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; the monoclonal antibody has the advantages that the monoclonal antibody can effectively recognize SV40LTA, the molecular weight is small, the immunogenicity is small, the solubility and stability are better, and the CDR region is longer; and a potential application value is provided for SV40LTA detection.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and immunology, and specifically relates to an alpaca-derived nano antibody 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 obtained from cell culture of kidney tissue of African green monkeys in the early 1960s. It is a DNA virus that can cause malignant transformation of normal cells of various tissue types in humans and animals in vitro, induce the formation of various tumors in transgenic mice, and is also related to the occurrence of many human tumors. It can induce cell transformation and has been widely used in basic research and the construction of genetic engineering vectors.

[0003] SV40 is a small non-enveloped virus particle with a diameter of 45nm and an icosahedral structure. The capsid of the SV40 virus is composed of three viral capsid proteins VP1, VP2 and VP3; inside the capsid is the viral DNA genome, which is wrapped by histones to form a structure similar to a nucleosome. The genome is a circular double-stranded DNA with a size of only 5243bp.

[0004] The temporal order of expression of the SV40 viral genome is quite strict, and it can be divided into early expression regions and late expression regions. Early primary transcripts are processed into two different early mRNAs, while late primary transcripts are processed into three different late mRNAs. The two early mRNAs encode large T antigens and small T antigens (i.e., tumor proteins or antigens), respectively. The proteins encoded by late mRNAs can be divided into three types of mRNAs, 16S, 18S, and 19S, according to their specific sedimentation coefficients, which encode VP1, VP2, and VP3 viral capsid proteins, respectively.

[0005] In 1993, scientists discovered immunoglobulin G in alpacas that naturally lacks part of the light chain and heavy chain constant regions but still has antigen binding ability, which is called heavy chain antibody (HCAbs). By analyzing and identifying the structure and sequence of this heavy chain antibody, it was found that its antigen binding region is only composed of variable region fragments. Therefore, the antigen recognition region fragment of the heavy chain antibody is called VHH, and on this basis, a single domain antibody (sdAb) containing only the VHH domain was developed. Its variable region is only composed of heavy chains. The diameter of the variable region protein is less than 10 nanometers, so it is also called a nanobody (Nb). In subsequent studies, this specific heavy chain antibody was also found in the blood of animals such as alpacas and sharks. Compared with other small molecule antibodies, nanobodies have the advantages of small molecular weight, strong penetration, easy expression, easy genetic modification, and easy binding to multiple epitopes.

[0006] The main advantages of nanoantibodies are: first, their size 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 using protein to immunize alpacas, camels and other animals. The non-immune library is prepared based on the structure of the constant region and variable region of the nanobody, retaining a certain constant region and randomly editing the variable region. When the capacity of the nanobody library reaches 10 7 When the above is achieved, specific nano-antibodies against antigens 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 is to provide 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 with 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, and provides potential application value for SV40LTA detection.

[0010] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an alpaca-derived nanoantibody 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 having an amino acid sequence as shown in SEQ ID NO:1, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:3, wherein the VHH comprises four framework regions of FR1-4, wherein 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 FR1 framework regions, positions 30-43 are FR2 framework regions, positions 60-91 are FR3 framework regions, positions 105-115 are FR4 framework regions, CDR1 is inserted between FR1 framework regions and FR2 framework regions, CDR2 is inserted between FR2 framework regions and FR3 framework regions, and CDR3 is inserted between FR3 framework regions and FR4 framework regions.

[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 comprises 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 nanoantibody 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 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 nano-antibody drugs for SV40LTA, collects peripheral blood after antigen immunization of alpacas, constructs antibody library, and uses phage display technology to screen and obtain high-affinity nano-antibodies that specifically bind to SV40LTA. The obtained alpaca-derived nano-antibodies or antigen-binding fragments thereof are nano-antibodies with high neutralizing activity, have strong binding ability with SV40LTA, can effectively detect SV40LTA, and have the advantages of small molecular weight, low immunogenicity, better solubility and stability, and a longer CDR region, providing potential application value for SV40LTA detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than 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: Line 1: Marker, Line 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; Line 1: Marker, Line 2-13: VHH nucleic acid fragments amplified from lymphocytes; Figure 3 This is a diagram showing the results of plate detection of 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 detection SV40LTA-VHH nanolibrary in Example 3 of the present invention; Figure 5 Schematic diagram of the prokaryotic expression and purification results of SV40LTA-VHH of Example 4 of the present invention; in the figure: Line 1: 16°C E. coli expression ultrasonic disrupted bacterial solution, Line 2: 16°C E. coli expression supernatant, Line 3: Marker, Line 4: 16°C E. coli expression precipitate, Line 5: E. coli expression purification flow-through, Line 6: E. coli purification wash, Line 7-10: E. coli purification elution; Figure 6 Schematic diagram of the purification results after renaturation of SV40LTA-VHH; in the figure: A is the antibody purified by nickel ion affinity chromatography; B is the antibody purified by gel filtration chromatography and SDS-PAGE. In the figure: Line 1: 16℃ E. coli expression precipitation, Line 2: Marker, Line 3: E. coli expression purification flow-through, Line 4: E. coli purification wash, Line 5-10: E. coli purification elution. DETAILED DESCRIPTION

[0024] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. The described embodiments are part of the embodiments of the present invention, rather than all of the 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 should understand that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail to highlight the purport 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 are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0028] Example 1: Expression and purification of target protein 1. Materials: Male alpaca (alpaca) 2 years old, in good physical condition, raised in the Alpaca Breeding Center of Shanxi Agricultural University. 293F cells, EcoRI and XhoI restriction endonucleases, lymphocyte separation solution, 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 value of alpaca serum in each group was 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] 3. Acquisition of target protein: SV40LTA preparation: Official website sequence information: NC_001669.1 (NCBI), P03070 (Uniprot). The protein is 708 amino acids in length and has a molecular weight of 81,624Da. 98% is located in the nucleus, has ATPase and DNA helicase activities, phosphorylates protein serine / threonine residues, ADP ribosylates and acetylates. It also activates host cell nuclear ribosomal genes, induces DNA synthesis, and modifies protein initiation synthesis. It is widely used in retrovirus production, gene expression and recombinant protein production.

[0031] The SV40LTA amino acid sequence used in the present invention was purchased from Abcam (https: / / www.abcam.com / en-us / targets / ltsv40 / 418755). The sequence is a mutation of the 531st and 549th amino acid sites of the original P03070 (Uniprot) sequence, with the 531st position mutated to F and the 549th position mutated to A; the mutation will 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 preparation. 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, collect 10ml of experimental alpaca blood before immunization, separate the serum and store it at -20℃ for later use. Dilute the purchased SV40 large T antigen SV40LTA 100μg with PBS to a final volume of 1ml, and inject it subcutaneously at multiple points to immunize the alpaca. For the first immunization, use 1ml of complete Freund's adjuvant emulsified for 5min. After that, immunization is performed every two weeks, using incomplete Freund's adjuvant. Collect alpaca blood and separate serum after the third immunization.

[0034] The diluted SV40LTA was added to the ELISA plate for coating, and blank wells (without any liquid) and negative wells (only coating solution) were set up. The plate was left to stand overnight at 4°C, and then the coating solution was discarded. After washing with PBS, 5% calf serum was added, and the plate was incubated at 37°C for 40 minutes. After blocking, the coated plate was washed, and the washing solution was dried with filter paper for later use. The serum after gradient dilution was used as the primary antibody, and the HRP-labeled goat anti-alpaca antibody was used as the secondary antibody and added to the ELISA plate pre-coated with SV40LTA for reaction. The pre-immune serum was used as a blank control. After the reaction, TMB-urea hydrogen peroxide solution was added to the colorimetric solution, and the color was developed at 37°C in the dark for 15 minutes, and then the stop solution was added to terminate the reaction. The absorbance value was measured within 15 minutes at a wavelength of 450nm by an enzyme reader to detect the immune effect of the alpaca.

[0035] The results of the SV40LTA antigen protein activity test are shown in Table 1: the unimmunized serum does not contain SV40LTA antibodies, while the positive reaction in the whole serum after immunization is very strong, and it still has a positive reaction after dilution 100 times. The results show that after immunization with SV40LTA, the alpaca produces anti-SV40LTA heavy chain antibodies, which can be used for subsequent library construction.

[0036] Table 1: Results of ELISA test on immune activity of alpaca serum 2. Isolation and library construction of alpaca peripheral blood lymphocytes (PBMC): On the 12th day after the fourth immunization, 50-60 mL of anticoagulated venous blood was collected from the jugular vein for peripheral blood lymphocyte (PBMC) isolation, mixed with PBS at a ratio of 1:1 and slowly added to the lymphocyte separation solution, centrifuged at 2000rpm and 20℃ for 20 minutes, and the PBMC layer liquid was aspirated. Total RNA was extracted from the isolated PBMCs according to the instructions. Using the extracted total RNA as a template, the Superscript II First-StrandSynthesis System for RT-PCR kit was used to synthesize cDNA with random primers oligo-dT primers. Using cDNA as a template, the first round of PCR experiments were performed using specific primers CALL001 and CALL002. The primer sequences for the first round of PCR reactions are shown in Table 2.

[0037] Table 2: Primers for the first round of PCR reaction The synthesized first-strand cDNA was used as a template for PCR amplification, and the reverse transcription product was divided into 30 reactions, with each PCR reaction system being 50 ul. 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: The gel recovery kit was used to recover and purify the 700 bp nucleic acid fragment. 1 μL of the gel recovery product was connected to the pMD19-T simple vector. The reaction system is shown in Table 5.

[0039] Table 5: Reaction system The recovered nucleic acid fragments were connected to the pMD19-T simple vector. The ligation product was transferred into DH5α competent cells, and a single clone was picked to determine the base sequence. The specific method is: instant vortex reaction system, react at 4°C overnight, heat shock the ligation product and DH5α competent cells, incubate on ice for 25 min, heat shock at 42°C for 90 s, incubate on ice for 4 min, add 400 μL of LB medium and incubate at 37°C and 200 rpm for 40 min, 50 μL of the transformed bacterial solution was evenly spread on the surface of LB solid medium containing AMP, and inverted and cultured at 37°C overnight. The next day, 20 single clone colonies were picked and inoculated into 5 ml LB liquid medium containing AMP for overnight culture, and then their base sequences were determined.

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

[0041] Table 6: Second round PCR primer sequences The number of lymphocytes isolated from the immunized alpaca was 3.8×10 7 The results of the first round of PCR were as follows: Figure 2 As shown in A, PCR amplified the VHH fragments of the traditional antibody (900 bp) and the heavy chain antibody (700 bp). The results of the second round of PCR are shown in Figure 2 As shown in B: The product size is about 351 bp, which is consistent with expectations.

[0042] The second round of PCR amplified the (VHHs) sequence of the nano-amplified heavy chain antibody, and recovered and purified the VHHs sequence with a size of about 351 bp. A total of 24 PCR reactions were performed in a 50 μl reaction system. The specific method is as follows: The second round of PCR primers are shown in Table 6, and the second round of PCR amplification reaction conditions are shown in Table 7. The PCR reaction system is cDNA 2 μl, primers 1 μl each, Taq Green PCRmix 25 μl, and deionized water 21 μl.

[0043] Table 7: Second round PCR amplification reaction conditions The VHHs fragment was connected to the plasmid pMES4 through the restriction enzyme sites EcoRI and EcoRV, and then transferred into the electrocompetent E. coli TG1 cells. The cultured bacterial solution was centrifuged and the supernatant was discarded, and the precipitate was resuspended in fresh culture medium. 100 μl of bacterial solution was taken to calculate the library capacity, and the remaining bacterial solution was spread on 20 2×YTAG solid culture plates. After overnight culture, the colonies were collected, which was 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 heated to 10 -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 as follows: the primary library bacterial solution is diluted in a gradient manner, with the dilution ranging 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°C 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 Pieces / 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 with the determined library capacity, cultured at 37°C overnight with shaking, and the bacterial liquid is collected for PCR identification. The PCR identification results and sequencing results are analyzed. The lengths of the fragments of the 34 bacterial liquid PCR samples are consistent with the VHH fragments. The insertion rate of the VHH fragment in the library is calculated to be 100%.

[0047] Example 4: Screening, identification and expression of nanobodies: 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, and it was constructed into the pET23a plasmid through the restriction endonuclease sites EcoRI and XhoI to construct an expression vector. The specific method is as follows: The colonies collected in the previous step were added with VCSM13 helper phage at a multiplicity of infection of 1:20. After overnight culture, the supernatant was centrifuged and PEG6000 / NaCl was added at a volume ratio of 1:4. After standing at 4°C for at least 1 hour, the supernatant was centrifuged for 30 minutes. The precipitate after centrifugation was resuspended in PBS to obtain the collected phage particles.

[0048] 1×10 of the collected phages were 10 Mix an equal volume of 5% (w / v) skim milk and add it to a 96-well plate coated with SV40LTA. Incubate at room temperature for 1 hour. Elute the specific phage with 0.2 M glycine, and then neutralize the eluted phage with Tris-HCl (pH 9.1). Infect E. coli TG1 cells with the eluted phage to amplify the phage single-chain antibody library. Perform the next round of panning with the same method as above, and perform 3 rounds of phage panning.

[0049] The screening eluate was mixed with the Escherichia coli culture in the early logarithmic growth stage, cultured in 2×YTAG liquid medium for 2 hours, helper phage was added at a ratio of cells: phage = 1:20, cultured overnight, the supernatant was transferred to a new centrifuge tube, 1 / 5 volume of PEG-NaCl was added, mixed and placed in a 4°C environment for 3 hours, centrifuged to remove the supernatant, the precipitate was resuspended in 1 ml PBS, centrifuged for 1 minute, 50 single colonies were randomly selected from the plate with the titer of the last round of screening eluate and inoculated in 1 ml 2×YTAG, shaken and cultured for 12 hours, inoculated in 2×YTAG at a 1% inoculum, cultured to the early logarithmic growth stage, helper phage was added at a ratio of cells: phage = 1:1, cultured overnight, centrifuged for 1 minute, the precipitate was resuspended in an equal volume of 2×YTAG, vigorously shaken and cultured for 12 hours, and the supernatant was collected after centrifugation for ELISA identification. Positive clone judgment criteria: the ratio (S / N) of the test sample A value (S) to the negative control A value (N) ≥ 2.1, the positive clones were sequenced using specific primers MP57 and GⅢ to obtain the sequence encoding VHHs in the plasmid. The core coding sequence of the nanobody was obtained by sequence determination.

[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: Take 1 μL of the constructed pET23a plasmid containing the target gene and add it to 50 μL of E. coli BL21 (DE3) competent cells. Place it on ice for 30 minutes, then heat shock it in a 42℃ water bath for 60 seconds. Place it on ice for 5 minutes, then add 450 μL of LB culture medium to the bacterial solution. Mix well and place it in a 37℃ shaker at 200 rpm for 1 hour to recover. Then take 200 μL of bacterial solution and spread it on the LB+Amp solid plate, invert the plate and place it in a 37℃ incubator for overnight culture. Pick a single clone from the overnight culture plate and inoculate it in 5 mL LB+Amp culture medium. After shaking and culturing at 37°C and 200 rpm for 8 hours, transfer all the bacterial liquid to 4L LB+Amp culture medium, culture to the logarithmic phase, add 1mM IPTG for induction, culture at 16°C overnight, collect the bacterial precipitate by centrifugation the next day, add an appropriate amount of 1×PBS to resuspend it, and perform ultrasonic disruption at 112.5 w, 3 seconds of disruption, and 5 seconds of rest. Then, centrifuge at 4°C and 12000 rpm for 20 minutes, and collect the supernatant and precipitate after disruption. Figure 5 As shown, SDS-PAGE detection showed that nanoantibodies were present in the supernatant after disruption, proving 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, purified by nickel ion affinity chromatography and gel filtration chromatography (SuperdexTM75Increase Hiload column (GE Healthcare)), and the purified protein was identified by SDS-PAGE. The specific method is: 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 determined 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 position of the protein band was consistent with the size of the target protein.

[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 the following SEQ ID NO: 8 (ESGGSLVQAGGSLRLSCTASQAFGR NAIN WYRQAPGEQREWVA TITSGGVTSYAPAVKG RFAISRDSAKNTGYLQMNSLKVEDTAVYYCNS LWGGGSGHIPTDY WGQGTEVTVSS); wherein: positions 1-25 are FR1 framework region, positions 30-43 are FR2 framework region, positions 60-91 are FR3 framework region, positions 105-115 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 (NAIN) is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 (TITSGGVTSYAPAVKG) is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 (LWGGGSGHIPTDY) 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 alternately with CDR1, CDR2 and CDR3 in sequence.

[0055] The amino acid sequence of the FR1 framework region is shown in SEQ ID NO:4 (ESGGSLVQAGGSLRLSCTASQAFGR), the amino acid sequence of the FR2 framework region is shown in SEQ ID NO:5 (WYRQAPGEQREWVA), the amino acid sequence of the FR3 framework region is shown in SEQ ID NO:6 (RFAISRDSAKNTGYLQMNSLKVEDTAVYYCNS), and the amino acid sequence of the FR4 framework region is shown in SEQ ID NO:7 (WGQGTEVTVSS).

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

[0057] Example 5: ELISA detection of nanobody activity: Using an ELISA plate pre-coated with SV40LTA, dilute the purified NB as the primary antibody, and use HRP-labeled llama as the secondary antibody to perform an ELISA reaction to detect the activity of the nanobody. After the reaction is completed, add TMB-hydrogen peroxide urea solution colorimetric solution, color at 37°C in the dark for 15 minutes, and then add the stop solution to terminate the reaction. The absorbance value is measured within 15 minutes at a wavelength of 450nm using an ELISA reader.

[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 The NTA chip was selected and SV40LTA was fixed on the chip through the NTA chip. The fixed amount was about 100 RU. The SV40LTA-VHH protein was diluted in multiple ratios with PBST buffer 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, which proves that SV40LTA-VHH nanobody can bind to SV40LTA with high affinity.

[0061] Since its discovery, nanobodies have become a research hotspot in the field of infectious diseases, tumors and immune diseases due to their unique properties compared to ordinary antibodies, such as high water solubility and conformational stability, strong antigen affinity, and easy in vitro expression and humanization modification, etc., combined with phage display technology. Nanobodies can achieve the purpose of preventing diseases by binding to key proteins of pathogenic microorganisms, controlling the source of infection or cutting off the transmission route.

[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 immune selection, identified a high-affinity anti-SV40LTA nanoantibody NB1, which can effectively bind to SV40LTA. Subsequently, multivalent nanoantibodies can be constructed based on NB1 to enhance the binding effect on SV40LTA. The present invention lays a foundation for the detection and application of SV40LTA by screening high-affinity anti-SV40LTA nanoantibodies.

[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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 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, 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 of 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 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 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 shown in the following SEQ ID NO:8, wherein: positions 1-25 are FR1 framework regions, positions 30-43 are FR2 framework regions, positions 60-91 are FR3 framework regions, positions 105-115 are FR4 framework regions, CDR1 is inserted between FR1 framework regions and FR2 framework regions, CDR2 is inserted between FR2 framework regions and FR3 framework regions, and CDR3 is inserted between FR3 framework regions and FR4 framework regions.

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 also 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 SV40LTA as described in any one of claims 1 to 3 and a pharmaceutically acceptable carrier and / or excipient.

10. Use of an alpaca-derived nanobody or an antigen-binding fragment thereof that specifically binds to simian vacuolating virus 40 large T antigen SV40LTA as claimed in any one of claims 1 to 3 in the preparation of a kit for detecting SV40LTA.

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