Alpaca-derived nano antibody specifically bound with simian vacuolar virus 40 large T antigen and application of alpaca-derived nano antibody

Abstract

The invention belongs to the technical field of molecular biology and immunology, and provides an alpaca-derived nano antibody specifically bound with a simian vacuolar virus 40 large T antigen SV40LTA and application of the alpaca-derived nano antibody. The invention relates to a heavy chain antibody, which has a heavy chain variable region VHH, the VHH comprises the following CDR: CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO: 3, the VHH comprises four frame regions of FR1-4, and FR1, FR2, FR3 and FR4 and CDR1, CDR2 and CDR3 are arranged in a staggered manner in sequence. The antibody is high in neutralizing activity and strong in binding capacity with SV40LTA, and the equilibrium dissociation constant is less than 0.1 nM; sV40LTA can be effectively identified, the molecular weight is small, the immunogenicity is small, the solubility and the stability are better, and the CDR region is longer; and a potential application value is provided for SV40LTA detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and immunology, and particularly relates to an alpaca-derived nanoantibody that specifically binds to simian vacuolating virus 40 large T antigen SV40LTA and an application thereof. Background Art

[0002] Simian vacuolating virus 40 (SV40) was first isolated in cell cultures from the kidney tissue of African green monkeys in the early 1960s. It is a DNA virus that can cause malignant transformation of normal cells from various human and animal tissue types in vitro, induce the formation of various tumors in transgenic mice, and has been implicated in the development of many human tumors. Its ability to induce cell transformation has been widely used in basic research and in the development of genetic engineering vectors.

[0003] SV40 is a small, non-enveloped virus particle with a diameter of 45 nm and an icosahedral structure. The SV40 capsid is composed of three viral coat proteins: VP1, VP2, and VP3. Within the capsid is the viral DNA genome, which is wrapped around histones to form a nucleosome-like structure. The genome is a circular, double-stranded DNA sequence of only 5243 base pairs.

[0004] The SV40 viral genome is expressed in a highly specific temporal order, distinguishing it from early and late expression regions. Early primary transcripts are processed into two distinct early mRNAs, while late primary transcripts are processed into three distinct late mRNAs. The two early mRNAs encode the large T antigen and the small T antigen (i.e., tumor proteins or antigens), respectively. Late mRNAs encode proteins that can be distinguished by their specific sedimentation coefficients into three types: 16S, 18S, and 19S mRNAs, encoding the VP1, VP2, and VP3 viral capsid proteins, respectively.

[0005] In 1993, scientists discovered immunoglobulin G proteins in alpacas that naturally lacked portions of the light and heavy chain constant regions but still possessed antigen-binding capacity. These proteins, known as heavy-chain antibodies (HCAbs), were identified. Analysis and sequence analysis of these HAbs revealed that their antigen-binding regions consisted solely of the variable region fragment. Consequently, this antigen-recognition region of HAbs was termed VHH. Based on this, single-domain antibodies (sdAbs) containing only the VHH domain were developed. Their variable regions consisted solely of the heavy chain, and with a diameter less than 10 nanometers, they were also called nanobodies (Nb). Subsequent studies have also found this specific type of HAb in the blood of animals such as alpacas and sharks. Compared to other small-molecule antibodies, nanobodies offer advantages such as small molecular weight, enhanced penetrance, ease of expression, ease of genetic modification, and ability to bind to multiple epitopes.

[0006] The main advantages of nanoantibodies are: first, their volume is 1 / 10 of that of ordinary antibodies. Due to their small size, they have strong penetrating power in animal tissues, can pass through human brain tissue, and can reach the inside of high-density tumors. Nanoantibodies can be used to treat certain tumors or brain diseases; second, they have good antigen specificity; third, they are easy to genetically modify and facilitate artificial modification to obtain antibodies against different pathogens; fourth, they are highly stable. Nanoantibodies can remain in the body longer than ordinary antibodies without being naturally decomposed, and their efficacy lasts longer. Nanoantibodies can even pass through the human stomach and remain effective.

[0007] Specific nanobodies are obtained by screening the phage library of nanobodies. The nanobody phage library is divided into immune library and non-immune library. The immune library is prepared by immunizing animals such as alpacas and camels with proteins. The non-immune library is prepared based on the structure of the constant and variable regions of nanobodies, retaining certain constant regions and randomly editing the variable regions. When the capacity of the nanobody library reaches 10 7 When the above is achieved, specific nanoantibodies against the antigen can be obtained. The use of non-immune libraries saves time and avoids the harm caused to animals by immunizing animals and collecting animal blood. Summary of the Invention

[0008] The object of the present invention is to provide an alpaca-derived nanoantibody that specifically binds to the simian vacuolating virus 40 large T antigen SV40LTA and its application; specifically, it provides an alpaca-derived nanoantibody or its antigen-binding fragment that binds to SV40LTA, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a preparation method thereof, a transformed cell, and a pharmaceutical composition comprising the above.

[0009] The alpaca-derived nanoantibody or its antigen-binding fragment of the present invention is a nanoantibody with high neutralizing activity, has a strong binding ability to SV40LTA, and can effectively identify SV40LTA. The nanoantibody has the advantages of small molecular weight, low immunogenicity, better solubility and stability, and a longer CDR region, providing potential application value for SV40LTA detection.

[0010] To achieve the above objectives, the present invention provides the following technical solution: an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40LTA, having a heavy chain variable region VHH, wherein the VHH comprises the following CDRs: a CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 3, wherein the VHH comprises four framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

[0011] Furthermore, the amino acid sequence of the FR1 framework region is shown in SEQ ID NO: 4, the amino acid sequence of the FR2 framework region is shown in SEQ ID NO: 5, the amino acid sequence of the FR3 framework region is shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 framework region is shown in SEQ ID NO: 7.

[0012] The amino acid sequence of the heavy chain variable region is shown in the following SEQ ID NO: 8; wherein: positions 1-25 are the FR1 framework region, positions 30-43 are the FR2 framework region, positions 61-92 are the FR3 framework region, and positions 113-123 are the FR4 framework region, CDR1 is inserted between the FR1 framework region and the FR2 framework region, CDR2 is inserted between the FR2 framework region and the FR3 framework region, and CDR3 is inserted between the FR3 framework region and the FR4 framework region.

[0013] The present invention also provides a polynucleotide encoding an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40LTA as described above, and the sequence of the polynucleotide is shown in SEQ ID NO:9.

[0014] The present invention also provides a nucleic acid construct comprising the above-mentioned polynucleotide.

[0015] An expression vector comprising the nucleic acid construct.

[0016] A transformed cell comprising the polynucleotide, nucleic acid construct or expression vector.

[0017] The nucleic acid construct further comprises at least one expression control element, such as a histidine tag, a stop codon, etc., operably linked to the polynucleotide.

[0018] The pharmaceutical composition contains the alpaca-derived nanobody or its antigen-binding fragment that specifically binds to SV40LTA and a pharmaceutical carrier.

[0019] The present invention also provides use of an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40LTA in preparing a kit for detecting SV40LTA.

[0020] The present invention establishes an ELISA method for detecting SV40LTA using nanobodies as detection antibodies, develops a kit, and realizes effective, rapid and convenient detection of the content of SV40LTA in samples, accumulating experience for establishing detection methods for other impurity proteins in biological products.

[0021] The present invention develops a nanoantibody drug targeting SV40 LTA. By immunizing alpacas with an antigen, peripheral blood is collected, an antibody library is constructed, and phage display technology is used to screen and obtain high-affinity nanoantibodies that specifically bind to SV40 LTA. The resulting alpaca-derived nanoantibodies or antigen-binding fragments thereof possess high neutralizing activity, strong binding to SV40 LTA, and effective detection of SV40 LTA. These nanoantibodies have the advantages of a small molecular weight, low immunogenicity, improved solubility and stability, and a long CDR region, offering potential applications for SV40 LTA detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] One or more embodiments are illustrated by way of example in the accompanying drawings, and such exemplary descriptions do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0023] Figure 1 This is a schematic diagram of the SV40LTA-SDS-PAGE identification results of Example 1 of the present invention; in the figure: 1: Marker, 2: SV40LTA; Figure 2 Schematic diagram of the VHH fragment amplification results of Example 2 of the present invention; in the figure: A is the result of the first round of PCR; B is the result of the second round of PCR; M: Marker, 1-12: VHH nucleic acid fragments amplified from lymphocytes; Figure 3 This is a diagram showing the results of plate-based detection of the SV40LTA-VHH nanolibrary capacity in Example 3 of the present invention; Figure 4 This is a diagram showing the library abundance results of the plate-based assay of the SV40LTA-VHH nanolibrary in Example 3 of the present invention; Figure 5 Schematic diagram of the prokaryotic expression and purification results of SV40LTA-VHH in Example 4 of the present invention; in the figure: 1: 16°C E. coli expression ultrasonic disrupted bacterial solution, 2: 16°C E. coli expression supernatant, 3: Marker, 4: 16°C E. coli expression precipitate, 5: E. coli expression purification flow-through, 6: E. coli purification wash, 7-10: E. coli purification elution; Figure 6 Schematic diagram of the purification results after renaturation of SV40LTA-VHH; Figure: A is the antibody purified by nickel ion affinity chromatography; B is the antibody purified by gel filtration chromatography and SDS-PAGE; Figure: 1: 16℃ E. coli expression precipitate, 2: Marker, 3: E. coli expression purification flow-through, 4: E. coli purification wash, 5-10: E. coli purification elution. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In addition, in order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art will understand that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention. The present invention is described in detail below.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0026] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0027] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0028] Example 1: Expression and purification of target protein 1. Materials: 2-year-old male alpacas in good condition, raised at the Alpaca Breeding Center of Shanxi Agricultural University. 293F cells, EcoRI and XhoI restriction endonucleases, lymphocyte separation medium, total RNA extraction kit, cDNA reverse transcription kit, Taq Green PCR Mix, PstI and BstEII restriction endonucleases.

[0029] 2. Statistical methods: In the immune effect detection experiment, the absorbance values of alpaca serum in each group were expressed as mean ± standard error (Means ± SE), and the data were analyzed by one-way analysis of variance using SPSS 16.0 statistical software.

[0030] III. Target Protein Acquisition: Preparation of SV40 LTA: Official website sequence information: NC_001669.1 (NCBI), P03070 (Uniprot). This protein consists of 708 amino acids with a molecular weight of 81,624 Da. 98% of it is localized in the nucleus. It possesses ATPase and DNA helicase activities, phosphorylating serine / threonine residues, ADP-ribosylating, and acetylating proteins. It also activates host cell nuclear ribosomal genes, induces DNA synthesis, and modifies the initiation of protein synthesis. It is widely used in retroviral production, gene expression, and recombinant protein production.

[0031] The SV40LTA amino acid sequence used in the present invention was purchased from Abcam. The sequence is obtained by mutating the amino acid positions 531 and 549 of the original P03070 (Uniprot) sequence, with position 531 mutated to F and position 549 mutated to A. These mutations enhance protein stability.

[0032] The amino acid sequence after mutation is shown in SEQ ID NO.10 in the sequence list, which is used as the amino acid sequence of the subsequent outsourced standard. Figure 1 The specific amino acid sequence is as follows: MDKVLNREESLQLMDLLGLERSAWGNIPLMRKAYLKKCKEFHPDKGGDEEKMKKMNTLYKKMEDGVKYAHQPDFGGFWDATEIPTYGTDEWEQWWNAFNEENLFCSEEMPSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLSFLSHAVFSNRTLACFAIYTTKEKAALLYKK IMEKYSVTFISRHNSYNHNILFFLTPHRHRVSAINNYAQKLCTFSFLICKGVNKEYLMYSALTRDPFSVIEESLPGGLKEHDFNPEEAEETKQVSWKLVTEYAMETKCDDVLLLLGMYLEFQYSFEMCLKCIKKEQPSHYKYHEKHYANAAIFADSKNQKTICQQAVDTVLAKKR VDSLQLTREQMLTNRFNDLLDRMDIMFGSTGSADIEEWMAGVAWLHCLLPKMDSVVYDFLKCMVYNIPKKRYWLFKGPIDSGKTTLAAALLELCGGKALNVNLPLDRLNFELGVAIDQFLVVFEDVKGTGGESRDLPSGQGINNLDNLRDYLDGSVKVNLEKKHLNKRTQIFPP GIVTMNEFSVPKTLQARFVKQIDFRAKDYLKHCLERSEFLLEKRIIQSGIALLLMLIWYRPVAEFAQSIQSRIVEWKERLDKEFSLSVYQKMKFNVAMGIGVLDWLRNSDDDDDEDSQENADKNEDGGEKNMEDSGHETGIDSQSQGSFQAPQSSQSVHDHNQPYHICRGFTCFKK PPTPPPEPET.

[0033] Example 2: Alpaca immunization and antibody library construction 1. Alpaca Immunization: Select adult male alpacas in good physical condition. Before immunization, collect 10 ml of blood from the alpaca. Separate the serum and store at -20°C for later use. Dilute 100 μg of purchased SV40 large T antigen (SV40 LTA) with PBS to a final volume of 1 ml and inject subcutaneously at multiple sites. For the first immunization, emulsify with 1 ml of complete Freund's adjuvant for 5 minutes. Immunizations are repeated every two weeks using incomplete Freund's adjuvant. After the third immunization, collect blood from the alpaca and separate the serum.

[0034] Diluted SV40LTA was added to an ELISA plate for coating. Blank wells (no liquid added) and negative wells (coating solution added alone) were set up and incubated at 4°C overnight. The coating solution was then discarded, and the plate was washed with PBS and incubated with 5% calf serum at 37°C for 40 minutes. After blocking, the plate was washed and the wash solution was blotted dry with filter paper for later use. Serially diluted serum was used as the primary antibody, and HRP-conjugated goat anti-alpaca antibody was used as the secondary antibody. Pre-immune serum served as a blank control. After the reaction, TMB-urea hydrogen peroxide solution was added as a colorimetric solution. The reaction was incubated at 37°C in the dark for 15 minutes, followed by termination with stop solution. The alpaca immune response was assessed by measuring absorbance at 450 nm for 15 minutes using a microplate reader.

[0035] The results of the SV40LTA antigen protein activity assay are shown in Table 1. While no SV40LTA antibodies were present in the unimmunized serum, the positive reaction in the whole serum following immunization was very strong, remaining positive even after a 100-fold dilution. These results demonstrate that SV40LTA heavy chain antibodies were produced in alpacas following immunization, and these antibodies can be used for subsequent library construction.

[0036] Table 1: Results of ELISA test on immune activity of alpaca serum 2. Isolation of Peripheral Blood Lymphocytes (PBMCs) and Library Construction from Alpacas: On day 12 after the fourth immunization, 50-60 mL of anticoagulated venous blood was collected from the jugular vein for peripheral blood lymphocyte (PBMC) isolation. The blood was mixed with PBS at a 1:1 ratio and slowly added to lymphocyte separation buffer. The mixture was centrifuged at 2000 rpm and 20°C for 20 minutes, and the PBMC layer was aspirated. Total RNA was extracted from the isolated PBMCs according to the manufacturer's instructions. cDNA was synthesized using the Superscript II First-Strand Synthesis System for RT-PCR kit with random oligo-dT primers using the extracted total RNA as a template. A first-round PCR reaction was performed using the cDNA as a template using the specific primers CALL001 and CALL002. The primer sequences for the first-round PCR reaction are shown in Table 2.

[0037] Table 2: Primers for the first round of PCR reaction PCR amplification was performed using the synthesized first-strand cDNA as a template. The reverse transcription product was divided into 30 reactions, with each PCR reaction system containing 50 μl. The PCR amplification reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0038] Table 3: PCR amplification reaction system Table 4: PCR amplification reaction conditions Gel excision and DNA fragment recovery: A gel recovery kit was used to recover and purify a 700 bp nucleic acid fragment. 1 μL of the gel recovery product was ligated to the pMD19-T simple vector. The reaction system is shown in Table 5.

[0039] Table 5: Reaction system The recovered nucleic acid fragment was ligated with the pMD19-T simple vector. The ligation product was transformed into DH5α competent cells, and a single colony was selected for base sequence determination. The specific method was as follows: vortex the reaction system briefly, incubate at 4°C overnight, heat shock the ligation product and DH5α competent cells, incubate on ice for 25 minutes, heat shock at 42°C for 90 seconds, incubate on ice for 4 minutes, add 400 μL of LB medium, and incubate at 37°C and 200 rpm for 40 minutes. 50 μL of the transformed bacterial suspension was evenly spread onto the surface of LB solid medium containing AMP and incubated inverted at 37°C overnight. The next day, 20 single colonies were selected and inoculated into 5 mL of LB liquid medium containing AMP for overnight culture, and their base sequence was determined.

[0040] Vector NTI software was used to analyze the sequencing results and design the second-round PCR primers VHH2-F and VHH2-R for library construction. The 700 bp fragment recovered from the first-round PCR was used as a template to amplify the VHH fragment using the sequencing-designed primers VHH2-F and VHH2-R. The second-round primer sequences are shown in Table 6.

[0041] Table 6: Second round PCR primer sequences The isolated alpaca immunized lymphocytes were detected by cell counting instrument and were 3.8×10 7 The two PCR products were detected by agarose gel electrophoresis. The results of the first round of PCR were as follows. Figure 2 As shown in A, PCR amplified the VHH fragments of traditional antibodies (900 bp) and heavy chain antibodies (700 bp). The results of the second round of PCR are shown in Figure 2 Shown in B: The product size is approximately 372 bp, consistent with expectations.

[0042] The second round of PCR amplified the nanoparticle heavy chain antibody (VHHs) sequence, recovering and purifying the VHHs sequence, which was approximately 372 bp in size. Twenty-four PCR reactions were performed in a 50 μl reaction volume. The second-round PCR primers and reaction conditions are shown in Table 5 and Table 7, respectively. The PCR reaction volume consisted of 2 μl of cDNA, 1 μl of each primer, 25 μl of Taq Green PCR Mix, and 21 μl of deionized water.

[0043] Table 7: Second round PCR amplification reaction conditions The VHHs fragments were ligated into the plasmid pMES4 using the restriction enzymes EcoRI and EcoRV, and then transformed into electrocompetent E. coli TG1 cells. The culture was centrifuged, the supernatant discarded, and the pellet resuspended in fresh culture medium. 100 μl of the culture was used to calculate the library volume, and the remaining culture was plated onto 20 2×YTAG solid culture plates. After overnight incubation, colonies were harvested to form the SV40LTA single-domain antibody library.

[0044] Example 3: Antibody library capacity and abundance detection (1) The method for determining the library capacity is as follows: the electroporated bacterial solution is -1 ~10 -8 Gradient dilution; 100 μl of each dilution was spread on a solid culture plate, two plates for each gradient, and cultured overnight at 25°C; the next day, the colonies on the gradient plates were statistically calculated for the reservoir capacity, and the results were as follows: Figure 3 The library capacity is 42÷(100×10 -5 )×30×10 3 = 1.26 × 10 9 indivual.

[0045] (2) The method for determining the abundance of the library is to perform a gradient dilution of the primary library solution from 10 -4 ~10 -10 ; Take 100 μl of bacterial solution from each dilution and spread it on a solid culture plate, and culture it at 25℃ overnight; the next day, count the colonies on the gradient plate and calculate the library abundance. Figure 4 The abundance of the library is 25÷(100×10 -10 )×10 3 =8.1×10 12 pcs / ml.

[0046] (3) The method for determining the insertion rate and insertion diversity of the VHH fragments in the library is as follows: After the library capacity is determined, 34 monoclonal colonies are randomly picked from the solid culture plate where the library capacity was determined, cultured with shaking at 37°C overnight, and the bacterial liquid is collected for PCR identification. Analysis of the PCR identification results and sequencing results showed that the lengths of the fragments in the 34 bacterial liquid PCR samples were consistent with the VHH fragments, and the insertion rate of the VHH fragments in the library was calculated to be 100%.

[0047] Example 4: Screening, Identification, and Expression of Nanobodies: The nucleotide sequence of the alpaca-derived nanobody shown in SEQ ID NO: 9 was followed by the coding sequence of a 6-histidine tag and the translation termination codon TGA. This was then inserted into the pET23a plasmid using the restriction endonuclease sites EcoRI and XhoI to construct an expression vector. The specific method is as follows: Add VCSM13 helper phage to the colonies collected in the previous step at a multiplicity of infection (MOI) of 1:20. After overnight incubation, centrifuge and remove the supernatant. Add PEG6000 / NaCl at a volume ratio of 1:4. Incubate at 4°C for at least 1 hour and then centrifuge for 30 minutes. Resuspend the pellet in PBS to obtain the collected phage particles.

[0048] 1×10 collected phages 10 An equal volume of 5% (w / v) skim milk was mixed and added to a 96-well plate coated with SV40 LTA. The plates were incubated at room temperature for 1 hour. Specific phages were eluted with 0.2 M glycine and then neutralized with Tris-HCl (pH 9.1). The eluted phages were used to infect E. coli TG1 cells and amplify the phage-encoded single-chain antibody library. The next round of panning was performed using the same method as above, for a total of three rounds.

[0049] The screening eluate was mixed with an Escherichia coli culture in the early logarithmic growth phase and cultured in 2×YTAG liquid medium for 2 h. Helper phage was added at a cell: phage ratio of 1:20 and cultured overnight. The supernatant was transferred to a new centrifuge tube, 1 / 5 volume of PEG-NaCl was added, mixed and placed at 4°C for 3 h, centrifuged to remove the supernatant, and the pellet was resuspended in 1 ml PBS and centrifuged for 1 min. 50 single colonies were randomly selected from the plate with the titer of the last round of screening eluate and inoculated into 1 ml 2×YTAG and cultured with shaking for 12 h. The colony was inoculated into 2×YTAG at a 1% inoculum size and cultured until the early logarithmic growth phase. Helper phage was added at a cell: phage ratio of 1:1 and cultured overnight. Centrifuged for 1 min, and the pellet was resuspended in an equal volume of 2×YTAG and cultured with vigorous shaking for 12 h. After centrifugation, the supernatant was collected for ELISA identification. Positive clones were identified by a ratio (S / N) of the A value of the test sample (S) to the A value of the negative control (N) ≥ 2.1. Plasmids of positive clones were sequenced using specific primers MP57 and GIII (primers shown in Table 8) to obtain the sequence encoding the VHHs in the plasmid. Sequencing revealed the core coding sequence of the Nanobody.

[0050] Table 8: Sequencing primer sequences The obtained nanobody core coding sequence (NB) was constructed into the pcoldI plasmid, and the coding sequence of 6 histidine tags and the translation termination codon TGA were connected to the nucleotide sequence of the alpaca-derived nanobody as shown in SEQ ID NO: 9. It was constructed into the pET23a plasmid through the restriction endonuclease sites EcoRI and XhoI to construct an expression vector.

[0051] Identification: Add 1 μL of the constructed pET23a plasmid containing the target gene to 50 μL of E. coli BL21 (DE3) competent cells. Place on ice for 30 minutes, then heat shock the culture in a 42°C water bath for 60 seconds. After 5 minutes on ice, add 450 μL of LB medium to the culture. Mix thoroughly and incubate at 37°C at 200 rpm for 1 hour. Then, spread 200 μL of the culture onto a solid LB+Amp plate. Place the plate upside down in a 37°C incubator and incubate overnight. Single colonies were picked from the overnight culture plates and inoculated into 5 mL of LB+Amp culture medium. After 8 hours of shaking culture at 37°C and 200 rpm, the entire bacterial culture was transferred to 4 L of LB+Amp culture medium. Cultured to the logarithmic phase, induced with 1 mM IPTG, and cultured overnight at 16°C. The next day, the bacterial pellet was collected by centrifugation and resuspended in an appropriate amount of 1× PBS. Ultrasonic disruption was performed at 112.5 w for 3 seconds and a 5-second pause. The supernatant and pellet were then collected after centrifugation at 4°C and 12,000 rpm for 20 minutes. Figure 5 As shown, SDS-PAGE detection showed that nanoantibodies were present in the supernatant after disruption, demonstrating that the nanoantibody construction could be expressed in a soluble form in Escherichia coli BL21 (DE3).

[0052] Purification: The obtained nanobody core coding sequence (NB) was constructed into the pcoldI plasmid and transfected into 293F cells for eukaryotic expression. The supernatant after cell culture was collected and purified by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM75Increase Hiload column (GE Healthcare)). The purified protein was identified by SDS-PAGE. The specific method is as follows: The supernatant was centrifuged to remove insoluble matter and then analyzed by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM75Increase Hiload column (GE Healthcare)). The results are as follows: Figure 6 As shown, the target peak was confirmed by SDS-PAGE to obtain a relatively pure nanobody. Nickel ion affinity chromatography ( Figure 6 A) and gel filtration chromatography ( Figure 6 B) The results show that a relatively pure target protein was obtained. The target peak was confirmed by SDS-PAGE, and the protein band position was consistent with the target protein size.

[0053] The antibody has a heavy chain variable region VHH, wherein the VHH comprises the following CDRs: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8 below (ESGGGLVQPGESLLLSCTVSGFMDY AAVA WFRQAPGKEREGVS CISSNSDATYYADSVKG RFTISRDNRKKQVYLQMSNLKPEDTAVYYCAA DTSITFNYYSGPLSPSNFDS WGQGTHVTVSS); wherein: positions 1-25 are FR1 framework region, positions 30-43 are FR2 framework region, positions 61-92 are FR3 framework region, positions 113-123 are FR4 framework region, CDR1 is inserted between FR1 framework region and FR2 framework region, CDR2 is inserted between FR2 framework region and FR3 framework region, and CDR3 is inserted between FR3 framework region and FR4 framework region.

[0054] The amino acid sequence of CDR1 (AAVA) is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 (CISSNSDATYYADSVKG) is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 (DTSITFNYYSGPLSPSNFDS) is shown in SEQ ID NO: 3. The VHH includes four framework regions of FR1-4, and the FR1, FR2, FR3 and FR4 are arranged in sequence alternately with CDR1, CDR2 and CDR3.

[0055] The amino acid sequence of the FR1 framework region is shown in SEQ ID NO:4 (ESGGGLVQPGESLLLSCTVSGFMDY), the amino acid sequence of the FR2 framework region is shown in SEQ ID NO:5 (WFRQAPGKEREGVS), the amino acid sequence of the FR3 framework region is shown in SEQ ID NO:6 (RFTISRDNRKKQVYLQMSNLKPEDTAVYYCAA), and the amino acid sequence of the FR4 framework region is shown in SEQ ID NO:7 (WGQGTHVTVSS).

[0056] The sequence of the polynucleotide encoding the alpaca-derived nanobody that specifically binds to SV40LTA is shown in SEQ ID NO: 9. Specifically, ATGGAAAGTGGTGGAGGCTTAGTTCAACCGGGTGAAAGTCTGTTACTGAGCTGTACCGTTTCTGGTTTTATGGATTACGCAGCGGTTGCATGGTTTCGTCAGGCTCCGGGTAAAGAACGCGAAGGCGTTAGCTGTATTAGCAGTAACAGCGATGCAACCTATTACGCGGATAGCGTTAAAGGCCGT TTTACCATCAGCCGTGATAACCGCAAGAAACAGGTCTACCTGCAGATGAGCAACCTGAAACCGGAGGATACCGCAGTTTTACTGCGCAGCGGATACCAGCATTACCTTTAACTACTATAGCGGTCCGCTGAGTCCGAGTAACTTTGATAGTTGGGGACAAGGTACCCACGTTACCGTTAGTAGC.

[0057] Example 5: ELISA assay for Nanobody activity: ELISA assays were performed using SV40LTA-precoated ELISA plates with diluted purified NB as the primary antibody and HRP-labeled llama as the secondary antibody. After the reaction, TMB-urea hydrogen peroxide solution was added as a colorimetric solution. The reaction was incubated at 37°C in the dark for 15 minutes, followed by termination with stop solution. Absorbance was measured at 450 nm using a microplate reader over 15 minutes.

[0058] The results of ELISA detection of specific binding of nanoantibodies to SV40LTA are shown in Table 9. The NB nanoantibody has a positive reaction with the SV40LTA protein, indicating that the obtained NB nanoantibody has good immune activity.

[0059] Table 9: Neutralization effect of nanobodies on SV40LTA Example 6: Antigen and Nanobody Affinity Experiment An NTA chip was used to immobilize SV40LTA on the chip. The immobilization amount was about 100 RU. The SV40LTA-VHH protein was diluted in PBST buffer in multiple ratios and loaded one by one from low concentration to high concentration.

[0060] The equilibrium dissociation constant (KD) between SV40LTA-VHH and SV40LTA is less than 0.1 nM, demonstrating that the SV40LTA-VHH nanobody can bind to SV40LTA with high affinity.

[0061] Since their discovery, nanobodies have become a research hotspot in the prevention and treatment of infectious diseases, tumors, and immune disorders due to their unique properties compared to conventional antibodies, such as high water solubility and conformational stability, strong antigen affinity, and ease of in vitro expression and humanization. Combined with phage display technology, these antibodies have become a research hotspot in the prevention and treatment of infectious diseases, tumors, and immune disorders. Nanobodies can prevent disease by binding to key proteins of pathogenic microorganisms, controlling the source of infection or interrupting transmission pathways.

[0062] The present invention uses SV40LTA to immunize alpacas and constructs a reservoir with a capacity of 1.26× 10 9 CFU / ml phage display antibody library. After three rounds of immunopanning, a high-affinity anti-SV40LTA nanobody, NB1, was identified. This nanobody effectively binds to SV40LTA. Subsequent multivalent nanobodies based on NB1 can be constructed to enhance binding to SV40LTA. This invention lays the foundation for the detection and application of SV40LTA using the high-affinity anti-SV40LTA nanobody screened.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. An alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40 LTA, comprising a heavy chain variable region VHH, characterized in that: The VHH comprises the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The VHH comprises four framework regions, FR1-4, and the FR1, FR2, FR3 and FR4 are arranged alternately with CDR1, CDR2 and CDR3 in sequence.

2. The alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40 LTA according to claim 1, characterized in that: The amino acid sequence of the FR1 framework region is shown in SEQ ID NO: 4, the amino acid sequence of the FR2 framework region is shown in SEQ ID NO: 5, the amino acid sequence of the FR3 framework region is shown in SEQ ID NO: 6, and the amino acid sequence of the FR4 framework region is shown in SEQ ID NO:

7.

3. The alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40 LTA according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is as follows SEQ ID NO: 8, wherein: positions 1-25 are FR1 framework region, positions 30-43 are FR2 framework region, positions 61-92 are FR3 framework region, positions 113-123 are FR4 framework region, CDR1 is inserted between FR1 framework region and FR2 framework region, CDR2 is inserted between FR2 framework region and FR3 framework region, and CDR3 is inserted between FR3 framework region and FR4 framework region.

4. A polynucleotide encoding an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40LTA as described in any one of claims 1 to 3, wherein the sequence of the polynucleotide is shown in SEQ ID NO:

9. A nucleic acid construct comprising the polynucleotide according to claim 4 . An expression vector comprising the nucleic acid construct according to claim 5 .

7. A transformed cell comprising the polynucleotide of claim 4, the nucleic acid construct of claim 5 or the expression vector of claim 6.

8. The nucleic acid construct according to claim 5, characterized in that: The nucleic acid construct further comprises at least one expression control element operably linked to the polynucleotide.

9. A pharmaceutical composition comprising an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40 LTA as claimed in any one of claims 1 to 3 and a pharmaceutically acceptable carrier and / or excipient.

10. Use of the alpaca-derived nanobody or antigen-binding fragment thereof that specifically binds to the simian vacuolating virus 40 large T antigen SV40 LTA according to any one of claims 1 to 3 in the preparation of a kit for detecting SV40 LTA.

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