Heavy chain and light chain gene variable region sequence of a lsdv orf123 monoclonal antibody, expression of immunoglobulin full gene sequence and application thereof

Abstract

The application relates to the field of biomedical technology, in particular to a heavy chain and light chain variable region gene sequence of a LSDV ORF123 monoclonal antibody, an expression immunoglobulin full gene sequence and application thereof, the application extracts total RNA of hybridoma cells (1G1-1G12) by hybridoma sequencing, takes mRNA in the total RNA as a template, carries out reverse transcription to obtain cDNA of corresponding antibody genes, and then uses specific PCR to obtain a heavy chain variable region (Variable region of heavy chain, V H ) and a light chain variable region (Variable region of light chain, V L ) of the corresponding antibody, and carries out cloning and sequencing analysis. Finally, the heavy chain and light chain variable region gene nucleotide sequences of a monoclonal antibody (1G1-1G12) targeting the LSDV ORF123 protein are obtained, and the application is realized. Sequence analysis of the heavy chain (V H ) and light chain antibody (V L ) of the above-mentioned monoclonal antibody shows that the V H and V L gene subtypes of 1G1-1G12 are as follows: the heavy chain is IgG type, and the light chain is kappa type.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the heavy chain and light chain variable region gene sequences of an LSDV ORF123 monoclonal antibody, the full gene sequence of expressed immunoglobulin, and their applications. Background Technology

[0002] Bovine lumpy skin disease (LSD) is an acute, subacute, and contagious infectious disease caused by bovine lumpyskin disease virus (LSDV). LSDV primarily causes fever, papules, or skin nodules in cattle, such as on the head, neck, shoulders, and udder. In severe cases, pustular lesions and necrotic scabs develop. LSD is a significant imported animal disease that has recently emerged in my country, causing substantial economic losses to the cattle industry and seriously impacting its healthy development. LSD was first discovered in Zambia in 1929, and subsequently spread rapidly to the Middle East, Eastern Europe, and Central Asia. However, due to the lack of effective treatments and specific vaccines, since 2019, LSD has been diagnosed in 14 provinces, municipalities, and autonomous regions across China, showing a gradually spreading trend, making prevention and control increasingly challenging.

[0003] LSDV differs significantly from vaccinia virus (CPXV), but is similar to sheep poxvirus (SPPV) and goat poxvirus (GTPV), belonging to the genus CaPV. Currently, our understanding of poxvirus genomes largely comes from studies on vaccinia virus (VACV), with extremely limited research specifically targeting the functional aspects of the LSDV genome. There are very few studies specifically addressing the biological functions of LSDV-encoded proteins. The LSDV ORF123 gene is 588 bp long, encoding 196 amino acids, and is a protein located in the outer membrane of the EEV. The ORF123 protein is an important structural protein of LSDV, but its specific function has not yet been clearly reported. Therefore, a deeper understanding of the biological function of the LSDV ORF123 protein will contribute to a more comprehensive understanding of the etiology and pathogenic mechanisms of LSDV. Literature review revealed that LSDV ORF123 shares a homologous sequence with A34R, the gene encoded by Vaccinavirus (VACV). The A34R gene encodes a type C lectin-like glycoprotein, a type II transmembrane protein, expressed late, and N-glycosylated and methylated, possessing a type I C lectin-like domain. VACV A34R protein has multiple functions, including involvement in EEV viral particle release and regulation of viral virulence. Currently, the structure and antigenic characteristics of LSDV ORF123 protein are not fully understood, requiring further investigation to elucidate its mechanism of action in LSDV infection of host cells.

[0004] In previous studies, we obtained the LSDV ORF123 protein through prokaryotic expression, purified it, and immunized BALB / c mice. After detecting mice that tested positive for the corresponding antibody, we isolated spleen cells and fused them with myeloma cells. Through screening in HAT selective medium, we ultimately screened a hybridoma cell line that stably secretes the LSDV ORF123 monoclonal antibody, named 1G1-1G12. Hybridoma technology can produce high-purity monoclonal antibodies and enable large-scale production. However, hybridoma cells generated during the hybridoma process may lose highly specific and active sequences, and the cell condition may affect the sustainability of subsequent research. Therefore, hybridoma sequencing is necessary. Hybridoma sequencing allows for the stable expression of recombinant proteins to obtain antibodies, eliminating the need for hybridoma cells as a carrier to preserve the antibody; instead, the antibody is preserved in the form of the base sequence, avoiding the risk of losing specific antibody characteristics. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the heavy and light chain variable region gene sequences of an LSDV ORF123 monoclonal antibody, the complete gene sequences of two expressed immunoglobulins, and their applications, thus solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A monoclonal antibody against LSDV ORF123, comprising a heavy chain and a light chain, characterized by:

[0008] The three complementarity-determining region (CDR) sequences of the light chain variable region are as follows:

[0009] CDR1(1G1-1G12): CAAAGCGTTGCACATAGGAGTGGAAACAGCTAC

[0010] CDR2(1G1-1G12): AAAGTTTCC

[0011] CDR3(1G1-1G12): TTTCAAGGTTCACATGTTCCGCTCACG

[0012] The three complementarity-determining region (CDR) sequences of the heavy chain variable region are as follows:

[0013] CDR1(1G1-1G12):GGGTACACATTTACTGACTATGAA

[0014] CDR2(1G1-1G12):ATGGATCCTGAAAGTGGTGGTACT

[0015] CDR3(1G1-1G12):ACAAGATCCTATTACTACGAAACCCTCTACTTTGACTAC

[0016] The amino acid sequence of the light chain variable region is as follows: V L (1G1-1G12); the amino acid sequence of the heavy chain variable region is shown in V. H As shown in (1G1-1G12).

[0017] Two full-length gene sequences expressing immunoglobulins, such as pCDNA3.4-V. L (1G1-1G12), pCDNA3.4-V H As shown in (1G1-1G12).

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] In this patent application, we utilize hybridoma sequencing to extract total RNA from hybridoma cells (1G1-1G12), then use the mRNA from the total RNA as a template to perform reverse transcription to obtain the cDNA of the corresponding antibody gene, and finally use specific PCR to obtain the variable region of the heavy chain (V) of the corresponding antibody. H ) and the variable region of light chain (V L We then performed cloning and sequencing analysis. Ultimately, we obtained the nucleotide sequences of the heavy and light chain variable regions of a monoclonal antibody (1G1-1G12) targeting the LSDV ORF123 protein, thus realizing this invention. The heavy chain (V...) of the above monoclonal cell line antibody... H ) and light chain antibodies (V L Sequence analysis revealed that the heavy chain gene is of the IgG type, and the light chain is of the κ type. To improve antibody affinity, codon optimization was performed, and the gene was fused with the constant region of a murine antibody while retaining the variable region. The chimeric antibody genome sequence was then synthesized and expressed through bioengineering. In vitro cell culture showed that this chimeric immunoglobulin exhibited good reactivity and exogenously expressed the heavy and light chains (V1 and V2) of the monoclonal antibody. H +V L It possesses the biological activity of neutralizing antibodies. This application discloses the complete gene sequence of the chimeric immunoglobulin and its applications, aiming to solve the technical problems of imperfect and incomplete veterinary detection, diagnosis, prevention, and treatment or drugs for LSDV serotypes. Attached Figure Description

[0020] Figure 1 This invention is intended to screen and validate the LSDV ORF 123 hybridoma cell line in Example 1 of this invention; wherein: (A) PCR amplification of the LSDV ORF 123 gene; (B) purification of the LSDV ORF 123 protein; (D) Western blot analysis of the antibody response characteristics and antibody titer determination in mouse ascites fluid of this monoclonal antibody hybridoma cell line.

[0021] Figure 2 HybSeq-HT, the antibody gene of the LSDV ORF123 hybridoma cell line in Example 2 of this invention. TM Sequencing analysis; including: (A) HybSeq-HT antibody for hybridoma cell lines. TM Sequencing flowchart; (B) 1G1-1G12 hybridoma cell line V H Statistical analysis of antibody gene sequence percentage (%); (C) 1G1-1G12 hybridoma cell line V L Statistical analysis of antibody gene sequence percentage (%); (D) 1G1-1G12 hybridoma cell line V HAntibody gene HybSeq-HT TM Sequencing and statistical analysis; (E)1G1-1G12 hybridoma cell line V L Antibody gene HybSeq-HT TM Sequencing and statistical analysis.

[0022] Figure 3 V is the 1G1-1G12 antibody gene chimeric plasmid in Example 3 of this invention. H and V L Gene map; where: (A) V of the 1G1-1G12 antibody gene chimeric plasmid H Gene map; (B) V of the 1G1-1G12 antibody gene chimeric plasmid L Gene map.

[0023] Figure 4 This study analyzed the reaction characteristics of the chimeric plasmid containing the 1G1-1G12 antibody gene from the hybridoma cell line in Example 4 of this invention. Western blot analysis was performed on the V1 of the exogenously expressed 1G1-1G12 antibody gene. H V L and V H +V L Reactivity.

[0024] Figure 5 In Example 5 of this invention, the antigen complementarity-determining region (CDR) for V L and V L Essential for the performance of biological functions; among which: (A) 1G1-1G12 hybridoma cell line V H and V L Schematic diagram of the amino acid sequence structure of CDR1, CDR2, and CDR3; (B, D, and F)V H and V L Schematic diagram of the constructed CDR1, CDR2, and CDR3 gene sequence missing; (C, E, and G) Western blot analysis V H +V L The construction lacks the immune activity response of CDR1, CDR2 and CDR3.

[0025] Figure 6 The LSDV ORF123 hybridoma cell line 1G1-1G12 monoclonal antibody in Example 6 of this invention has neutralizing antibody activity; wherein: (A and F) exogenously transfected LSDV ORF123 V H and V L Fluorescence observation showing inhibition of EGFP-LSDV and RFP-LSDV replication; (B and G) Western blot analysis of exogenously transfected LSDV ORF123V H and VL It can inhibit intracellular EGFP-LSDV and RFP-LSDV replication; (C and H) exogenous transfection of LSDV ORF123 V H and V L TCID that can inhibit intracellular EGFP-LSDV and RFP-LSDV replication 50 Assay analysis; (D and I) exogenous transfection with LSDV ORF123 V H and V L Cell flow cytometry analysis showing inhibition of intracellular EGFP-LSDV and RFP-LSDV replication; (E and J) exogenous transfection with LSDV ORF123 V H and V L Statistical analysis of the ability to inhibit intracellular EGFP-LSDV and RFP-LSDV replication.

[0026] Figure 7 The antibody gene (V) of the LSDV ORF123 hybridoma cell line 1G1-1G12, which is exogenously expressed in Example 7 of this invention. H +V L (A) Cell culture supernatant showed neutralizing antibody activity; (B) Exogenous transfection with LSDV ORF123 V H and V L (A) Culture supernatant inhibited intracellular LSDV replication in a gradient-dependent manner; (B) exogenous transfection with LSDV ORF123 V H and V L Fluorescence observation showing that culture supernatant inhibits intracellular EGFP-LSDV and RFP-LSDV replication; (C and D) exogenous transfection with LSDVORF123 V H and V L Culture supernatant can inhibit TCID of intracellular EGFP-LSDV and RFP-LSDV replication. 50 Titer determination. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Screening of a hybridoma cell line containing LSDV ORF123 monoclonal antibody

[0029] 1. Construction of pET28a-LSDV ORF123 recombinant plasmid

[0030] Primers were designed using SnapGene 6.0.2 software to amplify the LSDV ORF123 gene sequence, introducing the restriction enzyme sites BamHI and EcoRI, ORF123-For:5'-CAGCAAATGGGTCGC GGATCC GCCACCATGTTAGTTGATATTCCAAAGAGTGGAACTGAAAC-3' (underlined area is the BamHI restriction site), downstream primer ORF123-Rev: 5'-TTGTCGACGGAGCTC GAATTC GTGGTGGTGGTGGTGGTGGCTTCCTCCTCCAAAAAAAGATCTTACACAGTAATAGCTTCTC-3' (underlined part is the EcoRI restriction site). The above primers were sent to Genewiz Biotechnology Co., Ltd. for synthesis. Using the extracted LSDV / FJ / CHA / 2021 strain whole-genome DNA as a template, the amplification system was as follows: 2×PhantaMax Buffer 25μl, ddH2O 18μl, forward and reverse primers 2μl each, dNTP 1μl, PhantaMax Super-Fidelity DNA 1μl, and template 1μl. The reaction program was as follows: 95℃ for 3 min, 95℃ for 15 s, 58℃ for 72 s, for a total of 32 cycles, with a final extension of 5 min. The PCR product was recovered by 1% agarose gel electrophoresis. The MultiS One Step Cloning Kit clones the amplified fragments from gel recovery and the digested empty vector, transforming them into *E. coli* BL21(DE3) competent cells. The cells are plated on kanamycin-containing 2YT solid culture plates, and single colonies are picked for shaking and plasmid extraction. The samples are then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced positive recombinant plasmid is named pET28a-LSDV ORF123. Figure 1 A).

[0031] 2. Purification of pET28a-LSDV ORF123 recombinant protein induced expression

[0032] The recombinant plasmid pET28a-LSDV ORF123 was transformed into culture medium, and when the optical density value OD... 600When the protein concentration was 0.8–1.0, 1 mmol / L IPTG was added and expression was induced at 37°C for 12 h. The bacterial cells were collected by centrifugation, resuspended in PBS, and sonicated. After centrifugation again, the supernatant and precipitate were collected. The mixture was then mixed with 5× protein loading buffer, centrifuged at 1200 rpm for 1 min, boiled in a 100°C metal bath for 10 min, and after cooling, SDS-PAGE electrophoresis was performed. Finally, the protein expression pattern was analyzed by Coomassie brilliant blue staining. In this invention, LSDV ORF123 protein was expressed in the precipitate after purification following induction. Conventional inclusion body protein purification methods were used. Nickel column affinity chromatography was used for LSDV ORF123 protein purification. A small amount of the purified protein was taken and subjected to SDS-PAGE electrophoresis and staining again to compare the changes in protein purity. Figure 1 ).like Figure 1 As shown in B, we successfully purified and obtained the ORF123 protein (as indicated by the red arrow).

[0033] 3. Hybridoma cell line screening, preparation and titer determination of monoclonal antibodies

[0034] In the previous stage of this study, the prokaryotic expression plasmid pET28a-LSDV ORF123 was constructed. After induction expression and purification, the purified LSDV ORF123 protein was obtained. Figure 1 B, as shown by the red arrow). Mice were immunized with the purified protein, and spleen cells were fused with myeloma cells SP2 / 0. Cells were then screened in HAT selective medium, and a hybridoma cell line 1G1-1G12 that stably secretes a monoclonal antibody against LSDV ORF123 was selected. Figure 1 C).

[0035] The specific experimental procedure is as follows: The purified protein antigen was emulsified with Freund's adjuvant and immunized 6-8 week old female SPF-grade BALB / c mice for the first immunization, at a subcutaneous injection of 50 μg / mouse. A second immunization was performed at week 3, with the antigen mixed with Freund's incomplete adjuvant and injected subcutaneously at the same dose. A third and fourth immunization were performed at weeks 5 and 7, respectively, using the same dose and method. Finally, at week 9, a pulse immunization was performed, with the antigen mixed with PBS and injected subcutaneously at a dose of 50 μg / mouse. Approximately 15-21 days after the booster immunization, mouse spleen cells were aseptically collected. SP2 / 0 cells were fused with spleen cells, and after selection culture in HAT medium and two subcloning processes, positive cell lines were expanded and the culture supernatant was collected for Western blotting and ELISA screening. After multiple rounds of screening and verification, the 1G1-1G12 hybridoma cell lines with good reactivity and specificity were finally obtained. Figure 1 C).

[0036] Example 2: LSDV ORF123 hybridoma cell line 1G1-1G12 antibody gene V H and V L sequencing

[0037] Understanding the gene sequences corresponding to the antibodies expressed by hybridomas is a prerequisite for large-scale production and antibody engineering. To provide more comprehensive and reliable hybridoma cell line sequencing, the aforementioned LSDV ORF1231G1-1G12 hybridoma cells were sent to Anshengda Biotechnology Co., Ltd. for hybridoma antibody gene profiling sequencing (HybSeq-HT). TM The hybridoma antibody sequencing experimental procedure includes Trizol method for RNA extraction, reverse transcription of samples to obtain cDNA, amplification and acquisition of heavy and light chain variable region sequences, cloning into vectors for sequencing library construction and analysis. Figure 2 A). After sequencing, the sequencing results are compared with those in the IMGT database to extract antibody V. H and V L Information such as CDR1, CDR2, and CDR3 of the sequence. For each sample, the 10 sequences with the highest abundance percentage are selected for abundance analysis, and only the sequence ranking highest is considered the target gene sequence. For example... Figure 2 B, 2C, 2D, and 2E, Hybseq HT TM Hybridoma antibody genome sequencing results showed that the hybridoma cell line V H The abundance of variable regions is relatively homogeneous, with the highest abundance accounting for nearly 95%. Figure 2 B); V L The highest abundance value in the variable region is close to 42%. Figure 2 C). Meanwhile, after analyzing the highest abundance of V... H and V L Sequence alignment and subtype analysis showed that the heavy chain was of the IgG type. Figure 2 Type D), the light chain is type κ (D) Figure 2 E).

[0038] The specific experimental procedure is as follows:

[0039] RNA was extracted from hybridoma cell lines 1G1-1G12. The mRNA extracted from the hybridoma cells was then reverse transcribed into cDNA using the SuperScript.III First-Strand Synthesis System for RT-PCR kit. The light chain (V) of the anti-LSDV ORF123 antibody gene was amplified using universal specific primers. L ) or heavy chain variable region (V H ). This will contain the corresponding heavy chain variable region (V) H ) or light chain variable region (V LThe PCR product of the target fragment was subjected to 1% agarose gel electrophoresis, and the fragment was separated by gel excision. After purification of the target product using a gel recovery kit, the purity of the target fragment was identified by 1% agarose gel electrophoresis, and the corresponding heavy chain variable region (V) was recovered. H ) and light chain variable region (V L The clone was inserted into the empty pMD19-T sequencing vector and then subjected to Sanger sequencing. After sequencing, sequence alignment, homology library construction, and analysis were performed on the sequencing results.

[0040] Example 3: Hybridoma cell 1G1-1G12 antibody gene V H and V L Sequence abundance analysis

[0041] like Figure 3 By analyzing the heavy chain variable region (V) of the antibody gene in the LSDV ORF123 hybridoma cell line 1G1-1G12, H ) and light chain variable region (V L Sequencing and library construction analysis showed that the V antibody gene of this hybridoma cell line... H and V L It has high coverage and a single target gene sequence, especially V. H Through V H and V L For nucleotide sequence alignment, we selected V, which ranked first. H and V L The antibody gene sequence was obtained, and the gene was synthesized and constructed into the pcDNA3.4 empty vector for expressing the above-mentioned monoclonal antibody hybridoma cell antibody for subsequent antibody expression studies.

[0042] Example 4: Synthesis and Construction of Chimeric Plasmid for Antibody Gene of Hybridoma Cell Line 1G1-1G12

[0043] HybSeq-HT based on antibody genes from 1G1-1G12 hybridoma cell lines TM Sequencing amino acid sequence alignment results ( Figure 2 D-2E), we selected the heavy chain with the highest richness (Rank 1) (V). H ) and light chains (V L Genes were synthesized and constructed into the pCDNA3.4 empty vector, named pcDNA3.4-V. H -1G1-1G12(ORF123)( Figure 3 A) and pcDNA3.4-V L -1G1-1G12(ORF123)( Figure 3 B). To improve antibody affinity, codon optimization was performed, followed by fusion with the constant region of the murine antibody, while chimeric antibody synthesis retained the variable region (e.g., Figure 3 V of the 1G1-1G12 antibody gene chimeric plasmid H and V L Gene maps, such as Figure 4 V. For the above-mentioned LSDV ORF123 monoclonal antibody cell lines (1G1-1G12) H and V L Synthesized expression plasmids were transfected into HEK-293T cells for secretory expression. Figure 4 Western blotting results showed that the hybridoma monoclonal antibody only affected the variable region V. H or V L Co-expression (V) H +V L Only under certain conditions can highly specific target bands be detected. The above results indicate that we have successfully expressed the antibody gene sequence of LSDV ORF123 hybridoma cells 1G1-1G12, and the antigen-antibody reaction exhibits good specificity and sensitivity.

[0044] Example 5: Antigen complementary determinant region (CDR) for V H and V L Necessary for biological functions

[0045] The antigen complementarity-determining region (CDR) is located within the variable regions of the heavy and light chains. It is a crucial site for specific antigen recognition and is generally divided into three regions: CDR1, CDR2, and CDR3. Monoclonal antibody sequences of 1G1-1G12 can be obtained through hybridoma cell gene sequencing. Further analysis of the variable region V using the IgBLAST online analysis platform can further refine this sequence. H (CDR1, CDR2, and CDR3 are marked in light blue) and V L (CDR1, CDR2, and CDR3 are marked in purple) Different areas are labeled. Figure 5 Using the missing primers in Table 1, construct V... H △CDR1、V H △CDR2、V H △CDR3、V L △CDR1、V L △CDR2 and V L △CDR3 deletion plasmid. After successful sequencing, the V plasmid with the CDR region deleted will be... H and V L The plasmid was transfected into 293T cells alone. Cell culture supernatant was collected 24 hours later for later use. Western blotting results showed no specific bands. Figure 5 B). According to VH △CDR1、V H △CDR2、V H △CDR3、V L △CDR1、V L △CDR2、V L △CDR3 L and V H +V L Co-transfect cells in the form of [a specific transfection method] to express [a specific expression], while simultaneously [transfecting] V [a specific expression]. H +V L Set as a positive control, the Western blotting results showed V H and V L No specific target bands were observed in the absence of CDR1, CDR2, and CDR3. Figure 5 C). Additionally, V will also be included. H +V L ΔCDR1, V H +V L ΔCDR2, V H +V L ΔCDR3, V H ΔCDR1+V L V H ΔCDR2+V L V H ΔCDR3+V L and V H +V L Co-transfection expression, Western Blot results showed that regardless of V H and V L Neither of the two lacked CDRs (CDR1, CDR2, and CDR3), and no specific bands were observed. Figure 5 D-5G). Analysis of the above results shows that regions CDR1, CDR2, and CDR3 are V regions of 1G1-1G12. H +V L The key antigen complementarity-determining region (CDG) for antigen recognition directly affects the binding of antigens and antibodies.

[0046] Table 1 PCR primers

[0047]

[0048] Example 6: Exogenous expression of the heavy and light chains (V) of the antibody gene from LSDV ORF123 hybridoma cell lines 1G1-1G12. H +V L It has neutralizing antibody activity.

[0049] like Figure 6As shown, using fluorescent LSDV strains labeled with green fluorescent protein (EGFP) and red fluorescent protein (RFP) (EGFP-LSDV and RFP-LSDV), we observed V in exogenously transfected hybridoma cell lines 1G1-1G12. H +V L The fluorescence intensity of EGFP and RFP was significantly suppressed at 24h.pi, 48h.pi, and 72h.pi. Figure 6 (A and 6F). Simultaneously, the TCID values ​​of the cell culture supernatant collected at the corresponding time points... 50 The results show V H +V L Extracellular EGFP-LSDV and RFP-LSDV viral titers were significantly inhibited at 24h.pi, 48h.pi, and 72h.pi. Figure 6 C and 6H). Protein samples collected at different time points, as shown by Western blotting, were co-transfected with V. H +V L It can significantly inhibit the replication of EGFP-LSDV and RFP-LSDV viruses in cells. Figure 6 B and 6G). Next, we used flow cytometry to further validate the V expression of exogenous 1G1-1G12 antibody. H and V L Neutralizing antibody activity. For example... Figure 6 D, 6E, 6I, and 6J, cell flow cytometry analysis further validated the exogenous expression of the antibody gene sequence of the 1G1-1G12 hybridoma cell line (V). H +V L This significantly inhibited the replication of EGFP-LSDV and RFP-LSDV viruses. The above observations included fluorescence intensity, Western blot, and TCID. 50 Both the results of cell flow cytometry and other analyses indicated that the antibody gene sequence (V) of the exogenously transfected LSDV ORF123 hybridoma cell line 1G1-1G12 was positive. H +V L Both can effectively inhibit the replication of EGFP-LSDV and RFP-LSDV viruses, indicating that exogenous expression of the hybridoma cell line 1G1-1G12 antibody gene (V) through genetic engineering can effectively inhibit the replication of EGFP-LSDV and RFP-LSDV viruses. H +V L It has neutralizing antibody activity.

[0050] The specific experimental procedure is as follows: Vero cells were seeded into 12-well plates. When the cell density reached 70-80%, Vero cells were transfected into the 1G1-1G12 hybridoma cell line. H +V L1 μg of exogenous co-transfected V DNA was added, along with 3.41 μg of empty pcDNA and a positive control (untransfected plasmid). H and V L The virus strain used in the plasmid experiment was EGFP-LSDV (virus content 10). -5.5 / 0.1mL) and RFP-LSDV (RFP-LSDV viral load 10 -6.25 / 0.1mL), after plasmid transfection, inoculated 18-24h. The positive controls for the two experiments were EGFP-LSDV and RFP-LSDV, respectively, with fluorescence intensity observed and recorded at 24h, 48h, and 72h post-inoculation. In parallel experiments, 2.5μL of EGFP-LSDV (10-1 viral load) was inoculated into each well 18h post-transfection. -5.5 / 0.1mL and RFP-LSDV viral content 10 -6.25 / 0.1mL), and then at 24h, 48h, 72h and 96h after infection, 100μL of cell culture supernatant was taken from the corresponding cell samples to determine TCID. 50 The extracellular LSDV viral load was assessed, and viral growth curves were plotted. After cell transfection, LSDV-LSDV and LSDV-RFP samples were collected for Western blotting experiments to further assess intracellular LSDV viral replication.

[0051] Example 7: Exogenous expression of the antibody light chain and heavy chain genes (V) of LSDV ORF123 hybridoma cell lines 1G1-1G12 H +V L Cell culture supernatant has neutralizing antibody activity.

[0052] like Figure 7 As shown in Figure A, the cell culture supernatant containing the LSDV ORF1231G1-1G12 antibody gene inhibited intracellular LSDV replication in a dose-dependent manner. Simultaneously, fluorescence intensity observation indicated that 5 mL of the antibody gene (V...) H +V L The culture supernatant significantly suppressed the fluorescence intensity of the green fluorescently labeled strain (EGFP-LSDV) and the red fluorescently labeled strain (RFP-LSDV) at 24 h, 48 h, and 72 h. Figure 7 B). Meanwhile, such as Figure 7 C, compared with the control group, TCID 50 The test results also showed that 5ml of antibody gene (V H +V L The culture supernatant showed significant LSDV viral replication in cells at 24h, 48h, and 72h. Western blot, fluorescence intensity observation, and TCID assays were performed. 50The analysis results all indicate that the antibody gene sequence (V) of exogenously transfected LSDV ORF123 hybridoma cell lines 1G1-1G12 is positive. H +V L The cell culture supernatant effectively inhibited the replication of EGFP-LSDV and RFP-LSDV viruses, indicating that the light and heavy chain genes of the hybridoma cell line 1G1-1G12 expressed exogenously through genetic engineering have neutralizing antibody activity.

[0053] The specific experimental procedure is as follows: 293T cells were seeded in 100cm culture dishes and exogenously transfected with the 1G1-1G12 antibody gene sequence (VH+VL). 24 hours after transfection, the cell culture supernatant was collected, filtered through a 0.45μm filter membrane, and stored at -80°C for later use. 1mL, 2mL, 3mL, 4mL, and 5mL of the culture supernatant from the hybridoma cell line IC2IG12 were incubated with EGFP-LSDV and RFP-LSDV viruses at 37°C for 3 hours. Serum-free DMEM was used as a positive control. The cells were then seeded into 6-well Vero plates with a cell density of 80-90%. Since the EGFP-LSDV and RFP-LSDV virus strains contain recombinant EGFP and RFP tags, fluorescence intensity was observed at 24h, 48h, and 72h. Cell samples were collected at 72h for Western blotting. Simultaneously, 100 μL of cell culture supernatant was collected at 24 h, 48 h, 72 h, and 96 h to determine the viral titer, and its growth curve was plotted using Prism 8.0 software. In parallel experiments, protein samples were prepared for Western blotting 72 h after infection with different culture supernatants and equal amounts of virus.

[0054] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A monoclonal antibody against LSDV ORF123, comprising a heavy chain and a light chain, characterized in that: The nucleic acid sequences of the three complementarity-determining regions (CDRs) of the light chain variable region are as follows: CDR1 of 1G1-1G12: CAAAGCGTTGCACATAGGAGTGGAAACAGCTAC; CDR2 of 1G1-1G12: AAAGTTTCC; CDR3 of 1G1-1G12: TTTCAAGGTTCACATGTTCCGCTCACG; The nucleic acid sequences of the three complementarity-determining regions (CDRs) of the heavy chain variable region are as follows: CDR1 of 1G1-1G12: GGGTACACATTTACTGACTATGAA; CDR2 of 1G1-1G12: ATGGATCCTGAAAGTGGTGGTACT; CDR3 of 1G1-1G12: ACAAGATCCTATTACTACGAAACCCTCTACTTTGACTAC.

2. The LSDV ORF123 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is as follows: V of 1G1-1G12 L : DVLMTQSPLFLPVSLGDQASISCRSSQSVAHRSGNSYLEWYLQKPGQSPKLLISKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK The amino acid sequence of the heavy chain variable region is as follows: V of 1G1-1G12 H : QVQLQQSGAELVRPGASVTLSKASGYTFTDYEMHWVKQTPVHGLEWIGGMDPESGGTVYNQKFKGKATLTADKSSSTAYMEFRSLTSEDSAVYYCTRSYYYETLYFDYWGQGTTLTVSS.

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