Cattle TLR8 specific protein nano antibody and application thereof

Abstract

The invention discloses a bovine TLR8 specific protein nano antibody and application thereof, and belongs to the technical field of nano antibodies. According to the preparation method disclosed by the invention, three bovine TLR8 specific protein nano antibodies, namely, Nb-TLR8-4, Nb-TLR8-5 and Nb-TLR8-6, are successfully prepared; the prepared three nano antibodies are strong in specificity, high in purity, good in functional integrity and excellent in biological activity and reactogenicity; the provided three nano antibodies have different thermal stability, and the activity of Nb-TLR8-4 is stable before 60 DEG C and then gradually decreases; the activity of the Nb-TLR8-5 is stable before 50 DEG C, and is sharply reduced after 50 DEG C; the activity of the Nb-TLR8-6 is relatively low on the whole and is continuously reduced along with temperature rise, so that the Nb-TLR8-6 can be applied to different temperature environments and is beneficial to screening of nano antibodies adaptive to specific temperature conditions.

Description

Technical Field

[0001] This invention relates to the field of nanobody technology, and in particular to a bovine TLR8-specific protein nanobody and its applications. Background Technology

[0002] TLRs are an indispensable core component of the innate immune system. Their main functions include recognizing pathogen-associated molecular patterns (PRRs), promoting the activation of immune cells, and playing a crucial role in triggering immune responses. TLR7 and TLR8 are important members of the TLR family, primarily responsible for recognizing viral ssRNA and synthesizing ligands, forming a vital line of defense against viruses. When the body is attacked by a virus, TLR7 / 8 can be rapidly activated, then, through the MyD88-dependent signaling pathway, induce the expression of NF-κB and interferon, thereby promoting the release of pro-inflammatory factors (such as IL-6 and TNF-α) and initiating antiviral responses. However, overactivation or abnormal activation of TLR7 / 8 is closely related to antiviral immunity, autoimmune diseases, and cancer, while targeted regulation can be used for antiviral therapy or vaccine adjuvant development.

[0003] Classical antibodies have a structure with two heavy chains and two light chains, while HCAbs consist of only a single heavy chain and contain no light chain. The heavy chain of HCAbs (Heavychain Antibodies, HCAbs) comprises three globular domains, fewer than the four globular domains of classical antibodies, making HCAbs a unique single-domain antibody. The variable region of HCAbs is called the Variable Domain of the Heavy Chain of Heavy-Chain Antibodies (VHH), which can directly bind to the antigen and is independent of the light chain. This single-domain antibody is smaller and more stable than traditional antibodies, possessing unique functions and advantages, and is therefore called Nb.

[0004] Nanobodies (Nb) possess significant advantages: their molecular size is only about 1 / 10 that of ordinary antibodies, enabling them to penetrate tissues and cells more effectively and reach target areas that are difficult for traditional antibodies to access; furthermore, they maintain stable biological activity under extreme environments such as high temperatures, strong acids, or strong alkalis; they have strong affinity, can specifically bind to target molecules, and their preparation process is simple and low-cost. With these unique advantages, nanobodies show broad application prospects in disease detection, clinical treatment, and new drug development. Summary of the Invention

[0005] The purpose of this invention is to provide a bovine TLR8-specific protein nanobody and its application. The TLR8 nanobody provided by this invention has high specificity, high purity, good functional integrity, and excellent biological activity and reactivity, and can be used for the development of antiviral drugs, cancer therapeutics, and vaccine adjuvants.

[0006] To achieve the above objectives, the present invention provides a bovine TLR8-specific protein nanobody, comprising Nb-TLR8-4, Nb-TLR8-5, and Nb-TLR8-6; the amino acid sequence of Nb-TLR8-4 is shown in SEQ ID NO.5; the amino acid sequence of Nb-TLR8-5 is shown in SEQ ID NO.6; and the amino acid sequence of Nb-TLR8-6 is shown in SEQ ID NO.7.

[0007] A nucleic acid sequence encoding the above-mentioned bovine TLR8-specific protein nanobody.

[0008] An expression vector containing the above-mentioned nucleic acid sequence encoding a bovine TLR8-specific protein nanobody.

[0009] Preferably, the expression vector is modified from the pCANTAB5E-ccdb vector or the prokaryotic expression vector pET-30a(+).

[0010] A host cell containing the above expression vector, wherein the host cell is plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), or macrophages.

[0011] A TLR8 immunoassay kit, comprising the aforementioned bovine TLR8-specific protein nanobody.

[0012] Application of a bovine TLR8-specific protein nanobody as described above in the preparation of antiviral infection agents.

[0013] Application of a bovine TLR8-specific protein nanobody as described above in the preparation of cancer therapeutic drugs.

[0014] Application of a bovine TLR8-specific protein nanobody as described above in the preparation of vaccine adjuvants.

[0015] Therefore, the bovine TLR8-specific protein nanobody and its application provided by this invention have the following specific technical effects:

[0016] (1) The present invention successfully prepared three bovine TLR8-specific nanobodies, Nb-TLR8-4, Nb-TLR8-5 and Nb-TLR8-6; the three nanobodies prepared have high specificity, high purity, good functional integrity and excellent biological activity and reactivity.

[0017] (2) The three nanobodies provided by the present invention have different thermal stability. Nb-TLR8-4 has relatively stable activity before 60℃, and then gradually decreases; Nb-TLR8-5 has stable activity before 50℃, and then decreases sharply after 50℃; Nb-TLR8-6 has low overall activity, and continues to decrease with increasing temperature. It can be applied to different temperature environments and helps to screen nanobodies that are suitable for specific temperature conditions.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This refers to the antibody titer determination results of alpaca serum in Example 1 of the present invention;

[0021] Figure 2 This is the agarose gel electrophoresis result of the RNA obtained in Example 1 of the present invention;

[0022] Figure 3 These are the results of nested PCR amplification in Embodiment 1 of the present invention; where A represents the first round; B represents the second round; and M represents the DL 2000 Marker.

[0023] Figure 4 This is the result of phage display library capacity identification in Embodiment 1 of the present invention;

[0024] Figure 5 This is the result of phage display library abundance identification in Embodiment 1 of the present invention;

[0025] Figure 6 This is the positive rate identification result of the phage display library in Example 1 of the present invention; where M is DL2000 Marker; 1-48 are 48 single clones respectively;

[0026] Figure 7 This is the titration result of the M13K07 helper phage in Example 1 of the present invention;

[0027] Figure 8 This is the ELISA result of 96 monoclonal TLR8 proteins in Example 1 of this invention;

[0028] Figure 9 This is the Nb amino acid sequence alignment result in Example 1 of the present invention;

[0029] Figure 10 This is an electrophoresis diagram of the Nb gene amplification product in Example 2 of the present invention; where M is DL 500 Marker; 1 is Nb-TLR8-4; 2 is Nb-TLR8-5; 3 is Nb-TLR8-6; and 4 is blank control.

[0030] Figure 11 This is an electrophoresis image of the PCR product of a single clone of bacteria in Example 2 of the present invention; where M is DL 1000 Marker; 1 is Nb-TLR8-4 bacterial culture; 2 is Nb-TLR8-5 bacterial culture; 3 is Nb-TLR8-6 bacterial culture; and 4 is blank control.

[0031] Figure 12 This is an SDS-PAGE electrophoresis image of Nb expression detected in Example 2 of the present invention; where M is Protein Marker; 1 is the control (without IPTG); 2-3 are the supernatant and precipitate induced by Nb-TLR8-4, respectively; 4-5 are the supernatant and precipitate induced by Nb-TLR8-5, respectively; 6-7 are the supernatant and precipitate induced by Nb-TLR8-6, respectively.

[0032] Figure 13 This is an SDS-PAGE electrophoresis image of Nb purified in Example 2 of the present invention; where A is Nb-TLR8-4; B is Nb-TLR8-5; C is Nb-TLR8-6; M is Protein Marker; 1 is lysis buffer; 2 is flow buffer; 3-4 are denaturing lysis buffer washes; 5-6 are denaturing wash buffer washes; 7-9 are elution buffers;

[0033] Figure 14 These are the Western Blot identification results in Example 2 of this invention; where M is Protein Marker; 4 is purified Nb-TLR8-4; 5 is purified Nb-TLR8-5; and 6 is purified Nb-TLR8-6.

[0034] Figure 15 This refers to the Nb-specific detection results in Embodiment 3 of the present invention;

[0035] Figure 16 This is the thermal stability test result of Nb in Embodiment 3 of the present invention. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0039] All alpacas were provided by the alpaca breeding base of Shanxi Agricultural University. They were healthy, adult male alpacas that had not been vaccinated recently.

[0040] Example 1

[0041] The specific steps for preparing bovine TLR8-specific protein nanobodies are as follows:

[0042] S11. Immunizing Alpacas. Select healthy, adult male alpacas (60kg, 3 years old) that have not been vaccinated in the six months prior to immunization. Use purchased TLR8 protein as the antigen and administer immunization via subcutaneous injection at multiple sites. Inject 400μL of TLR8 antigen at a concentration of 1μg / μL (200μg / injection) subcutaneously into the alpaca's back. For the initial immunization, use complete Freund's adjuvant to dilute the antigen, emulsify it, and then inject it. For the second to fourth immunizations, use incomplete Freund's adjuvant to dilute the antigen, with each immunization spaced two weeks apart.

[0043] S12. Alpaca Antibody Titer Determination. On the fourth day after the final immunization in step S11, blood was collected intravenously and serum was separated. The titer of TLR8 protein-specific antibodies was detected using indirect ELISA, with pre-immunization serum serving as a control. The specific steps are as follows:

[0044] (1) Antigen coating: Using PBS as coating buffer, TLR8 protein was diluted to 2 μg / mL and added to the microplate at 100 μL / well. The plate was coated overnight at 4°C.

[0045] (2) Blocking: Discard the coating buffer, wash the plate 4 times with PBST, add blocking solution at 200 μL / well to block non-specific binding sites, and incubate at 37℃ for 1 h.

[0046] (3) Add alpaca serum: Discard the blocking solution, wash the plate 4 times with PBST, and serially dilute the alpaca serum sample with the blocking solution at a ratio of 100 μL / well. Incubate at 37°C for 1 h.

[0047] (4) Add enzyme-labeled antibody: Discard the antiserum, wash the plate 4 times with PBST, add HRP-labeled goat anti-alpaca IgG diluted to the working concentration at a rate of 100 μL / well, and incubate at 37°C for 1 h.

[0048] (5) Color development: Discard the enzyme-labeled antibody, wash the plate 4 times with PBST, add 100 μL of TMB substrate per well, and incubate at 37°C in the dark for 10 min.

[0049] (6) Termination: Add 50 μL / well of ELISA termination solution to terminate the colorimetric reaction, and measure the OD using a microplate reader. 450nm .

[0050] The results are as follows Figure 1 As shown, the titer reached 1:204800, which is significantly higher than the threshold standard required for library construction and fully meets the experimental requirements for subsequent nanobody library construction.

[0051] S13. Isolation of peripheral blood lymphocytes from alpacas.

[0052] (1) Anticoagulated blood was collected from the veins of the immunized alpacas two weeks after the fourth immunization.

[0053] (2) Gently mix and dilute the freshly collected anticoagulated blood with an equal volume of PBS by blowing and aspiration.

[0054] (3) Add the diluted anticoagulated blood to a 50mL centrifuge tube containing an equal volume of peripheral blood lymphocyte separation solution. Carefully and slowly spread the diluted anticoagulated blood on the upper layer of the separation solution using a Pasteur pipette. At this time, a clear dividing line will be observed.

[0055] (4) Centrifuge at 800 r / min for 20 min at room temperature, take the upper lymphocyte layer into a new 50 mL centrifuge tube, add 10 mL of PBS to wash, and centrifuge at 6000 r / min for 10 min at room temperature.

[0056] (5) Discard the supernatant and add 10 mL of PBS again to wash the precipitate. Centrifuge at 6000 r / min for 5 min.

[0057] (6) Discard the supernatant and resuspend the lymphocytes in 1 mL of PBS for later use.

[0058] S14. RNA was extracted from the lymphocytes resuspended in step S13 using the Trizol method. The quality of the obtained RNA was assessed using NANADROP 2000 and agarose gel electrophoresis, and high-quality RNA (such as...) was selected. Figure 2As shown, the 28S and 18S ribosomal RNA bands are clear, accompanied by a 5S band. The RNA concentration, A260 / A280, and A260 / A230 ratios are 240 ng / μL, 1.96, and 2.10, respectively, which are within the standard range. Reverse transcription was performed using a reverse transcription kit according to the attached instructions, and the obtained cDNA was stored at -20℃ for later use.

[0059] The concentration of cDNA, A260 / A280, and A260 / A230 were determined using a spectrophotometer, and the results were 1200 ng / μL, 1.90, and 2.00, respectively.

[0060] S15 and VHH fragment amplification.

[0061] (1) Design nested PCR primers for amplifying the VHH fragment (as shown in Table 1), and send the sequence information to a biotechnology company for primer synthesis.

[0062] Table 1. Primers for nested PCR amplification of the VHH fragment

[0063]

[0064] (2) After centrifuging the received primers, dilute them to 10mM with sterile ddH2O according to the attached instructions. Using the cDNA obtained in step S14 as a template, perform the first round of nested PCR amplification in the system shown in Table 2. The amplification program is as follows: 95℃ for 3 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0065] Table 2. First-round nested PCR amplification system

[0066]

[0067]

[0068] The PCR products were electrophoresed on a 1.5% agarose gel at 120V. The results are as follows. Figure 3 As shown in A, after cutting the target band around 700bp, the PCR product was purified by gel extraction using the kit according to the instructions. The obtained gel-extracted product was stored at -20℃ for later use.

[0069] (3) Using the purified first-round nested PCR gel product as a template, the reaction system was prepared according to Table 3 for the second round of nested PCR. The amplification program was the same as that for the first round of nested PCR. The PCR products were electrophoresed on a 1.5% agarose gel at 120V. The results are as follows: Figure 3As shown in B, the amplification product is the VHH fragment, which is approximately 350 bp in size, consistent with the expectation. After cutting out the target band, the PCR product was purified by gel extraction using the kit according to the attached instructions. 1 μL of the gel-extracted product was taken and the concentration was measured by spectrophotometer before being stored at -20℃ for later use.

[0070] Table 3. Second-round nested PCR system

[0071] PCR reagents Reagent dosage 2×MagicGreenTaqSuperMix 25μL VHH-F 3μL VHH-R 3μL Nested first-round PCR gel recovery products 2μL <![CDATA[ddH2O]]> 17μL

[0072] Construction of S16 recombinant plasmid pCANTAB-5E+VHH.

[0073] Take one vial of frozen pCANTAB-5E glycerol bacteria, use a sterile inoculation swab to streak the bacteria onto a 2×YTAG solid culture plate, and incubate at 37℃ for 12 hours. The next day, pick a single colony from the culture plate and inoculate it into 15mL×2YTAG medium, and incubate overnight at 37℃ and 220 rpm. Extract the plasmid pCANTAB5E-ccdb from the overnight culture according to the instructions of the Kangwei endotoxin-free plasmid extraction kit.

[0074] The second-round nested PCR gel-recovered product obtained in step S15 and the extracted empty vector pCANTAB5E-ccdb were digested with SfiI enzyme. The digestion reaction system is shown in Table 4. The reaction was carried out at 50℃ for 2 h. The digestion products were recovered using a gel recovery kit. Then, the gel-recovered VHH fragment and vector pCANTAB5E-ccdb were digested again using the same system and conditions (to ensure complete digestion and eliminate empty vectors). The digestion products were then recovered from the gel. Finally, the gel-recovered VHH fragment and vector pCANTAB5E-ccdb were ligated overnight at 16℃ using T4 DNA ligase.

[0075] Table 4. VHH fragment and pCANTAB5E-ccdb digestion system

[0076] Enzyme digestion reagent Reagent dosage Enzyme digestion reagent Reagent dosage 10×NEBuffer 25μL 10×NEBuffer 50μL SfiⅠ 20μL SfiⅠ 40μL VHH segment 20μg pCANTAB5e-ccdB 40μg <![CDATA[ddH2O]]> Up to 250 μL <![CDATA[ddH2O]]> Up to 500 μL

[0077] The ligation product was purified as follows:

[0078] The ligation product was placed in a 65°C water bath for 10 min to inactivate the enzymes. Then, 220 μL of DNA extraction buffer (phenol:chloroform:isoamyl alcohol volume ratio 25:24:1) was added, and the mixture was centrifuged at 18000 rpm for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube, and 22 μL of 3M NaAc (pH 5.2) and 500 μL of anhydrous ethanol (-20°C) were added. The mixture was then incubated at -80°C for 40 min. The mixture was centrifuged at 18000 rpm for 15 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of 75% ice-cold ethanol (-20°C). The mixture was centrifuged at 18000 rpm for 10 min at 4°C. The supernatant was discarded, and the mixture was air-dried in a clean bench for 10 min. The precipitate was then resuspended in 100 μL of deionized water and incubated at 4°C.

[0079] S17. Construct a phage display library.

[0080] 1 mL of ER2738 competent cells were fused with 100 μL of the ligation product purified in step S16 using an electroporator (electroporation parameters: 1800 V, 25 nF, 200 Ω, 5 ms). The fusion product was spread onto 20 90 mm diameter 2×YTAG solid culture plates and cultured overnight at 37 °C. The next day, the colonies were collected using 2×YT liquid medium, centrifuged at 2000 g for 5 min at 4 °C, the waste liquid was discarded, and the cells were resuspended in 8 mL of 2×YTAG liquid medium, thus successfully constructing the VHH phage display library.

[0081] Determine the volume: Take 100 μL of the electroporated bacterial culture and use a serial dilution method. First, add 100 μL of the bacterial culture to 900 μL of SOC medium and mix well to achieve a concentration of 10. -1 Then, take 100 μL of the bacterial culture and add it to another tube containing 900 μL of SOC medium, mix well, and the concentration will be 10. -2 Dilute sequentially, with the lowest concentration being 10. -8 The samples were coated on 2×YTAG plates, incubated overnight at 37°C, and then the volume was measured.

[0082] The results are as follows Figure 4 As shown, 10 -6 A total of 102 single-clone colonies were detected on the dilution plate, based on which the library volume was calculated to be 1.02 × 10⁻⁶. 8 CFU / mL. This result indicates that the constructed VHH phage display library has high diversity and abundance, providing a good foundation for subsequent antigen screening and antibody research.

[0083] Library abundance determination: Take 100 μL of primary library bacterial culture and determine the primary library abundance using the serial dilution method (the method is the same as for determining library volume).

[0084] The results are as follows Figure 5 As shown, 10 -8 There were 30 single-clone colonies on the dilution plate. The abundance of the phage display library was calculated to be 3.0 × 10⁻⁶. 10 CFU / mL. This provides a sufficient material basis for subsequent screening and functional studies, ensuring that multiple potential antibody sequences in the library can be covered during the screening process, thereby improving screening efficiency and success rate.

[0085] Recombination rate identification: 48 single-clone strains were randomly selected from the identification library volume gradient culture plate, cultured on 2×YTAG medium, and the library insertion rate was identified by bacterial PCR.

[0086] The results are as follows Figure 6 As shown, the insertion rate is 100%.

[0087] Diversity assessment: 96 monoclonal colonies that were identified as positive were sent to a biotechnology company for sequencing.

[0088] Sequencing results were compared with those obtained through NCBI analysis. The results showed that all sequences were highly homologous to alpaca VHH variants, but with diverse nucleotide compositions, fully demonstrating the diversity of the VHH gene. Experimental results confirmed that the constructed phage display library contained abundant VHH variants, providing high-quality gene resources for subsequent screening of specific antibodies. The library quality met experimental requirements.

[0089] Selection and identification of S18 and bovine TLR8 protein-specific Nb.

[0090] (1) Assisting in the determination of phage titers.

[0091] The titer of the prepared helper phage M13K07 was determined using the double-layer agar plate method. The phage was serially diluted 10-fold with 2×YT medium, and the dilution was taken as 10⁻⁶. -7 -10 -9 100 μL of each solution was used to infect 100 μL of ER2738 cells in the logarithmic growth phase at room temperature for 10 min. Then, 3 mL of 0.7% agarose at 47°C was added to each of the above dilutions, shaken well, and spread onto LB plates (preheated to 37°C). After solidification, the plates were incubated overnight at 37°C.

[0092] Count the number of plaques at each dilution and determine the titer. Results are as follows: Figure 7 As shown, in 10 9 260 plaques formed on the dilution culture plate. The titer of the helper phage was calculated to be 2.60 × 10⁻⁶. 12 The PFU / μL concentration indicates that the prepared helper phage M13K07 has extremely high activity and purity, which can fully meet the experimental requirements for subsequent phage library rescue.

[0093] (2) TLR8 protein-specific recombinant phage screening.

[0094] Immunotubes were coated with different concentrations of TLR8 protein (20 μg / mL, 10 μg / mL, and 5 μg / mL). Four rounds of specific phage panning were performed using the antigen-antibody binding property. After each round of panning, the phage infection efficiency and amplification titer were determined using a serial dilution method. The number of rescued phages was recorded as Input, and the number of eluted phages was recorded as Output. The recovery rate was calculated as Output / Input. The results are shown in Table 5. The phage recovery rate continuously increased after four rounds of panning, indicating that the specific VHH phages of TLR8 protein were continuously enriched.

[0095] Table 5. Enrichment of TLR8 protein by four-round phage washing.

[0096]

[0097] (3) Identification of specific, high-affinity positive phages.

[0098] After the phage elution from the fourth round of washing was used to infect ER2738 bacterial culture, it was serially diluted and plated. 96 single colonies were randomly selected and inoculated into 2×YTAG medium. After incubation at 37°C and 180 rpm for 8 h, 50 μL was aspirated from each well and transferred to fresh medium. The culture was then shaken until the OD value was reached. 600nm The concentration was 0.6. Helper phage was added, and the mixture was incubated at 37°C for 30 min with shaking. After incubation for 40 min with shaking, the mixture was centrifuged at 1800 rpm for 10 min. The supernatant was discarded, and the pellet was resuspended in each tube with 400 μL of 2×YTAK medium. The mixture was then incubated overnight at 37°C with shaking. The next day, the mixture was centrifuged at 5000 rpm for 10 min. 250 μL of phage supernatant was transferred from each tube to a new centrifuge tube, and 250 μL of blocking buffer was added to each tube. The mixture was then incubated at room temperature for 1 h for indirect ELISA detection.

[0099] Indirect ELISA test results as follows Figure 8 As shown in the figure. Positive clones with high binding affinity to TLR8 protein were selected and sent to Sangon Biotech for sequencing, successfully obtaining 20 valid sequences. A total of 3 valid TLR8 protein monoclonal clones were obtained. Analysis of the monoclonal clones yielded the following results: Figure 9 As shown, the amino acid sequences of the three monoclonal clones were found to be different. Based on the P / N value and absorbance, the VHH gene sequences of the three monoclonal clones were selected and named Nb-TLR8-4 (amino acid sequence as shown in SEQ ID NO.5), Nb-TLR8-5 (amino acid sequence as shown in SEQ ID NO.6), and Nb-TLR8-6 (amino acid sequence as shown in SEQ ID NO.7), respectively.

[0100] SEQ ID NO.5:

[0101] LQLVESGGGLVQAGGSLLRLSCAASGFSVDDYAIGWFRQAPGKEREGLSCISSG

[0102] NGITYYEDSIKGRFTVSSDNAKNTVYLQMNSLKPEDTAVYYCAAGNGVVGLL

[0103] GASYYYAEEYESWGQ GTQVTVSSAHHSEDPSS

[0104] SEQ ID NO.6:

[0105] LQLVESGGGSVEPGGSLRLSCTVSGFPFDEYVIGWFRQTPGKEREGILCISDKA

[0106] SHSDYSAEFIEGRFTAANDNAKNTAHLQMNNLKPEDTAVYYCAAIWGHYCSR

[0107] YDYNYKYWGQGTQVTVSSAHHSEDPSS

[0108] SEQ ID NO.7:

[0109] LQLVESGGGLVQTGGSLRLSCVTSGFTFDDRSIGWFRQAPGKERESVSCISITD

[0110] QKTYYPEAVKGRFTISRDNENHTVYLQMHNLKPEDTAVYYCAATPPNLGPCT

[0111] WFDLYQSWGQGTQVTVSSAHHSEDPSS

[0112] Example 2

[0113] The bovine TLR8-specific protein nanobody prepared in Example 1 was expressed, purified, and identified. The specific steps are as follows:

[0114] Construction of S21 and Nb prokaryotic expression recombinant plasmids.

[0115] Positive monoclonal bacteria obtained in Example 1 were selected, and plasmids were extracted and used as templates. The VHH gene was amplified using primers Nb-F (sequence shown in SEQ ID NO. 8) and Nb-R (sequence shown in SEQ ID NO. 9) according to the instructions accompanying the Taq enzyme package. The results are as follows: Figure 10 As shown, the amplified product exhibited a single specific band at approximately 400 bp, consistent with expectations, and was subsequently recovered via gel extraction. The prokaryotic expression vector pET-30a(+) was double-digested with BamHI and XhoI (digestion system and conditions followed the enzyme instructions). The VHH gene was ligated to the vector via seamless cloning, as shown in Table 6. After incubation at 50℃ for 1 h, the cells were transformed into DH5α competent cells and plated on LB / Kan plates. Two to three single colonies were picked from each plate for colony PCR identification. The results are shown below. Figure 11 As shown, the plasmid was extracted from the positive single-clone colony and sent to the company for sequencing. The sequence was compared with the target sequence using Megalign, indicating that the prokaryotic expression recombinant plasmid was successfully constructed and can be used for subsequent VHH gene expression and function studies.

[0116] SEQ ID NO.8:GCCATGGCTGATATCGGATCCTTGCAGCTCGTGGAGTC

[0117] SEQ ID NO.9:

[0118] GGTGGTGGTGGTGGTGCTCGAGGGAGCTGGGGTCTTCGCT

[0119] Table 6 Seamless Cloning System

[0120] Ligating reagents Reagent dosage Nb gene 10ng pET-30a(+) 50ng 2×SeamlessCloningMix 5μL <![CDATA[ddH2O]]> Up to 10 μL

[0121] Soluble expression and purification of S22 and Nb.

[0122] (1) The prokaryotic expression recombinant plasmid constructed in step S21 was transformed into BL21(DE3) competent cells and aseptically plated on LB / Kan plates and incubated overnight at 37°C. Single colonies were picked for PCR identification. Strains with positive results were inoculated into 100 mL of LB / Kan medium at a volume ratio of 1:100 and cultured in a shaker at 37°C and 220 r / min until the OD of the bacterial culture was reached. 600nmThe value was approximately 0.6. IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 16℃ and 180 rpm for 16 h. An empty vector was used as a control. After induction, the bacterial culture was collected, centrifuged at 7000 rpm for 10 min, and the supernatant was discarded. The liquid culture medium was washed with PBS, and the bacterial cells were resuspended in 20 mL of PBS. The cells were then sonicated on ice (4 sec, 6 sec intermittent, 100 W), centrifuged at 7000 rpm for 10 min at 4℃, and the supernatant was collected. 40 μL of the supernatant was then transferred to a sterile 1.5 mL centrifuge tube, 10 μL of 5× protein loading buffer was added, and the mixture was mixed by pipetting. The tube was then boiled in water at 100℃ for 10 min, centrifuged at 6000 rpm for 5 min, and the supernatant was collected for SDS-PAGE analysis to detect the expression of VHH protein.

[0123] The results are as follows Figure 12 As shown, Nb-TLR8-4, Nb-TLR8-5, and Nb-TLR8-6 are expressed in inclusion bodies.

[0124] (2) The successfully expressed Nb-TLR8-4, Nb-TLR8-5, and Nb-TLR8-6 proteins were purified using a His-tagged protein purification kit according to the accompanying instructions. The purified proteins were then analyzed by SDS-PAGE. The results are as follows: Figure 13 As shown, the target proteins appearing on the gel are all consistent with the expected size, indicating that Nb-TLR8-4, Nb-TLR8-5 and Nb-TLR8-6 proteins have been successfully purified.

[0125] The concentration of purified Nb protein was determined using the Bradford method with BSA as a standard. The results showed that the concentrations of Nb-TLR8-4 protein were 1.143 μg / μL, Nb-TLR8-5 protein were 0.818 μg / μL, and Nb-TLR8-6 protein were 0.848 μg / μL, indicating that all target proteins were effectively purified and their concentrations met the requirements for subsequent experiments.

[0126] (3) Using murine anti-His-tagged monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody, Western blot analysis was performed on the purified Nb-TLR8-4, Nb-TLR8-5, and Nb-TLR8-6 proteins. The results are as follows: Figure 14 As shown, the recombinant nanobody proteins exhibited clear, specific bands at their expected molecular weight positions, confirming that the purified nanobodies possess good biological activity and reactivity. The functional integrity of the target protein provides crucial experimental evidence for subsequent studies on its interaction with antigens and its immunological applications.

[0127] Example 3

[0128] The specificity and thermal stability of the purified Nb from Example 2 were identified, and the specific steps are as follows:

[0129] (1) Specific identification.

[0130] a. Coat 96-well microplates with TLR8 protein at a concentration of 5 μg / mL, 100 μL per well, and incubate overnight at 4°C. Use Nb-TLR8-4 / 5 / 6 protein and PBS as controls for coating the 96-well microplates.

[0131] b. Discard the antigen solution, wash the plate 5 times with PBST, add 100 μL of blocking solution to each well to block non-specific binding sites, and incubate at 37°C for 1 hour.

[0132] c. Discard the blocking solution, wash the plate 5 times with PBST, add 100 μL of twice diluted Nb to each well, and incubate at 37°C for 1 h.

[0133] d. Discard Nb, wash the plate 5 times with PBST, add 100 μL of mouse anti-His tag monoclonal antibody diluted 1:4000 to each well, and incubate at 37°C for 1 h.

[0134] e. Discard the mouse anti-His tag monoclonal antibody, wash the plate 5 times with PBST, add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:10000 to each well, and incubate at 37°C for 1 h.

[0135] f. Discard the enzyme-labeled secondary antibody, wash the plate 5 times with PBST, add 100 μL of TMB chromogenic substrate to each well, and incubate at 37°C in the dark for 10 min.

[0136] g. Termination of reaction. Add 50 μL of ELISA stop solution to each well to terminate the colorimetric reaction, and measure the OD using a microplate reader. 450nm .

[0137] The results are as follows Figure 15 As shown, the Nb-TLR8-5 nanobody exhibits significantly higher reactivity than other clones, and its titer level shows a stronger signal intensity in the detection system.

[0138] (2) Thermal stability assessment.

[0139] The purified nanobodies from Example 2 were diluted to a concentration of 100 μg / mL and then placed in water baths at 4°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C, respectively. After incubation for 2 hours, the nanobodies were allowed to equilibrate to room temperature, and then each nanobodies were diluted to 5 μg / mL. Next, the treated histones were serially diluted with 1% skim milk to a series of concentration gradients to serve as primary antibodies, which were then added to the corresponding wells of overnight-coated ELISA plates. The secondary antibody was a mouse anti-His-tagged monoclonal antibody diluted 1:5000, and the triclonal antibody was HRP-labeled goat anti-mouse IgG diluted 1:10000. The remaining antibody activity was determined using an indirect ELISA method.

[0140] The results are as follows Figure 16 As shown, Nb-TLR8-4 exhibited relatively stable activity up to 60℃, which gradually decreased thereafter; Nb-TLR8-5 showed stable activity up to 50℃, which decreased sharply after 50℃; and Nb-TLR8-6 showed generally low activity, which continued to decrease with increasing temperature. These results indicate significant differences in the thermal stability of different nanobodies, providing crucial activity assessment criteria for their application in different temperature environments (such as high-temperature or room-temperature scenarios), and aiding in the screening of nanobodies suitable for specific temperature conditions.

[0141] Therefore, this invention successfully prepared three bovine TLR8-specific nanobodies: Nb-TLR8-4, Nb-TLR8-5, and Nb-TLR8-6. The three nanobodies exhibit high specificity, high purity, good functional integrity, and excellent biological activity and reactivity. The three nanobodies possess different thermal stabilities: Nb-TLR8-4 shows relatively stable activity up to 60°C, which gradually decreases thereafter; Nb-TLR8-5 shows stable activity up to 50°C, which decreases sharply after 50°C; and Nb-TLR8-6 exhibits generally low activity, which continues to decrease with increasing temperature. This allows for application in different temperature environments and helps in screening nanobodies suitable for specific temperature conditions.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A bovine TLR8-specific protein nanobody, characterized in that: Including Nb-TLR8-4, Nb-TLR8-5 and Nb-TLR8-6; the amino acid sequence of Nb-TLR8-4 is shown in SEQ ID NO.5; the amino acid sequence of Nb-TLR8-5 is shown in SEQ ID NO.6; the amino acid sequence of Nb-TLR8-6 is shown in SEQ ID NO.

7.

2. The nucleic acid sequence encoding the bovine TLR8-specific protein nanobody of claim 1.

3. An expression vector, characterized in that: The expression vector contains the nucleic acid sequence of the bovine TLR8-specific protein nanobody as described in claim 2.

4. An expression vector according to claim 3, characterized in that: The expression vector was modified from the pCANTAB5E-ccdb vector or the prokaryotic expression vector pET-30a(+).

5. A host cell containing the expression vector according to claim 3 or 4, characterized in that, The host cells are plasmacytoid dendritic cells, myeloid dendritic cells, or macrophages.

6. A TLR8 immunoassay kit, characterized in that: The kit contains the bovine TLR8-specific protein nanobody as described in claim 1.

7. The use of the bovine TLR8-specific protein nanobody as described in claim 1 in the preparation of antiviral infection agents.

8. The use of the bovine TLR8-specific protein nanobody as described in claim 1 in the preparation of cancer therapeutic drugs.

9. The application of the bovine TLR8-specific protein nanobody as described in claim 1 in the preparation of vaccine adjuvants.

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