Hybridoma cell strain capable of secreting high-titer anti-PRRSV monoclonal antibody and application of hybridoma cell strain

Abstract

A hybridoma cell strain 28F6 capable of secreting a high-titer anti-PRRSV monoclonal antibody is prepared and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the hybridoma cell strain 28F6 is CCTCC NO: C2022373. The antibody titer in the cell culture supernatant of the hybridoma cell strain reaches 1: 640, the hybridoma ascites titer can reach 1: 4.096 * 10 < 5 > which is far higher than that of other prepared hybridoma cell strains, the monoclonal antibody secreted by the hybridoma cell strain 28F6, the PRRSV strain and the PRRSV vaccine attenuated strain obtained through passage have good reaction activity, and the monoclonal antibody and the PRRSV strain secreted by the hybridoma cell strain 28F6 have good immunogenicity. The probe has broad-spectrum reaction activity on the American type PRRSV and can be used for detecting the American type PRRSV.

Description

Technical Field

[0001] This application belongs to the field of biology, specifically to a hybridoma cell line that can secrete a high-titer anti-PRRSV monoclonal antibody and its application. Background Technology

[0002] PRRSV was first discovered in China in 1996. In 2006, HP-PRRSV became widespread in Chinese pig populations and has remained epidemic ever since. In recent years, my country has imported a large number of breeding pigs from the United States, Canada, and European countries, significantly increasing the spread of PRRSV. Furthermore, recombination between wild-type strains and between attenuated vaccine strains has led to the continuous emergence of new strains. This recombination not only results in new PRRSV genotypes but is also associated with increased virulence, making disease control difficult and causing huge economic losses to the global pig industry. It has now become one of the major diseases seriously affecting pig farming. Vaccination is the primary means of controlling PRRSV infection. Although various PRRS vaccines have been developed, due to the virus's tendency to mutate and escape host humoral immune responses, current PRRSV vaccines offer limited protection, providing complete protection only against specific genotypes. This means that conventional PRRS vaccines cannot meet the expected requirements in production practice. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) focuses on disease eradication. This involves continuously improving the biosecurity system of farms, strictly implementing comprehensive prevention and control measures such as immunization, monitoring and eradication, quarantine supervision, emergency response, and harmless disposal, and actively carrying out farm-wide or regional eradication efforts to reduce morbidity, reduce the epidemic's spread, and gradually achieve the goal of eradication. Therefore, specific, sensitive, rapid, and accurate detection methods are of great significance for the monitoring, eradication, and quarantine supervision of PRRS. Research on PRRSV monoclonal antibodies can provide materials for the establishment of rapid PRRSV detection methods and is one of the hot topics in PRRS research. Summary of the Invention

[0003] The purpose of this invention is to provide a hybridoma cell line that can secrete a high-titer anti-PRRSV monoclonal antibody and its application, with the aim of providing a cell line that can secrete a high-titer antibody.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A hybridoma cell line capable of secreting a high-titer anti-PRRSV monoclonal antibody, characterized in that: the hybridoma cell line is named 28F6, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022373, and deposited on December 15, 2022.

[0005] A monoclonal antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID No: 1; The amino acid sequence of the light chain is shown in SEQ ID No: 2.

[0006] The present invention also claims protection for the DNA encoding the monoclonal antibody, including the DNA encoding the heavy chain and the DNA encoding the light chain, characterized in that: The DNA sequence encoding the heavy chain is shown in SEQ ID No: 3; The DNA sequence encoding the light chain is shown in SEQ ID No: 4.

[0007] The present invention also claims protection for the use of the monoclonal antibody in establishing a PRRSV detection method for non-clinical laboratory studies.

[0008] The present invention also claims protection for the use of the monoclonal antibody in the preparation of an ELISA kit for PRRSV detection.

[0009] This invention also claims protection for the use of the monoclonal antibody in the preparation of colloidal gold test strips for PRRSV detection. Compared with the prior art, the present invention has the following features and beneficial effects: This invention uses PRRSV preserved in our laboratory as an antigen to prepare a highly specific and sensitive monoclonal antibody hybridoma cell line, 28F6. The antibody titer in the cell culture supernatant of this hybridoma cell line reaches 1:640, and the titer in the hybridoma ascites fluid reaches 1:4.096×10⁵, which is much higher than other hybridoma cell lines prepared. The monoclonal antibody secreted by hybridoma cell line 28F6 has good reactivity with PRRSV strains and attenuated PRRSV vaccine strains obtained through passage. It has broad-spectrum reactivity against American PRRSV and can be used for the detection of American PRRSV. Attached Figure Description

[0010] Figure 1 IPMA assay results of virus-carrying levels in Marc145 cells at different passages; Figure 2 PRRSV TCID in cell culture supernatants of each generation 50 Measurement results; Figure 3 RT-PCR results of PRRSV content in cell culture supernatants from different generations; Figure 4 RT-PCR electrophoresis images of PRRSV before and after purification; Figure 5 Results of mouse serum antibody titer detection; Figure 6IPMA assay results for screening positive hybridoma cell supernatant; Figure 7 shows the titer results of the culture supernatant for the 20A4 cell line. Figure 8 Results of titer assay for culture supernatant of 24A10 cell line; Figure 9 Results of titer assay for culture supernatant of 26D1 cell line; Figure 10 Results of titer assay in culture supernatant of 28F6 cell line; Figure 11 Results of ascites titer induced by 20A4 cell line; Figure 12 Results of ascites titer induced by 24A10 cell line; Figure 13 Results of ascites titer assay induced by 26D1 cell line; Figure 14 Results of ascites titer induced by 28F6 cell line; Figure 15 Results of SDS-PAGE of the superisotropic virus (a) and preliminary identification of the monoclonal antibody recognition protein by Western blot (b); Figure 16 NCBI BLAST protein sequence alignment results. Implementation

[0011] To make the technical means, innovative features, objectives and effects of this invention easier to understand, the invention will be further described below.

[0012] The embodiments described herein are specific implementations of the present invention, used to illustrate the concept of the invention, and are illustrative and exemplary, and should not be construed as limiting the implementation or scope of the invention. In addition to the embodiments described herein, those skilled in the art can employ other obvious technical solutions based on the content disclosed in the claims and specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein.

[0013] To prepare monoclonal antibodies against PRRSV, this application employed a strategy combining differential centrifugation and membrane ultrafiltration concentration to purify PRRSV. The purified whole PRRSV virus was used as the antigen to immunize female Balb / c mice, and the immunization effect was detected using a commercially available PRRSV ELISA antibody detection kit. When the mouse titer reached 1:103 or higher, mouse spleen cells were fused with SP2 / 0 cells under sterile conditions. Subsequently, hybridoma cells were screened using the IPMA method, while the PRRSV-infected monkey kidney cell line Marc145 cells served as a control for uninfected cell plates. Simultaneously, serum from PRRSV-immunized mice, serum from uninfected mice, culture supernatant from uninfected cell wells, and culture supernatant from SP2 / 0 cells were collected from each plate as controls. After color development, if the results from the control wells (PRRSV-immunized mice, uninfected mice, uninfected cell wells, and SP2 / 0 culture supernatant) were verified to be correct, subcloning was performed on wells in the infected cell plate where color development was observed, and the corresponding uninfected cell plate showed no color development. After three rounds of subcloning, four hybridoma cell lines capable of stably secreting anti-PRRSV monoclonal antibodies were successfully screened, with a positive cloning rate of 0.1%. These were named 20A4, 24A10, 26D1, and 28F6. The four selected monoclonal antibodies were expanded into large-scale cultures, cryopreserved, and then used in vivo ascites induction for mass production. The ascites titers ranged from 1:2.56×10⁴ to 1:4.096×10⁵, with hybridoma cell line 28F6 exhibiting the highest ascites titer at 4.096×10⁵. These cells were deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C2022373. Western spectroscopy was used to analyze the titers. Blot analysis of the four monoclonal antibodies revealed no bands for 24A10 and 28F6, suggesting they may recognize non-structural proteins or their recognition epitopes may be conformational epitopes, requiring further experimental confirmation. 20A4 showed a distinct band around 10 kDa, and 26D1 showed a distinct band between 35 and 45 kDa, indicating that 20A4 and 26D1 specifically recognize HP-PRRSV. Protein sequencing and comparison with the NCBI database showed that 20A4 recognizes PRRSV N protein, and 26D1 recognizes PRRSV GP5 protein. Recombinant plasmids were extracted from antibodies secreted by hybridoma cell line 28F6 and identified. The recombinant cloning plasmid pMD19-T-28F6VL / VH was positive after PCR and double enzyme digestion, and then sent to Shanghai Sangon Biotech for gene sequencing. The sequencing results are shown in SEQ ID No: 1 and SEQ ID No: 2. The VL (SEQ ID No: 2) fragment is approximately 320 bp in size, and the VH (SEQ ID No: 2) fragment is approximately 346 bp in size, consistent with the expected results.

[0014] Preparation of anti-PRRSV monoclonal antibody Specifically, this includes SP2 / 0 cell culture, preparation of feeder cells, preparation of spleen cells, cell fusion, medium exchange of fused cells, screening of positive clone wells, subcloning of positive hybridoma cells; large-scale preparation and preservation of anti-PRRSV monoclonal antibodies, cryopreservation and thawing of positive hybridoma cells, identification of monoclonal antibodies, determination of monoclonal antibody titer, and proteomic identification. Results and analysis of each step in section 1.

[0015] 2.1 PRRSV detection IPMA tests on the toxin content of Marc145 at different generations Marc145 cells initially infected with PRRSV were designated F0, cells from their first passaging were designated F1, and so on. Cell suspensions from each passaging were plated, and the virus-carrying status of each passage was identified using the IPMA method. The results are shown below. Figure 1 The results showed that the number of cells exhibiting staining gradually increased from F0 to F8 generations, meaning that the amount of virus carried by each generation of cells gradually increased.

[0016] 2.1.2 TCID50 determination in cell culture supernatant of each generation Marc145 cells initially infected with PRRSV were named F0, cells from their first passage were designated F1, and so on. The TCID50 of each collected culture supernatant was measured, and the results are as follows: Figure 2 The TCID50 of the cell culture supernatant from F0 to F8 generations showed an "S"-shaped increase, with the highest TCID50 in F5 generation, followed by a downward trend.

[0017] 2.1.3 RT-PCR detection of PRRSV content in cell culture supernatants of different generations To further determine the proliferation status of PRRSV in different generations, RT-PCR detection results are as follows: Figure 3Under the same conditions, as the number of cell passages increased, the brightness of the PRRSV ORF7 amplification band gradually increased, indicating that the PRRSV viral copy number in the cell culture supernatant gradually increased from F0 to F8 passages, and the increase was no longer significant after F5 passage. Combined with IPMA and TCID50 results, it was determined that the virus was harvested at F5 passage.

[0018] 2.2 Determination of TCID50 before and after PRRSV purification The results of the IPMA detection method are shown in Tables 1 and 2. The PRRSV TCID50 was calculated using the Karber method, which showed that the collected virus was 10⁻⁶.25 TCID50 / 100 μL, and after purification, it was 10⁻¹¹ TCID50 / 100 μL.

[0019] Table 1 PRRSV TCID before purification 50 Measurement Virus dilution <![CDATA[10 -1 ]]> <![CDATA[10 -2 ]]> <![CDATA[10 -3 ]]> <![CDATA[10 -4 ]]> <![CDATA[10 -5 ]]> <![CDATA[10 -6 ]]> <![CDATA[10 -7 ]]> <![CDATA[10 -8 ]]> <![CDATA[10 -9 ]]> <![CDATA[10 -10 ]]> <![CDATA[10 -11 ]]> color development hole 8 8 8 8 7 5 2 0 0 0 0 Non-coloring pores 0 0 0 0 1 3 6 8 8 8 8 Colorimetric ratio 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 5 / 8 2 / 8 0 0 0 0 Table 2. Determination of PRRSV TCID50 after purification Virus dilution <![CDATA[10 -1 ]]> <![CDATA[10 -2 ]]> <![CDATA[10 -3 ]]> <![CDATA[10 -4 ]]> <![CDATA[10 -5 ]]> <![CDATA[10 -6 ]]> <![CDATA[10 -7 ]]> <![CDATA[10 -8 ]]> <![CDATA[10 -9 ]]> <![CDATA[10 -10 ]]> <![CDATA[10 -11 ]]> color development hole 8 8 8 8 8 8 8 7 8 7 6 Non-coloring pores 0 0 0 0 0 0 0 1 0 1 2 Colorimetric ratio 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 8 / 8 7 / 8 6 / 8 2.3 RT-PCR identification results of PRRSV before and after purification RT-PCR results as follows Figure 4 A band of approximately 275 bp appeared in the 250 bp to 500 bp range, which is consistent with the expected size.

[0020] 2.4 Immunization Results in Mice Mice were immunized with PRRSV whole virus, and the titer of the antibodies was detected using a pre-pregnancy PRRSV ELISA antibody detection kit. A positive result was defined as P / N ≥ 2.1. Figure 5 The titer of mouse No. 2 reached 1:1.28×104, while that of mice No. 1 and No. 3 reached 1:6.4×103. Mouse No. 2 was selected for super-immunization and cell fusion was performed.

[0021] 2.5 Results of screening for positive hybridoma cells Hybridoma supernatant was inoculated onto infected and uninfected plates for incubation, with serum from immunized and non-immunized mice used as controls on each plate. After color development, wells in the infected plates that showed color development and the control group (uninfected plates) that did not show color development were selected for subcloning (e.g., ...). Figure 6 ).

[0022] 2.6 Monoclonal antibody subtype identification The results of identification using Proteintech's mouse monoclonal antibody subtype identification kit are shown in Table 3. The heavy chain type of 20A4, 24A10, and 28F6 is IgG1, the heavy chain type of 26D1 is IgG2a, and the light chain type of 20A4, 24A10, 26D1, and 28F6 is Kappa.

[0023] Table 3. Identification of heavy and light chain subtypes of PRRSV mAb Heavy chain / light chain type IgG1 IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda <![CDATA[20A4( D 450nm )]]> 2.890 0.053 0.053 0.052 0.051 0.052 1.749 0.056 <![CDATA[24A10( D 450nm )]]> 2.941 0.059 0.063 0.059 0.061 0.058 1.817 0.059 <![CDATA[26D1( D 450nm )]]> 0.058 3.004 0.058 0.056 0.058 0.055 2.926 0.054 <![CDATA[28F6( D 450nm )]]> 3.058 0.064 0.057 0.059 0.057 0.053 2.465 0.054 2.7 Monoclonal Antibody Titer Determination The culture supernatant of positive hybridoma cells and the induced ascites were serially diluted, and after IPMA staining, the cutoff point was set at the well where stained and unstained cells could not be clearly distinguished. The results showed that the titer of the culture supernatant of 20A4 was 160-fold. Figure 7 Its potency in inducing ascites is 2.56 × 10⁴ times ( Figure 11 The culture supernatant titer of 24A10 was 160 times higher. Figure 8 Its potency in inducing ascites is 2.56 × 10⁴ times ( Figure 12 The culture supernatant titer of 26D1 was 320 times higher. Figure 9 Its ascites-inducing potency is 1.024 × 10⁵ times ( Figure 13 The titer of the culture supernatant of 28F6 was 640 times higher. Figure 10 Its most potent potency in inducing ascites is 4.096 × 10⁵ times ( Figure 14 2.8 Western blot detection of proteins recognized by monoclonal antibodies Preliminary identification of the monoclonal antibody recognition protein was performed using Western blot, and the results are as follows: Figure 15 The results showed that no bands appeared for 24A10 and 28F6, suggesting they may recognize non-structural proteins or their recognition epitopes may be conformational epitopes, requiring further experimental confirmation. 20A4 showed a distinct band near 10 kDa, and 26D1 showed a distinct band between 35 and 45 kDa, indicating that 20A4 and 26D1 can specifically recognize HP-PRRSV. 2.9 Protein Spectroscopy Identification Results Based on the Western blot (WB) results, the proteins recognized by each monoclonal antibody were excised from the corresponding positions on an SDS-PAGE gel and recovered. These gels were then sent to Sangon Biotech (Shanghai) Co., Ltd. for protein identification. After obtaining the protein sequences, alignment analysis was performed using the NCBI / BLAST database. The results are as follows: Figure 16The comparison results showed that the protein identified by 20A4 had 100% similarity to the PRRSV N protein, thus identifying the protein identified by 20A4 as the PRRSV N protein; the protein identified by 26D1 had 100% similarity to the PRRSV GP5 protein, thus identifying the protein identified by 26D1 as the PRRSV GP5 protein.

[0024] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0025] SEQUENCE LISTING <110> Xinxiang University <120> A hybridoma cell line capable of secreting high-titer anti-PRRSV monoclonal antibodies and its applications <130> 20230517 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 106 <212> PRT <213> Artificial sequence <400> 1 Arg Lys Ile Glu Leu Lys Thr Gly Ser Gly Phe Thr Leu Pro Asn Ser 1 5 10 15 Ser Trp Gln Gln Cys Tyr Tyr Thr Ala Ala Asp Glu Ala Glu Met Ser 20 25 30 Ser Ile Thr Leu Ser Phe Ser Thr Gly Ser Gly Ser Gly Ser Phe Arg 35 40 45 Ala Pro Val Gly Ser Ala Leu Lys Ser Thr Asp Tyr Ile Trp Arg Lys 50 55 60 Pro Ser Thr Gly Ser Lys Gln Gln Tyr Trp His Met Tyr Ser Val Ser 65 70 75 80 Ser Asn Ala Ser Cys Thr Met Thr Val Lys Glu Gly Pro Ser Ala Ser 85 90 95 Met Ile Ala Pro Ser Gln Thr Leu Glu Ile 100 105 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <400> 1 Ser Val Thr Val Thr Thr Gly Gln Gly Trp Tyr Asp Leu Ala Lys Trp 1 5 10 15 Trp Arg Ala Cys Tyr Tyr Val Ser Ser Asp Gly Ser Thr Leu Ser His 20 25 30 Leu Glu Met Tyr Ala Thr Thr Ser Ser Lys Asp Val Thr Leu Ser Ala 35 40 45 Arg Asp Lys Phe Ile Gln Asn Tyr Lys Thr Asp Gly Ser Tyr Pro Asp 50 55 60 Ile Arg Gly Ile Trp Glu Leu Ser Lys Ile His Ser Gln Lys Val Trp 65 70 75 80 Ile Met Tyr Tyr Gly Thr Phe Leu Tyr Gly Ser Ala Lys Cys Ser Ile 85 90 95 Lys Val Ser Ala Gly Pro Lys Val Leu Glu Pro Gly Ser Glu Gln Leu 100 105 110 Gln Val <210> 3 <211> 346 <212> DNA <213> Artificial sequence <400> 1 gaggagacgg tgaccgtggt cccttggccc cagtagtcca aagccttcca ccatcttgca 60 caataataga ctgaagagtc cccagatgtc agactgtgga gttccatgta ggctgtggtg 120 gatgacttat ctacagtcaa gctggccctg tccttgaaaa tctggttgta cttagtatca 180 ccactataag gatcaatgcg tccgatccat tcaaggctct ttatatgact ttgcttaacc 240 cagatcatgt agtagccagt gaataagtaa ccagaagcct tacaggatat cttcactgaa 300 gccccaggtt tcaccaactc aggtccagac tcctgcagct ggacct 346 <210> 4 <211> 320 <212> DNA <213> Artificial sequence ccgtttgatt tccagcttgg tgcccgagcc gaacgtgagt gggttactac tccactgctg 60 gcagtaataa gtggcagcat cttcagcctc catgctgctg attgtgagag agaaagaggt 120 cccagaccca ctgccactga agcgagcagg gactccagaa gccaatttgg atgtgtcata 180 aatccatctt ttgggggagg tgcctgactt ctgttggtac cagtgcatgt aacttacact 240 tgagttggca ctgcaggtca tggtgacctt ctcccctgga gatgcagaca tgattgctgg 300 agactgggtg agctcaatga 320

Claims

1. A hybridoma cell line capable of secreting high titer anti-PRRSV monoclonal antibodies, characterized in that: The hybridoma cell line was named 28F6 and deposited in the China Center for Type Culture Collection (CCTCC). Its deposit number is: CCTCC NO: C2022373, and the deposit date is December 15, 2022.

2. The monoclonal antibody secreted by the hybridoma cell line according to claim 1, comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID No: 1; The amino acid sequence of the light chain is shown in SEQ ID No:

2.

3. A DNA encoding the monoclonal antibody according to claim 2, comprising a DNA encoding a heavy chain and a DNA encoding a light chain, characterized in that: The coding DNA sequence of the heavy chain is shown in SEQ ID No: 3; The coding DNA sequence of the light chain is shown in SEQ ID No:

4.

4. Use of the monoclonal antibody as claimed in claim 2 in establishing a PRRSV detection method, wherein the detection method is used for non-clinical laboratory research.

5. Use of the monoclonal antibody as claimed in claim 2 in preparing an ELISA kit for PRRSV detection.

6. Use of the monoclonal antibody as claimed in claim 2 in preparing colloidal gold test strips for PRRSV detection.

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