Preparation of canine bocavirus MVC non-structural protein NP1 monoclonal antibody

Abstract

The invention discloses a preparation method of a canine bocavirus MVC non-structural protein NP1 monoclonal antibody, which specifically comprises the following steps: constructing a prokaryotic expression vector PET-32a (+)-NP1 of NP1 protein, expressing and purifying by using Rosetta cells to obtain NP1 recombinant protein, immunizing a mouse, preparing and screening hybridoma B cells secreting the NP1 specific monoclonal antibody, and preparing the canine bocavirus MVC non-structural protein NP1 monoclonal antibody by using the hybridoma B cells to obtain the canine bocavirus MVC non-structural protein NP1 monoclonal antibody. And collecting ascites, and purifying through an affinity chromatography column to obtain the NP1 monoclonal antibody. The method has the advantages that 1, the positive rate of hybridoma cells is improved; 2, the screening process is simplified; and 3, the antibody is excellent in performance, high in specificity and high in sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation of a monoclonal antibody against the NP1 non-structural protein of canine bocavirus MVC. Background Technology

[0002] Large Bocavirus (MVC) is a linear single-stranded DNA virus belonging to the genus Bocavirus in the family Parvoviridae. Infection with MVC causes severe respiratory and gastrointestinal illnesses. The non-structural protein NP1 of MVC plays a crucial role in viral replication. As a protein unique to the Bocavirus genus, NP1 is essential for the accumulation of capsid mRNA and capsid proteins, and is necessary for MVC viral DNA replication. When NP1 protein is absent, MVC replication is reduced by approximately 320-fold. Studies have reported that when HBoV1 infectious clones or MVM infectious clones that do not express NP1 protein are transfected into cells, Southern blot analysis shows that when NP1 is not expressed, almost no single- or double-replicated DNA is produced. In addition, NP1 protein also plays multiple roles in the processing of viral precursor mRNA. It has been reported that the NP1 protein of both MVC and HBoV1 viruses promotes the transcription of mRNA precursors into mRNA and can promote the generation of VP and NS protein transcripts. Furthermore, in the mechanism by which MVC virus infects the host and induces an immune response in host cells, the cellular RNA processing factor CPSF6 (cleavage and polyadenylation specific factor 6) has been shown to interact with the NP1 protein of MVC and regulate the transcriptional activity of MVC virus mRNA.

[0003] The prevalence and spread of bocaviruses pose a significant threat to the health of humans and other mammals. Clinically, it causes gastrointestinal symptoms such as vomiting and diarrhea, as well as respiratory symptoms such as coughing and runny nose in humans and various animals. Minute virus of canines (MVC), belonging to the genus *Bocavirus* within the subfamily Parvoviruses, is widely present in serological evidence, with a global serological prevalence of 50%-70%. Infection can lead to abortion and stillbirth in pregnant dogs, and can cause mild gastroenteritis and respiratory diseases in puppies. Recent studies have shown that MVC infection is associated with diseases such as pneumonia, myocarditis, lymphadenitis, and hepatitis in large dogs. Therefore, the prevention and treatment of diseases caused by MVC infection are particularly urgent, but there is currently no effective vaccine. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a monoclonal antibody against the canine bocavirus MVC nonstructural protein NP1, specifically including the following steps:

[0005] I. Construction and identification of the prokaryotic expression vector PET-32a(+)-NP1 for NP1 protein

[0006] The NP1 gene of the MVC virus (GenBank NC 075119.1) was indexed in the GenBank database (F: 5'-GCGGATCCATGTCTACGAGACATATGAGCA AGA-GATCA-3'; R: 5'-GCGTCGACCTATTCGGAGGAGCCATCTAC CTCC-3'). The NP1 gene was amplified by PCR, and the prokaryotic expression vector pET32a(+) was double-digested with restriction endonucleases BamHI and SalI. The digestion reaction was carried out at 37℃ for 2 hours. The target gene and the digested plasmid were ligated by homologous recombination. After transformation, plating, bacterial selection, culture, and plasmid extraction, double digestion was performed for identification.

[0007] Specifically, the enzyme digestion products were recovered using an agarose gel DNA recovery kit. Next, the PCR-recovered products were ligated with the vector plasmid using the Clon ExpressII One Step Cloning Kit, and the ligation reaction was carried out at 37°C for 30 min. Finally, we obtained a recombinant prokaryotic expression vector with a His tag, named pET-32a(+)-NP1. Subsequently, these ligation products were transformed into the TOP10 strain. After ampicillin resistance selection culture, 2-3 single clones were selected and sent to the company for sequencing. The sequencing results were compared with the target fragment sequence. Clones with identical sequences were selected, expanded, and the plasmid was extracted and stored at -20°C for later use.

[0008] II. Induction and Purification of Recombinant Proteins

[0009] The recombinant plasmid pET-32a(+)-NP1 was transformed into Rosetta competent cells and plated for culture. Several single clones were picked from the plates and inoculated into LB medium containing ampicillin. Expression was induced at 37°C for 4 h using 1 mM IPTG. The bacterial pellet was collected and sonicated. The remaining sample was placed in a 100°C water bath for 5 min, followed by SDS-PAGE electrophoresis. After electrophoresis, Coomassie brilliant blue staining was performed and the stain was removed. The expression levels of the three selected clones were analyzed to determine the correct protein molecular weight. The clone with the highest expression level was selected to prepare glycerol-containing bacteria and stored for later use. The clone with the highest expression level was inoculated, cultured, and induced to express at high volume. The cells were collected by centrifugation (6000 rpm, 5 min). The cells were resuspended in 20 mM Tris buffer and analyzed by electrophoresis. The bacterial resuspension was then sonicated and centrifuged at high speed (4°C, 13000 rpm, 10 min). The supernatant was transferred to a new centrifuge tube for purification. The precipitate was dissolved overnight at 4°C with a suitable ratio of dissolving buffer (8M urea, PBS, pH 7.4) and stirred. After high-speed centrifugation (4°C, 13000 rpm, 10 min), the supernatant was collected for purification. The supernatant and precipitate were collected separately for electrophoresis detection. NP1 protein was mainly expressed in the supernatant. The supernatant was then loaded onto a Ni column pre-equilibrated with PBS, mixed, and incubated for 40 min. After incubation, the flow-through was collected, and the column was washed with PBS for 100 column volumes. The column was then washed again with PBS containing 20 mM imidazole for 20 column volumes. Elute the target protein with 5-10 mL of PBS containing 250 mM imidazole, collect the eluent, add 1 mL of eluent each time, and elute multiple times. Each time the eluent is collected, take 10 μL of the eluent and add it to 100 μL of Braford G250 to observe the color change. When protein is eluting, it will turn blue. Repeat adding 1 mL of eluent until no more protein is eluted. The collected protein solution is retained for SDS-PAGE analysis.

[0010] 3. BALB / c mice were immunized with purified recombinant protein, and 10 hybridoma cells that stably secreted anti-NP1 monoclonal antibodies were successfully obtained by hybridoma technology. The fusion cells were then injected into mice and ascites fluid was collected. The NP1 monoclonal antibody was obtained after purification by affinity chromatography.

[0011] Specifically, to induce an immune response in mice against a specific antigen (recombinant protein): For the first immunization, an equal volume of purified recombinant protein was thoroughly mixed with Freund's complete adjuvant and administered via multiple subcutaneous injections; three BALB / c mice were injected each time, with each mouse receiving 30 mL of recombinant protein. The interval between each immunization was 14 days, and a total of four immunizations were administered. Seven days after the third immunization, tail vein blood samples were collected from the mice to detect serum antibody titers. Mice with the highest titers were selected for cell fusion, and a pulse immunization was administered three days before fusion.

[0012] Hybridoma technology: 100 μL of immune solution was injected into the peritoneal cavity of mice. The mouse spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1. Hybridoma cells were obtained by screening with HAT medium. The cell supernatant was then used for preliminary screening by ELISA. The positive clone replicates were used for secondary screening at different dilutions. Finally, fusion cells that stably secrete NP1 monoclonal antibody were obtained.

[0013] In summary, the present invention has the following beneficial effects: 1. Improved positive rate of hybridoma cells: As an immunogen, the recombinant protein has a single stimulating component compared with the traditional whole virus immunogen, which can more accurately induce mice to produce specific antibodies against NP1 protein, thereby significantly improving the positive rate of hybridoma cells.

[0014] 2. Simplified screening process: Reduces tedious work, avoids complex screening steps in the preparation of monoclonal antibodies from whole viral immunogens, reduces a lot of tedious work in the screening process, reduces the uncertainty of screening results, and improves screening efficiency and success rate.

[0015] 3. Excellent antibody performance: The NP1 monoclonal antibody specifically recognizes the NP1 protein in MVC-infected WRD cells, exhibiting high specificity. The antibody also boasts high sensitivity, effectively detecting low concentrations of NP1 protein, thus improving the accuracy and reliability of detection.

[0016] 4. Laying the foundation for research: Identification of biological functions: It provides a material basis for the identification of the biological functions of the MVC virus NP1 protein, which helps to conduct in-depth research on the function of the NP1 protein and the interaction between the MVC virus NP1 protein and host cell proteins, and helps to reveal the pathogenic mechanism of the virus. Attached Figure Description

[0017] Figure 1 The operation flowchart of this invention;

[0018] Figure 2 Construction of the pET-32a(+)-NP1 prokaryotic expression vector;

[0019] M: DL10000 DNA Marker, Lane 1: pET-32a(+)-NP1 recombinant plasmid double-digested with BamHI and SalI.

[0020] Figure 3 Expression and purification of recombinant NP1, a non-structural protein of MVC;

[0021] A. Small-scale induction expression: Lane 1 and Lane 3: Recombinant protein before induction; Lane 2 and Lane 4: Samples after recombinant protein induction. B. Large-scale induction expression: Single clones with the highest expression levels were selected for large-scale induction. Lane 1: Recombinant protein sample before induction; Lane 2: Recombinant protein sample after large-scale induction. C. Cell lysis diagram: Cells were sonicated, centrifuged, and detected by SDS-PAGE electrophoresis. Lane 1: Represents the lysis supernatant; Lane 2: Represents the precipitate. NP1 recombinant protein was mainly expressed in the supernatant. D. The supernatant was purified using a Ni affinity chromatography column. The resulting recombinant protein was approximately 41.7 kDa, consistent with the size of the fusion protein. The molecular weight of the recombinant protein was 41.7 kDa, and the molecular weight of the carrier protein (uncleaved) was 20 kDa.

[0022] Figure 4 Identification of monoclonal antibodies against NP1, a non-structural protein of MVC;

[0023] AC. Western blot validation of the detection efficacy of NP1 monoclonal antibody. Lanes 1-12: Protein Maker, His-tagged antibody, NP1 monoclonal antibody of models 1C19, 1C19-1, 1H6, 1J22, 1J22-1, 2112, 2112-3, 3M17, 3M17-1, and 4E19. D. ELISA assay to detect the titer of NP1 monoclonal antibody.

[0024] Figure 5 Validation of the efficacy of monoclonal antibody against NP1, a non-structural protein of MVC;

[0025] A. Western blot to verify the detection efficacy of all types of NP1 monoclonal antibodies; B. Western blot to verify the detection efficacy of screened NP1 monoclonal antibodies and polyclonal antibodies; CB: Quantitative analysis of NP1 protein expression.

[0026] Figure 6 To observe the effects of NP1 monoclonal antibody and polyclonal antibody using immunofluorescence;

[0027] Immunofluorescence (IFA) assays were performed on WRD cells infected with MVC 24 hours prior to the detection of viral protein NP1 labeled with different types of NP1 monoclonal antibodies. The NP1 monoclonal antibodies were combined with green fluorescent secondary antibody. DAPI (blue) was used to label the cell nucleus. The Merge group shows the overlap between NP1 protein staining and the cell nucleus.

[0028] Figure 7 To identify the characteristics of NP1 monoclonal antibodies using immunoprecipitation.

[0029] Immunoprecipitation analysis of NP1 was performed using three different types of NP1 monoclonal antibodies (1C19, 1J22, and 2112). IgG-L represents the light chain of IgG antibody, and IgG-H represents the heavy chain of IgG antibody. Detailed Implementation

[0030] The following is in conjunction with the appendix Figure 1-7 The present invention will be further described in detail below with reference to the embodiments:

[0031] Example 1

[0032] 1. Construction and identification of recombinant plasmid pET-32a(+)-NP1

[0033] Using the infectious clone pI-MVC (GenBank accession number: NC_075119.1) of MVC as a template, the NP1 gene of approximately 570 bp was amplified. The pET-32a(+) vector was simultaneously digested with BamHI and SalI. Homologous recombination was performed between the target gene and the digested plasmid. After transformation, plating, bacterial picking, culture, and plasmid extraction, double enzyme digestion was performed for identification. The results are shown below. Figure 2 As shown in the figure, a clear target band appeared at around 570bp, and the sequencing was correct, indicating that the NP1 gene was successfully inserted into the pET-32a(+) vector, and the pET-32a(+)-NP1 plasmid was successfully constructed.

[0034] 2. Expression and purification of recombinant MVC nonstructural protein NP1

[0035] After successful double enzyme digestion verification of the recombinant expression plasmid, it was transformed into *E. coli* Rosetta and plated. Three single clones were selected from the plate and induced for small-scale expression with IPTG at a final concentration of 1 mM for 4 h at 37°C. The results are as follows: Figure 3 As shown in A); secondly, the clone with the highest expression level was selected for large-scale induction ( Figure 3 B); The supernatant and precipitate from the two inductions were analyzed by SDS-PAGE, and the results showed ( Figure 3C), the NP1 recombinant protein was mainly expressed in the supernatant. Finally, the recombinant protein was further purified using a Ni affinity chromatography column, and the SDS-PAGE results are shown below ( Figure 3 D) shows that we successfully obtained a recombinant NP1 protein with a size of approximately 42 kDa.

[0036] 3. Identification of monoclonal antibodies against the MVC nonstructural protein NP1

[0037] BALB / c mice were immunized with purified recombinant protein, and 10 stable hybridoma cell lines secreting anti-NP1 monoclonal antibodies were successfully obtained using hybridoma technology: 1C19, 1C19-1, 1J22, 1J22-1, 1H6, 2112, 2112-3, 3M17, 3M17-1, and 4E19. These hybridoma cells were then inoculated into mice at specific rates, and ascites fluid was collected for later use. After transfecting 293T cells with the NP1 recombinant protein and culturing for 48 hours, the cells were collected, lysed, and the protein was extracted as antigen (2 ng). Western blot analysis was performed using the collected mouse ascites fluid as the primary antibody. The results showed... Figure 4 A), except for 3M17-1, the detection limit of the other 9 NP1 monoclonal antibodies reached 2ng; in ELISA detection, the maximum amount of protein antigen coating was 10ng, and after serial dilution, the minimum coating amount was 0.02ng. The results showed that ( Figure 4 B) The coating amount of these 9 clones was less than 10 ng, which indicates that the NP1 monoclonal antibody was successfully prepared and can be used for the exogenous Western blot detection of NP1 protein.

[0038] 4. Western blot identification of NP1 monoclonal antibody specificity

[0039] The Western blot and ELISA experiments described above demonstrate that we have successfully prepared monoclonal antibodies. Next, we will further validate the efficacy of these nine NP1 monoclonal antibodies in MVC-infected WRD cells using a Western blot experiment at a dilution ratio of 1:1000. The Western blot results are shown below. Figure 5 As shown in A), all nine NP1 monoclonal antibodies specifically reacted with the lysate of MVC-infected WRD cells. The effects of 1C19, 1C19-1, 1J22-1, and 2112 monoclonal antibodies were compared with those of polyclonal antibodies. Western blot results were obtained. Figure 5 The results (BC) showed that the four selected NP1 monoclonal antibodies all had high specificity and could be used for the endogenous Western blot detection of NP1 protein.

[0040] 5. Observe the effect of NP1 monoclonal antibody by immunofluorescence.

[0041] This study further identified four NP1 monoclonal antibodies—1C19, 1C19-1, 1J22-1, and 2112—specifically in MVC-infected WRD cells using immunofluorescence assays. The immunofluorescence results are shown below (…). Figure 6 As shown in the figure, specific green fluorescence appeared in MVC-infected WRD cells, while no fluorescence was observed in the negative control WRD cells that were not infected with MVC. The above results indicate that the prepared NP1 monoclonal antibody can specifically recognize the NP1 protein in MVC-infected WRD cells. Therefore, all four NP1 monoclonal antibodies obtained by screening can be used for immunofluorescence detection and have strong reactivity.

[0042] 6. Immunoprecipitation to verify the activity of NP1 monoclonal antibody

[0043] To verify whether the prepared NP1 monoclonal antibody could be used for immunoprecipitation experiments, we collected WRD cells 48 h after MVC infection and added pre-chilled RIPA buffer containing protease inhibitors to lyse the WRD cells on ice for immunoprecipitation. The results are shown below. Figure 7 As shown in the figure, the prepared monoclonal antibody against the MVC nonstructural protein NP1 has strong specificity and can meet the requirements of immunoprecipitation experiments.

[0044] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Preparation of a monoclonal antibody against the nonstructural protein NP1 of canine bocavirus MVC, characterized in that: The following steps are involved:

1. Construction and identification of the prokaryotic expression vector PET-32a(+)-NP1 of NP1 protein The MVC virus NP1 gene sequence was used to design primers and amplify the NP1 gene using PCR. The prokaryotic expression vector pET32a(+) was then double-digested with restriction endonucleases BamHI and SalI. The target gene was then homologously recombined with the digested plasmid. After transformation, plating, picking, culturing, and plasmid extraction, double-digestion identification was performed. The primers are F: 5'-GCGGATCCATGTCTACGAGACATATGAGCAA GA-GATCA-3'; R: 5'-GCGTCGACCTATTCGGAGGAGCCATCTACC TCC-3'; Second, after successful double enzyme digestion verification of the recombinant expression plasmid, the plasmid was transformed into Escherichia coli Rosetta and plated. A single clone was selected from the plate and induced for small-scale expression with a final concentration of 1 mM IPTG at 37°C for 4 hours. Second, the clone with the highest expression was selected for large-scale induction and purification. The supernatants and precipitates of the two inductions were analyzed by SDS-PAGE, and samples were retained for electrophoresis detection. The NP1 recombinant protein was mainly expressed in the supernatant. Finally, the recombinant protein was further purified using a Ni affinity chromatography column, and a sample of the collected protein solution was retained for SDS-PAGE analysis to obtain the NP1 recombinant protein.

3. Use the purified recombinant protein to immunize BALB / c mice, and obtain hybridoma cells that stably secrete anti-NP1 monoclonal antibodies through hybridoma technology. Then use the fusion cells to inject mice and collect ascites. After purification through affinity chromatography columns, NP1 monoclonal antibodies are obtained.

2. The preparation of a monoclonal antibody against the nonstructural protein NP1 of canine bocavirus MVC according to claim 1, characterized in that: In the step 1, the enzyme digestion reaction system was placed at 37° C. for 2 h.

3. The preparation of a monoclonal antibody against the nonstructural protein NP1 of canine bocavirus MVC according to claim 1, characterized in that: In the step 1, homologous recombination ligation, transformation, plating, picking, culture and plasmid extraction are specifically performed by using an agarose gel DNA recovery kit to recover the above-mentioned enzyme digestion product, using a Clon Express II One Step Cloning Kit to recombinantly ligate the PCR recovered product with the vector plasmid, and placing the ligation reaction system at 37° C. for 30 minutes to obtain a recombinant prokaryotic expression vector with a His tag, named pET-32a(+)-NP1; subsequently, these ligation products are transformed into a TOP10 strain, and after ampicillin resistance screening and culture, sequencing is performed and the sequencing results are compared with the target fragment sequence. The clone strain with the same sequence is selected for expansion and culture, and the plasmid is extracted and stored in a -20° C. refrigerator.

4. The preparation of a monoclonal antibody against the nonstructural protein NP1 of canine bocavirus MVC according to claim 1, characterized in that: In the step 2, the specific operation of selecting the one with the highest expression level is to collect the bacterial precipitate and perform ultrasonic crushing, place the retained sample in a 100° C. water bath for 5 min, and then perform SDS-PAGE electrophoresis. After the electrophoresis, perform Coomassie Brilliant Blue staining and decolorization, analyze the expression of the three selected clones, judge whether the molecular weight of the protein is correct, select the clone with the highest expression level to prepare glycerol bacteria and save it for use; after selection, purification: after the expression is completed, centrifuge and collect the bacterial cells, resuspend the bacterial cells with a concentration of 20mMTris buffer and perform electrophoresis detection, then ultrasonically crush the bacterial resuspension and perform high-speed centrifugation, transfer the supernatant after centrifugation to a new centrifuge tube, purify it for use, dissolve the precipitate with a suitable ratio of dissolution buffer at 4° C. and stir overnight, and retain the supernatant after high-speed centrifugation for purification for use.

5. The preparation of a monoclonal antibody against the nonstructural protein NP1 of canine bocavirus MVC according to claim 1, characterized in that: In the step 2, the purification of NP1 protein by Ni affinity chromatography is specifically as follows: the supernatant and the precipitate are sampled separately for electrophoresis detection, and the NP1 protein is mainly expressed in the supernatant. Then, the supernatant is loaded onto a Ni column pre-equilibrated with PBS, mixed and incubated for 40 minutes, and after the incubation, the flow-through is collected, the chromatography column is washed with PBS for 100 column volumes, and then the chromatography column is washed with PBS containing 20mM imidazole for 20 column volumes, and the target protein is eluted with 5-10mL of PBS containing 250mM imidazole, and the eluate is collected. 1mL of eluate is added each time, and elution is performed multiple times. Each time the eluate is collected, 10uL of the eluate is added to 100μL Braford G250 to observe the color change. When protein is eluted, it will turn blue. 1mL of eluate is repeatedly added until no protein is eluted. The collected protein solution is sampled for SDS-PAGE analysis.

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