Nanometer antibody targeting hepatitis B core antigen as well as coding gene and application thereof

Abstract

The invention discloses a nano antibody targeting a hepatitis B core antigen as well as a coding gene and application thereof, and relates to the technical field of biological medicines. The nano antibody comprises an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, and can be specifically combined with HBcAg. The invention also relates to a coding nucleic acid, an expression vector, a host cell and a preparation method of the nano antibody, and application of the nano antibody in preparation of anti-hepatitis B virus drugs and diagnostic reagents. Experiments show that the nano antibody has high affinity of targeting HBcAg, can combine and destroy hepatitis B virus nucleocapsid assembly and inhibit secretion of virus proteins such as HBsAg, HBeAg and the like, and thus has good anti-hepatitis B virus activity.

Description

Technical Field

[0001] This invention relates to the field of nanobody technology, and in particular to a nanobody targeting the hepatitis B core antigen, its encoding gene, and its applications. Background Technology

[0002] Hepatitis B virus (HBV) infection is a major global public health problem. HBcAg is the nucleocapsid protein of HBV, which is crucial for the assembly and replication of viral particles. Unlike hepatitis B surface antigen (HBsAg) secreted into the bloodstream, HBcAg is mainly found inside viral particles and in the nuclei of infected hepatocytes, making it a key target for persistent viral infection and immune response.

[0003] Currently, the development of therapeutic antibodies against HBcAg, especially traditional monoclonal antibodies, is progressing slowly, mainly due to their large molecular weight, poor tissue penetration, and high production costs. Nanobodies, derived from single-domain heavy chain antibodies from camelids, possess advantages such as small molecular weight, high stability, strong penetration, and ease of large-scale production, making them ideal candidates for next-generation biopharmaceuticals.

[0004] However, efficiently screening nanobodies with high affinity and specificity for HBcAg and viral inhibitory function from a vast library of natural antibodies remains a significant challenge. Existing screening techniques are complex and time-consuming, and the antibodies obtained often fail to achieve a balance between binding activity and function. Therefore, there is an urgent need in this field for a method that can efficiently screen and obtain high-performance, specific nanobodies against HBcAg, as well as candidate molecules with clear therapeutic and diagnostic value obtained through this method. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a nanobody targeting the hepatitis B core antigen, its encoding gene, and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A nanobody targeting the hepatitis B core antigen, comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0007] Preferred: The nanobody described above is composed of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0008] A nanobody targeting the hepatitis B core antigen has at least 90% sequence identity with the above-mentioned amino acid sequence and can specifically bind to HBcAg.

[0009] An isolated nucleic acid molecule encoding a nanobody as described above.

[0010] An expression vector containing the aforementioned nucleic acid molecules.

[0011] Preferably, the expression vector described above is a prokaryotic expression vector, a yeast expression vector, an insect cell expression vector, or a eukaryotic cell expression vector, and the nucleic acid molecule is cloned between any restriction enzyme sites in the multiple cloning site (MCS) region of the vector.

[0012] A host cell containing the aforementioned expression vector.

[0013] A method for recombinantly preparing nanobodies as described above includes the following steps: a) Culture the above-mentioned host cells under suitable expression conditions; b) Isolate and purify the nanobody from the culture.

[0014] A recombinant nanobody is composed of the above-mentioned nanobody fused with a purified tag.

[0015] Preferably, the purification tag of the recombinant nanobody described above is a histidine tag.

[0016] A fusion protein comprising the aforementioned nanobody and a functional portion linked thereto.

[0017] A pharmaceutical composition comprising a therapeutically effective amount of a nanobody or fusion protein as described above, and a pharmaceutically acceptable carrier.

[0018] Preferred: The pharmaceutical composition described above is used to inhibit hepatitis B virus replication.

[0019] Preferably, according to the above-described pharmaceutical composition, the inhibition of hepatitis B virus replication is manifested in reducing the expression levels of Danscher particles, subviral particles, and secreted proteins of hepatitis B virus in HepG2.2.15 cells or AD38 cells; and reducing the expression levels of hepatitis B virus particles, subviral particles, and secreted proteins in HBV transgenic mice.

[0020] A diagnostic kit for hepatitis B virus infection, comprising nanobodies or fusion proteins as described above.

[0021] A method for screening anti-HBcAg nanobodies includes the following steps: S1: Prepare a natural alpaca nanobody library; S2: Using HBcAg as the target antigen, at least three rounds of biological screening are performed using phage display technology; S3: Perform monoclonal ELISA validation on the phage elution buffer after the last round of screening to identify positive clones that bind to HBcAg and whose OD450nm detection value is more than 2.1 higher than that of the negative control. S4: Sequencing the positive clones to obtain the coding sequence of the nanobody.

[0022] Preferred method: According to the above method, each round of screening in step S2 includes: incubating the phage library with an immunotube coated with HBcAg; washing away unbound phages; and eluting specifically bound phages with trypsin solution.

[0023] The beneficial effects of this invention are as follows: 1. This invention employs optimized phage display technology for three rounds of biological screening, combined with highly sensitive monoclonal ELISA verification, to ensure rapid and accurate screening of high-affinity clones.

[0024] 2. This invention was verified by multiple binding assays, including ELISA and ITC (isothermal titration microcalorimetry). The nanobody obtained by this invention can specifically bind to HBcAg and has almost no cross-reactivity with the negative control (BSA). Sequencing analysis further confirmed its unique amino acid sequence, providing a structural basis for high affinity binding.

[0025] 3. In this invention, the target gene was cloned into the pET-28a(+) vector and successfully expressed in soluble form in Escherichia coli Shuffle T7 strain. Through the established nickel column affinity chromatography purification process, high-purity nanobody protein was obtained, with molecular weight consistent with the predicted size, providing sufficient material support for subsequent functional studies and application development.

[0026] 4. The cytotoxicity experiments conducted by the CCK-8 assay showed that even at high concentrations, the nanobody of the present invention did not exhibit significant toxicity to liver-derived cell lines such as AD38 and HepG2.2.15.

[0027] 5. In AD38 and HepG2.2.15 cell lines, the nanobodies (NbA and NbB) of the present invention significantly reduced the levels of HBsAg and HBeAg in the culture supernatant in a dose-dependent manner, directly demonstrating their strong biological function in inhibiting hepatitis B virus replication and showing clear potential for therapeutic development. Attached Figure Description

[0028] Figure 1This is a graph showing the ELISA verification results of the nanobody screening in this invention; Figure 2 This is a diagram showing the results of the enzyme-linked immunosorbent assay (ELISA) of this invention. Figure 3 This is a diagram showing the results of the first part of the ITC experiment of this invention; Figure 4 This is a diagram showing the results of the second part of the ITC experiment of this invention; Figure 5 This is a graph showing the results of the CCK-8 assay for cytotoxicity in this invention. Figure 6 This is a schematic diagram illustrating the antiviral effect of NbA in the AD38 and HepG2.2.15 cell lines of the present invention; Figure 7 This diagram illustrates the antiviral effect of NbB in the AD38 and HepG2.2.15 cell lines of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0030] Example 1: A nanobody specifically targeting hepatitis B core antigen: 1. Phage display technology was used to screen for hepatitis B core antigen-specific nanobodies using a natural alpaca nanobody library. After screening, the obtained proteins were further screened for specificity using enzyme-linked immunosorbent assay (ELISA), followed by deep sequencing to obtain target clones for monoclonal isolation and then large-scale production. The specific screening method is as follows: 2.1 First round of screening 2.1.1 Remove the screening antigen from the -80℃ freezer, thaw it on ice, and aliquot it into 100 µg tubes; 2.1.2 Coat the screening antigen-coated immunosorbent tubes (50 µg / tube, coating solution: CBS, pH 9.6, 2 ml / tube) (CBS (pH 9.6): Na2CO3 3.03 g, NaHCO3 6.0 g, add to 1000 ml sterile water, mix thoroughly to dissolve, store at 4°C), and slowly rotate overnight at 4°C; 2.1.3 Discard the liquid in the overnight coated immunotubes, add 2 ml of PBS buffer and wash the immunotubes 3 times at room temperature, rotating for 5 min each time; 2.1.4 Add 2 ml of blocking solution (3% BSA) and incubate at room temperature by rotation for 2 h; 2.1.5 Discard the liquid in the sealed immunosorbent tube and add 2 ml of PBST buffer to wash the immunosorbent tube 3 times at room temperature, rotating for 5 min each time; 2.1.6 Discard the washing buffer in the immunofluorescence tube, add 2 ml of PBS, and calculate and add the prepared phage library according to the following formula (the volume of the library added depends on the titer of the phage library). This is used as the input phage library for the first round of screening. Incubate at room temperature by rotation for 1 h.

[0031] Where V is the volume of added phage (µl), and Tlibrary is the phage titer; 2.1.7 Discard the liquid in the immunoassay tube, add 2 ml of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer and wash the immunoassay tube 20 times at room temperature, rotating for 5 min each time; 2.1.8 Discard the liquid in the immunosorbent assay tube, remove as much residual liquid as possible, add 1 ml of 0.25 mg / ml Trypsin solution, and elute by rotating at room temperature for 30 min; 2.1.9 Add 10 μl of 10% AEBSF to stop elution, and transfer the solution in the immunoassay tube to a new 1.5 ml centrifuge tube, which is the first round of phage screening elution solution.

[0032] 2.2 First-round phage eluent titer detection 2.2.1 The TG1 strain stored at -80℃ was streaked on 2×YT solid medium (without resistance) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium and incubated overnight at 37℃. 2.2.2 Take 500 μl of overnight culture and transfer it to 5 ml of 2×YT liquid medium. Incubate at 37℃ and 250 rpm for about 45 min-60 min until the OD600 is 0.5-0.55. 2.2.3 Take 10 μl of the first round of phage elution buffer and serially dilute it 10-fold in a 1.5 ml centrifuge tube. Repeat this process 12 times. Specifically, take 10 μl of the first round of phage elution buffer and dilute it to 100 μl. Mix well and then take another 10 μl of the buffer and dilute it to 100 μl. Continue this process until you have a total of 12 serial dilutions to 10⁻¹². Shake well to mix. 2.2.4 Add 90 μl of TG1 bacterial culture to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min; 2.2.5 Take 5 μl from each dilution centrifuge tube and add it to 2×YT solid medium (Amp), and incubate overnight at 37°C upside down; 2.2.6 The statistical panel clearly distinguishes the number of single colonies at different dilutions. The number of phage particles per milliliter of phage solution, i.e., the phage library titer, is calculated using the following formula:

[0033] Where T is the phage titer (unit: pfu / ml), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0034] 2.3 Amplification of the first round of phage elution buffer 2.3.1 The TG1 strain stored at -80℃ was streaked onto 2×YT solid medium (without resistance) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium and incubated overnight at 37℃. 2.3.2 Take 500 μl of overnight culture and transfer it to 5 ml of 2×YT liquid medium. Incubate at 37℃ and 250 rpm for about 45 min-60 min until the OD600 value is 0.5-0.55. 2.3.3 Add 500 μl of the phage elution buffer obtained after the first round of screening to the bacterial culture with an OD600 of 0.5-0.55 (store the remaining elution buffer at 4°C). 2.3.4 Continue culturing at 37℃ and 250 rpm for 30 min; 2.3.5 Spread all bacterial cultures evenly onto 10 2xYT medium (10cm x 10cm) plates containing 100 μg / ml Amp and 2% agarose, and incubate overnight at 37°C; 2.3.6 Take the culture plate that has been incubated overnight, add 1 ml of 2×YT liquid medium to each surface of the culture plate, gently scrape off the colonies from the square plate with a spreader and collect all the bacterial solution into a 15 ml centrifuge tube, which is the amplified bacterial sub-library. At the same time, use a spectrophotometer to measure the OD600 value of the bacterial solution, which is the OD600 value of the elution buffer bacterial library. Take out the corresponding volume of the library (the calculation method is shown in 2.3.7) and add glycerol to a final concentration of 20%, which is the first round of bacterial library.

[0035] 2.3.7 Calculate the corresponding bacterial volume of the eluted bacterial library using the following formula, and transfer it to 100 ml of 2×YT liquid medium (100 μg / ml Amp) to achieve an initial OD600 of 0.1:

[0036] Where V is the volume of the transferred bacterial solution (in μl), and OD600 is the OD600 of the constructed elution bacterial library; 2.3.8 Incubate at 37℃ and 250 rpm until the bacterial OD600 reaches 0.5-0.55; 2.3.9 Calculate and add helper phage M13K07 according to the following formula to make the ratio of bacteria to phages = 1:20.

[0037] Where V is the volume (in ml) of helper phage added. The titer of the helper phage used is given; OD 600 is the absorbance of the bacterial culture at 600 nm; culture continued at 37°C and 250 rpm for 30 min. 2.3.11 Add Kanamycin to a final concentration of 50 μg / ml and incubate overnight at 30℃ and 250 rpm.

[0038] 2.4 First round of phage purification 2.4.1 Transfer the overnight culture to a new 50 ml centrifuge tube and centrifuge at 4000 rpm for 10 min at 4°C; 2.4.2 Transfer the supernatant after centrifugation to a new 50 ml centrifuge tube, add 1 / 4 volume of 20% PEG / 2.5M NaCl pre-cooled at 4℃, mix thoroughly, and place on ice for 30 min; 2.4.3 Centrifuge at 4000 rpm and 4℃ for 20 min, discard the supernatant, and invert on paper for 2 min; 2.4.4 Add 1 ml of PBS to resuspend the precipitate, transfer the resuspended solution to a new 1.5 ml centrifuge tube, and centrifuge at 13000 rpm and 4°C for 20 min; 2.4.5 Transfer the supernatant after centrifugation to a new 1.5 ml centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix well and place on ice for 10 min; 2.4.6 Centrifuge at 13000 rpm, 4℃ for 10 min, discard the supernatant, and resuspend the precipitate in 1 ml PBS; 2.4.7 Centrifuge at 13000 rpm, 4℃ for 2 min, and transfer the supernatant to a new 1.5 ml centrifuge tube. This is the first round of phage sub-liquid selection. Aliquot 100 μl / tube and store at -80℃ for long-term storage. For short-term storage (1-2 weeks), store at -20℃. 2.4.8 First-round screening of phage sub-library titer detection, the method is the same as 2.2.

[0039] 2.5 Second round of screening: The screening method is the same as in 2.1. The input phage is 0.25 ml of the phage sub-library obtained from the first round of screening, which is used as the input phage library for the second round of screening to obtain the phage elution buffer for the second round of screening.

[0040] 2.6 The method for detecting the titer of the second round of phage eluent is the same as in 2.2.

[0041] 2.7 The amplification and purification methods for the second round of elution buffer are the same as those in 2.3-2.4, to obtain the second round of phage sub-libraries for screening.

[0042] 2.8 Second round of screening phage sub-library titer detection, the method is the same as 2.2.

[0043] 2.9 Third round of screening (selected based on the second round of screening) The screening method is the same as 2.1. The input phage is 0.25 ml of the phage sub-library obtained from the second round of screening, which is used as the input phage library for the third round of screening to obtain the phage elution buffer for the second round of screening.

[0044] 2.10 The method for detecting the titer of the third round of phage eluent is the same as in 2.2.

[0045] 2.11 Monoclonal ELISA Detection 2.11.1 The TG1 strain stored at -80℃ was streaked onto 2×YT solid medium (without resistance) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium and incubated overnight at 37℃. 2.11.2 Take 500 μl of overnight culture and transfer it to 5 ml of 2×YT liquid medium. Incubate at 37℃ and 250 rpm for about 45 min-60 min until the OD600 value is 0.5-0.55. 2.11.3 Take 10 μl of phage elution buffer after the third round of screening and serially dilute it 10-fold in a 1.5 ml centrifuge tube. Repeat this process 12 times. Specifically, take 10 μl of the phage library and dilute it to 100 μl, then take another 10 μl of the phage library and dilute it to 100 μl, and so on, for a total of 12 serial dilutions. Shake well to mix. 2.11.4 Add 90 μl of bacterial suspension with an OD600 value of 0.5-0.55 to each dilution centrifuge tube and mix well; 2.11.5 Continue culturing at 37℃ and 250 rpm for 30 min; 2.11.6 Spread the bacterial culture evenly onto a solid culture medium plate containing 100 μg / ml Amp and incubate overnight at 37°C; 2.11.7 Randomly pick single colonies from the overnight culture medium plates and place them into sterile 96-well cell culture plates (P1-P2). Add 200 µl of 2×YT medium (100 µg / ml Amp) to each well and incubate at 37°C overnight. 2.11.8 Take 2 µl of the overnight culture and transfer it to a new 96-well cell culture plate with 200 µl of 2×YT liquid medium (100 µg / mlAmp) per well. Incubate at 37 °C for 3 h. Store the overnight culture at 4 °C before transfer. 2.11.9 Calculate and add helper phage M13K07 to each well according to the following formula to achieve a bacterial count: phage count ratio of 1:20.

[0046] Where V is the volume (in ml) of helper phage added. The titer of the helper phage used; 2.11.10 Incubate at 37 ℃ for 30 min, then incubate at 30 ℃ overnight; 2.11.11 After overnight incubation, the 96-well culture plate was centrifuged at 4000 rpm for 10 min at 4 ℃ and stored at 4 ℃ for later use; 2.11.12 Coat the screening antigen onto the ELISA plate (1 ng / μl, coating solution is CBS pH 9.6, 100 μl / well), and simultaneously coat with BSA as a control, and incubate overnight at 4°C; 2.11.13 Discard the liquid in the overnight coated microplate, add 200 µL of PBS buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time; 2.11.14 Add 200 μl of blocking buffer (3% BSA) to each well to block the microplate and incubate at room temperature for 1 h; 2.11.15 Discard the blocking solution, add 200 µL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time; 2.11.16 Add 120 µl of 3% BSA to each well, followed by 80 µl of the supernatant from centrifugation in step 2.11.11, and incubate at room temperature for 2 h; 2.11.17 Discard the liquid in the microplate, add 200 µl of PBST buffer to each well and wash 3 times, 10 min each time; 2.11.18 Add M13 Bacteriophage Antibody (HRP) and Mouse Mab, diluted 1:40000 in blocking buffer to each well, 100 μl / well, and incubate at room temperature for 1 h; 2.11.19 Discard the liquid in the ELISA plate, add 200 µl of PBST buffer to each well and wash 3 times, 10 min each time; 2.11.20 Add 100 µl of TMB single-component colorimetric solution to each well, incubate in the dark for 2-3 min, then add 100 µl of 1M HCl to each well to stop the reaction. Read the OD 450 nm value using a microplate reader, record and save the data.

[0047] 2.12 Secondary ELISA validation of positive clones To rule out false positive results, clones initially identified as positive were subjected to a second ELISA verification, using the same method as in section 2.9.

[0048] 2.13 Sequencing of positive clones Positive monoclonal antibodies were selected based on ELISA test data and secondary validation data. Take 5 µl of positive clone bacterial culture from the monoclonal ELISA plate (2.11.7) and inoculate it into 1 ml of 2×YT medium (100 µg / ml Amp). Incubate at 37°C and 250 rpm until the OD600 reaches 0.8-1.0 (approximately 6-8 h). Sequencing is performed on 0.5 ml of the bacterial culture, and the remaining bacterial culture is stored at 4°C. Simultaneously, the third round of elution buffer is used for full-sequence deep sequencing.

[0049] 2.14 Sequence Analysis The sequenced sequences were analyzed using Snap Gene software for sequence alignment, and the antibody sequences were translated into amino acids using Snap Gene software.

[0050] like Figure 1 The results showed that, according to the phage ELISA results, 39 out of 48 single colonies had a ratio exceeding 3 compared to the negative control, with 15 of these negative control colonies having a ratio exceeding 10, the highest reaching a 65-fold difference. This indirectly suggests that these sequences may have a high affinity for HBsAg. Subsequently, the top 5 sequences were sequenced, and the sequencing results were compared with the full-length deep sequencing results. The 5 sequences with the strongest affinity were selected to synthesize a plasmid.

[0051] The five amino acid sequences are as follows:

[0052] Expression and purification of HBcAg-specific nanobodies 1. The five sequences obtained from sequencing were subcloned into the pet-28a(+) vector, with the restriction enzyme site being N. de I and X hoI. Transform the recombinant plasmid into the *E. coli* Shuffle T7 expression strain, select single clones and inoculate them into 2 mL LB-Kana medium, incubate overnight at 37°C and 250 rpm; then inoculate 1% (V / V) into 400 mL LB-Kana medium (in a 1000 mL shake flask), incubate at 37°C and 250 rpm until the OD 600 is approximately 0.6; (3) Add IPTG to a final concentration of 0.3 mM, and induce overnight at 19°C and 170 rpm; (4) Centrifuge (round bottom centrifuge tube) at 4000 rpm for 20 min, discard the supernatant and collect the bacterial cells; (5) Take the above bacteria and resuspend them in binding buffer (PBS, 150 mM NaCl, 10 mM imidazole); use 40 mL of binding buffer for every 400 mL of bacterial solution; (6) Ultrasonic disruption of bacterial cells; ultrasonic conditions: 25-35 min, 4 s intervals with 6 s intervals, 38% power; (7) Centrifuge at 12000 rpm at 4℃ for 20 min, take the supernatant and filter it through a 0.45 μm filter membrane for purification.

[0053] Purification of anti-hepatitis B core antigen nanobodies: (1) All reagents used in the purification process need to be filtered through a 0.45 μm filter membrane in advance to prevent column blockage; (2) Add 5 column volumes of ultrapure water to rinse the Ni column; (3) Add 5 column volumes of binding buffer (PBS, 150 mM NaCl, 10 mM imidazole) to equilibrate the column; (4) Add the membrane-coated sample into the Ni column and collect the eluent; (5) Add 5 column volumes to the buffer leveling column; (6) Elute the nanobody sequentially with binding buffers containing 40 mM, 60 mM, 90 mM and 400 mM imidazole, and collect the eluent; (7) Thoroughly wash the column with 5 mL of 400 mM imidazole solution; (8) Add 5 column volumes of buffer to clean (equilibrate) the Ni column; (9) Clean the Ni column with 5 column volumes of ultrapure water; (10) Preserve the column with 20% ethanol.

[0054] See results Figure 2 As can be seen, the molecular weight of the nanobody is consistent with the predicted size.

[0055] The specificity of the expressed and purified nanobodies was detected by ELISA, and the results are shown in the figure. Figure 3 The figure shows an ELISA plate coated with HBcAg recombinant antigen, with bovine serum albumin (BSA) as a control. It can be seen that the nanobody of this invention can specifically bind to the hepatitis B core antigen, and exhibits strong specificity.

[0056] The specificity of the above nanobodies was tested, and the results are shown in [the table below]. Figure 4 .

[0057] Method: First, five nanobodies were labeled with HRP tags. Then, HBcAg recombinant antigen and BSA were coated on an ELISA plate. The labeled nanobodies (HRP) were then used to bind to the antigens on the ELISA plate, and finally, TMB single-component chromogenic solution was used for color development.

[0058] 1. Coat the plates one night in advance (100 ng / well, with BSA as a control (negative) 100 μl / well, and set up a positive control (HBcAg-His coating, with HRP-labeled antibody against His tag for color development). 2. Wash with 200 μl PBS per well 3 times for 10 min each time; Block with 3.5% BSA for 2 h, then wash 8 times with PBST; 4. Dilute Nb-HRP 10,000 times (5% BSA), add 100 μL to each well and incubate at room temperature for 2 h; 5. Wash 8 times with PBST; 6. Add 100 μl of TMB colorimetric solution and incubate at 37°C for 30 min; 7. Add 100 μl of 0.5 M H2SO4 to terminate the reaction.

[0059] Verification of antiviral activity at the cellular level: Methods: CCK-8 cytotoxicity assay, such as... Figure 5 As shown; 1. Cell counting; 2. Seed cells: 10,000 cells per well (100 μL). Set up experimental, control, and blank groups, and add PBS around the cells (to prevent evaporation). (Experimental group: cells, culture medium, Nb. Control group: cells, culture medium. Blank group: culture medium.)

[0060] 3. After plating, incubate in an incubator for 24 h until the cells adhere well. Aspirate the culture medium, add different concentrations of Nb (detoxified) to each well, add fresh culture medium, and incubate for 24 h. 4. Add 10 μl of CCK-8 to each well, mix well (to prevent air bubbles), and incubate for 1-2 hours; 5. Measure OD 450.

[0061] Antiviral experiments, such as Figure 6 , Figure 7 As shown; Experimental methods: Plate formation: AD38 and HepG2.2.15 were seeded evenly in 12-well plates and incubated at 37°C for 24 h. After 24 h, the culture medium in the culture dish was replaced, and different concentrations of nanobodies (A, B) were added to the new culture medium in a gradient. After culturing for another 72 h, the supernatant was collected, and the expression of HBsAg and HBeAg in the supernatant was detected.

[0062] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A nanobody targeting the hepatitis B core antigen, characterized in that, It contains an amino acid sequence selected from or composed of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

2. A nanobody targeting the hepatitis B core antigen, characterized in that, It has at least 90% sequence identity with the amino acid sequence described in claim 2 and is capable of specifically binding to HBcAg.

3. A nucleic acid molecule encoding an isolated nanobody as described in claim 2.

4. An expression vector comprising the nucleic acid molecule of claim 3; wherein the nucleic acid molecule is a prokaryotic expression vector, a yeast expression vector, an insect cell expression vector, or a eukaryotic cell expression vector, and the nucleic acid molecule is cloned between any restriction enzyme sites in the multiple cloning site region of the vector.

5. A host cell comprising the expression vector of claim 4.

6. A method for recombinantly preparing nanobodies as described in any one of claims 1-2, characterized in that, Includes the following steps: a) Culture the host cells of claim 5 under conditions suitable for expression; b) Isolate and purify the nanobody from the culture.

7. A recombinant nanobody, comprising the nanobody of any one of claims 1-2 fused with a purified tag; wherein the purified tag is a histidine tag.

8. A fusion protein comprising the nanobody of any one of claims 2 and the functional portion thereto.

9. A pharmaceutical composition comprising a therapeutically effective amount of the nanobody of claim 1 or a therapeutically effective amount of the fusion protein of claim 8, and a pharmaceutically acceptable carrier, which inhibits the expression and secretion of hepatitis B virus particles by interfering with or reducing the expression level of HBcAg in vivo.

10. A diagnostic kit for hepatitis B virus infection, comprising the nanobody of claim 1 and the fusion protein of claim 8.

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