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] The invention belongs to the field of virus detection, and in particular relates to a monoclonal antibody for specifically detecting rotavirus, and a rotavirus detection kit containing the antibody. Background Art

[0002] Rotavirus (RV) belongs to the Reoviridae family and the genus Rotavirus. It was first discovered by Bishop et al. in 1973 and was named rotavirus because it resembles wheel spokes under an electron microscope. The complete rotavirus particle has a three-layer capsid. Its genome is composed of 11 double-stranded RNA segments. Each gene segment encodes a protein. Gene segment 6 is responsible for encoding the inner shell structural protein VP6. VP6 protein is the most abundant protein in rotavirus and is a group (subgroup) antigen protein of the virus. It plays an important role in the assembly process of viral particles. It acts as a physical receptor in the assembly of the inner shell protein genome RNA and the entry of the outer shell protein into the cell. At the same time, the presence of VP6 protein is essential for the transcription of the virus. Therefore, VP6 is widely used in the detection of rotavirus.

[0003] Because rotavirus is an RNA virus with high variability and multiple serotypes (G type and P type), and different serotypes have different infectivity to different hosts, the results show that porcine rotaviruses in groups A, B, and C are all prevalent to varying degrees, but RVA is only detected in pigs less than 10 weeks old, and its genotypes are G2, G5, and G9. It is difficult to distinguish this disease from diarrheal diseases caused by other bacteria and viruses clinically, and accurate diagnosis is of great significance for the treatment of infection with this virus. At present, the kit for detecting rotavirus is mainly used for clinical detection, and is mainly used to detect rotavirus antigens. There is no kit for detecting RV antibodies. Therefore, it is necessary to develop a colloidal gold test strip for rapid, convenient, and specific detection of rotavirus antibodies. In addition, there are currently commercial monoclonal antibodies for VP6 protein, but the antigens prepared are mostly obtained by animal immunization of fully human rotavirus, and the binding level with porcine rotavirus is not high, and large-scale use is limited due to the supply of commercial antibodies and high prices. Summary of the invention

[0004] In order to solve the above problems, the present invention further optimizes the antigenic epitope on the basis of previous research (CN110470829A), and screens and obtains a rotavirus structural protein VP6 with antigenicity. The gene sequence of the recombinant protein VP6 is shown in SEQ ID NO.1 (GSKDARDKIGGSGNDFQTGIGGSVENARGGARESQRNGIAPQSEGSSFDANSSDYIENWNLQNRRQGGNRSQPAHDNG SNAPKANIGRFSFPRVINSHAGSDGATTGSPNMTPAVANLGSFPQGGAIPVGPVFPPMPNWTEGSLITNYSPSREDN).

[0005] Furthermore, the present application uses the above fusion protein as an immunogen to screen and obtain a monoclonal antibody FK14 that can specifically recognize porcine rotavirus, wherein 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;

[0006] Further, 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;

[0007] 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.

[0008] Furthermore, the present invention provides an isolated nucleic acid encoding the above monoclonal antibody.

[0009] Furthermore, the present invention provides an expression vector comprising the isolated nucleic acid described above.

[0010] Furthermore, the present invention provides a host cell comprising the above-mentioned isolated nucleic acid or the above-mentioned expression vector.

[0011] Furthermore, the present invention provides an antibody-drug conjugate, comprising: the antibody or antigen-binding fragment thereof prepared by the above method, or the above antibody or antigen-binding fragment thereof; and a ligand, preferably, the ligand is selected from a radioactive isotope, a fluorescent group and a delivery vector.

[0012] Furthermore, the present invention provides a pharmaceutical composition, comprising: the monoclonal antibody prepared by the above method, or the above antibody-drug conjugate; and a pharmaceutically acceptable carrier.

[0013] Furthermore, the present invention provides a use of the monoclonal antibody prepared by the above method, or the above isolated nucleic acid, or the above antibody-drug conjugate or the above pharmaceutical composition in the preparation of a drug for preventing, treating and / or diagnosing a disease caused by rotavirus.

[0014] Furthermore, the present invention provides a kit for in vitro detection of rotavirus or rotavirus VP6 protein, characterized in that it comprises the monoclonal antibody prepared by the above method, or the above isolated nucleic acid.

[0015] Preferably, the anti-antibody or antigen-binding fragment thereof is labeled with a marker.

[0016] Preferably, the label is selected from an enzyme, a chemiluminescent group and an isotope group.

[0017] Furthermore, the present invention provides the use 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 in vitro detection of rotavirus or rotavirus VP6 protein, wherein the application is for non-disease diagnosis purposes, and the samples may be various samples such as water samples, food, and environment.

[0018] Beneficial Effects

[0019] The present invention targets the epidemic strain of porcine rotavirus, refers to the gene sequence of VP6 protein published in GenBank, further optimizes the antigenic epitope, obtains a recombinant VP6 protein with antigenicity for animal immunization, and successfully screens out a monoclonal antibody that can specifically identify porcine rotavirus. The screened monoclonal antibody can effectively identify the epidemic strain, has a broad spectrum of recognition, and is superior to commercially available antibodies. At the same time, it has a high antigen recognition target and low non-specificity. In addition, the sequence background is clear, and it can be scaled up and produced, and has good commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is the electrophoresis diagram of the VP6 gene sequence fragment recovered by enzyme digestion and the pET-32a(+) empty vector, wherein M is a 5000bp Marker, 1 and 2 are pET-32a(+) empty vector fragments; 3 and 4 are VP6 gene sequence fragments recovered by enzyme digestion.

[0021] Figure 2 This is the SDS-PAGE electrophoresis result of VP6 recombinant protein;

[0022] Figure 3This is the western blot result after the target protein is expressed and purified, where M is the protein Maker, 1 is the negative control, and 2 is the recombinant protein

[0023] Figure 4 The results of rotavirus immunofluorescence test. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 Construction, expression and activity identification of rotavirus rVP6 recombinant gene

[0026] With reference to the gene sequence of VP6 protein published in GenBank, a gene sequence with antigenicity was constructed, and the amino acid sequence, theoretical molecular weight and isoelectric point expressed by the recombinant sequence were analyzed using DNAstar software, and the obtained antigen epitopes were reorganized and analyzed ( Figure 1 ), the gene sequence of the recombinant protein rVP6 is shown in SEQ ID NO. 1. The recombinant gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0027] The VP6 gene sequence fragment was connected to the pET-32a(+) empty vector using T4 ligase to construct the pET-28a(+)-r VP6 recombinant plasmid, which was transformed into Escherichia coli BL21(DE3), spread on LB solid medium containing kanamycin antibody and cultured overnight at 37°C, and then several single colonies were picked for expansion culture. The bacterial liquid PCR and plasmid double enzyme digestion and sequencing identification were consistent with expectations. The primers used in PCR were T7 universal primers.

[0028] Inoculate 5 mL of correctly sequenced bacterial culture containing the pET-32a(+)-VP6 recombinant plasmid into 250 mL of LB liquid culture medium containing ampicillin, place in a 37°C shaking incubator, and shake at 250 rpm. When the bacteria are cultured to an OD of 0.6, add 1 mmol / L IPTG to induce expression. Take 1 mL of the bacterial culture before induction, 1h, 2h, 3h, 4h, 6h, and 8h after induction, centrifuge at 12000 rpm for 10 min, discard the supernatant, resuspend the precipitate with 40 μL of 1×PBS, perform SDS-PAGE electrophoresis, and analyze the optimal induction time. Figure 2 As shown, the SDS-PAGE electrophoresis results showed that the rotavirus Vp6 protein was successfully expressed in Escherichia coli.

[0029] The induced bacterial solution was centrifuged at 12000rmp for 15min, the supernatant was discarded, the precipitate was resuspended in 15mL of 1×PBS, repeated freeze-thaw for 3 times, ultrasonically disrupted for 20min (ultrasonic time 2s, pause time 9.9s), centrifuged at 12000rmp for 15min, and the supernatant and precipitate were collected separately. The precipitate was resuspended in 10mL of 8moL urea, centrifuged at 12000rmp for 15min, and the supernatant was collected. The solubility of the expressed product was analyzed by SDS-PAGE electrophoresis. The expressed recombinant protein was purified using a His affinity chromatography column, and the eluate samples from each section were collected. Figure 3 As shown, the immunoreactivity of the recombinant protein with mouse anti-rotavirus serum was verified by Western Blot.

[0030] Example 2 Preparation of rotavirus rVP6 monoclonal antibody

[0031] The monoclonal antibodies were prepared and screened using routine operations in the art, which are briefly as follows: 3 8-12 week old BALB / c mice were taken and intraperitoneally immunized with purified recombinant PRVVP6 protein plus an equal amount of Freund's complete adjuvant, with 34 μg for each mouse for the first immunization; 20 days later, Freund's incomplete adjuvant was added for immunization, with the same dose as before; 15 days later, the antigen was intraperitoneally injected, with the same dose as before; 3 days later, the mouse spleen cells were taken for cell fusion.

[0032] The indirect ELISA was used to screen and identify mice with the highest titer. The following is summarized as follows: take purified PRV VP6 and dilute it with antigen diluent (preferably 1:120), add it to the ELISA plate, 100μl / well, and flick or vibrate the plate to evenly distribute the antigen. Seal the coated ELISA plate with plastic wrap, keep it at 4℃ overnight, discard the liquid in the ELISA plate, and wash it three times with washing solution. Add 300μl of blocking solution (PBS containing 0.5% polyvinyl alcohol) to each well, block it at 37℃ for 2h, wash it three times, pat it dry, and put it in a 4℃ refrigerator for later use. Dilute the negative and positive sera with blocking solution (preferably 1:600), add them to each reaction well, do 2 wells in parallel for each sample, 100μl in each well, and set up a blank control at the same time, and act at 37℃ for 60min. Wash it three times and pat it dry. Dilute goat anti-mouse enzyme-labeled antibody to working concentration (1:40000 dilution) with blocking solution (5% PVA in PBS), 100 μl per well, 37℃ for 1 hour, wash 3 times, pat dry. Add 100 μl of newly prepared OPD-H2O2 substrate colorimetric solution to each well, color at room temperature in the dark for 15 minutes, 2 mol / L H2SO4 per well 50 μl to terminate the reaction. Use an automatic enzyme-labeled analyzer to measure the light absorption (OD492) value of each well at a wavelength of 492 nm. Do 2 wells in parallel for each sample each time and take the average value.

[0033] One day before fusion, select two healthy BALB / c female mice and kill them by cervical dislocation, soak and disinfect them in 75% alcohol for 5 minutes, use a disposable sterile syringe to draw 5-6ml of HAT selection culture medium containing 20% ​​fetal bovine serum and inject it into the mouse's peritoneal cavity, rub the mouse's abdomen for 1-2 minutes, then use a syringe to suck out the culture medium in the peritoneal cavity (containing macrophages), add it to the prepared HAT culture medium, add the cell suspension to a 96-well cell culture plate, 0.1ml per well, culture in a 37℃, saturated humidity, 5% CO2 incubator for 18-24 hours, observe the cell growth status for use. Take the immunized BALB / c mice, remove the eyeballs and bleed them, separate the serum for positive antibodies; after killing the mice by cervical dislocation, separate the spleen cell suspension and transfer it to a 10ml centrifuge tube, centrifuge at 1000rpm for 10 minutes, discard the supernatant, replenish the basal culture medium to 2ml, resuspend the cells, and count them. Take the spleen cells of immune mice and myeloma cells SP2 / 0 and mix them in the ratio of 8:1, add them to a 15ml centrifuge tube, centrifuge at 1000rpm for 10min, discard the supernatant; tap the bottom of the tube with your fingers to mix the two cells thoroughly; centrifuge and wash them, and centrifuge again. Suspend the precipitated cells lightly in 20ml of pre-warmed HAT selection culture medium containing 20% ​​fetal bovine serum, mix them evenly, add them to the 96-well cell culture plate with feeder cells, 100μl per well, and move the culture plate to a 37℃, 5% CO2 saturated humidity incubator for culture. After the fusion, the culture medium of the cells was replaced every 3 days. When the hybridoma cells covered 1 / 4-1 / 3 of the bottom of the well, 100ul of the cell culture supernatant was taken 3-4 days after the medium was replaced. The specific antibody was detected by the indirect ELISA method (as above) with the purified recombinant PRV VP6 protein as the antigen without dilution. The cell cloned by liquid phase limiting dilution method: after 4 cloning operations, when the positive rate of all cloned cell wells was 100%, it was determined that the hybridoma cell line FK14 secreting monoclonal antibodies had been obtained. The selected FK14 was frozen and revived and inoculated into the peritoneal cavity of BALB / C mice for expansion culture, and the ascites was collected for standby use. The titer of hybridoma cell culture supernatant and monoclonal antibody ascites was detected by ELISA method, that is, the hybridoma cell culture supernatant was diluted from 1:100 to 1:12800, and the ascites was diluted from 1:100 to 1:204800 to establish ELISA. In addition to the hybridoma cell culture supernatant dilution and ascites dilution, standard negative and positive serum, the ascites of SP2 / 0 cells should also be used as a control. The specific judgment criteria are: negative serum OD value <0.2, positive serum OD value>1.0, test sample OD value>0.2, P / N>2.1, the maximum dilution of hybridoma cell culture supernatant and ascites is the ELISA titer of hybridoma cell culture supernatant and ascites. The results showed that the titer of FK14 hybridoma cell culture supernatant was 1:1600, and the titer of ascites was 1:1×10 7, with strong affinity.

[0034] The screened FK14 monoclonal antibody was sent to a sequencing company for sequencing, and the results showed that the monoclonal antibody FK14 contained 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 was: QVQLQQPGAVQSGPPGASVLSCKASGSTYTNYGMHWMKQPGKGLEWIGEIDTGQSITNYTEEFKDKATLTISLEASAYLQLTSLTSEATYFCARSLAMDGWGGTSLVTVSA (SEQ ID NO.2);

[0035] The amino acid sequence of the light chain variable region is: MESQTSVFVPLTLSVTIGQPIVMTQSQKFMSTSLRDRVCVTCKASQLGSDGSVAWYQKKPQ RPGQKPLIYSASKLRYSGVPDRTDFTLKDFTLAISNVAEDLGFCQQYNTHFPLTFGAGTKLL ELK (SEQ ID NO.3)

[0036] Further, the heavy chain variable region has a HCDR1 as shown in SEQ ID NO:4 (NYGMH), a HCDR2 as shown in SEQ ID NO:5 (EIDTGQSITNYTEEFKD), and a HCDR3 as shown in SEQ ID NO:6 (SLAMDG);

[0037] 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).

[0038] Example 3 Rotavirus immunofluorescence detection and specific detection thereof

[0039] Take virus samples (swine rotavirus SD23 strain (G2 serotype) and SC07 strain (G9 serotype), isolated and preserved by our research group), dilute 10 times with DMEM maintenance solution with a final concentration of trypsin of 10μg / mL, digest for 1h in a 37℃ water bath, and use PBS as a control sample. Dilute the digested virus solution tenfold, inoculate MA104 cells for infection for 90min, discard the virus solution after infection, add DMEM culture solution containing 2μg / mL trypsin, 200μL / well, and culture in an incubator for 18-24h. Refer to the "Veterinary Immunology Experiment Guide" for indirect immunofluorescence experiment. The specific operation process is as follows:

[0040] (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 4°C pre-cooled anhydrous methanol, 100 μL / well, fix at room temperature for 5 min, discard the fixative, and wash twice with PBS buffer, 5 min each time.

[0041] (2) Blocking: Add 100 μL / well of the blocking solution prepared on site, block for 1 h at room temperature, discard the blocking solution, and wash three times with PBS buffer.

[0042] (3) Primary antibody incubation: 50 μL of FK14 hybridoma cell culture supernatant diluted with antibody diluent (1:800) was added to each well and incubated at 4°C overnight.

[0043] (4) Secondary antibody incubation: discard the primary antibody, wash twice with PBS buffer, add goat anti-mouse IgG-FITC (1:2000) diluted with antibody diluent, 50 μL / well, and incubate at room temperature in the dark for 1 h.

[0044] (5) Discard the secondary antibody, wash the cells five times with PBS, and then observe the infected cells for fluorescence under a fluorescence microscope. Count and take photos to record the results of the immunofluorescence experiment.

[0045] The results are as follows: Figure 4 As shown, most cells in the cell culture inoculated with rotavirus showed green flashing fluorescence in their cytoplasm and cell membrane; no reaction signal was observed in the cell culture without virus inoculation. The above-screened monoclonal antibodies can effectively identify porcine rotavirus, providing a basis for the subsequent location and prevention and control of the virus.

[0046] Indirect ELISA was selected to determine the cross reaction between hybridoma cell culture fluid and common porcine diarrhea viruses (porcine transmissible gastroenteritis virus TH-98 strain, porcine epidemic diarrhea virus SH01 strain, porcine pseudorabies virus JS27 strain, porcine reproductive and respiratory syndrome virus JX145 strain, all isolated and preserved by this research group). The specific operation is as described above, and healthy mouse serum is used as a negative control. The results are shown in Table 1. There is no reaction signal between the above viruses and the FK14 monoclonal antibody screened in this application, which proves that the FK14 monoclonal antibody prepared in this application has good species specificity and can be used for rapid determination of clinical pathogens.

[0047] Table 1 Specificity analysis of FK14 monoclonal antibody

[0048]

[0049] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. 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 present invention should be within the protection scope of the present invention.

Claims

1. An antigenic rotavirus structural protein VP6, characterized in that The gene sequence of the recombinant protein VP6 is shown in SEQ ID NO.

1.

2. A monoclonal antibody that specifically recognizes rotavirus structural protein VP6, characterized in that 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.

3. A nucleic acid molecule encoding the rotavirus structural protein VP6 according to claim 1 or the monoclonal antibody according to claim 2. An expression vector comprising the nucleic acid molecule according to claim 3 .

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

6. A composition comprising: the rotavirus structural protein VP6 according to claim 1 or the monoclonal antibody according to claim 2, and a ligand.

7. Use of the rotavirus structural protein VP6 according to claim 1, the monoclonal antibody according to claim 2, the nucleic acid molecule according to claim 2, the host cell according to claim 4, and / or the composition according to claim 6 in the preparation of a medicament for preventing, treating and / or diagnosing a disease caused by rotavirus.

8. A kit for in vitro detection of rotavirus or rotavirus VP6 protein, characterized in that: Comprising the rotavirus structural protein VP6 according to claim 1, the monoclonal antibody according to claim 2, the nucleic acid molecule according to claim 2, the host cell according to claim 4, and / or the composition according to claim 6.

9. Use of the rotavirus structural protein VP6 described in claim 1, the monoclonal antibody described in claim 2, the nucleic acid molecule described in claim 2, the host cell described in claim 4, the composition described in claim 6 and / or the kit described in claim 8 for in vitro detection of rotavirus or rotavirus VP6 protein, wherein the use is non-disease diagnosis.

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