Anti-dalban virus gn protein nanobody, bispecific antibody and application thereof

Abstract

This invention discloses an anti-Dabie Banda virus Gn protein nanobody, a bispecific antibody, and their applications, belonging to the field of biodetection technology. This invention constructs an alpaca natural nanobody library, using recombinantly expressed Gn protein as the antigen. Through multiple rounds of gradient screening using phage display technology, three nanobodies—G15, G20, and G40—that specifically bind to the Dabie Banda virus Gn protein are obtained. All three antibodies exhibit excellent antigen-binding specificity and virus-neutralizing activity; among them, G40 shows the most prominent neutralizing effect. The bispecific antibody G40-C1q, constructed based on G40, combines Gn protein and human C1q protein, achieving a synergistic effect of antiviral activity and inhibition of inflammatory damage. It can be used for the development of drugs for the prevention and treatment of Dabie Banda virus infection and for high-specificity detection reagents, providing candidate antibody drugs for the treatment of fever with thrombocytopenia syndrome, and providing a reliable tool for virus-related research, disease diagnosis, and clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an anti-Dabie Banda virus Gn protein nanobody, a bispecific antibody, and their applications. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is caused by Dabie Bandar virus (Dabie Bandar virus). Bandavirus dabieense Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly lethal tick-borne infectious disease. It is prevalent in spring and summer, with ticks as the primary vector and a risk of human-to-human transmission. SFTS is clinically characterized by fever, thrombocytopenia, and leukopenia. Severe cases can lead to multiple organ failure and disseminated intravascular coagulation, posing a serious public health threat. In 2017, the World Health Organization (WHO) listed it as one of the top ten priority infectious diseases requiring research and development. Currently, there are no approved specific antiviral drugs for SFTS. Clinical treatment mainly focuses on symptomatic measures such as fluid replacement to maintain electrolyte balance, prevention of secondary infections, and organ support, which cannot fundamentally stop viral infection and the progression to severe illness. There is a significant unmet need for effective clinical treatment.

[0003] Clearly identifying the core pathogenic mechanism of the disease-causing pathogen and identifying key targets for the development of effective drugs are crucial prerequisites for overcoming the clinical treatment challenges of SFTS. Dabie Banda virus (DBV) belongs to the genus Bandavirus of the family Leukopiviridae in the order Bunyavirales. It is an enveloped, single-stranded, negative-sense RNA virus. The viral particles are spherical or elliptical, with a diameter of 80-100 nm, and its lipid bilayer envelope is a core structural feature. The DBV genome consists of three independent RNA segments: S, M, and L. The M segment encodes the Gn protein, a type I transmembrane protein with a molecular weight of approximately 61 kDa. After maturation, it integrates into the viral envelope surface and plays a decisive role in viral recognition of host cell receptors, mediating membrane fusion and invasion. It is a core candidate molecule for DBV therapeutic targets.

[0004] The core role of Gn protein in viral infection has been further validated by both structural biology and clinical studies. Structural biology studies have confirmed that Gn protein head subdomains II and III contain a wide range of dominant neutralizing antigenic epitopes, effectively inducing the body to produce high-titer specific neutralizing antibodies. Clinical studies have further confirmed that high levels of Gn protein-specific neutralizing antibodies can be detected in the serum of DBV infection survivors, while the levels of these antibodies are significantly absent or extremely low in fatal cases, suggesting a significant positive correlation between the production of Gn-specific neutralizing antibodies and patient clinical prognosis. Based on the clear neutralizing target value of Gn protein and its correlation with clinical prognosis, the development of neutralizing antibodies targeting Gn protein has become a mainstream direction in the development of effective treatments for SFTS.

[0005] Despite the well-defined targets, the development of antibodies targeting Gn proteins still faces multiple technical challenges and has not yet achieved clinical translation. On the one hand, traditional IgG antibodies, with a molecular weight of approximately 150 kDa, are too large, easily creating steric hindrance with adjacent Gn / Gc glycoproteins on the viral envelope, hindering their efficient binding to neutralizing epitopes on the viral surface. On the other hand, some core neutralizing epitopes are highly conformationally dependent, only briefly exposed during the dynamic movement of viral spike proteins and membrane fusion, making it difficult for traditional antibodies to recognize these occult epitopes. Furthermore, most reported neutralizing antibodies suffer from narrow protective spectrums, insufficient cross-neutralizing activity against different regional / genotype strains, and are susceptible to immune escape due to viral mutations, limiting their clinical application value. These inherent defects of traditional IgG antibodies severely restrict the development progress and clinical translation of neutralizing antibodies targeting Gn proteins, necessitating the development of novel antibody forms to overcome these technical bottlenecks.

[0006] Camel serum immunoglobulins contain a special class of heavy-chain antibodies lacking light chains. The antigen-binding function of these antibodies is accomplished solely by a single variable heavy domain (VHH) of the heavy chain. This fragment, after gene cloning and prokaryotic expression, can exist stably and is known as a nanobody. Nanobodies possess unique advantages unmatched by traditional antibodies: small molecular weight, structural stability, good solubility, strong tissue penetration, and a flat antigen-binding interface, enabling them to recognize occult epitopes that are difficult for traditional antibodies to access. Furthermore, nanobodies are easily genetically engineered and mass-produced, exhibit low immunogenicity, and possess stable physicochemical properties, demonstrating good drug-like properties and clinical translational potential in antiviral, tumor, and inflammatory disease research. The unique advantages of nanobodies provide a novel technological pathway to overcome the limitations of traditional antibodies in the treatment of SFTS, and related research has become a hot topic in this field.

[0007] For example, Chinese patent CN 120535616 A discloses a nanobody NbG7-4 that can bind to SFTSV Gn, clarifying its core CDR region sequence for antigen binding, completing humanization modification, and verifying its neutralizing activity at the cellular level, providing a certain technical foundation for the development of SFTS-targeting nanobodies. However, this technical solution still has obvious technical limitations: First, it only focuses on research on single-strain nanobodies and has not systematically screened multiple candidate nanobodies targeting different antigenic epitopes with high affinity, making it difficult to cope with the risk of immune escape caused by viral mutations; Second, it has not systematically completed a comprehensive evaluation of key druggability indicators such as antibody binding specificity, affinity, and broad-spectrum neutralizing activity, limiting its clinical translation potential; Third, it only verified the basic viral neutralizing activity of the antibody and did not conduct functional design for the inflammation problem mediated by abnormal overactivation of the complement system during the severe SFTS process.

[0008] In summary, there is currently a lack of specific treatments for SFTS caused by DBV infection. Existing antibody development targeting the Gn protein is either limited by the inherent defects of traditional IgG antibodies or suffers from technical limitations such as single-function limitations and incomplete drugability evaluation. There are no reports of bifunctional nanobody drugs that possess both high-affinity virus-neutralizing activity and synergistic inhibition of severe inflammatory pathways. Therefore, developing high-affinity nanobodies targeting the DBV Gn protein, and further constructing bispecific antibodies that can simultaneously block viral infection and severe inflammation, is crucial for filling the technological gap in the clinical treatment of SFTS and meeting unmet clinical needs. Summary of the Invention

[0009] This invention aims to address the shortcomings of existing technologies by providing a nanobody, a bispecific antibody, and related applications against Dabie Banda virus Gn protein. The nanobody provided by this invention exhibits high specificity and excellent neutralizing activity. The bispecific antibody constructed based on this nanobody can simultaneously achieve antiviral and anti-inflammatory effects. It can not only be used to prepare drugs for the prevention and / or treatment of Dabie Banda virus infection but also for the detection of the virus and Gn protein, providing a new technical solution for the clinical prevention and treatment of fever with thrombocytopenia syndrome.

[0010] The present invention is achieved as follows: The present invention first provides a nanobody against Dabie Banda virus Gn protein, wherein the nanobody is a VHH fragment, the VHH fragment retains the activity of specifically binding to Dabie Banda virus Gn protein, and its amino acid sequence is selected from any one of the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14.

[0011] In a preferred embodiment of the present invention, the VHH fragment is a specific nanobody G15, G20, or G40 targeting the Gn protein of Dabie Bandar virus. All three antibodies have been experimentally verified to possess excellent Gn protein binding specificity and target binding affinity. Specifically: the amino acid sequence of nanobody G15 is shown in SEQ ID NO.10, and its encoding nucleotide sequence is shown in SEQ ID NO.9; the amino acid sequence of nanobody G20 is shown in SEQ ID NO.12, and its encoding nucleotide sequence is shown in SEQ ID NO.11; the amino acid sequence of nanobody G40 is shown in SEQ ID NO.14, and its encoding nucleotide sequence is shown in SEQ ID NO.13.

[0012] The present invention also provides a bispecific nanobody, the bispecific nanobody comprising a first functional domain, a second functional domain, and a flexible linker connecting the first functional domain and the second functional domain; wherein, the first functional domain is the aforementioned nanobody against Dabie Banda virus Gn protein, and the second functional domain is a nanobody that specifically binds to human C1q protein.

[0013] Furthermore, the flexible linker is composed of the amino acid sequence shown in (GGGGS)n, where n is an integer from 1 to 5; preferably, n=3, in which case the amino acid sequence of the flexible linker is as shown in SEQ ID NO.24.

[0014] (GGGGS) repeating units constitute peptide segments that are classic flexible linkers in antibody engineering and fusion protein construction. The glycine residues, lacking side chains, possess extremely high conformational freedom, endowing the linker with excellent flexibility; the serine residues, with hydroxyl groups, significantly enhance the linker's water solubility and reduce the risk of fusion protein aggregation. Within a repeat range of 1 to 5 times, these linkers can stably achieve flexible isolation and connection between two functional domains, particularly suitable for the fusion expression of the anti-Gn protein nanobody described in this invention with other functional domains. They do not significantly interfere with the correct folding of the antigen-binding domains at both ends or their target binding activity, and do not have a substantial adverse impact on the core biological function of the fusion protein.

[0015] In a preferred embodiment of the present invention, the amino acid sequence of the bispecific nanobody is shown in SEQ ID NO. 26, and its encoding nucleotide sequence is shown in SEQ ID NO. 25.

[0016] The present invention also provides a nucleic acid molecule for encoding the aforementioned nanobody, or encoding the aforementioned bispecific nanobody.

[0017] The present invention further provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0018] The present invention also provides an engineered host cell, wherein the engineered host cell comprises the above-mentioned recombinant expression vector, or wherein the above-mentioned nucleic acid molecule is integrated into its genome; preferably, the host cell is a prokaryotic host cell or a eukaryotic host cell, and more preferably any one of Escherichia coli, Expi-293F cells, and HEK-293T cells.

[0019] The present invention also provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, excipient, or diluent; wherein the active ingredient is the aforementioned nanobody and / or bispecific nanobody; the pharmaceutical composition is a pharmaceutical preparation for the prevention and / or treatment of fever with thrombocytopenia syndrome caused by Dabie Banda virus infection.

[0020] This invention also provides the application of the aforementioned nanobodies, bispecific nanobodies, or pharmaceutical compositions, wherein the application is in the preparation of Dabie Bandar virus-related prevention and control products, the products comprising:

[0021] (1) Drugs used for the prevention and / or treatment of Dabie Bandar virus infection;

[0022] (2) Reagents / kits for detecting Dabie Bandar virus Gn protein or for detecting Dabie Bandar virus.

[0023] Beneficial effects:

[0024] 1. This application successfully obtained three nanobodies, G15, G20, and G40, specifically targeting the DBV Gn protein by constructing a large-capacity alpaca natural nanobody library and conducting multiple rounds of gradient biological screening. All three antibodies can specifically recognize and bind to the Gn protein, and they showed significant inhibitory activity in in vitro virus neutralization experiments. Among them, the neutralization effect of the G40 nanobody was particularly outstanding, and it has excellent potential to become a core antiviral candidate molecule. Compared with traditional IgG antibodies, the nanobodies obtained in this application are composed of only a single heavy chain variable region, which has the outstanding advantages of small molecular weight and simple structure. It can effectively avoid the steric hindrance caused by adjacent glycoproteins on the viral envelope, and accurately bind to conformation-dependent epitopes that are difficult for traditional antibodies to access, which greatly improves the efficiency and specificity of antigen binding.

[0025] 2. This application designs and constructs a bispecific nanobody that simultaneously targets Gn protein and human C1q protein. While retaining the highly efficient virus-neutralizing activity of the G40 nanobody, this bispecific antibody adds the function of regulating the complement system. It can block viral adsorption, invasion of host cells, and inhibition of viral replication and proliferation, while simultaneously inhibiting abnormal overactivation of the classical complement pathway, reducing the release of inflammatory factors and tissue / organ damage, thus achieving a dual synergistic effect of antiviral and anti-inflammatory action. In vitro functional experiments show that the virus neutralization inhibition rate of this bispecific antibody is further improved compared to single nanobody, exhibiting superior antiviral effects and providing a novel treatment strategy for critically ill patients with fever and thrombocytopenia syndrome.

[0026] 3. The G40 nanobody recombinant eukaryotic expression plasmid disclosed in this application can achieve stable and efficient secretory expression of the target antibody under standardized transfection and cell culture conditions, and can continuously obtain high-titer functional antibodies. It not only facilitates the establishment of unified antibody preparation process standards, but also effectively reduces the risk of batch differences in the process of large-scale production, and provides a stable and reliable source of raw materials for subsequent batch preparation of antibodies, preclinical research and development of related products.

[0027] 4. The anti-Gn protein nanobody obtained in this application has excellent detection application value. This type of antibody has high binding specificity and low background interference. Immunofluorescence experiments have confirmed that it can recognize the native conformation of Gn protein in cells, and Western blot experiments have confirmed that it can recognize denatured Gn protein. It is suitable for various routine laboratory detection scenarios such as immunoblotting and immunofluorescence. At the same time, this nanobody can be used as a core functional raw material to further develop rapid detection reagents or kits for Dabie Bandar virus, achieving high sensitivity and high specificity detection of DBV in clinical samples. This provides efficient and reliable technical tool support for the early rapid diagnosis of fever with thrombocytopenia syndrome, epidemiological monitoring in epidemic areas, and precise prevention and control. Attached Figure Description

[0028] Figure 1 Electrophoresis diagrams of heavy chain antibody and VHH gene amplification are shown. In the diagram, A represents the heavy chain antibody gene amplification result, lane M is the DL2000 Marker, and lane 1 is the heavy chain antibody gene amplification product from the first round of PCR. In the diagram, B represents the VHH gene amplification result, lane M is the DL2000 Marker, and lane 1 is the VHH gene amplification product from the second round of PCR.

[0029] Figure 2 Phage-ELISA binding activity assay results for anti-Gn protein nanobody monoclonal antibodies;

[0030] Figure 3The results are as follows: SDS-PAGE validation of eukaryotic expression and purification of nanobodies. In A, the SDS-PAGE validation results of eukaryotic expression of nanobodies are shown, with lane M being Protein Marker, lane 1 being 293F cell negative supernatant, lane 2 being G15 nanobodies expression supernatant, lane 3 being G20 nanobodies expression supernatant, and lane 4 being G40 nanobodies expression supernatant. In B, the purification validation results of G40 nanobodies are shown, with lane M being Protein Marker, lane 1 being unpurified G40 nanobodies supernatant, lane 2 being flow-through buffer, lane 3 being 10 mM imidazole elution buffer, lane 4 being 20 mM imidazole elution buffer, lane 5 being 50 mM imidazole elution buffer, lane 6 being 100 mM imidazole elution buffer, and lane 7 being 200 mM imidazole elution buffer.

[0031] Figure 4 The results of Western blot specific binding validation of anti-Gn protein nanobodies are shown. Lane M is the Protein Marker and lane 1 is the incubation group of recombinant Gn protein + G40 nanobodies.

[0032] Figure 5 The results are from the immunofluorescence assay of the G40 nanobody.

[0033] Figure 6 The results show the relative binding affinity of anti-Gn protein nanobodies determined by the ammonium thiocyanate method.

[0034] Figure 7 The results of SDS-PAGE analysis of the purified G40-C1q bispecific nanobody are shown below. Lane M is the pre-stained protein molecular weight standard, lane 1 is the unpurified cell supernatant of G40-C1q bispecific antibody, lane 2 is the flow-through buffer, lane 3 is 10 mM imidazole elution buffer, lane 4 is 20 mM imidazole elution buffer, lane 5 is 50 mM imidazole elution buffer, lane 6 is 100 mM imidazole elution buffer, and lane 7 is 200 mM imidazole elution buffer.

[0035] Figure 8 The results show the in vitro neutralizing and inhibitory activity of the anti-DBV nanobody and the bispecific antibody. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0037] Example 1

[0038] I. Construction of Natural Nanobody Libraries

[0039] 1) Obtaining the single heavy chain variable region (VHH) gene

[0040] Forty healthy adult alpacas were randomly selected, and 5 mL of peripheral blood was collected from each. At room temperature, the peripheral blood was slowly added along the tube wall to the surface of 5 mL of pre-cooled lymphocyte separation medium. The mixture was centrifuged at 1000 rpm for 30 min at room temperature. The system was separated from top to bottom into plasma, lymphocytes, lymphocyte separation medium, and erythrocyte layer. The lymphocyte layer was carefully transferred to a sterile centrifuge tube, and 10 mL of cell washing buffer (pH 7.4 PBS buffer containing 2% inactivated fetal bovine serum) was added. After mixing by pipetting, the mixture was centrifuged at 800 rpm for 10 min, and the supernatant was discarded. The washing operation was repeated three times, and the supernatant was discarded. The lymphocyte pellet was resuspended in 0.5 mL of pH 7.4 PBS buffer.

[0041] Total RNA was extracted from lymphocytes using the Trizol method. Using the obtained RNA as a template, genomic DNA was removed and cDNA was prepared by reverse transcription. The genomic DNA removal reaction system is shown in Table 1 below.

[0042] Table 1. Genomic DNA Removal Reaction System

[0043] ;

[0044] After gently mixing the above system, incubate at 42°C for 2 min to complete the removal of genomic DNA. Then, add 5 μL of 4×HiScript IV qRT SuperMix to the system for reverse transcription: incubate at 37°C for 15 min, then at 85°C for 5 sec to terminate the reaction. The resulting cDNA product is aliquoted and stored at -80°C for later use.

[0045] The VHH gene, a variable region of the heavy chain antibody, was specifically amplified using two rounds of nested PCR. The specific procedures are as follows:

[0046] First round of PCR amplification: Using cDNA obtained from reverse transcription as a template, heavy chain antibody-related genes were amplified using primers CALL001 (5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO.1) and CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID NO.2). The PCR amplification system is shown in Table 2 below:

[0047] Table 2. PCR amplification system for heavy chain antibody genes

[0048] ;

[0049] PCR reaction program: 95℃ pre-denaturation for 3 min, 1 cycle; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 30 sec, 30 cycles in total; 72℃ final extension for 5 min, 1 cycle.

[0050] The amplification products were identified by agarose gel electrophoresis, yielding a 900 bp fragment encoding the common alpaca antibody gene and a 700 bp target fragment of the heavy chain antibody gene. Figure 1 (A) The target fragment of about 700 bp was recovered by gel cutting, eluted with RNase-free ddH2O, and stored at -20℃ for later use.

[0051] Using the heavy chain antibody gene fragment recovered from the first round of PCR as a template, specific amplification primers with Sfi I restriction sites were designed based on the conserved sequences of the FR1 and FR4 regions of the alpaca heavy chain antibody variable region. The primer sequences are as follows:

[0052] VHH-F1: 5'-TCGC GGCCCAGGCGGCC ATGGCCCAGGTGCAGCTCGTGGAG-3' (SEQ ID NO.3);

[0053] VHH-F2: 5'-TCGC GGCCCAGGCGGCC ATGGCCCAGTTGCAGCTCGTGGAG-3' (SEQ ID NO.4);

[0054] VHH-R1: 5'-CGAGT GGCCGGCCTGGCC GGGGTCTTCGCTGTGGTGCGC-3' (SEQ ID NO.5);

[0055] VHH-R2: 5'-CGAGT GGCCGGCCTGGCC TTGTGGTTTTGGTGTCTTGGGTTC-3' (SEQ ID NO. 6);

[0056] The Sfi I restriction sites introduced at both ends of the primers are used for subsequent vector ligation and library construction; the second-round PCR amplification system is shown in Table 3 below:

[0057] Table 3. VHH gene PCR amplification system

[0058] ;

[0059] The PCR reaction procedure is the same as the first round of PCR amplification reaction procedure.

[0060] The amplification product was identified by agarose gel electrophoresis, yielding a target band of the VHH gene approximately 450 bp in size. Figure 1The target fragment (B in the sample) carries Sfi I restriction sites at both ends. The fragment is purified by gel extraction, stored at -20°C, and used for subsequent library construction.

[0061] 2) Preparation of nanobody libraries by electroconversion method

[0062] The phage vector pComb3XTT plasmid was extracted, and the VHH target gene fragment and the pComb3XTT circular plasmid were digested with Sfi I restriction endonuclease. The digestion reaction system was as follows: 1 μL Sfi I, 5 μL 10×NE Buffer, 1 μg pComb3XTT plasmid / VHH gene, and RNase-free ddH2O to a final volume of 50 μL; the digestion reaction was incubated at 50℃ for 3 h.

[0063] The enzyme digestion products were identified by agarose gel electrophoresis. After enzyme digestion, the pComb3XTT plasmid formed a vector backbone fragment of 3319 bp and a smaller fragment of 1447 bp. The larger vector backbone fragment was recovered by gel excision, and the VHH gene fragment after enzyme digestion was also recovered and purified for later use.

[0064] The VHH gene fragment purified by enzyme digestion and linearized pComb3XTT vector was recombinantly ligated using T4 ligase at a molar ratio of insert fragment to vector of 10:1. The ligation reaction system was prepared on ice (see Table 4), mixed well, and ligated overnight at 16°C.

[0065] Table 4. Vector-VHH gene ligation reaction system

[0066] ;

[0067] After the ligation reaction was completed, the recombinant ligation product was recovered and added to the electrotransformed competent cells XL1-Blue. After gentle pipetting and mixing, the cells were transferred to a pre-cooled electroporation cuvette and placed on ice for 5 min. The cells were then transformed using an electroporator with the following parameters: voltage 2400 V, capacitance 25 μF, and resistance 200 Ω.

[0068] Immediately after electroporation, 300 μL of preheated (37°C) SOC liquid medium (20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 0.186 g KCl, 4.07 g MgCl2·6H2O, 4.93 g MgSO4·7H2O, ddH2O to a final volume of 1 L, autoclaved, and then 2 mL of 1 M glucose solution) was added to the electroporation vessel. The bacterial culture was mixed thoroughly and incubated at 37°C and 200 rpm for 1 h for recovery.

[0069] The revived bacterial culture was evenly spread onto LB solid selection plates containing ampicillin (Amp, 100 μg / mL) resistance and incubated overnight at 37°C with the plates inverted. After incubation, all colonies on the plates were washed away with 2 mL of LB liquid medium containing ampicillin, and the bacterial culture was collected. 10 μL of the bacterial culture was serially diluted and spread onto LB+Amp solid plates, and incubated overnight at 37°C with the plates inverted for library capacity calculation. The remaining bacterial culture was used for expansion culture and subsequent phage-mediated phage display screening.

[0070] After gradient dilution and plate culture, the calculated capacity of the constructed natural nanobody library in this study was approximately 2.48 × 10⁻⁶. 8 cfu / mL. Forty-eight single colonies were randomly selected from the library plate and identified by PCR using universal primers for the pComb3XTT vector. The primer sequences are as follows:

[0071] pComb3XTT-F: 5'-AAGACAGCTATCGCGATTGCAG-3' (SEQ ID NO. 7);

[0072] pComb3XTT-R: 5'-GAAGCGTAGTCCGGAACGTC-3' (SEQ ID NO. 8);

[0073] The bacterial culture PCR amplification system is shown in Table 5 below:

[0074] Table 5. Bacterial PCR Amplification System

[0075] ;

[0076] PCR reaction program: 94℃ pre-denaturation for 1 min, 1 cycle; 98℃ denaturation for 5 sec, 60℃ annealing for 5 sec, 72℃ extension for 5 sec, 30 cycles in total; 72℃ final extension for 1 min, 1 cycle.

[0077] The library insertion positivity rate was tested, and the results showed that clones that amplified a target band of about 700 bp were positive clones. 45 out of 48 clones were positive clones, and the library positivity rate was about 94%.

[0078] II. Screening of Gn protein nanobodies against Dabie Banda virus

[0079] 1) Amplification and titer determination of helper phage VCSM13

[0080] XL1-Blue monoclonal antibodies were inoculated into LB liquid medium containing 10 μg / mL tetracycline (Tet) and cultured at 37℃ and 200 rpm until the logarithmic growth phase.

[0081] Perform 10-fold serial dilutions of the helper phage VCSM13 stock solution, and take 10 -4 10 -5 10 -6 10 μL of diluted phage solution was mixed with 200 μL of XL1-Blue bacterial solution in the logarithmic growth phase and incubated at 37°C for 15 min to allow the phage to infect the host bacteria.

[0082] Add 4 mL of preheated Top Agar (1.6 g tryptone, 1 g yeast extract, 0.5 g NaCl, 0.7 g Agar, ddH2O to 100 mL, autoclaved) to the mixture, mix quickly and immediately pour onto a preheated LB agar plate at 37°C. Tilt the plate to spread the Top Agar evenly. Let stand at room temperature for 5 min until the Top Agar is completely solidified, then invert the plate and incubate overnight at 37°C.

[0083] The next day, the plates were observed. Circular, transparent, independent phage plaques were visible against a hazy bacterial moss background. A single, uniformly shaped independent phage plaque was selected and inoculated into 100 mL of LB liquid medium (LB+Kana) containing 50 μg / mL kanamycin (Kana). The medium was then incubated overnight at 37°C with shaking at 200 rpm.

[0084] The overnight culture was centrifuged at 4°C and 9000 rpm for 10 min, and the supernatant containing bacteriophages was collected.

[0085] Add 1 / 6 volume of PEG-8000 / NaCl solution (20 g PEG-8000, 14.6 g NaCl, ddH2O to 100 mL) to the supernatant, mix by inverting, and let stand overnight at 4°C to precipitate the phage particles.

[0086] The next day, the mixture was centrifuged at 12,000 rpm for 10 min at 4°C. After centrifugation, a white phage precipitate the size of a fingerprint was visible on the tube wall. The supernatant was discarded. The precipitate was resuspended in 1 mL of sterile TBS buffer (pH 7.4), and then 200 μL of PEG-8000 / NaCl solution was added. The mixture was inverted and incubated on ice for 1 h to complete the secondary purification. The mixture was centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was discarded, and the phage precipitate was gently resuspended in 200 μL of sterile TBS buffer. The mixture was then stored at 4°C for a short period.

[0087] Select XL1-Blue single clones and inoculate them into LB+Tet liquid medium. Incubate at 37°C and 200 rpm with shaking until the logarithmic growth phase, and set aside for later use.

[0088] The purified phage was serially diluted 10-fold using sterile TBS buffer, with the dilution range being 10⁻¹⁰. -6 ~10 -12 Take 10 μL of each phage dilution and mix it with 200 μL of XL1-Blue bacterial culture in the logarithmic growth phase. Incubate at 37°C for 15 min to allow the phage to fully infect the host bacteria. Add 4 mL of preheated Top Agar (42°C) to each tube of mixture, mix quickly, and immediately spread it on a preheated LB agar plate (37°C). Tilt the plate to spread the Top Agar evenly. Let it stand at room temperature for 5 min until the agar is completely solidified, then invert the plate and incubate overnight at 37°C.

[0089] The following day, observe the plates and select plates with approximately 100 plaques for counting. Calculate the phage titer using the following formula: Phage titer (pfu / mL) = Number of plaques on plate × Dilution factor × 100, where pfu is the plaque-forming unit. The final auxiliary phage titer should be ≥1 × 10⁻⁶. 11 pfu / mL.

[0090] 1) Construction of phage library: Helper phage VCSM13 was added to the constructed bacterial library to control the multiple of infection (MOI) at 20:1. After static infection and shaking culture, the phage library was enriched and purified twice using PEG / NaCl solution (20 g PEG-8000, 14.6 g NaCl, ddH2O to 100 mL, filtered for sterilization). After serial dilution, the library titer was measured to obtain a nanobody phage display library that can be used for affinity screening.

[0091] 2) Biopanning: A three-round gradient biopanning method was used to progressively enrich high-affinity specific antibodies. Recombinant Gn protein (coding sequence from the S gene of strain DBV E-JS-2013-24, GenBank accession number: AMY99382) was diluted to 100 µg / mL using 0.05 M, pH 9.6 NaHCO3 buffer, and 50 µL / well was added to a 96-well microplate and incubated overnight at 4°C. The next day, the coating solution was discarded, and 300 μL of 5% BSA blocking buffer prepared with TBST buffer (pH 7.4, containing 0.05% Tween-20 TBS) was added to each well. The plate was blocked at 37°C for 2 h to block non-specific sites. The blocking solution was discarded, and the plate was washed twice with TBST for later use. Mix 100 μL of phage display library with 900 µL of TBST buffer to achieve a 10-fold dilution pretreatment. Add 100 µL / well to an ELISA plate and incubate with gentle shaking at 50 rpm for 1 h to allow the phage-displayed nanobodies to fully bind to the solid-phase Gn protein. Discard any unbound phage solution from the wells and wash the plate five times with TBST buffer to remove unbound phage. Add 100 µL of pH 2.2 Gly-HCl elution buffer (15.014 g Glycine, 0.1 g BSA, ddH2O to a final volume of 100 mL) to each well and incubate with gentle shaking at 50 rpm for 15 min at room temperature to elute the phage bound to the Gn protein. Adjust the elution buffer to neutral with 0.2 M, pH 9.1 Tris-HCl buffer. Use 20 μL of the elution buffer for titer detection, and use the remaining elution buffer for subsequent amplification.

[0092] The screening rigor was gradually increased during the three rounds of screening: the Gn protein coating concentration was decreased sequentially, while the number of washes was increased and the Tween-20 concentration in the TBST buffer was increased to enhance the specificity screening effect. Specific parameters and screening results are shown in Table 6.

[0093] Second round of selection: Gn protein coating concentration was reduced to 60 μg / mL, TBST washing times were increased to 8 times, and Tween-20 concentration was increased to 0.1%;

[0094] Third round of selection: Gn protein coating concentration was reduced to 20 μg / mL, TBST washing times were increased to 10 times, and Tween-20 concentration was increased to 0.2%.

[0095] Table 6. Results of three rounds of bio-patch of nanobody phage libraries

[0096] ;

[0097] 3) Elution of bacteriophages for amplification: 50 µL of Escherichia coli XL1-Blue bacterial culture was inoculated into LB liquid medium (LB+Tet) containing 10 μg / mL tetracycline at a ratio of 1:100. After incubation at 37°C and 200 rpm to the logarithmic growth phase, the first round of eluted bacteriophages was added. The culture was incubated at 37°C for 30 min with static incubation and then incubated at 37°C and 200 rpm with shaking for 1 h. The cells were collected by centrifugation at 8000 rpm for 15 min, resuspended in LB medium and cultured (37°C, 200 rpm) to the logarithmic growth phase. Helper bacteriophage VCSM13 was added again for infection for 1 h. Ampicillin was added to a final concentration of 100 μg / mL and kanamycin to a final concentration of 50 μg / mL. The culture was then incubated overnight at 37°C and 200 rpm with shaking. The following day, the phage was centrifuged at 8000 rpm for 15 min at 4℃, and the supernatant was collected. 1 / 5 volume of PEG-8000 / NaCl solution was added, and the phage was precipitated overnight at 4℃. The next day, the phage was centrifuged at 8000 rpm for 8 min at 4℃, the supernatant was discarded, the precipitate was resuspended in 10 mL of TBS buffer, and purified again by precipitation on ice with 1 / 5 volume of PEG-8000 / NaCl. The phage was centrifuged at 8000 rpm for 15 min at 4℃, the supernatant was discarded, the precipitate was resuspended in 1 mL of TBS buffer, and centrifuged at 10000 rpm for 2 min at 4℃. The supernatant was collected, which was the first round of amplification product. 20 µL of the amplification product was used to determine the titer, and the remaining product was used for the next round of biopanning. The second and third rounds of phage amplification were repeated using the same procedure.

[0098] 4) Phage-ELISA identification: 96 single colonies were randomly selected from the third-round screening plates and inoculated into 1 mL of LB+Amp+Glu liquid medium. The culture was carried out at 37°C and 220 rpm until the logarithmic phase. Helper phages were added at a ratio of 1:20, and the culture was incubated at 37°C for 30 min with shaking. After 1 h of incubation at 37°C and 200 rpm with shaking, the culture was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in 1 mL of LB+Amp+Kana liquid medium and incubated overnight at 37°C and 200 rpm with shaking. The following day, the sample was centrifuged at 8000 rpm for 10 min at 4°C, the precipitate was discarded, and the supernatant was used for phage-ELISA experiments. Gn protein was diluted to 5 μg / mL with 0.05 M, pH 9.6 NaHCO3 buffer, and 100 μL / well was used to coat a 96-well microplate overnight at 4°C. The next day, the plate was blocked with 5% BSA-TBST blocking buffer at 37°C for 2 h, and washed 3 times with TBST. 100 µL of monoclonal phage supernatant was added to each well, and the plate was incubated at 37°C and 50 rpm for 2 h. After washing 10 times with TBST buffer, 100 µL of HRP-labeled mouse M13 phage capsid protein antibody diluted 1:5000 with TBST was added, and the plate was incubated at 37°C in the dark for 2 h. After washing again, TMB chromogenic buffer was added, and the reaction was developed at room temperature for 10 min. 70 µL of stop solution was added to terminate the reaction, and the absorbance (OD) was measured at 450 nm. 450 ).

[0099] The experiment was set up with wells without phage supernatant as blank negative controls, and the OD of the wells to be tested was used as the control. 450 Value (P) / Mean OD of negative control 450 A value (N) ≥ 2.1 was used as the criterion for positive clones. The results showed that 74 out of 96 randomly selected clones were positive, with a positive rate of approximately 77%. Figure 2 Positive clones were sequenced, and duplicate clones were removed through sequence alignment, ultimately yielding 24 unique VHH gene sequences for anti-Gn protein nanobodies. Combining sequence diversity with Phage-ELISA binding signal intensity, three nanobodies with optimal affinity were selected: G15, G20, and G40. Their VHH gene coding sequences are as follows:

[0100] The G15 nanobody encodes the nucleotide sequence (SEQ ID NO.9):

[0101] CAATTGCAGCTTGTGGAAAGCGGCGGGGGCTTGGTGCAATTTGGTGGTAGCTTGCGGCTTTCTTGCGCGGCTTCTGGAAGGACACGCTCTAGGTACGGCATGGTGTGGTTTAGGCAAGTGCCAGGAAAGGAACGAGAGTTTGTAGCCGGAATCACTTGGAGTGGCGGCAGTACAATTTATGCCTGCTCCGTTAAGGGCAGGTTCAACATCTTCAGGGATAACTTCAAAAATGCCGTGTACCTGCAGATGAATAGTCTCAAGCCTGAGGATACGGCCCTCTACTATTGTGCTGCAAACACAAGAACTTGGGCTATTGTTCGCGGGATAGGAGAATACGATCTGTGGGGGGGAGGAACTCAAGTCACAGTGTCATCT;

[0102] G15 nanobody amino acid sequence (SEQ ID NO.10):

[0103] QLQLVESGGGLVQFGGSLRLSCAASGRTRSRYGMVWFRQVPGKEREFVAGITWSGGSTIYACSVKGRFNIFRDNFKNAVYLQMNSLKPEDTALYYCAANTRTWAIVRGIGEYDLWGGGTQVTVSS;

[0104] G20 nanobody encoding nucleotide sequence (SEQ ID NO.11):

[0105] CAGTTGCAGCTCGCGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGGATCTAGGCAGTTTTATTCCATAGCCTGGTTCCGGCAGGCCCCAGGGAAGGAGCGCGAGGCGGTCTCATGTATTAGTGTTAATGGTGGCGCCACAGACTATGCGGCCTCCGTGAAGGGCCGATTCACCATTTCCAGAGACCACGCCAAGAACACGGTGTATCTGCAAATGGACGGCCTGAAACCTGAGGACACAGCCGTTTATTTCTGTGCAGCCGGCGAAGGGGGATACTATGGTGGTAGTACAACATGTCCCGCCTTCAGGTATCGCGAAGCCTGGGGCCAGGGCACTCAGGTCACTGTCTCCTCA;

[0106] G20 nanobody amino acid sequence (SEQ ID NO.12):

[0107] QLQLAESGGGLVQPGGSLRLSCVASGGSRQFYSIAWFRQAPGKEREAVSCISVNGGATDYAASVKGRFTISRDHAKNTVYLQMDGLKPEDTAVYFCAAGEGGYYGGSTTCPAFRYREAWGQGTQVTVSS;

[0108] G40 nanobody coding nucleotide sequence (SEQ ID NO.13):

[0109] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCGCCTGTGCAGCCTCTGGATTCACTTTGGATTATTATGTCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGCGCGAGGGGGTCTCATGTGTTAGTACTTTTGATAGTAGCACAGACTATGCAGACTCCGTGAA GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGACTACAGGGCTCTTTGTCCTTACGATAGCGACTCCGATGGGGAGTATGAGTATGACCACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA;

[0110] G40 nanobody amino acid sequence (SEQ ID NO.14):

[0111] QLQLVESGGGLVQPGGSLRLACAASGFTLDYYVIGWFRQAPGKEREGVSCVSTFDSSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADYRALCPYDSDSDGEYEYDHWGQGTQVTVSS;

[0112] The three nanobodies mentioned above were used for subsequent recombinant expression and in vitro functional verification.

[0113] III. Eukaryotic Expression and Purification of Anti-Dabie Banda Virus Gn Protein Nanobody

[0114] 1) Obtaining the target gene of nanobody

[0115] Sequencing-verified G15, G20, and G40 positive monoclonal strains were selected and inoculated into LB liquid medium (LB+Amp) containing ampicillin (Amp) and cultured at 37°C and 200 rpm for 16 h. Plasmids were extracted from each strain using a plasmid miniprep kit. After determining the plasmid concentration, the plasmid concentration was diluted to below 50 ng / µL with RNase-free ddH2O and used as PCR amplification templates.

[0116] Taking the construction of the G40 antibody recombinant expression plasmid as an example, specific amplification primers were designed, and a recombinant sequence homologous to the end of the linearized vector (underlined) was added to the 5' end of the primers. The primer sequences are as follows:

[0117] G40-F: 5'- TTGCACTTGTCACGAATTCA ATGGCCCAGTTGCAGCTCG-3' (SEQ ID NO. 15);

[0118] G40-R: 5'- GGGTCTTCGCTGTGGTGCGC TGAGGAGACGGTGACCTGGG-3' (SEQ ID NO. 16).

[0119] The G15 and G20 antibody genes were amplified using the same homologous recombination strategy, and corresponding primers were designed accordingly. The PCR amplification system is shown in Table 7 below:

[0120] Table 7. PCR amplification system of nanobody VHH gene

[0121] ;

[0122] PCR reaction procedure: 95℃ pre-denaturation for 3 min, 1 cycle; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 30 sec, 30 cycles in total; 72℃ final extension for 5 min, 1 cycle. A VHH antibody gene target band of approximately 432 bp was amplified. After verifying the band was single and of the correct size by agarose gel electrophoresis, the target fragment was excised and purified.

[0123] 2) Preparation of eukaryotic expression linearization vectors

[0124] This embodiment uses the pCMV-hIL2-His eukaryotic expression plasmid. The core elements of the vector include the human cytomegalovirus (CMV) promoter, the human interleukin-2 (hIL2) signal peptide sequence, the C-terminal 6×His tag, and the ampicillin resistance gene (AmpR).

[0125] The circular plasmid was linearized and amplified using reverse PCR. The reverse PCR primer sequences were as follows:

[0126] pCDNA-F: 5'-GCGCACCACAGCGAAGAC-3' (SEQ ID NO. 17);

[0127] pCDNA-R: 5'-TGAATTCGTGACAAGTGCAAGAC-3' (SEQ ID NO. 18);

[0128] The reverse PCR amplification system is shown in Table 8. The reaction program is as follows: 98℃ pre-denaturation for 30 sec, 1 cycle; 98℃ denaturation for 10 sec, 60℃ annealing for 10 sec, 72℃ extension for 3 min 30 sec, 30 cycles in total; 72℃ final extension for 5 min, 1 cycle.

[0129] Table 8. Reverse PCR linearized vector amplification system

[0130] ;

[0131] The amplification product was verified by agarose gel electrophoresis, and a linearized plasmid band of approximately 6651 bp was obtained. The band was single and the correct size. The linearized vector fragment was recovered and purified by gel excision for later use.

[0132] 3) Homologous recombination to construct recombinant expression plasmids

[0133] The VHH gene PCR product and the linearized vector PCR product were digested with Dpn I to remove template plasmid interference. The Dpn I digestion system consisted of 17 μL of PCR product, 2 μL of 10×CutSmart Buffer, and 1 μL of Dpn I restriction enzyme, for a total volume of 20 μL. The digestion conditions were incubation at 37°C for 2 h.

[0134] After digestion, the target band was recovered and purified again by agarose gel electrophoresis. The homologous recombination reaction system was prepared on ice: 133 ng linearized vector, 18 ng VHH antibody gene, 4 μL 5×CE II Buffer, 2 μL Exnase II, and RNase-free ddH2O was added to make up to 20 μL.

[0135] Homologous recombination reaction conditions: Incubate at 37℃ for 30 min, then immediately cool on ice for 5 min. Transform the recombinant product into DH5α chemocompetent cells, pick single colonies for colony PCR verification and sequencing. Strains whose sequencing results completely match the target VHH gene sequence are positive recombinant strains. Expand the positive strains, extract the recombinant expression plasmid using a plasmid large-scale extraction kit, and store at -20℃ for later use.

[0136] 4) Eukaryotic expression of anti-nanobodies

[0137] Expi-293F suspension cells revived in liquid nitrogen were cultured in a constant temperature shaker at 37°C, 8% CO2, and 125 rpm. After 2-3 passages, when the cells were stable and viable at ≥95%, they were used for transfection experiments.

[0138] On the day of transfection, Expi-293F cells were loaded at a rate of 3 × 10⁻⁶. 6Cells were seeded at a density of ≥95% in shake flasks containing 50 mL of serum-free 293F expression medium. Transfection complex preparation: 50 µg of recombinant expression plasmid and 150 µL of PEI 40000 were added to 5 mL of serum-free 293F medium, mixed thoroughly by pipetting, and incubated at room temperature for 10–15 min to form the plasmid-transfection reagent complex.

[0139] Add the transfection complex to the cells to be transfected, mix gently, and incubate at 37°C, 8% CO2, and 125 rpm in a constant temperature shaker. Simultaneously, set up a negative control group transfected with an empty vector plasmid and a positive control group transfected with a known high-expression recombinant plasmid.

[0140] Cell density and viability were monitored daily for 5-7 days after transfection. Cell culture was collected when cell viability was below 60% for subsequent antibody purification.

[0141] 5) Expression verification and purification of nanobodies

[0142] Expression validation: The collected cell culture was centrifuged at 1500 rpm for 10 min at 4℃. The supernatant was filtered through a 0.45 µm filter to obtain a crude extract containing secretory nanobodies. 40 µL of the crude extract was taken, and 10 µL of 5× Protein Loading Buffer was added. The mixture was pipetted and stirred, and the protein was boiled at 98℃ for 10 min. The samples were separated by SDS-PAGE electrophoresis. After electrophoresis, the samples were stained with Coomassie Brilliant Blue and destained to verify the secretory expression of the nanobodies.

[0143] Purification: Affinity purification was performed using Ni-NTA agarose gel (Ni Beads) based on the C-terminal 6×His tag. The specific steps are as follows:

[0144] After mixing the Ni-NTA agarose gel, the nickel beads were washed three times with PBS buffer to remove the protective solution. The pretreated nickel beads were then thoroughly mixed with the filtered cell culture supernatant and incubated overnight at 4°C on a shaker to allow the target protein to specifically bind to the nickel beads. The next day, the mixture was centrifuged at 1000 rpm for 1 min, and the supernatant was collected as flow-through buffer for subsequent SDS-PAGE validation. The nickel beads were thoroughly washed with 10 mL of 10 mM imidazole solution and centrifuged at 1000 rpm for 1 min. The wash buffer was collected for electrophoresis validation. Subsequently, a gradient elution was performed with 20 mM, 50 mM, 100 mM, and 200 mM imidazole solutions (prepared with PBS buffer at pH 7.4). The eluents of each concentration were collected, and the optimal elution conditions were determined by electrophoresis. The target protein eluent was transferred in batches to protein ultrafiltration tubes with a 3 kDa cutoff. The tubes were concentrated by centrifugation at 4°C and 5000 rpm for 15 min, and the buffer system was replaced with PBS buffer. 10 µL of the purified protein was taken and its purity was identified by SDS-PAGE electrophoresis.

[0145] The results of SDS-PAGE validation of nanobody eukaryotic expression are as follows: Figure 3 As shown in Figure A, the three nanobodies G15, G20, and G40 are all secretory expression, with a protein molecular weight of approximately 16 kDa, consistent with the expected size. After Ni-NTA affinity purification, the G40 nanobody began elution at a concentration of 20 mM imidazole. The purified G40 antibody protein band was single, without obvious impurities, and its purity met the requirements for subsequent functional experiments. Figure 3 (B in the middle).

[0146] IV. Functional Validation of Anti-Gn Protein Nanobodies

[0147] 1) Western blot specific binding verification

[0148] Recombinant Gn protein was subjected to SDS-PAGE electrophoresis. After electrophoresis, a 0.45 μm PVDF membrane matching the gel size was cut. The PVDF membrane was activated in methanol for 3 min and then placed in transfer equilibration buffer. The gel and PVDF membrane were placed on a transfer sponge in sequence, and air bubbles between the gel and membrane were carefully removed using a roller. Transfer was performed at 200 mA for 100 min. After transfer, the PVDF membrane was removed and washed twice with TBST buffer for 5 min each time. Blocking was performed at room temperature using 5% skim milk for 2 h. After blocking, the membrane was washed twice with TBST buffer for 5 min each time. 5 mL of G40 nanobody solution diluted to a final concentration of 1 μg / mL with TBST buffer containing 1% BSA was added to the incubation box containing the PVDF membrane, and incubated overnight at 4°C. The following day, the primary antibody solution was discarded, and the membrane was washed 5 times with TBST buffer at 100 rpm for 5 min each time. HRP-labeled rabbit anti-camel VHH antibody diluted 1:5000 with TBST buffer was added, and the membrane was incubated at room temperature for 1 h. After incubation, the secondary antibody solution was discarded, and the membrane was washed 5 times with TBST buffer at 100 rpm for 5 min each time. The membrane was then developed and photographed using ECL chemiluminescence solution.

[0149] The results are as follows Figure 4 As shown, a single bright, specific band was observed at approximately 51 kDa in the G40 nanobody incubation group, completely consistent with the expected molecular weight of the recombinant Gn protein, with no other non-specific bands. The G15 and G20 nanobodies also showed the same specific binding results as G40. These results indicate that the three anti-Gn protein nanobodies, G15, G20, and G40, can specifically recognize and bind to the recombinant Gn protein.

[0150] 2) Immunofluorescence (IF) verification of nanobody recognition and binding to intracellular Gn protein.

[0151] 2.1) Construction and preparation of recombinant adenovirus rAdv5-Gn

[0152] The recombinant adenovirus rAdv5-Gn used in this experiment (the viral genome carries an EGFP fluorescent tag and a Gn protein expression cassette) was constructed using a dual plasmid system (pDC316-Gn shuttle plasmid and pBHGlox(delta)E13Cre packaging plasmid) co-transfected. Specific information and procedures are as follows:

[0153] (I) Core Construction Information

[0154] The target gene is the Gn gene (GenBank accession number: KY362350.1, which is consistent with the sequence of the antigen protein used for immunization) with the signal peptide and transmembrane region removed. Its nucleotide sequence is shown in SEQ ID NO.19 and the encoding amino acid sequence is shown in SEQ ID NO.20.

[0155] Cloned gene (Gn with signal peptide and transmembrane region removed) nucleotide sequence (SEQ ID NO.19):

[0156]

[0157] Amino acid sequence (SEQ ID NO.20):

[0158] DTGPIICAGPIHSNKSADIPHLLGYSEKICQIDRLIHVSSWLRNHSQFQGYVGQRGGRSQVSYYPAENSYSRWSGLLSPCDADWLGMLVVKKAKGSDMIVPGPSYKGK VFFERPTFDGYVGWGCGSGKSRTESGELCSSDSGTTSSGLLPSDRVLWIGDVACQPMTPIPEETFLELKSFSQSEFPDICKIDGIVFNQCEGSLPQPFDVAWMDVGHSH KIIMREHKTKWVQESSSKDFVCYKEGTGPCSESEEKTCKTSGSCRGDMQFCKVAGCEHGEEASEAKCRCSLVHKPGEVVVSYGGMRVRPKCYGFSRMMATLEVNEPEQ RIGQCTGCHLECISGGVRLITLTSELKSATVCASHFCSSATSGKKSTEIQFHSGSLVGKTAIHVKGALVDGTEFTFEGSCMFPDGCDAVDCTFCREFLKNPQCYPAKK;

[0159] (II) Specific Construction Operation Process

[0160] 1. Construction of the shuttle vector pDC316-Gene

[0161] The above-mentioned Gn target gene (GeneTF) was artificially synthesized, and the target gene was cloned into the EcoRI and SalI restriction sites of the pDC316 vector by double enzyme digestion to obtain the recombinant shuttle vector pDC316-Gene.

[0162] 2. Extraction and identification of recombinant plasmids

[0163] The recombinant plasmid pDC316-Gene and the packaging plasmid pBHGlox(delta)E13Cre were extracted using a plasmid extraction kit. After extraction, the plasmids were analyzed by agarose gel electrophoresis to verify the quality of plasmid extraction.

[0164] 3. Recombinant adenovirus packaging

[0165] 1) Inoculate 1×10⁻⁶ cells / mL into a 10 cm diameter culture dish. 6 One HEK293 cell was cultured until the cells covered 80-90% of the bottom of the dish, and then set aside.

[0166] 2) Transfection was performed using lipo2000 liposomes: 4 μg of pDC316-Gene plasmid and 6 μg of pBHGlox(delta)E13Cre plasmid were added to 0.5 mL of serum-free DMEM medium; at the same time, 20 μL of lipo2000 was added to 0.5 mL of serum-free DMEM medium and incubated at room temperature for 10 min.

[0167] 3) Add the plasmid dilution solution dropwise to the liposome dilution solution, mix gently, and let stand at room temperature for 15 min to prepare the transfection complex.

[0168] 4) Slowly add the transfection complex to the HEK293 cell culture dish, gently shake well, and incubate at 37°C in a 5% CO2 incubator; 6 h after transfection, replace with DMEM complete medium containing 10% fetal bovine serum (FBS) and continue culturing.

[0169] 5) When cells exhibit significant cytopathic effect (CPE) and more than 50% of cells detach from the cell wall, collect the cells for virus lysis and isolation. The specific steps are as follows:

[0170] a. Collect the diseased cells into a clean 15 mL centrifuge tube;

[0171] b. Centrifuge at 1500 g for 5 min, discard part of the supernatant, and retain 1 mL of supernatant;

[0172] c. Place the centrifuge tube between a dry ice bath and a 37°C water bath and freeze-thaw three times rapidly. Then centrifuge at 2000 g for 15 min and collect the supernatant, which is the adenovirus solution obtained initially.

[0173] 4. Scale-up culture of recombinant adenovirus rAd-Gene

[0174] 1) Inoculate 3 × 10⁶ cells / mL into a 10 cm diameter culture dish. 6 HEK293 cells were cultured until they adhered to the culture vessel.

[0175] 2) Passage the cells at a ratio of 1:5 until 200 culture dishes are obtained.

[0176] 3) Take an appropriate amount of the adenovirus cell lysis supernatant obtained above and add it to HEK293 cells with a confluence of 95% for viral infection and amplification.

[0177] 4) When obvious CPE phenomenon is observed in the cells, collect the cell supernatant and cell precipitate respectively.

[0178] 5) After washing the cell pellet three times with PBS buffer, resuspend it in PBS buffer at 1 / 10 of the volume of the cell culture supernatant. Then freeze and thaw it three times between -80℃ and 37℃, centrifuge at 5000 g for 10 min, and collect the supernatant for later use.

[0179] 2.2) Cell seeding, adenovirus infection, and immunofluorescence staining

[0180] 1) Cell seeding: HEK-293T cells in logarithmic growth phase were seeded at a rate of 1×10⁻⁶ cells / year. 5 Cells / well were seeded at a density that was pre-placed with sterile smears in 6-well plates, DMEM medium containing 10% FBS was added, and the plates were incubated at 37°C in a 5% CO2 incubator. Adenovirus infection was then performed when the cell confluence reached 60%-70%.

[0181] 2) Adenovirus infection: Based on preliminary experimental results, the optimal infection MOI for HEK293T cells was determined to be 10. The prepared rAdv5-Gn adenovirus solution was diluted with serum-free DMEM medium to adjust the multiplicity of infection (MOI) to 10. The original medium in each well was discarded, and 1 mL of virus dilution was added to each well. The cells were incubated at 37°C for 2 h. The virus solution was then removed, and DMEM medium containing 10% FBS was added. The cells were cultured for another 24 h.

[0182] 3) Immunofluorescence staining: After infection, discard the culture medium in the wells, wash the cells three times with PBS for 5 minutes each time; complete the staining according to the following steps:

[0183] ① Fixation: Add 1 mL of 4% paraformaldehyde to each well, let stand at room temperature for 20 min, and wash with PBS 3 times, 5 min each time;

[0184] ② Permeability testing: Add 1 mL of 0.2% Triton X-100 solution to each well, incubate at room temperature for 10 min, and wash three times with PBS for 5 min each time;

[0185] ③ Blocking: Add 1 mL of 5% BSA blocking solution prepared with PBS to each well and let stand at room temperature for 1 h;

[0186] ④ Primary antibody incubation: Discard the blocking solution. Add 500 μL of G40 nanobody diluted 1:500 with PBS containing 1% BSA to each well of the experimental group (G40 group) (final concentration 2 μg / mL). Add an equal volume of PBS containing 1% BSA to the negative control group (Ctrl group) instead of the primary antibody. Place the 6-well plate in a humidified chamber and incubate overnight at 4°C. The next day, gently wash the cells 3 times with pre-cooled PBS for 5 min each time.

[0187] ⑤ Secondary antibody incubation: Add 500 μL of CoraLite®594-labeled anti-6×His-tagged fluorescent secondary antibody diluted 1:500 with PBS containing 1% BSA to each well, incubate at room temperature in the dark for 1 h, and rinse 3 times with pre-cooled PBS in the dark for 5 min each time.

[0188] ⑥ Cell nuclear staining and microscopic examination: Add 1 mL of ready-to-use DAPI staining solution to each well, incubate at room temperature in the dark for 3 min, rinse 3 times with pre-cooled PBS in the dark for 5 min each time; take out the cell slides, mount them with anti-fluorescence quenching mounting medium, and take pictures using a laser confocal fluorescence microscope.

[0189] The results are as follows Figure 5 As shown, both the negative control group (Ctrl group) and the experimental group (G40 group) showed bright green GFP fluorescence, indicating that the recombinant adenovirus rAdv5-Gn successfully infected HEK-293T cells and the target gene was successfully delivered; clear blue nuclear staining was visible in the DAPI channel, and the cell morphology was intact.

[0190] In the negative control group (Ctrl group), the Gn channel showed no specific red fluorescence signal, with only very low background fluorescence. In the experimental group (G40 group), the Gn channel showed a clear red fluorescence signal, and the fluorescence signal was mainly located in the cell membrane region, consistent with the membrane localization characteristics of Gn protein. The Merge channel showed a co-localization effect of red fluorescence, green fluorescence and blue nuclear fluorescence.

[0191] The above results indicate that the G40 nanobody prepared in this study can specifically recognize Gn protein in its native intracellular conformation without significant non-specific binding, and can be used for immunofluorescence qualitative and localization detection of Gn protein.

[0192] 3) Determination of relative binding affinity of anti-Gn protein nanobodies using the ammonium thiocyanate method

[0193] The binding affinity of nanobodies to Gn proteins was assessed using an ammonium thiocyanate (NH4SCN) elution ELISA method. This method induces complex dissociation by disrupting the non-covalent interaction between antigen and antibody with NH4SCN. The higher the affinity between the antibody and the antigen, the stronger the tolerance of the complex to high concentrations of NH4SCN, and the higher the residual binding signal under the same dissociation conditions. Based on this, the relative binding affinity of the antibody can be determined. The specific operation is as follows:

[0194] 1. Plate coating: Dilute Gn protein to 1 μg / mL with 0.05 M, pH 9.6 carbonate coating buffer, add 100 μL / well to a 96-well plate, and incubate overnight at 4°C in a humidified chamber.

[0195] 2. Blocking: The next day, discard the coating solution, wash the plate 3 times with 300 μL / well TBST buffer, 5 min each time; add 300 μL 5% BSA-TBST blocking solution to each well, and block in a humidified chamber at 37℃ for 2 h; discard the blocking solution, wash the plate 3 times with TBST, and set aside.

[0196] 3. Antibody binding: Dilute G15, G20, and G40 nanobodies to a final concentration of 1 μg / mL with TBST buffer and add 100 μL / well to the microplate. At the same time, set up a negative control group (Control group, using unrelated camel-derived nanobodies of the same concentration to replace anti-Gn nanobodies), with 3 replicates per group, and incubate at 37°C for 1 h.

[0197] 4. Dissociation treatment: Discard the antibody solution in the wells, wash the plate 3 times with 100 μL / well TBST, 5 min each time; add 100 μL of NH4SCN solution (prepared with TBST buffer) with concentration gradients of 0 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M and 5 M to each well, and incubate in a humidified chamber at 37°C for 30 min to induce dissociation of antigen-antibody complexes.

[0198] 5. Signal Detection: Discard the NH4SCN solution, wash the plate three times with 300 μL / well TBST for 5 min each time; add 100 μL of HRP-labeled rabbit anti-camel VHH antibody diluted 1:5000 with TBST to each well, and incubate at 37℃ in a humidified chamber for 1 h; discard the secondary antibody solution, wash the plate three times with TBST for 5 min each time; add 60 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 15 min; stop the chromogenic reaction by adding 60 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance (OD) of each well at 450 nm using a microplate reader. 450 ).

[0199] The results are as follows Figure 6 As shown, under the condition of no NH4SCN dissociation agent (0 M), the OD values ​​of experimental groups G15, G20, and G40 are... 450 The values ​​were all significantly higher than those in the negative control group, with the OD values ​​in the G40 group being significantly higher. 450 The highest value was 2.1, and groups G15 and G20 also showed strong positive signals, indicating that all three nanobodies screened in this study could specifically bind to Gn protein. As the NH4SCN concentration gradually increased from 0 M to 3 M, the antigen-antibody complex gradually dissociated, and the OD values ​​of the three antibodies increased. 450 The values ​​all showed a downward trend; among them, the OD of the G40 antibody was... 450The rate of decrease in binding intensity was relatively gradual. At the same NH4SCN concentration, the residual binding signal in group G40 was significantly higher than that in groups G15 and G20. These results indicate that among the three nanobodies, the G40-Gn protein binding complex exhibited the strongest stability and the highest binding affinity. Based on these affinity measurements, this study selected the G40 nanobodily with the optimal binding affinity as the core functional component for subsequent bispecific antibody construction.

[0200] V. Construction, Expression and Purification of G40-C1q Bispecific Nanobody

[0201] In the progression of severe febrile thrombocytopenic syndrome (SFTS), Dabie banda virus (DBV) infection not only directly damages target cells but also induces abnormal overactivation of the complement system, producing large amounts of pro-inflammatory effector molecules such as C3a and C5a, which in turn triggers a cytokine storm, ultimately leading to multiple organ dysfunction and is one of the core pathological mechanisms of patient death. In 2020, Laursen et al. reported a nanobody, C1qNb75, that specifically targets the human C1q protein and efficiently blocks abnormal activation of the classical complement pathway, significantly inhibiting tissue inflammatory damage mediated by excessive complement activation.

[0202] The C1qNb75 nanobody encodes the nucleotide sequence (SEQ ID NO.21):

[0203] CAGGTACAACTCGTAGAGACAGGGGGAGGTCTCGTCCAGGCAGGAGGAAGTCTTCGATTGTCTTGTGCCGCTAGCGGCCGAACTTTCAACAACGACGTCATGGCTTGGTTCAGACAAGCTCCTGGCACAGAGCGCGAGTTCGTTGCTCTGATAACCGCTGGAGGAGGCACCCACTATGCCGACTCAGT GAAGGGTCGGTTTGTGATCAGCCGAGACAACGACAAGAACATGGCCTACCTCCAGATGAACTCCCTGAAGTCTGAGGACAGCAATCTACTACTGTGGAGCCGACGAAAACCCTCCCGGCTGGCCTTCTCGATGGAGCAGTGCCTACGATTACTGGGGACAAGGCACACAGGTGACTGTATCTAGT;

[0204] C1qNb75 nanobody amino acid sequence (SEQ ID NO.22):

[0205] QVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTEREFVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIYYCGADENPPGWPSRWSSAYDYWGQGTQVTVSS;

[0206] Based on the above research, this study designed and constructed a bispecific nanobody G40-C1q that simultaneously targets DBV Gn protein and human C1q protein: the N-terminus of the antibody is a high-affinity nanobody G40 targeting DBV Gn protein, which is connected in the middle by a flexible linker (GGGGS)3, and the C-terminus is a nanobody C1qNb75 targeting human C1q.

[0207] The nucleotide sequence encoded by the flexible linker (GGGGS)3 (SEQ ID NO.23):

[0208] GGAGGAGGAGGTAGCGGCGGAGGAGGGTCTGGCGGCGGTGGATCT;

[0209] The 3 amino acid sequence of the flexible linker (GGGGS) (SEQ ID NO.24):

[0210] GGGGSGGGGSGGGGS;

[0211] This bispecific antibody can simultaneously achieve two functions: on the one hand, it specifically binds to the DBV Gn protein at the G40 end, blocking the adsorption and invasion of the virus into the host cell and inhibiting viral replication and proliferation; on the other hand, it specifically binds to C1q at the C1qNb75 end, inhibiting abnormal overactivation of the complement system, alleviating cascade inflammatory damage and tissue and organ lesions caused by abnormal release of inflammatory factors, and achieving a synergistic therapeutic effect of antiviral and anti-inflammatory treatment.

[0212] The method for constructing the G40-C1q bispecific antibody recombinant expression plasmid is as follows:

[0213] Target gene synthesis and amplification: The C1qNb75 nanobody encoding gene and the flexible linker (GGGGS)3 encoding sequence were artificially synthesized; using the synthesized gene fragment as a template, specific amplification primers with homologous arms were designed, and the flexible linker-C1qNb75 fusion gene fragment was obtained by PCR amplification. The PCR amplification system and procedure were the same as those in Part III, the VHH gene amplification system.

[0214] Vector linearization: Using the pCMV-hIL2-G40-His recombinant expression plasmid constructed in this study as a template, linearization amplification was performed by inverse PCR, so that the linearized vector ends with homologous arms matching the ends of the flexible linker-C1qNb75 fragment; after verifying the correct band size by agarose gel electrophoresis, the linearized vector fragment was excised and purified.

[0215] Homologous recombination and plasmid verification: A homologous recombination reaction system was prepared by mixing the linearized vector and the flexible linker-C1qNb75 target fragment at a molar ratio of 1:3 and incubating at 37°C for 30 min to complete the recombination. The recombination product was transformed into DH5α chemocompetent cells, plated on LB agar plates containing ampicillin, and incubated overnight at 37°C with the plates inverted. Single colonies were picked for colony PCR verification and Sanger sequencing. Strains whose sequencing results completely matched the expected sequence were identified as positive recombinant strains. Positive strains were expanded and cultured, and endotoxin-free plasmids were extracted and stored at -20°C for later use.

[0216] Eukaryotic expression of G40-C1q bispecific antibody:

[0217] Bispecific antibodies were expressed using the same eukaryotic expression system as for single nanobodies: stable Expi-293F suspension cells with ≥95% viability were expressed at a concentration of 3 × 10⁻⁶ cells / cells. 6 Cells were seeded at a density of 1 / mL in serum-free 293F expression medium. A transfection complex was prepared in every 1 mL of cell culture system, consisting of 1 µg of G40-C1q plasmid and 3 µL of PEI 40000 transfection reagent. This complex was added dropwise to the cell suspension and incubated in a shaker at 37°C, 8% CO2, and 125 rpm. Cell viability was monitored daily after transfection. When cell viability fell below 60%, all cell culture was collected for subsequent antibody purification.

[0218] Purification and Identification of G40-C1q Bispecific Antibody

[0219] The collected cell cultures were centrifuged at 4℃ and 1500 rpm for 10 min. The supernatant was filtered through a 0.45 µm sterile filter and purified by affinity chromatography using a Ni-NTA agarose gel based on the C-terminal 6×His tag. The purification procedure was the same as the nanobody purification method in Part III of this manual: nickel bead binding, washing with 10 mM imidazole to remove contaminating proteins, elution with a 20-200 mM imidazole gradient, ultrafiltration concentration, and buffer replacement to obtain the purified G40-C1q bispecific antibody. Samples from each stage were subjected to 12% SDS-PAGE electrophoresis, and the purification effect was verified by Coomassie brilliant blue staining.

[0220] Experimental results: The purification and identification results of the G40-C1q bispecific antibody are as follows: Figure 7As shown in the diagram. Lane 1 contains the unpurified cell culture supernatant, where a clear target protein band is visible at approximately 38 kDa, consistent with the expected theoretical molecular weight of the G40-C1q bispecific antibody. Lane 2 is the flow-through buffer, showing no clear target protein band, indicating high binding efficiency of the target protein to the nickel beads. Lane 3 is the 10 mM imidazole wash buffer, showing only a small amount of extraneous protein bands, with no loss of the target protein. Lanes 4-7 are gradient imidazole elution buffers, where the target protein can be efficiently eluted at a concentration of 20 mM imidazole. The elution product bands are single and without obvious extraneous bands, indicating that the purified G40-C1q bispecific antibody has high purity and meets the requirements for subsequent in vitro functional validation experiments.

[0221] The G40-C1q bispecific antibody encodes the nucleotide sequence (SEQ ID NO.25):

[0222] CAGTTGCAGCTCGCGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAGGATCTAGGCAGTTTTATTCCATAGCCTGGTTCCGGCAGGCCCCAGGGAAGGAGCGCGAGGCGGTCTCATGTATTAGTGTTAATGGTGGCGCCACAGACTATGCGGCCTCCGTGAAGGGCCGATTCACCATTTCCAGAGACCACGCCAAGAACACGGTGTATCTGCAAATGGACGGCCTGAAACCTGAGGACACAGCCGTTTATTTCTGTGCAGCCGGCGAAGGGGGATACTATGGTGGTAGTACAACATGTCCCGCCTTCAGGTATCGCGAAGCCTGGGGCCAGGGCACTCAGGTCACTGTCTCCTCAGGAGGAGGAGGTAGCGGCGGAGGAGGGTCTGGCGGCGGTGGATCTCAGGTACAACTCGTAGAGACAGGGGGAGGTCTCGTCCAGGCAGGAGGAAGTCTTCGATTGTCTTGTGCCGCTAGCGGCCGAACTTTCAACAACGACGTCATGGCTTGGTTCAGACAAGCTCCTGGCACAGAGCGCGAGTTCGTTGCTCTGATAACCGCTGGAGGAGGCACCCACTATGCCGACTCAGTGAAGGGTCGGTTTGTGATCAGCCGAGACAACGACAAGAACATGGCCTACCTCCAGATGAACTCCCTGAAGTCTGAGGACACAGCAATCTACTACTGTGGAGCCGACGAAAACCCTCCCGGCTGGCCTTCTCGATGGAGCAGTGCCTACGATTACTGGGGACAAGGCACACAGGTGACTGTATCTAGT;

[0223] Amino acid sequence of the G40-C1q bispecific antibody (SEQ ID NO.26): <N

[0224] QLQLAESGGGLVQPGGSLRLSCVASGGSRQFYSIAWFRQAPGKEREAVSCISVNGGATDYAASVKGRFTISRDHAKNTVYLQMDGLKPEDTAVYFCAAGEGGYYGGSTTCPAFRYREAWGQGTQVTVSSGGGGSG GGGSGGGGSQVQLVETGGGLVQAGGSLRLSCAASGRTFNNDVMAWFRQAPGTEREFVALITAGGGTHYADSVKGRFVISRDNDKNMAYLQMNSLKSEDTAIYYCGADENPPGWPSRWSSAYDYWGQGTQVTVSS;

[0225] VI. Virus Neutralization Experiment

[0226] The Dabie Bandar virus (DBV) strain used in this experiment was E-JS-2013-24. The complete genome sequence of this strain has been submitted to the GenBank database, accession number AMY99382.

[0227] Logarithmic growth phase Vero cells (African green monkey kidney cells) were digested with 0.25% trypsin-EDTA digestion solution, then resuspended in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin antibiotics, and the cell density was adjusted to 1×10⁻⁶ cells / year. 5 Cells / mL were seeded at 100 μL / well in 96-well cell culture plates and incubated at 37°C, 5% CO2 for 24 h until the cell count per well reached approximately 1×10⁶ cells / mL. 4 One backup.

[0228] Prepare DMEM cell maintenance medium containing 2% FBS and 1% penicillin-streptomycin antibiotics. Use this medium to dilute G15, G20, and G40 monoclonal antibodies and G40-C1q bispecific antibody to a final concentration of 50 µg / mL; simultaneously, dilute Dabie Bandar virus (DBV) strain to 1×10⁻⁶. 4 TCID 50 / mL.

[0229] Take a sterile, enzyme-free EP tube and add the diluted DBV virus solution and antibody dilution solution at a 1:1 volume ratio. Gently pipette to mix, and incubate at 37°C with 5% CO2 for 1 hour to complete the antigen-antibody neutralization reaction. Discard the original complete cell culture medium in the 96-well plate, and gently wash the cells once with sterile PBS buffer to ensure that no culture medium residue remains in the wells to avoid interfering with the experimental results.

[0230] Add the corresponding mixtures to the 96-well plates according to the following groups, with 3 replicates for each sample. The experiment is independently repeated 3 times to ensure the reliability and statistical significance of the results:

[0231] 1. Experimental group: Add 200 μL of the above-mentioned neutralized and incubated antibody-virus mixture to each well;

[0232] 2. Blank cell control group: Add 200 μL of DMEM cell maintenance medium to each well. It is virus-free and antibody-free, and is used to eliminate the interference of cell metabolism and culture medium components on the experimental results.

[0233] 3. DBV virus control group (positive control): Add 100 μL of diluted DBV virus solution and 100 μL of LMEM cell maintenance medium to each well. The final virus titer is consistent with that of the experimental group, serving as a positive reference for normal viral infection.

[0234] Incubate the inoculated 96-well plates at 37°C with 5% CO2 for 2 h to allow the virus to fully adsorb onto Vero cells. Discard all cell supernatant from the wells, gently wash the cells once with sterile PBS buffer, add 200 μL of fresh DMEM cell maintenance medium to each well, and continue incubating at 37°C with 5% CO2 for 24 h to allow the virus to replicate fully.

[0235] After culture, the 96-well plate was placed in a -80°C ultra-low temperature freezer for 30 min, then thawed in a 37°C water bath. This freeze-thaw cycle was repeated three times to fully lyse the cells and release the intracellular virus. The lysis buffer in each well was pipetted to mix thoroughly and transferred to sterile EP tubes. The tubes were centrifuged at 12,000 rpm for 2 min, and the supernatant was collected.

[0236] Following the instructions for the DBV viral RNA extraction kit, total DBV viral RNA was extracted from the supernatant. Absolute quantification of viral nucleic acid was performed using a one-step real-time quantitative RT-PCR (qRT-PCR) kit, targeting the DBV S gene (Gn protein coding region). The viral neutralization and inhibition efficiency of each antibody group was calculated using the viral nucleic acid copy number of the control group as a reference.

[0237] Virus neutralization inhibition rate (%) = (1 - average viral nucleic acid copy number in experimental group / average viral nucleic acid copy number in control group) × 100%;

[0238] Experimental results are as follows Figure 8As shown, all three single nanobodies exhibited significant DBV neutralizing activity. Among them, the neutralization inhibition rate of G15 antibody was 65%, G20 antibody was 94.7%, and G40 antibody reached 96.8%. However, the G40-C1q bispecific antibody showed the best neutralizing activity, with an inhibition rate as high as 98%, which was significantly better than that of the single nanobodies. This indicates that bispecific antibodies have greater application potential in the fight against DBV infection.

[0239] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the protection scope of the present invention.

Claims

1. A Nanobody against Dabie Bandavirus Gn protein, characterized in that, The nanobody is a VHH fragment, and the amino acid sequence of the VHH fragment is selected from any one of the amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.

14.

2. The nanobody against Dabie Bandar virus Gn protein as described in claim 1, characterized in that, The amino acid sequence of the VHH fragment is shown in SEQ ID NO.

14.

3. A bispecific nanobody, characterized in that, The bispecific nanobody comprises a first functional domain, a second functional domain, and a flexible linker connecting the first functional domain and the second functional domain. The flexible linker consists of an amino acid sequence represented by (GGGGS)n, where n is an integer from 1 to 5; The first functional domain is the nanobody against Dabie Bandar virus Gn protein as described in claim 2, and the second functional domain is a nanobody that specifically binds to human C1q protein.

4. The bispecific nanobody as described in claim 3, characterized in that, The amino acid sequence of the bispecific nanobody is shown in SEQ ID NO.

26.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody of claim 1 or the bispecific nanobody of claim 4.

6. The nucleic acid molecule as described in claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is the sequence shown in SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 or SEQ ID NO.

25.

7. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. An engineered host cell, characterized in that, It comprises the recombinant expression vector of claim 7, or the nucleic acid molecule of claim 6 integrated into the genome.

9. A pharmaceutical composition, characterized in that, It comprises an active ingredient, as well as a pharmaceutically acceptable carrier, excipient, or diluent; the active ingredient is the nanobody of claim 1 or the bispecific nanobody of claim 4.

10. The application of the nanobody of claim 1 or the bispecific nanobody of claim 4 in the preparation of Dabie Bandar virus prevention and control products, characterized in that, The products include: (1) Drugs used for the prevention and / or treatment of Dabie Bandar virus infection; (2) Reagents for detecting Dabie Bandar virus.

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