Monoclonal antibody for specifically detecting rotavirus and application thereof

Abstract

The invention provides a specific monoclonal antibody for detecting rotavirus and application of the specific monoclonal antibody, aiming at an epidemic strain of porcine rotavirus, reference to a gene sequence of VP6 protein published by GenBank, antigen epitopes are further optimized, and purified recombinant VP6 protein with antigenicity is obtained for animal immunization. The monoclonal antibody capable of specifically recognizing the porcine rotavirus is successfully screened out. The monoclonal antibody obtained through screening can effectively recognize epidemic strains, has broad-spectrum recognition, is superior to commercially available antibodies, and has high antigen recognition target performance and low non-specificity. And the sequence background is clear, large-scale amplification and production can be carried out, and the method has good commercial value.

Description

Technical Field

[0001] This invention belongs to the field of virus detection, specifically relating to a monoclonal antibody for the specific detection of rotavirus, and a rotavirus detection kit containing the antibody. Background Technology

[0002] Rotavirus (RV) belongs to the family Reoviridae and the genus Rotavirus. It was first discovered in 1973 by Bishop et al., and is named for its resemblance to wheel spokes under an electron microscope. A complete rotavirus particle has a three-layered capsid, and its genome consists of 11 double-stranded RNA segments, each encoding a protein. Segment 6 encodes the inner capsid structural protein VP6. VP6 is the most abundant protein in rotavirus and is a group (subgroup) antigen protein that plays a crucial role in viral particle assembly. It acts as a physical receptor in the assembly of the inner capsid protein genome RNA and in the entry of the outer capsid protein into the cell. Furthermore, the presence of VP6 is essential for viral transcription; therefore, VP6 is widely used in the detection of rotavirus.

[0003] Because rotavirus is an RNA virus with high variability and multiple serotypes (G and P), and different serotypes have varying infectivity to different hosts, results showed that rotaviruses in groups A, B, and C of pigs were prevalent to varying degrees. However, RVA was only detected in pigs under 10 weeks of age, with genotypes G2, G5, and G9. This disease is difficult to distinguish clinically from other diarrheal diseases caused by bacteria and viruses, making accurate diagnosis crucial for treating rotavirus infection. Currently, rotavirus detection kits are mainly for clinical use and primarily detect rotavirus antigens; there are no kits for detecting RV antibodies. Therefore, it is necessary to develop a rapid, convenient, and specific colloidal gold test strip for detecting rotavirus antibodies. Furthermore, while commercially available monoclonal antibodies against the VP6 protein exist, their antigens are mostly obtained through animal immunization with fully human rotavirus, resulting in low binding levels with porcine rotavirus. Large-scale use is also limited due to supply issues and high prices of commercially available antibodies. Summary of the Invention

[0004] To address the aforementioned issues, this invention, based on previous research (CN110470829A), further optimized the antigenic epitopes and screened for an antigenic rotavirus structural protein VP6. The gene sequence of the recombinant protein VP6 is shown in SEQ ID NO.1 (GSKDARDKIGGSGNDFQTGIGGSVENARGGARESQRNGIAPQSEGSSFDANSSDYIENWNLQNRRQGGNRSQPAHDNGSNAPKANIGRFSFPRVINSHAGSDGATTGSPNMTPAVANLGSFPQGGAIPVGPVFPPMPNWTEGSLITNYSPSREDN).

[0005] Furthermore, using the aforementioned fusion protein as an immunogen, this application screened and obtained a monoclonal antibody FK14 that can specifically recognize porcine rotavirus. The monoclonal antibody FK14 comprises at least one heavy chain variable region and at least one light chain variable region; wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3. Furthermore, the heavy chain variable region has HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6; The light chain variable region has LCDR1 as shown in SEQ ID NO:7, LCDR2 as shown in SEQ ID NO:8, and LCDR3 as shown in SEQ ID NO:9.

[0006] Furthermore, the present invention provides an isolated nucleic acid that encodes the aforementioned monoclonal antibody.

[0007] Furthermore, the present invention provides an expression vector comprising the nucleic acid isolated according to the above.

[0008] Furthermore, the present invention provides a host cell comprising the isolated nucleic acid described above, or the expression vector described above.

[0009] Furthermore, the present invention provides an antibody-drug conjugate comprising: an antibody or an antigen-binding fragment thereof prepared by the above method, or the above antibody or an antigen-binding fragment thereof; and a ligand, preferably, the ligand being selected from a radioactive isotope, a fluorescent group, and a delivery carrier.

[0010] Furthermore, the present invention provides a pharmaceutical composition comprising: a monoclonal antibody prepared by the above method, or an antibody-drug conjugate as described above; and a pharmaceutically acceptable carrier.

[0011] Furthermore, the present invention provides the use of the monoclonal antibody prepared by the above method, or the isolated nucleic acid, or the antibody-drug conjugate, or the pharmaceutical composition described above, in the preparation of a medicament for the prevention, treatment, and / or diagnosis of diseases caused by rotavirus.

[0012] Furthermore, the present invention provides a kit for in vitro detection of rotavirus or rotavirus VP6 protein, characterized in that it comprises a monoclonal antibody prepared by the above method or the isolated nucleic acid described above.

[0013] Preferably, the anti-antibody or its antigen-binding fragment is labeled with a marker.

[0014] Preferably, the marker is selected from enzymes, chemiluminescent groups, and isotope groups.

[0015] Furthermore, the present invention provides the application of the above-mentioned monoclonal antibody, or the above-mentioned isolated nucleic acid, or the above-mentioned antibody-drug conjugate, or the above-mentioned kit in the in vitro detection of rotavirus or rotavirus VP6 protein. The application is for non-disease diagnosis purposes, and the samples can be various samples such as water samples, food, and environmental samples.

[0016] Beneficial effects This invention targets prevalent strains of porcine rotavirus. Referring to the gene sequence of the VP6 protein published in GenBank, the antigenic epitopes were further optimized to obtain a purified recombinant VP6 protein with antigenicity. Animal immunization with this protein successfully screened for monoclonal antibodies that specifically recognize porcine rotavirus. The obtained monoclonal antibody effectively recognizes prevalent strains, exhibits broad-spectrum recognition, and is superior to commercially available antibodies. It also demonstrates high antigenicity and low nonspecificity. Furthermore, its clear sequence background allows for large-scale production and demonstrates significant commercial value. Attached Figure Description

[0017] Figure 1 Electrophoresis images of the VP6 gene sequence fragment recovered by enzyme digestion and the pET-32a(+) empty vector, where M is a 5000bp marker, 1 and 2 are pET-32a(+) empty vector fragments, and 3 and 4 are VP6 gene sequence fragments recovered by enzyme digestion.

[0018] Figure 2 The results of SDS-PAGE electrophoresis of recombinant VP6 protein; Figure 3 The results of Western blot analysis after purification of the target protein are shown, where M is the protein marker, 1 is the negative control, and 2 is the recombinant protein. Figure 4 This is the result of rotavirus immunofluorescence detection. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: Construction, expression, and activity identification of the rotavirus rVP6 recombinant gene Based on the published VP6 protein gene sequence in GenBank, an antigenic gene sequence was constructed. Software was used to analyze the amino acid sequence, theoretical molecular weight, and isoelectric point of the recombinant sequence. The obtained antigenic epitopes were then re-analyzed. Figure 1 The gene sequence of the recombinant protein rVP6 was obtained as shown in SEQ ID NO.1. The recombinant gene sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0021] The VP6 gene sequence fragment was ligated to the empty vector pET-32a(+) using T4 ligase to construct the pET-28a(+)-r VP6 recombinant plasmid. This plasmid was transformed into *E. coli* BL21(DE3) and plated on LB agar containing kanamycin antibody. The culture was incubated overnight at 37°C. Several single colonies were then picked for expansion. Colony PCR, plasmid double digestion, and sequencing were performed for identification, and the results were consistent with expectations. The PCR primers used were universal T7 primers.

[0022] Five mL of bacterial culture containing the correctly sequenced pET-32a(+)-VP6 recombinant plasmid was inoculated into 250 mL of LB broth containing ampicillin and incubated at 37°C with shaking at 250 rpm. When the bacterial OD reached 0.6, 1 mmol / L IPTG was added to induce expression. One mL of bacterial culture was collected before induction and at 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h after induction. The cultures were centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 40 μL of 1×PBS for SDS-PAGE electrophoresis to analyze the optimal induction time. Figure 2 As shown, SDS-PAGE electrophoresis results indicate that the rotavirus Vp6 protein was successfully expressed in Escherichia coli.

[0023] The induced bacterial culture was centrifuged at 12000 rpm for 15 min, the supernatant was discarded, and the precipitate was resuspended in 15 mL of 1×PBS. The mixture was subjected to three freeze-thaw cycles, followed by sonication for 20 min (2 s sonication time, 9.9 s pause), and centrifuged at 12000 rpm for 15 min. The supernatant and precipitate were collected separately. The precipitate was resuspended in 10 mL of 8 mol urea, centrifuged at 12000 rpm for 15 min, and the supernatant was collected. SDS-PAGE electrophoresis was used to analyze the solubility of the expression product. The expressed recombinant protein was purified using a His affinity chromatography column, and the eluent samples from each fraction were collected. Figure 3 As shown, the immunoreactivity of the recombinant protein with mouse anti-rotavirus serum was verified by Western blotting.

[0024] Example 2: Preparation of Rotavirus rVP6 Monoclonal Antibody The screening monoclonal antibody was prepared using standard procedures in the field, as briefly described below: Three 8-12 week old BALB / c mice were immunized intraperitoneally with purified recombinant PRVVP6 protein and an equal amount of Freund's complete adjuvant, with each mouse receiving 34 μg of the initial immunization; 20 days later, the mice were immunized with Freund's incomplete adjuvant at the same dose; 15 days later, the mice were injected intraperitoneally with the same dose; and 3 days later, mouse spleen cells were collected for cell fusion.

[0025] The following steps outline the screening and identification of mice with the highest titers using indirect ELISA: Purified PRV VP6 was serially diluted with antigen diluent (preferably 1:120) and added to an ELISA plate at 100 μl / well. The plate was gently tapped or shaken to ensure even antigen distribution. The coated ELISA plate was sealed with plastic wrap and incubated overnight at 4°C. The liquid in the ELISA plate was discarded, and the plate was washed three times with washing buffer. 300 μl of blocking buffer (PBS containing 0.5% polyvinyl alcohol) was added to each well, and the plate was blocked at 37°C for 2 hours. The plate was then washed three times, patted dry, and stored at 4°C for later use. Negative and positive sera were serially diluted with blocking buffer (preferably 1:600) and added to each well. Each sample was tested in duplicate, with 100 μl per well. A blank control was also included. The plates were incubated at 37°C for 60 minutes. The plates were washed three times and patted dry. Dilute goat anti-mouse enzyme-labeled antibody to the working concentration (1:40000 dilution) using blocking buffer (5% PVA in PBS), 100 μl per well, incubate at 37°C for 1 h, wash 3 times, and blot dry. Add 100 μl of freshly prepared OPD-H2O2 substrate chromogenic solution to each well, incubate at room temperature in the dark for 15 min, and terminate the reaction with 50 μl of 2 mol / L H2SO4 per well. Measure the absorbance (OD492) of each well at 492 nm using an automated microplate reader. Perform duplicate runs for each sample and take the average value.

[0026] One day prior to fusion, two healthy female BALB / c mice were euthanized by cervical dislocation. The mice were disinfected by immersion in 75% alcohol for 5 minutes. Using a sterile disposable syringe, 5-6 ml of HAT selection medium containing 20% ​​fetal bovine serum was injected into the peritoneal cavity of the mice. The abdomen was massaged for 1-2 minutes. The culture medium (containing macrophages) was then aspirated from the peritoneal cavity and added to prepared HAT medium. The cell suspension was then added to 0.1 ml per well of a 96-well cell culture plate and incubated at 37°C, saturated humidity, and 5% CO2 for 18-24 hours. Cell growth was then observed before use. Immunized BALB / c mice were excised by removing the eyeballs to extract blood, and serum was separated for positive antibody use. After euthanizing the mice by cervical dislocation, the spleen cell suspension was transferred to a 10 ml centrifuge tube, centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and basal culture medium was added to a final volume of 2 ml. The cells were resuspended and counted. Spleen cells from immunized mice and SP2 / 0 myeloma cells were mixed at a ratio of 8:1 and added to a 15ml centrifuge tube. The mixture was centrifuged at 1000rpm for 10min, and the supernatant was discarded. The bottom of the tube was gently tapped with a finger to thoroughly mix the two cell types. After centrifugation and washing, the cells were centrifuged again. The cell pellet was gently resuspended in 20ml of pre-warmed HAT selection medium containing 20% ​​fetal bovine serum, mixed thoroughly, and added to 100μl per well of a 96-well cell culture plate containing feeder cells. The culture plate was then transferred to a 37°C, 5% CO2 saturated humidity incubator for incubation. After fusion, the culture medium was changed every 3 days. When the hybridoma cells filled 1 / 4 to 1 / 3 of the well, 100 μL of cell culture supernatant was collected 3-4 days after the medium change. Without dilution, specific antibodies were detected using an indirect ELISA method (as described above) with purified recombinant PRV VP6 protein as the antigen. Cell cloning was performed using a liquid-phase limiting dilution method: after 4 cloning operations, until the positive rate of all cloned cell wells was 100%, the hybridoma cell line FK14 secreting monoclonal antibodies was confirmed. The selected FK14 cells were cryopreserved, thawed, and then inoculated into the peritoneal cavity of BALB / c mice for expanded culture. Ascites fluid was collected for later use. The titers of hybridoma cell culture supernatant and ascites fluid containing monoclonal antibodies were determined using an ELISA method. Specifically, the hybridoma cell culture supernatant was serially diluted from 1:100 to 1:12800, and the obtained ascites fluid was serially diluted from 1:100 to 1:204800. The ELISA was established using these methods. In addition to the hybridoma cell culture supernatant and ascites fluid dilutions, standard negative and positive sera, ascites fluid from SP2 / 0 cells was used as a control. The specific criteria were: negative serum OD value <0.2, positive serum OD value >1.0, test sample OD value >0.2, and P / N >2.1. The maximum dilution factor for the hybridoma cell culture supernatant and ascites fluid was defined as the ELISA titer of the hybridoma cell culture supernatant and ascites fluid.The results showed that the titer of the FK14 hybridoma cell culture supernatant was 1:1600, and the titer of the ascites fluid was 1:1×10. 7 It has a strong affinity.

[0027] The selected FK14 monoclonal antibody was sent to a sequencing company for sequencing. The results showed that the monoclonal antibody FK14 contains at least one heavy chain variable region and at least one light chain variable region; wherein the amino acid sequence of the heavy chain variable region is: QVQLQQPGAVQSGPPGASVLSCKASGSTYTNYGMHWMKQPGKGLEWIGEIDTGQSITNYTEEFKDKATLTISLEASAYLQLTSLTSEATYFCARSLAMDGWGGTSLVTVSA (SEQ ID NO.2). The amino acid sequence of the light chain variable region is: MESQTSVFVPLTLSVTIGQPIVMTQSQKFMSTSLRDRVCVTCKASQLGSDGSVAWYQKKPQRPGQKPLIYSASKLRYSGVPDRTDFTLKDFTLAISNVAEDLGFCQQYNTHFPLTFGAGTKLLELK (SEQ ID NO.3) Furthermore, the heavy chain variable region has HCDR1 as shown in SEQ ID NO:4 (NYGMH), HCDR2 as shown in SEQ ID NO:5 (EIDTGQSITNYTEEFKD), and HCDR3 as shown in SEQ ID NO:6 (SLAMDG). The light chain variable region has LCDR1 as shown in SEQ ID NO:7 (KASQLGSDGSVA), LCDR2 as shown in SEQ ID NO:8 (SASKLRYS), and LCDR3 as shown in SEQ ID NO:9 (QQYNTHFPLT).

[0028] Example 3: Rotavirus Immunofluorescence Detection and Specificity Detection Viral samples (porcine rotavirus strains SD23 (G2 serotype) and SC07 (G9 serotype), isolated and preserved by our research group) were collected and diluted 10-fold with DMEM maintenance medium containing trypsin at a final concentration of 10 μg / mL. The samples were then digested in a water bath at 37 ℃ for 1 h, with PBS used as a control. The digested virus solution was serially diluted 10-fold and inoculated into MA104 cells for infection for 90 min. After infection, the virus solution was discarded, and 200 μL / well of DMEM culture medium containing 2 μg / mL trypsin was added. The cells were incubated for 18-24 h. Indirect immunofluorescence experiments were then performed according to the "Veterinary Immunology Experimental Guide," with the specific procedure as follows: (1) Antigen fixation: Discard the culture medium in the 96-well plate, slowly add PBS to wash away the residual culture medium, discard the PBS, add 100 μL of anhydrous methanol pre-cooled at 4 ℃ / well, fix at room temperature for 5 min, discard the fixative, and wash twice with PBS buffer, 5 min each time.

[0029] (2) Blocking: Add 100 μL of the blocking solution prepared on site to each well and block at room temperature for 1 h. Discard the blocking solution and wash three times with PBS buffer.

[0030] (3) Primary antibody incubation: Add 50 μL of FK14 hybridoma cell culture supernatant diluted with antibody diluent (1:800) to each well and incubate overnight at 4 °C.

[0031] (4) Secondary antibody incubation: Discard the primary antibody, wash twice with PBS buffer, add 50 μL / well of goat anti-mouse IgG-FITC diluted with antibody diluent, and incubate at room temperature in the dark for 1 h.

[0032] (5) Discard the secondary antibody, wash five times with PBS, and observe the infected cells under a fluorescence microscope to see if they are fluorescent. Count and photograph the results of the immunofluorescence experiment.

[0033] The results are as follows: Figure 4 As shown, most cells in cell cultures inoculated with rotavirus exhibited bright green fluorescence in their cytoplasm and cell membrane; no reaction signal was observed in uninoculated cell cultures. The monoclonal antibodies screened above can effectively recognize porcine rotavirus, providing a foundation for subsequent virus localization and control.

[0034] Indirect ELISA was used to determine the cross-reactivity between hybridoma cell culture medium and common clinical porcine diarrhea viruses (swine transmissible gastroenteritis virus TH-98 strain, porcine epidemic diarrhea virus SH01 strain, porcine pseudorabies virus JS27 strain, and porcine reproductive and respiratory syndrome virus JX145 strain, all isolated and preserved by our research group). The specific procedures were as described above, with healthy mouse serum serving as a negative control. The results are shown in Table 1. No reaction signal was observed between the above viruses and the FK14 monoclonal antibody screened in this application, confirming that the FK14 monoclonal antibody prepared in this application has good species specificity and can be used for rapid identification of clinical pathogens.

[0035] Table 1. Specificity analysis of FK14 monoclonal antibody

[0036] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that, The antibody or antigen-binding fragment comprises light chain variable regions LCDR1, LCDR2, LCDR3 and heavy chain variable regions HCDR1, HCDR2, HCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is shown in SEQ ID NO:8, the amino acid sequence of LCDR3 is shown in SEQ ID NO:9, the amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

6.

2. A nucleic acid molecule encoding an antibody or antigen-binding fragment as described in claim 1.

3. An expression vector comprising the nucleic acid molecule according to claim 2.

4. A host cell comprising the nucleic acid molecule according to claim 2, or the expression vector according to claim 3.

5. A composition comprising: the antibody or antigen-binding fragment of claim 1.

6. The use of the antibody or antigen-binding fragment of claim 1 in the in vitro detection of rotavirus or rotavirus VP6 protein, wherein the use is not for disease diagnosis.

Images