Anti-swine astrovirus type 5 pa stv5 monoclonal antibody, primer, hybridoma cell strain, preparation and application

Abstract

The application relates to an anti-swine astrovirus type V PAstV5 monoclonal antibody, a primer, a hybridoma cell strain, preparation and application, and belongs to the technical field of swine astrovirus detection. The preparation method of the hybridoma cell strain for preparing the anti-swine astrovirus type V PAstV5 monoclonal antibody comprises the following steps: 1) immunizing a mouse by using a pET32a-PAstV5-Cap-1 fusion protein; 2) taking the spleen cells of the immunized mouse in step 1) and SP2 / 0 cells to prepare hybridoma cells; 3) screening the hybridoma cells prepared in step 2) to obtain positive hybridoma cells; and 4) subcloning the positive hybridoma cells screened in step 3) to obtain the hybridoma cell strain. The cell strain can stably secrete the antibody, the ELISA titer of the prepared PAstV5 monoclonal antibody can reach more than 500,000 times, and the prepared PAstV5 monoclonal antibody can be applied to the detection of PAstV5 in ELISA, WB and IFA.

Description

Technical Field

[0001] This invention relates to the field of porcine astrovirus detection technology, and more specifically, to a monoclonal antibody against porcine astrovirus type 5 (PAstV5), its primers, preparation method, and application. Background Technology

[0002] Porcine astrovirus (PAstV) was first observed by Bridger using electron microscopy in fecal samples from 3-week-old piglets with diarrhea. PAstV infection in pigs can cause clinical symptoms such as diarrhea, vomiting, dehydration, and growth retardation. PAstV often causes acute gastroenteritis in pigs through co-infection with coronaviruses, caliciviruses, rotaviruses, and other enteroviruses. Some scholars believe that PAstV is one of the main pathogens causing viral diarrhea outbreaks in piglets in recent years, particularly severe in pre-weaning piglets, and has had a significant impact on the pig farming industry.

[0003] Astroviruses are non-enveloped, single-stranded, positive-sense RNA viruses with a genome length of 6.4–7.8 kb and a viral particle diameter of approximately 28–30 nm. Astroviruses consist of 5' and 3' untranslated regions (UTRs), three open reading frames (ORF1a, ORF1b, and ORF2), and poly(A). ORF1a and ORF1b are located at the 5' end of the genome, with a seven-nucleotide (AAAAAAC) ribosomal frameshift signal (RFS) at the ORF1a / b junction; this sequence is highly conserved. The entire ORF1 encodes the non-structural proteins nsp1a and nsp1ab, which are processed by proteolysis into smaller proteins, including RNA-dependent RNA polymerase, serine proteases, viral genome-associated proteins (VPg), and several other proteins with unknown functions. ORF2 encodes a structural protein (capsid protein CP). In all astroviruses, the N-terminus of the capsid protein is more conserved than the C-terminus because all astrovirus capsid proteins contain a region rich in basic amino acids (arginine and lysine) at the N-terminus, which is thought to interact with the genomic RNA inside the virus.

[0004] Astroviruses exhibit genotypicity and variability, and are capable of gene recombination, giving them considerable ability to adapt to hosts and spread across species, posing a potential zoonotic risk. Gene recombination can occur within the ORF1a region. In addition, the overlapping region of ORF1b and ORF2 is a high-frequency region for homologous recombination, containing a relatively stable hairpin structure, where recombination mainly occurs.

[0005] Jonassen et al. found that human, feline astrovirus, and PAstV are genetically most closely related, suggesting interspecies cross-transmission of astroviruses during evolution, possibly through transmission from pigs to cats, then from cats to humans, or through an intermediate host. Ulloa et al. found partial gene recombination in human astrovirus and PAstV in diarrhea samples from people and pigs living in the same house, suggesting possible interspecies cross-transmission of astroviruses. The multihostility, genetic diversity, recombination, interspecies transmission, and adaptation to new hosts of astroviruses make them potential emerging zoonotic pathogens. Therefore, timely detection and control of porcine astroviruses have significant public health value.

[0006] Currently, five genotypes of porcine astrovirus (PoAstV1-5) have been discovered. Sequence analysis shows that there are significant differences between the five genotypes, with only about 40% homology among their genomes. This reflects the huge genetic differences among PoAstVs, which leads to very different detection methods for these five porcine astroviruses, and there is little mutual reference between them.

[0007] Regarding antibody detection of porcine astrovirus, there are many studies on the detection of porcine astrovirus type 1. For example, the paper "Truncation Expression of Porcine Astrovirus Type 1 Capsid Protein and Preparation of Polyclonal Antibodies" (Chinese Journal of Preventive Veterinary Medicine, Wang Weiyi et al., 2021.4) discloses a method for preparing polyclonal antibodies against porcine astrovirus type 1. The method designs primers based on the gene sequence of the Cs (aa420-aa669) Cap protein of PAstV-GX1 strain, amplifies the fragment (Cs) by PCR, clones it into the pET-32a(+) prokaryotic expression vector, constructs the recombinant expression plasmid pET32a-Cs, transforms it into E. coli BL21 competent cells, and after induction at 37℃ for 6 h with IPTG at a final concentration of 1 mmol / L, the expression product is detected by SDS-PAGE. The results show that the target protein appears at approximately 47 kDa, and the protein is expressed in a soluble form. The recombinant Cs protein was purified by Ni column and emulsified. Six-week-old BALB / c mice were immunized with 100 μg / mouse. Blood was collected 5 days after the third immunization to separate serum. Western blot and indirect immunofluorescence detection results showed that the obtained polyclonal antibody against the hypervariable region of the Cap protein could specifically recognize the viral protein expressed by PAstV-GX1 infected PK15 cells.

[0008] However, antibody testing for other types of porcine astrovirus has not been reported, especially for porcine astrovirus type 5, which is more difficult to detect, mainly due to the structure of porcine astrovirus type 5.

[0009] In 2011, a novel PAstV strain was discovered in fecal samples from the cecum of adult pigs at a Canadian pig farm. This PAstV could not be detected using universal viral primers because it lacked the highly conserved s2m stem-loop structure found in many viruses. Phylogenetic analysis revealed that it formed a unique lineage within the mammalian viridae family. This PAstV shared only 25%-30% amino acid homology with the classic PoAstV-1 and exhibited a certain genetic distance from the other three PAstV genotypes. Therefore, it was named PAstV-5.

[0010] The first PASTV-5 (KP747574) sequence in China was detected in healthy domestic piglets in 2015. Furthermore, the three earliest PoAstV-5 strains discovered in China are not distributed along the same phylogenetic clade, exhibiting a relatively complex distribution, indicating diverse and complex genetic characteristics of PoAstV-5. Therefore, developing an enzyme-labeled antibody with good serum-specific binding efficacy and high titer is of great significance. Summary of the Invention

[0011] To improve the specific binding effect of the antibody to PAstV5 serum, this application provides a monoclonal antibody against porcine astrovirus type 5 PAstV5, its primers, preparation method, and application.

[0012] A PCR primer for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5), the nucleotide sequence of which is as follows:

[0013] P1:5'-ggatccgggtctggcaatcttgagattgaaggt-3';

[0014] P2:5'-aagcttaggattcatgttccaatttgtaaactgccatgtggcgcg-3'.

[0015] A monoclonal antibody against porcine astrovirus type 5 (PAstV5) was produced by a hybridoma cell line with accession number CCTCC N0:C2022393.

[0016] A hybridoma cell line for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5) is disclosed, with accession number CCTCC N0:C2022393.

[0017] A method for preparing a hybridoma cell line for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5) includes the following steps:

[0018] 1) Mice were immunized with the pET32a-PAstV5-Cap-1 fusion protein;

[0019] 2) Take spleen cells from the immunized mice in step 1) and fuse them with SP2 / 0 cells to prepare hybridoma cells;

[0020] 3) The hybridoma cells obtained in step 2) are screened to obtain positive hybridoma cells;

[0021] 4) Subcloning of the positive hybridoma cells obtained in step 3) yields the monoclonal antibody cell line.

[0022] In step 3), the indirect ELISA method is used for screening.

[0023] The pET32a-PAstV5-Cap-1 fusion protein was obtained by amplification, cloning, recombination, fusion, and purification using the primers described above.

[0024] When performing PCR amplification using the above primers, piglet feces were used as a template. Piglet feces contain porcine astrovirus type 5. The PCR reaction system consisted of: Primer STAR 25 μL, DNA template (cDNA) 2 μL, upstream primer (10 μmol / L) 1 μL, downstream primer (10 μmol / L) 1 μL, and double-distilled water 21 μL. The total volume was 50 μL. The PCR reaction program was: 98℃ for 5 min, 98℃ for 30 s, 61.6℃ for 30 s, and 72℃ for 1 min, for a total of 35 cycles; followed by a final extension at 72℃ for 10 min.

[0025] When cloning PCR amplification products, the amplification products are cultured to obtain single clones. Universal primers are then used for colony PCR identification to obtain positive clones.

[0026] The universal primer sequences are as follows:

[0027] P1: 5'-cgaacgccagcacatggaca-3';

[0028] P2: 5'-tgctagttattgctcagcgg-3';

[0029] The PCR reaction system is as follows:

[0030] rTaq 12.5uL, DNA template (bacterial culture) 2uL, upstream primer (10μmol / L) 0.5uL, downstream primer (10μmol / L) 0.5uL, double-distilled water 9.5uL. Total volume 25uL.

[0031] The PCR reaction program was 94℃ for 5 min, 94℃ for 30 s, 61.6℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0032] During recombination, the positive clones obtained above were expanded and cultured, and the plasmid pET32a-PAstV5-Cap-1 was extracted. After identification by bacterial PCR, sequencing was performed. The correctly sequenced positive plasmid pET32a-PAstV5-Cap-1 was transformed into Escherichia coli competent cells BL21(DE3) and identified by PCR to obtain the recombinant expression strain pET32a-PAstV5-Cap-1 / BL21(DE3).

[0033] During fusion, the recombinant expression strain was induced to obtain the desired fusion protein. The inducer (operon) used was IPTG, with a final concentration of 100 mmol / L. Induction conditions were 37°C and 220 rpm. The resulting fusion protein had a density of 38 kDa.

[0034] During purification, sonication was used to break down the induced protein.

[0035] A method for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5) involves inducing and proliferating a hybridoma cell line with accession number CCTCCN0:C2022393 in the peritoneal cavity of mice to obtain the monoclonal antibody against porcine astrovirus type 5 (PAstV5).

[0036] Application of a monoclonal antibody against porcine astrovirus type 5 (PAstV5) in the detection of porcine astrovirus type 5.

[0037] The application is ELISA detection, WB detection, or IFA detection.

[0038] The application is the preparation of an indirect ELISA detection kit for porcine astrovirus type 5 (PAstV5), which contains the aforementioned anti-PAstV5 monoclonal antibody.

[0039] In summary, this application has the following beneficial effects:

[0040] The purified PAstV5 Cap protein expressed in this invention reacts with PAstV5-positive serum, exhibiting good immunogenicity. Immunizing mice with the purified protein and screening for monoclonal antibodies revealed that hybridoma cell lines stably secreted antibodies. The monoclonal antibody prepared in this invention achieves an ELISA titer exceeding 500,000 times and can be applied to the detection of PAstV5 in ELISA, Western blotting, and in vitro anabolism (IFA). Attached Figure Description

[0041] Figure 1 This is an electrophoresis image of the PCR amplification of the 421-921bp fragment of the PAstV5 Cap protein from Example 1.

[0042] Figure 2This is a Coomassie brilliant blue staining image for the identification of pET32a-PAstV5-Cap protein induced expression in Example 3. Lane M represents the protein molecular weight standard; lane 1 is the pET32a-BL21(DE3) empty vector induction control; lane 2 is the whole cell culture of pET32a-PAstV5-Cap-1 / BL21(DE3) before protein induction; and lane 3 is the pET32a-PAstV5-Cap-1 / BL21(DE3) cell lysate induced by IPTG.

[0043] Figure 3 This is the Western blot analysis of the fusion protein expression in *E. coli* in Example 4. Lane M represents the protein molecular weight standard, lane 1 is the pET32a-BL21(DE3) empty vector control, and lane 2 is the pET32a-PAstV5-Cap-1 / BL21(DE3) bacterial lysate.

[0044] Figure 4 This is a Coomassie brilliant blue staining image used in Example 5 to analyze the expression of the fusion protein. Lane M represents the protein molecular weight standard, lane 1 is the pET32a-BL21(DE3) empty vector control, lane 2 is the precipitate, lane 3 is the supernatant, and lane 4 is the whole bacteria.

[0045] Figure 5 This is the expression form of the fusion protein analyzed by Western blot in Example 5. Lane M is the protein molecular weight standard, lane 1 is the pET32a-BL21(DE3) empty vector control, lane 2 is the precipitate, and lane 3 is the supernatant.

[0046] Figure 6 This is the fusion protein before and after purification as analyzed by SDS-PAGE in Example 5. Lane M is the protein molecular weight standard, lane 1 is the pET32a-BL21(DE3) empty vector control, lane 2 is the fusion protein before purification, and lane 3 is the fusion protein after purification.

[0047] Figure 7 This is the fusion protein before and after purification as analyzed by SDS-PAGE in Example 6. Lane M represents the protein molecular weight standard, lane 1 is the pET32a-BL21(DE3) empty vector control, lane 2 is the fusion protein before purification, and lane 3 is the fusion protein after purification.

[0048] Figure 8 This is the result of the indirect immunofluorescence method used in Example 6 to identify the monoclonal antibody. In this diagram, b represents the blue fluorescence (nucleus), and a represents the green fluorescence (PAstV5 Cap protein).

[0049] Figure 9This is the result of the indirect immunofluorescence method used in Example 6 to identify the monoclonal antibody. In this diagram, b represents the blue fluorescence (nucleus), and a represents the green fluorescence (PAstV5 Cap protein).

[0050] Figure 10 The cross-validation assay using the indirect immunofluorescence method in Example 6 verifies the specificity of the monoclonal antibody. Here, b represents blue fluorescence, and a represents green fluorescence.

[0051] Figure 11 The Western blot method in Example 6 was used to verify the specificity of the monoclonal antibody.

[0052] Figure 12 , Figure 13 The antigenicity, hydrophilicity, and intracellular and extracellular distribution prediction of the PAstV5 Cap protein in Example 1 were analyzed. The prediction showed that the distribution was entirely extracellular and there was no transmembrane helical region. Detailed Implementation

[0053] The present application will be further described in detail below with reference to specific embodiments.

[0054] PAstV5 Cap protein is the only structural protein and a major factor determining viral virulence, cell tropism, and pathogenicity. As the only structural protein of astroviruses, the cap protein has good immunogenicity and its N-terminus is highly conserved, making it a target region for serological detection.

[0055] Example 1 (PCR amplification of the PAstV5-Cap-1 gene)

[0056] The gene sequence of PAstV5-Cap-1 was obtained from NCBI. Based on expression analysis using plasmid pET32a and the gene sequence, BamHI and HindIII were selected as restriction enzyme sites, and the primer sequences were designed as follows:

[0057] P1:5'-ggatccgggtctggcaatcttgagattgaaggt-3';

[0058] P2:5'-aagcttaggattcatgttccaatttgtaaactgccatgtggcgcg-3';

[0059] Using feces from piglets under 2 months old collected in Henan Province during the winter and spring of 2021 as a template, PCR amplification was performed using the primers described above. The PCR reaction system was as follows:

[0060] Primer STAR 25uL, DNA template (cDNA) 2uL, upstream primer (10μmol / L) 1uL, downstream primer (10μmol / L) 1uL, double-distilled water 21uL. Total volume 50uL.

[0061] The PCR reaction program was as follows: 98℃ for 5 min, 98℃ for 30 s, 61.6℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 10 min.

[0062] After the reaction was complete, agarose gel electrophoresis was performed, and the PCR products were recovered using a gel extraction kit, yielding a 501 bp band. The results are as follows: Figure 1 As shown.

[0063] Figure 1 This is an electrophoresis image of the PCR amplification of the 421-921bp fragment of the PAstV5 Cap protein. Lane 1 is the DNA standard 2K plus II Marker, lane 2 is the negative control, and lane 3 contains the 421-921bp fragment of the PAstV5 Cap protein. This indicates that the correctly sized fragment was successfully obtained.

[0064] Example 2 (Construction of recombinant expression vector and expression strain)

[0065] The PAstV5-Cap-1 gene and plasmid pET32a obtained in Example 1 were double-digested, recovered from gel, ligated, transformed, and plated on LB / Amp plates. They were then incubated upside down at 37°C. Single clones were picked from the LB / Amp plates, shaken, and identified by PCR using universal primers, yielding the positive clone pET32a-PAstV5-Cap-1.

[0066] The universal primer sequences are as follows:

[0067] P1: 5'-cgaacgccagcacatggaca-3';

[0068] P2: 5'-tgctagttattgctcagcgg-3';

[0069] The PCR reaction system is as follows:

[0070] rTaq 12.5uL, DNA template (bacterial culture) 2uL, upstream primer (10μmol / L) 0.5uL, downstream primer (10μmol / L) 0.5uL, double-distilled water 9.5uL. Total volume 25uL.

[0071] The PCR reaction program was 94℃ for 5 min, 94℃ for 30 s, 61.6℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0072] Positive clone pET32a-PAstV5-Cap-1 was expanded and cultured, plasmid pET32a-PAstV5-Cap-1 was extracted, and bacterial culture was identified by PCR and then sequenced.

[0073] The correctly sequenced positive plasmid pET32a-PAstV5-Cap-1 was transformed into E. coli competent cells BL21(DE3) and identified by PCR to obtain the recombinant expression strain pET32a-PAstV5-Cap-1 / BL21(DE3).

[0074] Example 3 (Induced Expression of Fusion Protein)

[0075] A fusion protein of approximately 38 kDa was obtained by inducing recombinant expression strain pET32a-PAstV5-Cap-1 / BL21(DE3) with IPTG at a final concentration of 100 mmol / L at 37 °C and 220 rpm, which was in line with expectations. No protein band was obtained from the empty vector. Figure 3 As shown.

[0076] Example 4 (Western blot verification)

[0077] To further validate the expression of the fusion protein, Western blot analysis was performed on the fusion protein to identify the His expression tag.

[0078] After induction of expression, bacterial cells were collected, boiled, and then subjected to SDS-PAGE. A control group with an empty vector was also included. Antibody incubation was performed after 75 min of PVDF membrane transfer. The PVDF membrane was then removed and blocked with 5% skim milk at 37°C for 2 h. The PVDF membrane was then removed again and incubated overnight at 4°C with a mouse His-tag monoclonal antibody diluted 1:5000 with 5% skim milk. The membrane was washed four times with PBST for 5 min each time. Development and exposure were then performed. Results are as follows: Figure 4 As shown, this indicates that the expressed protein is the target protein.

[0079] Example 5 (Identification of Fusion Protein Expression Form and Protein Purification)

[0080] After induction of expression, bacterial cells were collected, resuspended in PBS, and lysed by sonication. The supernatant and precipitate were then separated, and the expression pattern was analyzed by SDS-PAGE and Western blot. The expression product was verified to be inclusion body expression, as shown in the results below. Figure 5 As shown, inclusion body purification is therefore performed.

[0081] After protein induction, the protein was sonicated and centrifuged at 12000g for 10 min at 4℃. The supernatant was discarded, and the precipitate was resuspended in TRIS Buffer. Centrifugation and supernatant discarding were repeated twice until the inclusion body precipitate was a clean, milky white color. The supernatant was discarded, and 8 mol / L urea denaturing solution was added. The mixture was incubated overnight at 4℃ with shaking to dissolve the inclusion bodies. Centrifugation was then performed at 12000g for 30 min at 4℃. The supernatant was poured into a boiled dialysis bag, and leaks were checked. The dialysis bag was then placed in a graduated cylinder containing 1 L of 6 mol / L urea refolding solution and stirred at 400 rpm for 6 h at 4℃ using a magnetic stirrer. The solution was then replaced with 4 mol / L urea refolding solution, and the mixture was stirred at 400 rpm for 6 h at 4℃ using a magnetic stirrer. Then, replace the urea refolding solution with a concentration of 2 mol / L and stir at 400 rpm using a magnetic stirrer for 6 hours at 4°C; then replace the urea refolding solution with a concentration of 0 mol / L and stir at 400 rpm using a magnetic stirrer for 6 hours at 4°C. Then replace the TRIS buffer and stir at 400 rpm using a magnetic stirrer for 6 hours at 4°C.

[0082] Take an appropriate amount of dialysis protein, add 5× protein electrophoresis SDS buffer, boil the sample, and analyze it by SDS-PAGE. The results are as follows: Figure 6 As shown, a fusion protein with high purity was obtained.

[0083] Example 6 (Preparation of monoclonal antibody for hybridoma cell line 2A11)

[0084] 6.1 Animal Immunization

[0085] 1) First immunization: The purified pET32a-PAstV5-Cap-1 protein was emulsified with an equal amount of Freund's complete adjuvant as an antigen and injected subcutaneously into mice at a dose of 25 μg / mouse. The mice were 6-week-old female BALA / c mice.

[0086] 2) Second immunization: 14 days after the first immunization, the purified pET32a-PAstV5-Cap-1 protein was emulsified with an equal amount of Freund's incomplete adjuvant and then injected subcutaneously into female mice at a dose of 25 μg / mouse.

[0087] 3) Third immunization: 14 days after the second immunization, the purified pET32a-PAstV5-Cap-1 protein was emulsified with an equal amount of Freund's incomplete adjuvant as an antigen and then subcutaneously injected into female mice at a dose of 25 μg / mouse.

[0088] 4) Seven days later, inject the purified protein into the peritoneum at a dose of 25 μg / animal.

[0089] 5) Prepare for cell fusion 3 days later.

[0090] 6.2 Cell Fusion

[0091] 6.2.1 Culture of SP2 / 0 cells:

[0092] One week before fusion, the frozen SP2 / 0 cells were rapidly thawed in a 37°C water bath, centrifuged at 600 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum and transferred to T75 culture flasks. The cells were cultured in a 5% CO2 incubator at 37°C. When the cells reached 90% confluence, the cells were dispersed and centrifuged at 600 rpm for 10 min. The cells were then expanded to two T75 culture flasks. Once the cells reached a full and clear state, the medium was replaced with fresh medium to prepare for fusion.

[0093] 6.2.2 Preparation of feeder layer cells:

[0094] The day before fusion, two healthy, non-immune BALA / c mice were taken, and blood was collected by enucleation in a sterile table to obtain negative control blood. After euthanasia, the mice were immersed in 75% alcohol and irradiated with ultraviolet light for 15 minutes.

[0095] Beforehand, wrap a layer of plastic wrap on a foam board. Remove the mouse and fix its limbs with a 10mL syringe needle. Use curved forceps to lift the skin at the linea alba on the mouse's abdomen, make a small incision with ophthalmic scissors, and peel back the abdominal skin with forceps to separate the skin from the peritoneum, exposing the peritoneum. Disinfect the peritoneal surface with an alcohol swab. Pour 30mL of HAT culture medium into a petri dish, attach a 10mL syringe needle, and draw up the HAT culture medium.

[0096] Gently lift the peritoneum with forceps, slowly inject the culture medium into the peritoneal cavity using a syringe, and then aspirate the fluid from the peritoneal cavity. Avoid aspirating the mucous membranes and adipose tissue of any organs in the peritoneal cavity. After rinsing the mouse's peritoneal cavity, centrifuge at 1000 rpm for 10 minutes.

[0097] Discard the supernatant, resuspend the cells in HAT medium, and seed the cells into 10 wells of 96-well plates at a concentration of 100 μL / well. Incubate overnight in an incubator (37°C, 5% CO2).

[0098] 6.2.3 Preparation of immunized mouse spleen cells:

[0099] Three days after booster immunization, experimental mice were taken, and blood was collected by enucleation in a sterile table to obtain positive blood. After euthanasia, the mice were immersed in 75% alcohol and irradiated with ultraviolet light for 30 minutes.

[0100] Beforehand, wrap a layer of plastic wrap on a foam board, remove the mouse, and fix its limbs with a 10mL syringe needle. Use curved forceps to lift the skin at the linea alba on the mouse's abdomen upwards, and use ophthalmic scissors to cut open the skin and peritoneum. The spleen is located roughly on the upper left side of the mouse, near the back; locate the spleen and bluntly dissect it.

[0101] The spleen was washed in a culture dish containing serum-free DMEM, and the connective tissue on the spleen was removed. A single-cell sieve was placed in a culture dish containing serum-free DMEM, and the spleen was ground into single, dispersed cells using the plunger of a 10 mL syringe. The ground spleen cell solution was transferred to a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, and the cells were washed twice with DMEM for later use.

[0102] 6.2.4 Cell Fusion

[0103] Take two bottles of SP2 / 0 cells (T75) in good growth condition, disperse the cells, centrifuge them in a 50mL sterile centrifuge tube at 1000rpm / min for 10min, discard the supernatant, wash the cells again with serum-free DMEM medium, repeat the centrifugation twice, and discard the supernatant.

[0104] Transfer individual, dispersed spleen cells to a 50 mL centrifuge tube containing SP2 / 0 cells, centrifuge at 1000 rpm for 10 min, and discard the supernatant. Gently tap the bottom of the centrifuge tube to mix the spleen cells and SP2 / 0 cells evenly, and then place the centrifuge tube in a 200 mL beaker containing 37°C water.

[0105] Preheat 1 mL of PEG1420 fusion agent in a 37°C water bath, then add it dropwise at a rate of 1 drop per second to the mixture of spleen cells and SP2 / 0 cells, rotating the centrifuge tube continuously to ensure thorough mixing. After addition, allow the mixture to stand for 90 seconds, then add serum-free DMEM medium to terminate the fusion. Add 1 mL dropwise over the first minute, 2 mL over the second minute, and 3 mL over the third minute, increasing the volume to 30 mL over 10 minutes. Centrifuge the fusion mixture at 1000 rpm for 10 minutes, discard the supernatant, and resuspend the pellet in 300 mL of HAT selection medium containing feeder cells. Mix well and plate 100 μL into each well of a 96-well cell culture plate containing feeder cells. Incubate at 37°C in a 5% CO2 cell culture incubator.

[0106] Three days after fusion, cell growth was observed. When obvious cell clusters appeared and a large number of non-fused cells died, half of the medium was replaced with HT medium. When the cells grew to 1 / 10 of the well volume, a second half-volume medium replacement was performed. The titer of the cell supernatant was detected the next day using an indirect ELISA method.

[0107] 6.3 Screening and subcloning of positive hybridoma cell lines

[0108] pET32a-PAstV5-Cap-1 protein and pET32a empty vector-induced product were diluted to 2 μg / mL with carbonate solution and plated into 96-well microplates (100 μL per well). The plates were incubated at 37°C for 2 h. The coating solution was discarded, and the plates were washed with PBST and blotted dry. 150 μL of 5% BSA blocking solution was added to each well, and the plates were incubated at 37°C for 1 h. The blocking solution was then discarded, and the plates were washed with PBST and blotted dry. Mouse serum was diluted with PBS at ratios of 1 / 100, 1 / 1000, 1 / 100000, 1 / 500000, and 1 / 1000000, respectively. A negative mouse serum control was also included (100 μL per well). The plates were incubated at 37°C for 1 h. Discard the primary antibody, wash once with PBST, blot dry the ELISA plate, add 100 μL of HRP-goat anti-mouse IgG secondary antibody diluted in 1 / 100002% skim milk to each well, and incubate at 37°C for 1 h. Discard the secondary antibody, wash four times, blot dry the ELISA plate, add 100 μL of TMB single-component chromogenic solution to each well for color development, and incubate for 10 min. Add 50 μL of 2M H2SO4 to each well to stop the color development.

[0109] The OD value of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0110] Cell wells with high titers (the titer criteria can be set according to the actual situation) are replenished with fluid and retested the next day. Hybridoma cell lines with high titers are selected for subcloning.

[0111] Before subcloning, two blank mice were used to prepare feeder cells in pre-cooled HT medium, with 20 mL of culture medium prepared for each cell line. Positive clones were subcloned using the limiting dilution method. After subcloning, the single-clone wells were detected by ELISA. Cells with high detection titers were subcloned again to screen for stable secreting monoclonal clones and expand the culture. Finally, a stable secreting monoclonal antibody cell line was obtained and named 2A11.

[0112] The preservation information for this monoclonal antibody cell line is as follows:

[0113] Preservator: Henan Agricultural University

[0114] Date of preservation: December 24, 2022

[0115] Culture name (classification): Hybridoma cell line PAstV5-Cap-1-2A11

[0116] Accession number: CCTCC N0:C2022393; Name of depositary institution: China Center for Type Culture Collection; Address of the depository: China Center for Type Cultures of Microorganisms, Wuhan University, No. 16 Luojia Mountain Road, Wuchang District, Wuhan, Hubei Province.

[0117] 6.4 Western Blot Method for Identification of Monoclonal Antibodies

[0118] Take the purified protein solution, add 5×SDS loading buffer, mix well and boil for 10 min. Take 10 μL of sample and perform SDS-PAGE electrophoresis on a 12.5% ​​polyacrylamide gel. After electrophoresis, perform membrane transfer. Lay the filter paper, PVDF membrane, gel and filter paper flat in sequence, gently roll to avoid air bubbles, and transfer at 360mA for 70 min.

[0119] The PVDF membrane was incubated in 5% skim milk at room temperature for 1 hour. The blocking solution was discarded, and the PVDF membrane was immersed in the supernatant of the selected positive monoclonal antibodies and incubated at room temperature on a shaker at 40 rpm for 1 hour. The primary antibody was discarded, and the membrane was washed four times with TBST for 5 minutes each time.

[0120] The PVDF membrane was then immersed in 2% skim milk powder containing commercially available HRP-goat anti-mouse IgG (1:5000) and incubated at room temperature on a shaker at 40 rpm for 1 hour. It was washed four times with TBST, 5 minutes each time. ECL chromogenic reagent was added for development, and the membrane was then developed using a developing instrument. The results are as follows: Figure 7 As shown, the prepared monoclonal antibody can specifically recognize the PAstV5 Cap protein.

[0121] 6.5 Identification of Monoclonal Antibodies by Indirect Immunofluorescence Method

[0122] PK cells were cultured in 6-well plates. When the cells reached 70% confluence, the cell culture medium was discarded, and the cells were washed twice with autoclaved sterile PBS. PAstV5 was seeded onto the PK cells and incubated in a 5% CO2, 37°C incubator for 2 hours for adsorption. The cells were gently shaken every 30 minutes and washed twice with autoclaved sterile PBS to remove unadsorbed virus particles. Then, 10 mL of LDMMEM was added, and the cells were incubated for 72 hours. After this period, an indirect immunofluorescence assay was performed as follows:

[0123] (1) Fixation: Wash cells gently twice with PBST, add 1 mL of 4% PFA (paraformaldehyde) to each well and fix at room temperature for 30 min.

[0124] (2) Membrane disruption: Discard PFA, wash once with PBST, then add 300 μL of PBS solution containing 0.1% Triton X-100 and incubate at room temperature for 15 min.

[0125] (3) Blocking: Discard the solution, wash once with PBST, then add 300 μL of PBST solution containing 5% BSA to block the cells, and incubate at room temperature for 1 h.

[0126] (4) Primary antibody: Discard the blocking solution, wash the cells 4 times with PBST, add 300 μL of the supernatant of the cell line that recognizes PAstV5 protein, and incubate at room temperature for 1 h.

[0127] (5) Secondary antibody: Discard the primary antibody, wash the cells 4 times with PBST, add 300 μL of FITC-goat anti-mouse IgG antibody (1 / 500 dilution), and incubate at room temperature in the dark for 1 h.

[0128] (6) Nucleus staining: Discard the secondary antibody, wash the cells 4 times with PBST, add 4',6-diamidino-2-phenylindole (DAPI) to stain the cell nuclei at room temperature for 10 min, discard the DAPI, and wash 3 times with PBST.

[0129] (7) Observation results: The stained cells were observed using a fluorescence microscope. Blue fluorescence represented the nucleus, and green fluorescence represented the PAstV protein. The results are as follows: Figure 8 As shown, PAstV5 can be detected.

[0130] 6.6 Crossover assay to verify the specificity of monoclonal antibodies

[0131] PAstV5 was cultured in PK cells in DMEM medium (with 4 μg / mL trypsin added), and Seneca virus A (SVA) was cultured in PK cells in DMEM containing 10% serum. Monolayers of PK cells were infected with PAstV5 and SVA, respectively. After 2 hours of virus adsorption, the culture medium was discarded, maintenance medium was added, and cell growth was observed. An uninfected group served as a control.

[0132] When the cells began to show signs of cytopathic effect, the supernatant was discarded, the cells were collected, lysed with cell lysis buffer, and then subjected to SDS-PAGE electrophoresis after adding 5×SDS loading buffer. After transfer to a membrane and blocking, the cells were then subjected to Western blotting using the monoclonal antibody prepared above as the primary antibody to verify whether the monoclonal antibody could recognize PAstV5 and SVA. The results are as follows: Figure 10 As shown, monoclonal antibody 2A11 reacts strongly with PAstV5 but does not react with SVA.

[0133] The same test wells were set up. When cells began to show cytopathic effects, 4% PFA was added for fixation. The monoclonal antibody prepared above was used as the primary antibody for indirect immunofluorescence assay to verify whether there was cross-reactivity between the monoclonal antibody and PAstV5 and SVA. The results are as follows: Figure 9As shown, cells infected with PAstV5 produced obvious fluorescence, while cells infected with SVA did not react, indicating that the monoclonal antibody has strong specificity.

[0134] Example 7 (Application)

[0135] The hybridoma cell lines obtained above were cultured in a culture medium to induce the secretion of monoclonal antibodies against porcine astrovirus type 5 (PAstV5), which were then purified and separated. The culture medium can be an existing medium or a medium prepared using the feeder cells described above.

[0136] In other embodiments, the ascites can also be produced using a mouse ascites production method: one week in advance, an ascites inducer is injected into the peritoneal cavity of mice, followed by the injection of the above-mentioned hybridoma cell line in the logarithmic growth phase into the peritoneal cavity of mice to induce proliferation. After 7-15 days, the ascites is extracted, centrifuged, and purified to obtain the product.

[0137] In practical use, monoclonal antibodies can be prepared into detection kits. Preferably, the present invention prepares them into an indirect ELISA detection kit for porcine astrovirus type 5 (PAstV5). The method can refer to the methods in the prior art.

[0138] Experimental Example

[0139] The antigenicity, hydrophilicity, and intracellular and extracellular distribution of the PAstV5 Cap protein in Example 1 were analyzed, and the results are as follows: Figure 12 and Figure 13 As shown, all regions are extracellular regions, with no transmembrane helical regions.

[0140] This invention involves preparing a fusion protein of porcine astrovirus type 5 (PAstV5) Cap protein, purifying the protein, and identifying the immunogenicity of the prokaryotically expressed Cap protein using Western blotting (WB). Mice were immunized with the purified PAstV5 protein, and their spleen cells were fused with SP2 / 0 cells. Positive hybridoma cell lines were screened using indirect enzyme-linked immunosorbent assay (ELISA), and the reactivity of monoclonal antibodies with PAstV5 protein was identified using WB and indirect immunofluorescence assay (IFA). The stability of antibodies secreted by different passages of hybridoma cell lines was analyzed by measuring antibody titers.

[0141] The purified PAstV5 Cap protein expressed in this experiment reacts with PAstV5-positive serum, exhibiting good immunogenicity. Immunizing mice with the purified protein and screening for monoclonal antibodies showed that hybridoma cell lines stably secreted antibodies. Results indicate that the monoclonal antibody prepared in this invention specifically recognizes the N-terminal region (aa141-307) of the porcine astrovirus type 5 Cap protein. The monoclonal antibody prepared in this invention achieves an ELISA titer exceeding 500,000-fold and can be applied to the detection of PAstV5 in ELISA, Western blotting, and in vitro anabolism (IFA).

Claims

1. A hybridoma cell line for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5), characterized in that, The accession number is CCTCC N0:C2022393.

2. A monoclonal antibody against porcine astrovirus type 5 (PAstV5), characterized in that, Produced from a hybridoma cell line with accession number CCTCC N0:C2022393.

3. A method for preparing a monoclonal antibody against porcine astrovirus type 5 (PAstV5), characterized in that, A monoclonal antibody against porcine astrovirus type 5 PAstV5 was produced using a hybridoma cell line with accession number CCTCC N0:C2022393.

4. The method for preparing the anti-porcine astrovirus type 5 PAstV5 monoclonal antibody according to claim 3, characterized in that, The hybridoma cell line was cultured to secrete a monoclonal antibody against porcine astrovirus type 5 (PAstV5), which was then purified and separated to obtain the antibody.

5. The method for preparing the anti-porcine astrovirus type 5 PAstV5 monoclonal antibody according to claim 3, characterized in that, The hybridoma cell line was induced and proliferated in the peritoneal cavity of mice, and the ascites fluid was separated and purified to obtain the monoclonal antibody against porcine astrovirus type 5 PAstV5.

6. The application of the anti-porcine astrovirus type 5 (PAstV5) monoclonal antibody as described in claim 2 in the preparation of an indirect ELISA detection kit for porcine astrovirus type 5 (PAstV5), characterized in that, This kit contains the aforementioned monoclonal antibody against porcine astrovirus type 5 (PAstV5).

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