was then determined by quantitative real-time PCR (qPCR).

[0082] The degree of mRNA degradation in the transcripts was analyzed by agarose gel electrophoresis and quantitative real-time PCR, thereby evaluating the in vitro transcription conditions and library quality. Results ( Figure 5 The results showed that the mRNA molecules in the library were not severely degraded, and the number reached 10. 12 .

[0083] Example 5: Verifying the quality of the nanobody synthesis library using fluorescent proteins To verify the usability and screening efficiency of the constructed synthetic nanobody library (SEQ ID NO.32) in the ribosome display system, thermostable green protein (TGP) was selected as the model target to carry out the nanobody screening and verification experiment.

[0084] TGP was biotinylated using the BirA enzyme-catalyzed ligation method, and the labeling efficiency was verified by gel migration assay. Figure 6a, b). After further purification by molecular sieve chromatography, biotinylated TGP protein was obtained for subsequent screening (for specific procedures, please refer to the literature: Cai H, Yao H, Li T, et al. An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification[J]. Communications biology, 2020, 3(1): 753.).

[0085] The mRNA library obtained in Example 4 was translated in vitro using a commercially available cell-free translation kit (PUREfrex 2.1 kit, Cat. PF213-0.25-EX, Genefrontier, Chiba, Japan). This allowed the nascent peptide chains of the nanobody to form a stable ribosome-mRNA-nascent peptide chain ternary complex with the ribosome and its encoding mRNA under the influence of a ribosomal stationary sequence. After ribosome display, the resulting complex was enriched using streptavidin magnetic beads coated with biotinylated TGP (DynabeadsMyone Streptavidin T1, Invitrogen, Cat. 65601), and the mRNA bound to the complex was eluted. After the obtained mRNA was reverse transcribed into cDNA using primer RT_Primer (CTTCAGTTGCCGCTTTCTTTCTTG; SEQ ID NO.37), the cDNA molecules were absolutely quantified using quantitative real-time PCR with primers qPCR_RD_5'_for / qPCR_RD_5'_rev and qPCR_RD_tolA_3'_for / qPCR_RD_tolA_3'_rev.

[0086] The results showed that approximately 10 could be obtained. 8 The ratio of the values ​​obtained by quantifying the cDNA molecules using 5′ and 3′ primers was approximately 1:1. Figure 6 c) indicates that the mRNA did not undergo significant degradation during the screening process, and the constructed library is suitable for ribosome display screening.

[0087] The obtained cDNA was further amplified by PCR using primers FX_for (ATATGCTCTTCTAGTCAAGTACAGTTAGTAGAAAGTGGTGGTGG; SEQ ID NO.38) and FX_rev (TATAGCTCTTCATGCTGCGCTATGATGATGATGATGATGCTC; SEQ ID NO.39), and the resulting DNA was ligated into the phage display vector pDX-init by enzyme digestion. The ligation product was then transformed into E. coli SS320 competent cells by electroporation to construct a phage display library. Two rounds of phage display screening were performed targeting TGP.

[0088] Specifically, the obtained phage display library was first cultured to the logarithmic growth phase and then infected with M13KO7helper phage (NEB, Cat. N0315S) to assemble complete phage particles. Amplification was performed by overnight incubation at 37°C with shaking. The phage particles were then precipitated using 2.5 M NaCl / 20% PEG6000 solution and resuspended in PBS buffer to obtain phages suitable for phage display screening.

[0089] In the first round of phage display, 50 nM biotinylated TGP was combined with 10 12 After mixing the Pfu / mL phage library, the mixture was incubated at room temperature for 20 minutes, followed by incubation for 10 minutes in a 96-well plate coated with neutral streptavidin. Utilizing the specific binding of biotin to neutral streptavidin, phages capable of binding TGP were immobilized on the 96-well plate. After washing to remove irrelevant phages, the plate was eluted with 0.25 mg / mL trypsin to obtain the enriched phages.

[0090] In the second round of phage display, phages were incubated with 50 nM biotinylated TGP in solution, followed by incubation with streptavidin magnetic beads (Dynabeads Myone Streptavidin C1, Invitrogen, Cat. 65001) for 10 minutes to immobilize TGP-binding phages onto the beads. Irrelevant phages were then removed by washing. Simultaneously, the phage-bound magnetic beads were incubated with 5 μM unlabeled TGP at room temperature for 3 minutes to competitively bind loosely to nanobodies. After washing, the enriched phages were obtained by enzymatic digestion with 0.25 mg / mL trypsin.

[0091] Compared with the negative control protein, the enrichment of TGP in the library constructed in this invention increased significantly with each screening round. Figure 6c) indicates that the library contains nanobody sequences that can specifically recognize the target protein.

[0092] After the demonstration, single clones were isolated, and the ability of the synthesized nanobodies to bind to TGP was detected using fluorescent molecular sieve (FSEC). Specifically, the enriched nanobody sequences were subcloned into the expression vector pSb-init with a C-terminal Myc tag using the FX cloning method. The product was then transformed into E. coli MC1061, and single clones were screened and isolated on plates containing 25 μg / mL chloramphenicol. Single clones were then randomly selected and induced to express using the method described in Example 1, followed by periplasmic extraction. The periplasmic solution was mixed with TGP protein and incubated on ice for 30 minutes. The fluorescence signal of TGP at 482 / 508 nm was detected using the FSEC method with a Sepax Zenix C SEC-300 column. By comparing the peak positions of TGP in different samples, the ability of the nanobodies to form stable complexes with TGP was analyzed.

[0093] The results showed that among 10 randomly tested monoclonal antibodies, 5 nanobodies were able to cause a forward shift in the TGP elution position. Figure 6 d), indicating that it can form a complex with TGP.

[0094] Sanger sequencing analysis of the obtained positive nanobodies showed that the sequences were different from each other, indicating that the obtained nanobodies had low sequence redundancy and the positive clone ratio was about 50%.

[0095] Furthermore, to determine the binding affinity of the screened nanobodies to TGP, biotinylated TGP was diluted to a final concentration of 2 μg / mL and immobilized on the surface of a streptavidin-conjugated sensor (ForteBio, Cat 18-5019). After baseline equilibration in a PBS-T buffer system, a binding reaction was initiated with 50 nM nanobodies. The dissociation process was then measured, and the kinetic data were fitted using a 1:1 binding model. The results showed that all five obtained nanobodies exhibited nanomolar affinity, with the highest affinity being approximately 7 nM (…). Figure 6 e, f).

[0096] In summary, the synthetic nanobody library designed and constructed in this invention can operate stably in a combined ribosome display and phage display screening system, and has the advantages of high library integrity, high screening efficiency, and low sequence redundancy. Furthermore, it can efficiently obtain nanobody molecules with nanomolar affinity for target proteins.

[0097] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for constructing a synthetic nanobody library, characterized in that, Includes the following steps: S1. The sequence encoding the nanobody shown in SEQ ID NO.8 was seamlessly cloned into the ribosome display vector pRDV5 to obtain the basic vector pRDV5-Nb shown in SEQ ID NO.

10. Then, using the obtained vector as a template, different functional modules of the nanobody coding region were amplified using high-fidelity DNA polymerase to obtain fragment 1 shown in SEQ ID No.11, fragment 2 shown in SEQ ID No.12, and fragment 3 shown in SEQ ID No.

13. S2. A single-stranded DNA containing randomized sequences of CDR1, CDR2 and CDR3 is synthesized using a trinucleotide primer synthesis method. The single-stranded DNA contains sequences that overlap with adjacent frame region fragments at both ends. The single-stranded DNA includes rCDR1 shown in SEQ ID NO.14, rCDR2 shown in SEQ ID NO.15 and rCDR3 shown in SEQ ID NO.

16. The amino acid sequence of CDR1 and its adjacent region is GaTbcdefMg, where the bolded letters are randomized sites. The randomization design is as follows: a: F(80), R(20); b: F(80), I(20); c: S(57), R(15), Y(10), G(9), N(6), W(3); d: S(35), R(20), E(11), Y(10), Q(9), L / G(6), W(3); e: Y(30), R(20), E(15), Q(10), S / H / G / N / A(5); f: A(43), R(24), E(20), Y(10), W(3); g: A / E / G(16); R / H(15), Y(10), N / M / T / W(3), where the numbers in parentheses represent the proportion of each amino acid. The amino acid sequence of the CDR and its adjacent region 2 is AIXSXGhBTZ, where the bolded letters are randomized sites. The randomization design is as follows: X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); h: S(40), R(20), E(20), Y(20); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3), where the numbers in parentheses represent the proportion of each amino acid. The amino acid sequence of CDR3 and its adjacent regions is ZVBi XGXBYZ, where the bolded letters are randomized sites. The randomization design is as follows: Z: H(13), K / R / H / E / T / A / Y(12), V / L / I / F / W(3); X: N / S / T / A / Y(10.6), R / E(6), K / H / D / Q(5), W(3), V / L / I / M / F / G(2); B: S / T / Y(10), G(9), K / R / H / E / N / Q(8), W(3), V / L / I / M / F(2); i: I(50), K(18), Y / D(15), G(2), where the numbers in parentheses represent the proportion of each amino acid. S3. Assemble fragment 1 from S1 with rCDR1 from S2 to obtain functional module F-CDR1 containing CDR1 as shown in SEQ ID No.

28. Then assemble fragment 2 from S1 with rCDR2 from S2 to obtain functional module F-CDR2 containing CDR2 as shown in SEQ ID No.

29. Then assemble fragment 3 from S1 with rCDR3 from S2 to obtain functional module F-CDR3 containing CDR3 as shown in SEQ ID No.

30. Connect F-CDR1 and F-CDR2 to form an intermediate product containing the CDR1-CDR2 region via a directional enzyme digestion and ligation method mediated by Type IIS restriction endonuclease. The intermediate product was amplified and then ligated with F-CDR3 to finally obtain a complete DNA fragment containing the T7 promoter, ribosome binding site, nanobody open reading frame, spacer region and TolA ribosome stationary sequence, as shown in SEQ ID NO.31; S4. The DNA product obtained in S3 is digested with BspQI to remove non-target or reverse complementary fragments, and the target fragment is purified to obtain the desired synthetic nanobody library.

2. The method for constructing a synthetic nanobody library according to claim 1, characterized in that, The sequence of the nanobody described in S1 is shown in SEQ ID NO.7, and it is able to form a stable complex with MBP-PrAc and Fab8D3-2.

3. The method for constructing a synthetic nanobody library according to claim 2, characterized in that, The nanobody was obtained by site randomization design of the consensus nanobody framework sequence shown in SEQ ID NO.

6.

4. The method for constructing a synthetic nanobody library according to claim 1, characterized in that, The high-fidelity DNA polymerase described in S1 includes Phusion high-fidelity DNA polymerase or PrimeSTAR Max DNA polymerase.

5. The method for constructing a synthetic nanobody library according to claim 1, characterized in that, Before assembly of S3, fragments one, two and three described in S1 need to be separated and purified by agarose gel electrophoresis.

6. The method for constructing a synthetic nanobody library according to claim 1, characterized in that, The assembly described in S3 was performed using a multi-primer overlap extension PCR method.

7. The method for constructing a synthetic nanobody library according to claim 1, characterized in that, The Type IIS restriction endonucleases described in S3 include BsaI or BbsI.

8. A synthetic nanobody library prepared by the construction method according to any one of claims 1-7, characterized in that, The sequence of the synthetic nanobody library is shown in SEQ ID NO.

32.

9. The application of the synthetic nanobody library according to claim 8 in screening specific nanobody molecules, characterized in that, The screening method is a combination of ribosome display and phage display.

10. The application of the synthetic nanobody library according to claim 8 in constructing an in vitro nanobody high-throughput screening platform, characterized in that, The platform is used for the discovery of target antigen-specific nanobodies.

Images