G6 type bovine rotavirus nanobody and application thereof

Abstract

This invention discloses a G6 bovine rotavirus nanobody and its application, belonging to the field of nanobody technology. The purpose of this invention is to provide a G6 bovine rotavirus nanobody. This invention provides a bovine rotavirus nanobody, namely nanobody 5A. The amino acid sequence of CDR1 of nanobody 5A is shown in SEQ ID NO. 9, the amino acid sequence of CDR2 of nanobody 5A is shown in SEQ ID NO. 10, and the amino acid sequence of CDR3 of nanobody 5A is shown in SEQ ID NO. 11. This provides a novel and efficient strategy for the prevention and control of bovine rotavirus.

Description

Technical Field

[0001] This invention belongs to the field of nanobody technology, specifically relating to a G6 bovine rotavirus nanobody and its applications. Background Technology

[0002] Calf diarrhea is a significant disease threatening the healthy development of animal husbandry, especially in newborn calves, where it has a high morbidity and mortality rate. The disease is characterized by acute watery diarrhea, dehydration, and electrolyte imbalance. Without timely intervention, calf mortality can reach over 90%, and it significantly increases the risk of secondary bacterial infections (such as E. coli and Salmonella), severely impacting calf survival rates and economic benefits. The primary cause of calf diarrhea is infection by pathogenic microorganisms, among which bovine rotavirus (BRV) is the main pathogen causing calf diarrhea.

[0003] BRV (Breast Virus Respiratory Virus) belongs to the genus Rotavirus in the family Reoviridae. It causes severe diarrhea by damaging the epithelial cells of the small intestinal villi, interfering with nutrient absorption. This virus is highly contagious; it resides in the intestines of infected cattle and is excreted in feces, contaminating the environment. Healthy cattle can become infected upon contact with contaminated environments, and outbreaks are more likely to occur during cold seasons and in poorly hygienic farming conditions. Currently, treatment primarily involves glucose-glycine or glucose-amino acid solutions, and intravenous injection of glucose saline and sodium bicarbonate solutions to prevent dehydration and desalination. Antibiotics are ineffective against the virus, only alleviating secondary infections. Prevention and control primarily involve vaccination (such as maternal and active immunization) and hygiene management. However, existing vaccines suffer from insufficient serotype coverage (e.g., prevalent strains like G6 and G10), maternal antibody interference, and viral mutations, making it difficult to provide long-lasting and broad-spectrum protection. Therefore, there is an urgent need to develop new and highly effective prevention and control strategies. Summary of the Invention

[0004] The purpose of this invention is to provide a nanobody for detecting G6 bovine rotavirus.

[0005] This invention provides a bovine rotavirus nanobody, wherein the nanobody is nanobody 5A, the amino acid sequence of CDR1 of nanobody 5A is shown in SEQ ID NO.9, the amino acid sequence of CDR2 of nanobody 5A is shown in SEQ ID NO.10, and the amino acid sequence of CDR3 of nanobody 5A is shown in SEQ ID NO.11.

[0006] Further specifying, the amino acid sequence of nanobody 5A is shown in SEQ ID NO.7.

[0007] Further specifying, the amino acid sequence of frame region FR1 is shown in SDQ ID NO.12, the amino acid sequence of FR2 is shown in SDQ ID NO.13, the amino acid sequence of FR3 is shown in SDQ ID NO.14, and the amino acid sequence of FR4 is shown in SDQ ID NO.15.

[0008] The present invention provides a drug for treating bovine rotavirus, the drug containing the above-mentioned bovine rotavirus nanobody.

[0009] The present invention provides a nucleotide sequence encoding the above-mentioned rotavirus nanobody nucleotide sequence or its complementary sequence.

[0010] The present invention provides an expression vector containing the above-described nucleotide sequence.

[0011] The present invention provides a host cell containing the above-described expression vector.

[0012] This invention provides the application of the above-mentioned bovine rotavirus nanobody in the preparation of a kit for detecting bovine rotavirus or a drug for treating bovine rotavirus infection.

[0013] This invention provides the application of the above-mentioned expression vector in the preparation of a kit for detecting bovine rotavirus or a drug for treating bovine rotavirus infection.

[0014] This invention provides the use of the above-mentioned host cells in the preparation of a kit for detecting bovine rotavirus or a drug for treating bovine rotavirus infection.

[0015] This invention provides a kit for detecting bovine rotavirus, wherein the kit is an ELISA kit, the bovine rotavirus nanobody described in any one of claims 1-3 is used as a capture antibody, and BRV-VP6 is used as a detection antibody.

[0016] Beneficial effects: Nanobody 5A showed obvious lesions starting from a 512-fold dilution, FRNT 50 =512, exhibiting high neutralizing activity; when the antigen is diluted to a high dilution of 1:6400 and the antibody concentration is as low as 0.312 μg / mL, its OD... 450 The value was still significantly higher than the negative control, indicating that the target antigen could still be effectively detected under these conditions, fully demonstrating the excellent detection sensitivity of nanobody 5A. It showed no cross-reactivity with other non-target components (G10 BRV, BPIV3, BVDV, PPRV, IBRV, and BCoV), exhibiting high specificity. Based on checkerboard titration analysis, a 1:1600 antigen coating concentration and a 0.625 μg / mL nanobody 5A concentration are recommended as optimized working conditions for subsequent applications. This combination ensures high signal intensity (OD).450 While ≈ 0.98), the cost-effectiveness of antibody use is also taken into account. Attached Figure Description

[0017] Figure 1 Image showing the results of alpaca serum titer testing;

[0018] Figure 2 Figure 1 shows the construction results of the anti-bovine rotavirus nanobody phage library; Figure 2 shows the results of the first round of nested PCR amplification; Figure 3 shows the results of the second round of nested PCR amplification; Figure 4 shows the results of phage vector enzyme digestion; Figure 5 shows the results of the second round of nested PCR enzyme digestion; Figure 6 shows the results of bacterial culture identification PCR.

[0019] Figure 3 ELISA results of crude extracts from anti-bovine rotavirus nanobody strains after three rounds of screening; A shows the positive rates of the first, second, and third rounds of screening; B shows the positive rates of inter-batch and intra-batch replicates of positive strains from the third round of screening.

[0020] Figure 4 Figures showing the expression, purification, and identification results of anti-bovine rotavirus nanobodies; A is the SDS-PAGE result, 1 is the uninduced supernatant of 5A, 2 is the induced supernatant of 5A, 3 is the liquid after elution with 5A flow-through solution, 4 is the liquid after washing with 40mM imidazole, 5 is the liquid after washing with 60mM imidazole, 6 is the liquid after washing with 100mM imidazole, and 7-11 are the liquids after washing with 250mM imidazole; B is the ELISA identification result; C is the IFA identification result.

[0021] Figure 5 Figure showing the results of neutralizing titer assay for anti-bovine rotavirus nanobodies;

[0022] Figure 6 Image showing the specific detection results of anti-bovine rotavirus nanobodies;

[0023] Figure 7 Image showing the sensitivity detection results of anti-bovine rotavirus nanobodies;

[0024] Figure 8 The results of the application of anti-bovine rotavirus nanobody sandwich ELISA. Detailed Implementation

[0025] G6 type BRV (BRV-C73 strain) and G10 type BRV (BRV-BLR strain) are described in the article "Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening. Virol Sin. 2024 Sep 28:S1995-820X(24)00149-4.doi: 10.1016 / j.virs.2024.09.010".

[0026] Example 1. Construction of an alpaca immune and phage display library

[0027] 1.1 Alpaca Immunity

[0028] Positive immunization: The BRV-C73 strain previously isolated in our laboratory was amplified, 0.3% formaldehyde was added, and the mixture was inactivated at 37°C for 72 hours. This inactivated strain was used as the immunogen. The viral titer before antigen inactivation was 1×10⁻⁶. 8.0 TCID 50 / ml, after inactivation, prepare immunogen. For the first immunization, use 1ml of inactivated antigen and an equal volume of Freund's complete adjuvant emulsified. For subsequent immunizations, use 1ml of inactivated antigen and an equal volume of Freund's incomplete adjuvant emulsified. Immunize once every two weeks for a total of four immunizations. Each alpaca is immunized with 2ml each time. Two weeks later, collect venous blood from the alpaca.

[0029] Negative: Unimmunized alpaca venous blood was used as a negative control.

[0030] 1.2 Construction of Phage Library 50 ml of alpaca venous blood obtained through antigen immunization was collected. Peripheral blood lymphocytes were extracted using a peripheral blood lymphocyte extraction kit, followed by total RNA extraction using chloroform extraction. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. Primers VHH-F1 and VHH-R1 were designed based on the conserved regions at both ends of the VHH sequence. Using cDNA as a template, and the amplified product as a template, second-round PCR primers Pb3-VHH-FR1-F and Pb3-VHH-FR4-R were designed based on the conserved regions FR1 and FR4 in the alpaca VHH sequence and the restriction enzyme sites of the pComb3 vector. Nested PCR was performed, and the resulting product was the cDNA library. Primer sequences are shown in Table 1. The phage was ligated into the phage vector pComb 3, and the ligation product was then transformed into competent TG1 cells by electroporation. The bacterial culture was serially diluted 10-fold, and 100 μL of each dilution was plated onto 2×YTAG plates with two replicates for each dilution. The culture was incubated overnight at 37°C. The next day, the library volume was calculated based on the colony growth at each dilution, and 22 single colonies were randomly selected for colony PCR to verify the insertion of the VHH gene. All colonies were then collected, added to glycerol, and stored at -80°C to obtain the phage library.

[0031] Table 1 Primers required for constructing the anti-bovine rotavirus nanobody phage library and primers required for constructing the VHH-C73 prokaryotic expression plasmid.

[0032]

[0033] Note: W = A or T, R = A or G, Y = C or T, M = A or C.

[0034] The results showed that the serum titer of alpacas was highest two weeks after the fourth immunization. Figure 1 Lymphocytes were isolated from alpaca venous blood two weeks after the fourth immunization, and total RNA was extracted from the lymphocytes, reverse transcribed, and then amplified by nested PCR. The first-round amplification product was approximately 750 bp. Figure 2 In the A section, the second-round amplification product is around 400bp. Figure 2 The B in the sequence improved the specificity of the VHH sequence. Subsequently, the phage vector (pComb3) and the second round of amplification products were digested with SifⅠ enzyme. Figure 2 The C and D molecules were then ligated into a phage vector using T4 DNA ligase, resulting in a library with a volume of 2.4 × 10⁻⁶. 7 A phage display library of PFU / ml was used to randomly select single colonies. Colony PCR was performed to detect the positivity of recombinant phages. A target band at 750bp was considered positive, and below 750bp was considered negative. The results of the bacterial culture PCR showed (…). Figure 2Of the 22 single colonies randomly selected in the E) sample, 20 were positive, with a positive rate of 90.90%. Sequencing of the positive colonies revealed that the amino acid sequences of these 20 positive bacteria were different, indicating the rich diversity of the phage library.

[0035] Example 2. Screening and enrichment of anti-bovine rotavirus nanobodies

[0036] Purified BRV was used to coat immunotubes (2 ml / tube) at a concentration of 1:100 using CBS as the coating solution. The next day, unbound viruses were removed by washing with PBS, and the tubes were blocked for 2 hours with MPBS containing 2% skim milk powder to block non-specific binding sites on the surface of the immunotubes and reduce non-targeted phage adsorption in subsequent steps. The library of bacteria was inoculated into 2×YTAG medium and cultured until the logarithmic growth phase (OD200). 600 =0.4), KM13 helper phage was added to infect the host bacteria. After centrifugation, the medium was replaced with 2×YTAK medium (containing kanamycin and ampicillin), and cultured overnight at 30°C with shaking to induce phage secretion. The next day, after centrifugation to remove bacterial cells, the supernatant was concentrated by PEG / NaCl precipitation, followed by resuspension with PBS and blocking with MPBS to reduce nonspecific background. The treated phage was co-incubated with BRV-coated immunosorbent tubes, and unbound or weakly bound phages were removed by rigorous washing (ten washes with alternating PBST and PBS). Subsequently, specifically bound phages were eluted with 100 mM triethylamine (TEA) solution. TEA achieves gentle elution by disrupting the electrostatic or hydrogen bonding between protein and phage, avoiding damage to phage activity. After elution, the phages were neutralized with Tris-HCl (pH=7.4) and then used to infect TG1 Escherichia coli (OD1) in the logarithmic growth phase. 600 =0.4), and after centrifugation and resuspending the bacterial cells, the infected bacterial solution was plated on 2×YTAG solid medium and incubated overnight at 30°C to isolate single colonies. The plate colonies were collected and resuspended on 2×YT medium, glycerol (final concentration 15%) was added, and the mixture was aliquoted and frozen at -70°C to obtain the primary library (named VHH-C73-1). The above operations were repeated to screen the primary and secondary libraries, for a total of three rounds of screening.

[0037] The results, as shown in Table 2, indicate that after three rounds of screening, the output number of each round increases with the number of screenings. The output number of the first round is 1.32 × 10⁻⁶. 6 PFU / ml, increased to 1.29×10 in the second round. 7 PFU / ml, increased to 2.94×10 in the third round. 9 PFU / ml, and the output count in each round using coated CBS as a negative control did not show significant changes, all remaining within 10. 4The enrichment data showed that the enrichment degree from the second round of screening to the first round was 0.09, indicating a good enrichment effect. The enrichment degree from the third round of screening to the second round was 0.1, indicating a good enrichment effect. Moreover, the output number of the third round of screening increased by about 100 times compared with the second round. Therefore, screening was stopped and preliminary identification of the Chinese library bacteria in the third round of screening was started.

[0038] Table 2 Results of three rounds of screening for anti-bovine rotavirus nanobodies

[0039]

[0040] Example 3. Specificity identification of crude extract of anti-bovine rotavirus nanobody

[0041] The specificity of the strains output from each round of screening was preliminarily identified by ELISA. Eighty monoclonal strains were picked from each output plate from each round of screening and inoculated into three 96-well deep-well plates. Each well contained 200 μL of 2×YTAG medium and was incubated at 30°C with shaking. After 8 hours, 20 μL of bacterial culture was transferred from each well and inoculated into 180 μL of 2×YTAG medium, and incubated at 37°C with shaking. The remaining bacterial culture on the original plates was diluted with 60 μL of 60% glycerol to a final concentration of 15%, and the culture was stored at -80°C. After 1 hour of incubation with shaking, KM13 helper phage was added to each well, and the plates were incubated at 37°C for 30 minutes, followed by shaking at 37°C for 40 minutes. The plates were centrifuged at 1800 × g for 10 minutes, the supernatant was discarded, and the pellet was resuspended in 400 μL of 2×YTAK medium in each well and incubated overnight with shaking at 30°C. The following day, the deep-well plate was centrifuged at 5000 × g for 10 minutes. 250 μL of phage supernatant was transferred from each well to a new deep-well plate, and 200 μL of blocking buffer (PBS buffer containing 5% BSA) was added to each well. The plate was incubated at 37°C for 1 hour, ready for indirect ELISA detection. Purified BRV was used to coat 96-well microplates at a concentration of 1:100, 100 μL per well, and incubated overnight at 4°C. A CBS-coated microplate was also used as a negative control. The following day, the coating liquid was discarded, and 200 μL of 5% BSA was added to each well, which was then blocked at 37°C for 1 hour. Discard the blocking buffer from the wells. Add 100 μL of phage supernatant treated with the blocking buffer to each well as primary antibody. Incubate at 37°C for 1 hour. Wash the plate three times with PBST. Add 100 μL of HRP-M13 Antibody (1:10000 dilution) to each well and incubate at 37°C for 1 hour. Wash the plate three times with PBST. Add 100 μL of chromogenic substrate to each well and incubate in the dark for 15 minutes. Then add 100 μL of stop solution to each well to terminate the reaction. Read the OD values ​​using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 630 nm. Analyze the ELISA results and determine the positive well numbers.

[0042] The results showed that 80 single colonies were picked from the plates output from the first round of screening to prepare crude nanobody extracts. ELISA detection (Figure 3A) revealed 2 positive bacteria, with a positive rate of approximately 2%. 80 single colonies were picked from the plates output from the second round of screening to prepare crude nanobody extracts. ELISA detection (…) Figure 3 In step A), 59 positive bacteria were found, with a positive rate of approximately 73.7%. Eighty single colonies were picked from the plates output from the third round of screening to prepare crude nanobody extracts, which were then analyzed by ELISA. Figure 3In section A), 79 positive bacteria were found, with a positive rate of approximately 98%. Subsequently, inter-batch and intra-batch replicates were performed on the crude nanobody extract of the third round of exported bacteria. The results of inter-batch and intra-batch replicates (…) Figure 3 B) The results were the same as the initial test. Subsequently, the positive bacteria in the third round of output were sequenced, and the positive bacteria with better ELISA identification results were selected for expression identification.

[0043] Example 4. Expression, purification and identification of anti-bovine rotavirus nanobodies

[0044] Glyceryl bacteria corresponding to the positive wells were inoculated into 1 ml of 2 × YTAG medium and cultured at 37°C with shaking. The culture was then sent to a sequencing company for sequencing. After the sequencing results were returned, they were analyzed and combined with the preliminary specificity identification results of ELISA to determine the prokaryotic expression clone. Glyceryl bacteria of the strain to be cloned were inoculated into 5 ml of 2 × YTAG medium and cultured at 37°C with shaking for 12 h. Plasmids were extracted using a plasmid mini-extraction kit as template plasmids for prokaryotic expression. Primers for prokaryotic expression (Pcold-VHH-BRV-F, Pcold-VHH-BRV-R, see Table 1) were then designed based on the sequence information to amplify the VHH sequence of the nanobody and ligate it into the pCold-6×his prokaryotic expression vector via infusion to construct recombinant plasmids for prokaryotic expression of the nanobody. The recombinant plasmids were transformed into the BL21(DE3) strain to obtain the corresponding nanobody expression strain and induced at 16°C with shaking for 20 h. The expression of the nanobody was verified by SDS-PAGE, and the nanobody expressed in the supernatant was purified for further verification.

[0045] Protein purification: Inoculate 100 mL of LB medium with engineered bacteria containing the target plasmid. Incubate at 37°C and 220 rpm with shaking until the bacterial culture reaches OD500. 600 Reach a concentration of 0.4-0.6. Add IPTG to the culture to a final concentration of 0.5 mM. Adjust the temperature to 16℃ and the rotation speed to 200 rpm, inducing expression for 14-16 hours. After induction, aliquot the bacterial culture into 50 mL centrifuge tubes. Centrifuge at 12,000 g for 5 minutes at 4℃, collect the bacterial pellet, and carefully discard the supernatant. Add 10 mL of Lysis Buffer to each bacterial pellet. Resuspend the bacterial pellet thoroughly using a vortex mixer or pipette. Place the resuspended bacterial culture on ice and sonicate to disrupt the protein.

[0046] Sonication parameters: 3 seconds on, 5 seconds off, total duration 20 minutes or until the bacterial suspension becomes clear. Centrifuge the sonicated lysate at 4°C and 12,000g for 15-20 minutes. After centrifugation, carefully transfer the supernatant (containing soluble target protein) to a new centrifuge tube. Filter the supernatant using a 0.45 μm filter membrane to remove residual small particles and prevent column clogging. Take 3 mL of nickel column affinity packing and equilibrate with Lysis Buffer for at least 5-10 column volumes. Mix the filtered supernatant obtained in the previous step with the equilibrated nickel column packing at 4°C. Place on a rotary shaker or mixer and gently mix for 1-2 hours to ensure the target protein fully binds to the packing. Repack the packing into the column and collect the flow-through.

[0047] Wash the column sequentially with wash buffers containing different concentrations of imidazole to remove non-specifically bound proteins. Wash with 40 mM imidazole wash buffer, then with 60 mM imidazole wash buffer, and finally with 100 mM imidazole wash buffer. Elute the target protein with elution buffer containing 250 mM imidazole, collecting 1 mL of eluent per tube for a total of 3-5 tubes. Immediately perform SDS-PAGE electrophoresis to analyze the eluent fractions to determine which tubes contain high-purity target protein.

[0048] For ELISA identification, purified BRV was coated onto 96-well microplates at a concentration of 1:100, 100 μL per well, and incubated overnight at 4°C. CBS coating was used as a negative control. The next day, the coating solution was discarded, and 200 μL of 5% BSA was added to each well for blocking at 37°C for 1 hour. The blocking solution was then discarded, and 100 μL of purified nanobody was added to each well as the primary antibody. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and then 100 μL of HRP-Anti-Llama (SinoBiological, 1:10000) was added to each well for incubation at 37°C for 1 hour. The plates were washed three times with PBST. 100 μL of chromogenic substrate was added to each well, and the plates were incubated in the dark for 15 minutes. The reaction was then stopped by adding 100 μL of stop solution to each well. OD values ​​were read using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 630 nm. Analyze the ELISA results and determine the positive well numbers. The rest is the same.

[0049] Indirect immunofluorescence (IFA) identification was performed by first seeding Marc-145 cells into 96-well plates and culturing them for 48 hours, followed by inoculation with G6 type BRV (BRV-C73 strain). After obvious lesions appeared, the cells were fixed with pre-cooled anhydrous ethanol at -20°C for 30 minutes. The cells were washed with PBS to remove the fixative residue, and then 200 μL of blocking buffer (PBS buffer solution containing 5% BSA) was added and incubated at 37°C for 1 hour. Purified nanobody was added and incubated overnight at 4°C. The next day, His-Tag monoclonal antibody (Proteintech, 1:1000) was added and incubated at 37°C for 1 hour. Finally, FITC-labeled goat anti-mouse IgG was added and incubated at 37°C for 1 hour. After washing, the fluorescence signal was observed under a fluorescence microscope.

[0050] The results showed that the obtained 5A nanobody sequence was cloned into the pcold-6×His vector for expression verification, and the SDS-PAGE results were as follows ( Figure 4 As shown in A), the obtained nanobody molecular weight is around 15 kDa, and the nanobody expression level is high. ELISA identification results ( Figure 4 B) shows that, compared with negative and positive sera from alpacas, the purified nanobody 5A can specifically bind to G6 type BRV (BRV-C73 strain); IFA identification results ( Figure 4 As shown in C), the purified nanobody 5A can specifically bind to G6 type BRV (BRV-C73 strain) and exhibits a specific fluorescent signal, while no obvious fluorescent signal is observed in the cellular background.

[0051] 5A amino acid sequence: (SEQ ID NO.7) QGVQAQVQLVESGGGSVQPGGSLILSCAASESLSNSYITAWWRQGPGKQREFVASIFGHGSTKYAGFAEGRFTISRDNAKSTVYLQMNNLEPEDTAVYRCAIGQTNFGYDWWGQGTQVTVSA;

[0052] 5A nucleotide sequence: (SEQ ID NO.8) CAAGGTGTCCAGGCTCAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTGATACTCTCCTGTGCGGCCTCTGAAAGCCTCTCGAATAGTTATATCACGGCCTGGTGGCGCCAGGGCCCGGGGAAGCAGCGCGAGTTCGTCGCTTCGATTTTTGGTCATGGAA GTACAAAGTATGCGGGCTTCGCGGAGGGTCGATTCACCATCTCCAGAGACAACGCCAAAAGCACGGTGTATTTGCAAATGAACAACCTGGAGCCTGAGGACACGGCCGTCTATCGCTGTGCGATCGGGCAGACCAATTTCGGGTATGACTGGTGGGGCCAGGGGACCCAGGTCACCGTCTCCGCA;

[0053] 5A Complementarity Determining Zone:

[0054] CDR1: ASESLNSYIT (SEQ ID NO.9)

[0055] CDR2:IFGHGSTKYA (SEQ ID NO.10)

[0056] CDR3: AIGQTNFGYDW (SEQ ID NO.11)

[0057] 5A Framework Area:

[0058] FR1: QGVQAQVQLVESGGGSVQPGGSLILSCA (SEQ ID NO.12)

[0059] FR2:AWWRQGPGKQREFVAS (SEQ ID NO.13)

[0060] FR3: GFAEGRFTISRDNAKSTVYLQMNNLEPEDTAVYRC (SEQ ID NO.14)

[0061] FR4: WGQGTQVTVSA (SEQ ID NO. 15).

[0062] Example 5. Application of anti-bovine rotavirus nanobodies

[0063] Neutralization activity assay: G6 BRV was treated with 50 μg / ml trypsin solution at 37°C for 1 hour; the purified nanobody was serially diluted 2-fold starting from 1:4, using serum-free DMEM maintenance medium containing 10 μg trypsin; the trypsin-treated virus was diluted to 1000 TCID. 50 The diluent was serum-free DMEM containing 10 μg / ml trypsin. An equal volume of the diluted virus was added to the serially diluted nanobody sample, shaken at 75 rpm for 5 min, and neutralized at 37°C for 1 hour. Marc-145 cells cultured in 96-well cells for 48 hours were washed twice with 100 μL / well PBS. The serum-virus mixture was added to the 96-well cells, with two replicates per sample, added sequentially according to the nanobody dilution factor from highest to lowest. Adsorption was performed at 37°C for 1 hour. The adsorption buffer was discarded, and the cells were washed once with 100 μL / well PBS. Serum-free DMEM maintenance medium containing 10 μg / ml trypsin was added, and the cells were incubated at 37°C. CPE was observed daily. The endpoint was determined after 5 days of culture, and the neutralizing antibody titer was calculated using the Reed-Muench method.

[0064] Specificity identification: Different pathogens—G6 BRV (BRV-C73 strain), G10 BRV (BRV-BLR strain), bovine parainfluenza virus type 3 (BPIV3), bovine epidemic diarrhea virus (BVDV), small ruminant disease virus (PPRV), bovine infectious rhinotracheitis virus (IBRV), and bovine coronavirus (BCoV)—were coated onto 96-well ELISA plates at a concentration of 1:16, with 100 μL per well. The plates were incubated overnight at 4°C. A CBS-coated ELISA plate was used as a negative control. The next day, the coating liquid was discarded, and 200 μL of 5% BSA was added to each well, which was then blocked at 37°C for 1 hour. Discard the blocking solution in each well. Add the nanobody to be tested to each well and incubate at 37°C for 1 hour. Wash the plate three times with PBST. Add 100 μL of HRP-Anti-Llama (Sino Biological, 1:10000, dilution 1:10000) to each well and incubate at 37°C for 1 hour. Wash the plate three times with PBST. Add 100 μL of chromogenic substrate to each well and incubate in the dark for 15 minutes. Then add 100 μL of stop solution to each well to terminate the reaction. Read the OD values ​​using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 630 nm.

[0065] Sensitivity determination: G6 type BRV (BRV-C73 strain) was serially diluted and coated onto 96-well microplates at concentrations of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, with 100 μL per well. The plates were incubated overnight at 4°C. A CBS-coated microplate was used as a negative control. The next day, the coating liquid was discarded, and 200 μL of 5% BSA was added to each well, which was then blocked at 37°C for 1 hour. Discard the blocking solution in each well. Add different concentrations of the nanobody 5A to be tested (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.312 μg / mL, 0.156 μg / mL, and 0 μg / mL) to each well. Incubate at 37°C for 1 hour, then wash three times with PBST. Add 100 μL of HRP-Anti-Llama (Sino Biological, 1:10000, dilution 1:10000) to each well and incubate at 37°C for 1 hour, then wash three times with PBST. Add 100 μL of chromogenic substrate to each well and incubate in the dark for 15 minutes. Finally, add 100 μL of stop solution to each well to terminate the reaction. Read the OD values ​​using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 630 nm.

[0066] Establishment of the sandwich ELISA method: Purified nanobody 5A was diluted to a concentration of 100 ng / 100 μL / well as the capture antibody and coated overnight at 4 °C. Then, 200 μL of blocking buffer (PBS buffer containing 5% BSA) was added and incubated at 37 °C for 1 hour. Next, 50 μL of the test antigen (purified BRV-C73 strain) was added to each well and incubated at 37 °C for 1 hour. Then, rabbit anti-BRV-VP6 (100 ng / well, VP6 was obtained as a serum antibody from rabbits injected with the antigen) was added and incubated at 37 °C for 1 hour. HRP-Anti-rabbit (Thermo, 1:10000) was added and incubated at 37 °C for 1 hour. 100 μL of TMB chromogenic solution was added to each well and reacted in the dark for 15 minutes (positive wells showed a blue color). Immediately afterward, 100 μL of stop solution was added to terminate the reaction. OD values ​​were read using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 630 nm.

[0067] The results showed that the purified anti-bovine rotavirus nanobody underwent a neutralization test, such as... Figure 5 The results showed that 5A nanobody exhibited obvious lesions starting from a 512-fold dilution, FRNT 50 =512, exhibiting high neutralizing activity.

[0068] The prepared anti-bovine rotavirus nanobody 5A was specifically identified using ELISA, and the results are as follows: Figure 6 As shown in the figure. Nanobody 5A showed a specific response to G6 bovine rotavirus (BRV-C73 strain), but no significant reactivity was detected against G10 BRV or other pathogens. Sensitivity testing of nanobody 5A yielded the following results: Figure 7 As shown, it exhibits a high level of specificity across a wide range of antigen dilutions and antibody concentration gradients, OD 450 The absorbance value showed a clear dose-dependent relationship with both antigen and antibody concentrations. Even when the antigen was diluted to a high dilution of 1:6400 and the antibody concentration was as low as 0.312 μg / mL, its OD value remained relatively stable. 450 The OD values ​​were still significantly higher than those of the negative control, indicating that the target antigen could still be effectively detected under these conditions, fully demonstrating the excellent detection sensitivity of nanobody 5A. Furthermore, the reaction system showed extremely low background interference, and the OD values ​​of all CBS-coated negative control wells remained stable at around 0.10, indicating that the antibody had no cross-reactivity with other non-target components and possessed high specificity. Based on the checkerboard titration results, it is recommended that a 1:1600 antigen coating concentration and a 0.625 μg / mL nanobody 5A concentration be used as optimized working conditions in subsequent applications. This combination can ensure high signal intensity (OD... 450 While ≈ 0.98), the cost-effectiveness of antibody use is also taken into account.

[0069] Figure 8 This indicates that BRV-G6 is the detection well for the BRV-C73 strain of the antigen to be tested, and its OD... 450 The value was 0.88, indicating a clear positive signal. The OD of the DMEM negative control well was... 450 The value was 0.14, indicating a low background level, demonstrating that nanobody 5A effectively captured the BRV-C73 antigen and successfully formed a "capture-detection" complex with the subsequently added rabbit anti-BRV-VP6 detection antibody and enzyme-labeled secondary antibody system, thus producing a specific colorimetric reaction. The low negative control value indicates weak non-specific binding and a clean background in the experimental system. These results confirm that nanobody 5A possesses good capture ability and specificity in a sandwich ELISA system and can serve as an effective capture antibody for detecting G6 bovine rotavirus.

Claims

1. A nanobody against G6 bovine rotavirus, characterized in that, The nanobody is nanobody 5A. The amino acid sequence of CDR1 of nanobody 5A is shown in SEQ ID NO.9, the amino acid sequence of CDR2 of nanobody 5A is shown in SEQ ID NO.10, and the amino acid sequence of CDR3 of nanobody 5A is shown in SEQ ID NO.

11.

2. The anti-G6 bovine rotavirus nanobody according to claim 1, characterized in that, The amino acid sequence of nanobody 5A is shown in SEQ ID NO.7; the amino acid sequence of frame region FR1 is shown in SDQ ID NO.12, the amino acid sequence of FR2 is shown in SDQ ID NO.13, the amino acid sequence of FR3 is shown in SDQ ID NO.14, and the amino acid sequence of FR4 is shown in SDQ ID NO.

15.

3. A drug for treating G6 bovine rotavirus, characterized in that, The drug contains the anti-G6 bovine rotavirus nanobody as described in claim 1.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encoding the anti-G6 bovine rotavirus nanobody as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5.

7. The use of the anti-G6 bovine rotavirus nanobody according to claim 1 or 2 in the preparation of a kit for detecting G6 bovine rotavirus or a medicament for treating G6 bovine rotavirus infection.

8. The use of the expression vector according to claim 5 in the preparation of a kit for detecting G6 bovine rotavirus or a drug for treating G6 bovine rotavirus infection.

9. The use of the host cell described in claim 6 in the preparation of a kit for detecting G6 bovine rotavirus or a medicament for treating G6 bovine rotavirus infection.

10. A kit for detecting G6 bovine rotavirus, characterized in that, The kit is an ELISA kit, with the G6 bovine rotavirus nanobody as described in claim 1 or 2 as the capture antibody and BRV-VP6 as the detection antibody.

Images