Heavy and light chain variable region gene sequences of three LSDV ORF29 monoclonal antibodies, complete gene sequences of two expressed immunoglobulins, and their applications.

Abstract

The present application relates to the biomedical technology field, specifically to three heavy chain and light chain variable region gene sequences of LSDV ORF29 monoclonal antibody, two kinds of expression immunoglobulin full gene sequences and application thereof, the present application uses hybridoma sequencing, extracts hybridoma cell (1C1-4D11, 3C4-2E9 and 1E1-2C11) total RNA, then mRNA in total RNA is used as a template, reverse transcription is carried out to obtain the cDNA of corresponding antibody gene, then specific PCR is used to obtain the heavy chain variable region (Variable region of heavy chain, V H ) and light chain variable region (Variable region of light chain, V L ) of corresponding antibody, and cloning and sequencing analysis are carried out. Finally, the heavy chain and light chain gene nucleotide sequences of three monoclonal antibodies (1C1-4D11, 3C4-2E9 and 1E1-2C11) targeting LSDV ORF29 protein are obtained, and the present application is realized. Sequence analysis of the heavy chain (V H ) and light chain antibody (V L ) of the above three monoclonal antibodies shows that the gene subtype of 1C1-4D11, 3C4-2E9 and 1C1-2C11 is IgG type, and the light chain is κ type.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the heavy and light chain variable region gene sequences of three LSDV ORF29 monoclonal antibodies, the complete gene sequences of two expressed immunoglobulins, 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 related to 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 ORF29 protein is an important structural protein of LSDV, but its specific function has not yet been clearly reported. Therefore, elucidating the biological function of the LSDV ORF29 protein will help deepen our understanding of the etiology and pathogenic mechanisms of LSDV. Current research indicates that as a member of the poxviridae family, the LSDV ORF29 protein is a membrane protein with a structure and function similar to the vaccinia virus F15 protein, playing a crucial role in the packaging of envelope proteins and viral particles.

[0004] In previous studies, LSDV ORF29 protein was obtained through prokaryotic expression, purified, and used to immunize BALB / c mice. Mice showing positive results for the corresponding antibody were then isolated from spleen cells and fused with myeloma cells. After screening in HAT selective medium, three hybridoma cell lines—1C1-4D11, 3C4-2E9, and 1E1-2C11—were identified that stably secrete LSDV ORF29 monoclonal antibodies. Hybridoma technology can produce high-purity monoclonal antibodies and enable large-scale production. However, hybridoma cells generated during the 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 after obtaining the antibody sequence, enabling researchers and manufacturers to preserve the antibody in the form of the base sequence rather than using hybridoma cells as a carrier, thus avoiding the risk of losing specific antibodies. 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 three LSDV ORF29 monoclonal antibodies, 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] Three LSDV ORF29 monoclonal antibodies, comprising heavy and light chains, are characterized by:

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

[0009] CDR1(1C1-4D11):CAGAATGTGGGTACAAAT

[0010] CDR2(1C1-4D11): TCGGCATCC

[0011] CDR3(1C1-4D11): CAACAATATAACAGCTTTCCGCTCACG

[0012] CDR1(1E1-2C11):CAGAATGTGGGTACAAAT

[0013] CDR2(1E1-2C11): TCGACATCC

[0014] CDR3(1E1-2C11): CAACAATATAACAGCTTTCCGCTCACG

[0015] CDR1(3C4-2E9):CAGAATGTGGATACTAAT

[0016] CDR2(3C4-2E9): TCGGCATCC

[0017] CDR3(3C4-2E9):CAGCAATATAACAGCTATCCGCTCACG

[0018] The three complementarity-determining region (CDR) sequences of the heavy chain variable region are as follows:

[0019] CDR1(1C1-4D11):GGCTACACCTTCACAAGCTACTAT

[0020] CDR2(1C1-4D11):ATTTATCCTGGAAGTGTTAATGCT

[0021] CDR3(1C1-4D11): GCGAACTACGGTAGTAGTGACTTCGATGTC

[0022] CDR1(1E1-2C11):GGCTACACCTTCACAAGCTACTAT

[0023] CDR2(1E1-2C11):ATTTACCCTGGAAGTGTTAATGCT

[0024] CDR3(1E1-2C11): GCGAACTACGGTAGTAGTGACTTCGATGTC

[0025] CDR1(3C4-2E9):GGCTACACCTTCACATCCTACTAT

[0026] CDR2(3C4-2E9):ATTTATCCTGGAAATGTTAATACT

[0027] CDR3(3C4-2E9): GCGAACTACGGTAGTAGGGACTTCGATATC

[0028] The amino acid sequence of the light chain variable region is as follows: V L (1C1-4D11), V L (1E1-2C11), V L (3C4-2E9); the amino acid sequence of the heavy chain variable region is shown in V.H (1C1-4D11), V H (1E1-2C11), V H As shown in (3C4-2E9).

[0029] Two full-length gene sequences expressing immunoglobulins, such as pCDNA3.4-V. L (1C1-4D11), pCDNA3.4-V H (1C1-4D11), pCDNA3.4-V L (3C4-2E9), pCDNA3.4-V H As shown in (3C4-2E9).

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] In this patent application, we utilize hybridoma sequencing to extract total RNA from hybridoma cells (1C1-4D11, 3C4-2E9, and 1E1-2C11). Then, using the mRNA from the total RNA as a template, we perform reverse transcription to obtain the cDNA of the corresponding antibody gene. Finally, we 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 heavy and light chain nucleotide sequences of three monoclonal antibodies (1C1-4D11, 3C4-2E9, and 1E1-2C11) targeting the LSDV ORF29 protein, thus realizing this invention. The heavy chain (V...) of the above three monoclonal antibodies... H ) and light chain antibodies (V L Sequence analysis revealed that the genotypes of 1C14-D11, 3C4-2E9, and 1E1-2C11 are all IgG, and their light chains are all κ. Based on sequence similarity comparison, we ultimately selected the heavy and light chains of two hybridoma antibodies, 1C1-4D11 and 3C4-2E9, which have significant nucleotide sequence differences, for gene synthesis. To improve antibody affinity, codon optimization was performed, and the chimeric antibody was fused with the constant region of a murine antibody while retaining the variable region. The chimeric antibody full-gene sequence was then synthesized and expressed through bioengineering. In vitro cell culture showed that this chimeric immunoglobulin exhibited good reactivity and exogenously expressed IgG. H +V L It possesses neutralizing antibody biological activity. This application discloses the complete gene sequence of the chimeric immunoglobulin and its applications, aiming to solve the technical problems of imperfect and incomplete detection, diagnosis, prevention, and treatment or drugs for LSDV serotypes. Attached Figure Description

[0032] Figure 1 This invention was used to screen and verify the LSDV ORF29 hybridoma cell line in Example 1 of the present invention; wherein: (A) PCR amplification of the LSDV ORF29 gene; (B) induced expression of pET28a-LSDV ORF29 prokaryotic plasmid; (C) purification of LSDV ORF29 protein; (D) Western blot analysis of the reaction characteristics and antibody titers of the ascites fluid of the three monoclonal antibody hybridoma cell lines.

[0033] Figure 2 HybSeq-HT, representing the antibody genes of the three LSDV ORF29 hybridoma cell lines in Example 2 of this invention. TM Sequencing analysis; including: (A) HybSeq-HT of antibody genes from three LSDV ORF29 hybridoma cell lines. TM Sequencing abundance statistical analysis; (B) Antibody V from three LSDV ORF29 hybridoma cell lines H HybSeq-HT gene TM Sequencing statistics; (C) Antibody V from three LSDVORF29 hybridoma cell lines L HybSeq-HT gene TM Sequencing statistics.

[0034] Figure 3 The V antibody gene of the three LSDV ORF29 hybridoma cell lines in Example 3 of this invention. H and V L Sequence alignment analysis; among which: (A) V of antibody genes of three LSDV ORF29 hybridoma cell lines H (B) V-series alignment analysis of antibody genes from three LSDV ORF29 hybridoma cell lines. L Results of gene nucleotide sequence alignment analysis.

[0035] Figure 4 This invention relates to the synthesis and construction of chimeric plasmids for the antibody genes of hybridoma cell lines 1C1-4D11 and 3C4-2E9 in Example 4 of this invention; wherein: (A) V of the antibody genes of 1C1-4D11 and 3C4-2E9 H Gene amino acid sequence alignment analysis; (B) V of 1C1-4D11 and 3C4-2E9 antibody genes L (C) Gene amino acid sequence alignment analysis; (D) Antibody gene structure diagram; (E) Western blot analysis of the V of antibody genes 1C1-4D11 and 3C4-2E9. H and V L Reactivity analysis;

[0036] Figure 5 V is the 1C1-4D11 antibody gene chimeric plasmid in Example 4 of this invention. H and V L Gene map; in which: (A) V of the 1C1-4D11 antibody gene chimeric plasmid H Gene map; (B) V of the 1C1-4D11 antibody gene chimeric plasmid L Gene map.

[0037] Figure 6 V, the chimeric plasmid of the 3C4-2E9 antibody gene in Example 4 of the invention. H and V L Gene map; where: (A) V of the 3C4-2E9 antibody gene chimeric plasmid H Gene map; (B) V of the 3C4-2E9 antibody gene chimeric plasmid L Gene map.

[0038] Figure 7 In Example 5 of this invention, the antigen complementarity-determining region (CDR) for V H and V L Essential for the performance of biological functions; among them: (A) 3C4-2E9 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 and V L The construction of antigen-antibody binding activity analysis was performed without CDR1, CDR2, and CDR3.

[0039] Figure 8 The antibody gene V of the LSDV ORF29 hybridoma monoclonal antibody in Example 6 of this invention. H and V L It possesses neutralizing antibody activity; among which: (A) LSDV ORF29 hybridoma monoclonal antibody gene V H and V L (B) Fluorescent observation of neutralizing EGFP-LSDV viral replication; LSDV ORF29 hybridoma monoclonal antibody gene V H and V L Western blot analysis of neutralizing intracellular EGFP-LSDV replication; (C) LSDV ORF29 hybridoma monoclonal antibody gene V H and V LTCIDs that neutralize extracellular EGFP-LSDV viral replication 50 Determination; (D)LSDV ORF29 hybridoma monoclonal antibody gene V H and V L Fluorescent observation of neutralizing RFP-LSDV viral replication; (E) LSDV ORF29 hybridoma monoclonal antibody gene V H and V L Western blot analysis of neutralizing intracellular RFP-LSDV replication; (F) LSDV ORF29 hybridoma monoclonal antibody gene V H and V L TCID that neutralizes extracellular RFP-LSDV viral replication 50 Measurement; Detailed Implementation

[0040] 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.

[0041] Example 1: Screening of three LSDV ORF29 monoclonal antibody hybridoma cells

[0042] 1. Construction of pET28a-ORF29 recombinant plasmid

[0043] Primers were designed using SnapGene 6.0.2 software to amplify the LSDV ORF29 gene sequence, introducing the restriction enzyme sites BamHI and EcoRI. The upstream primer was LSDV ORF29-For:5'-CAGCAAATGGGTCGC. GGATCC GCCACCATGTGGTCCTTATTTTTTTCAAAACCTCCATCTG-3' (underlined area is the BamHI restriction site), downstream primer LSDV ORF29-Rev: 5'-TTGTCGACGGAGCTC GAATTCGTGGTGGTGGTGGTGGTGGCTTCCTCCTCCCAGCACTGTATTTTTTTTGTCTGACCAATCTC-3' (underlined part is the EcoRI restriction site). The above primer pair was synthesized by Genewiz Biotechnology Co., Ltd. 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, template 1μl. The reaction program was as follows: 95℃ for 3 min, 95℃ for 15 s, 58℃ for 15 s, 72℃ for 75 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 the gel and ligates them with the enzyme-digested empty vector. The clones are then transformed into *E. coli* BL21(DE3) competent cells, plated on kanamycin-containing 2YT solid culture plates, and single colonies are picked for shaking and plasmid extraction. The resulting samples are sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced positive recombinant plasmid is named pET28a-LSDV ORF29. Figure 1 A).

[0044] 2. Purification of pET28a-LSDV ORF29 recombinant protein induced expression

[0045] The recombinant plasmid pET28a-LSDV ORF29 was transformed into culture medium, and when the optical density value OD28a was reached... 600 When 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 a second centrifugation, the supernatant and precipitate were collected. This mixture was then combined 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, subjected to SDS-PAGE electrophoresis. Finally, the protein expression pattern was analyzed by Coomassie brilliant blue staining. Figure 1 As shown in Figure B, LSDV ORF29 protein is expressed in the precipitate. Then, using standard inclusion body protein purification methods, nickel column affinity chromatography was employed for LSDV ORF29 protein purification. A small amount of the purified protein was then subjected to SDS-PAGE electrophoresis and staining again to compare changes in protein purity. Figure 1 As shown in Figure C, we successfully purified and obtained the LSDV ORF29 protein.

[0046] 3. Hybridoma cell line screening, preparation and titer determination of monoclonal antibodies

[0047] In the previous stage of this study, the LSDV ORF29 target protein was obtained by constructing the prokaryotic expression plasmid pET28a-ORF29, followed by induction expression and purification. The purified protein was used to immunize mice, and spleen cells were fused with myeloma cells SP2 / 0. After screening in HAT selective medium, three hybridoma cell lines that stably secreted monoclonal antibodies against ORF29 were selected: 1C1-4D11, 1E1-2D11, and 3C4-2E9. Figure 1 D).

[0048] 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 intraperitoneally 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. Three hybridoma cell lines with good specificity (1C1-4D11, 1E1-2D11, and 3C4-2E9) were selected. Figure 1 D).

[0049] Example 2: Sequencing of Antibody Genes from Three Monoclonal Antibody Hybridoma Cells

[0050] 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 three hybridoma cell lines mentioned above (1C1-4D11, 1E1-2D11, and 3C4-2E9) were sent to Anshengda Biotechnology Co., Ltd. for hybridoma antibody gene profiling sequencing (HybSeq-HT). TM The hybridoma antibody sequencing experimental procedure includes RNA extraction using the Trizol method, reverse transcription of the sample to obtain cDNA, amplification to obtain the heavy and light chain variable region sequences, cloning into a vector, and sequencing. After sequencing, the sequencing results are compared with the IMGT database to extract CDR1, CDR2, and CDR3 information of the antibody sequence. For each sample, the 10 sequences with the highest abundance are selected for abundance analysis, and only the sequences ranked highest are considered as target gene sequences. Figure 2A, 2B, and 2C, Hybseq HT TM Hybridoma antibody genome sequencing results showed that the variable region (V) of the three monoclonal antibodies... H and V L The abundance value was high, approaching 80%, and the differences among the three antibodies were low, indicating that the three antibody genes had high coverage and the target gene sequence was singular. Figure 2 A). Sequence alignment results showed that the subtype identification results indicated that all heavy chains were of the IgG type ( Figure 2 B), the light chains are all κ-type ( Figure 2 C).

[0051] The specific experimental procedure is as follows:

[0052] Subsequently, using the SuperScript.III First-Strand Synthesis System for RT-PCR kit, mRNA extracted from three hybridoma cell lines (1C1-4D11, 3C4-2E9, and 1E1-2C11) was reverse transcribed into cDNA. The light or heavy chain variable region (V region) of the anti-LSDV ORF29 antibody gene was then amplified using universal specific primers. H and V L ). This will contain the corresponding heavy chain variable region (V) H ) or light chain variable region (V L The PCR product of the fragment was subjected to 1% agarose gel electrophoresis, and the target 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. The recovered heavy chain variable region and light chain variable region were cloned into the empty sequencing vector pMD19-T, and then Sanger sequencing was performed. After sequencing, sequence alignment and homology analysis were performed on the sequencing results.

[0053] Example 3: Antibody V from three hybridoma cells H and V L Gene sequence alignment

[0054] like Figure 3 The heavy chain variable region (V) of the antibody gene of three LSDV ORF29 hybridoma cell lines (1C1-4D11, 1E1-2D11 and 3C4-2E9) was analyzed. H ) and light chain variable region (V L Sequencing analysis showed that the V gene of the three antibody strains... H and V L It has high coverage and targets a single gene sequence. (Through V) H and V LBased on nucleotide sequence alignment, we selected the antibody gene sequences of two LSDV ORF29 hybridoma cell lines, 1C1-4D11 and 3C4-2E9, which had significant sequence differences. We synthesized the genes and constructed them into the pCDNA3.4 empty vector to express the hybridoma cell antibodies of the two monoclonal antibodies mentioned above for subsequent research.

[0055] Example 4: Synthesis and Construction of Chimeric Plasmids for Antibody Genes from Hybridoma Cell Lines 1C1-4D11 and 3C4-2E9

[0056] HybSeq-HT based on antibody genes of 1C1-4D11 and 3C4-2E9 tumor cell lines TM Sequencing amino acid sequence alignment results ( Figure 4 In our analysis (A-4B), we found that 1C1-4D11 and 3C4-2E9 showed little difference in their amino acid sequences and high homology. Based on the sequence similarity comparison results, we ultimately selected the heavy and light chains of the two hybridoma antibodies, 1C14-D11 and 3C4-2E9, for gene synthesis and constructed them into the pCDNA3.4 empty vector, named pCDNA3.4-V. H -1C1-4D11(ORF29)( Figure 5 A) pCDNA3.4-V L -1C1-4D11(ORF29)( Figure 5 B) pCDNA3.4-V H -3C4-2E9(ORF29)( Figure 6 A) and pCDNA3.4-VL-3C4-2E9(ORF29) Figure 6 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 4 C). V of the 1C14-D11 antibody gene chimeric plasmid H and V L Gene maps such as Figure 5 V of the 3C4-2E9 antibody gene chimeric plasmid H and V L Gene maps such as Figure 6 V H and V L Synthesized expression plasmids were transfected into HEK-293T cells for secretory expression. Figure 4 D. Western blotting results showed that the two monoclonal antibodies only had the variable region V. H or V L and co-expression V H +VL The presence of specific target bands in both cases indicates that we have successfully expressed the antibody gene sequences of two monoclonal hybridoma strains, 1C1-4D11 and 3C4-2E9, and that the antigen-antibody reaction exhibits good specificity and sensitivity.

[0057] Example 5: Antigen complementary determinant region (CDR) for V L and V L Necessary for biological functions

[0058] The antigen complementarity-determining (CDR) region, located within the variable regions of the heavy and light chains, is a crucial site for specific antigen recognition and is generally divided into three regions: CDR1, CDR2, and CDR3. The 3C42E9 monoclonal antibody sequence can be obtained through hybridoma cell gene sequencing, and further analysis of the variable region V... H and V L Different areas are labeled (yellow for CDR1, red for CDR2, dark green for CDR3) Figure 7 A). 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 7 C). According to V H +V L ΔCDR1, V H +V L ΔCDR2, V H +V L ΔCDR3, V H ΔCD1+V L V H ΔCDR2+V L and V H ΔCDR3+V L Co-transfect cells in the form of V to express the expression, while simultaneously expressing V H +V L As a positive control, Western blotting results showed no specific target band. Figure 7 E). Additionally, V will also be included. HΔCDR1+V L ΔCDR1, V H ΔCDR2+V L ΔCDR2 and V H ΔCDR3+V L ΔCDR3 co-transfection expression, Western blot results showed no specific bands in any of the three. Figure 7 G). The analysis of the above results shows that the CDR region is a key target site for antigen recognition and can directly affect the binding of antigens.

[0059] Table 1 PCR Primers

[0060]

[0061]

[0062] Example 6: Antibody genes (V) of hybridoma cell lines 1C1-4D11 and 3C4-2E9 H +V L It has neutralizing antibody activity.

[0063] like Figure 8 As shown, using fluorescent LSDV strains (EGFP-LSDV and RFP-LSDV) labeled with green fluorescent protein (EGFP) and red fluorescent protein (RFP), we observed exogenous transfection of V... H +V L (1C1-4D11) and V H +V L (3C4-2E9) can significantly inhibit the fluorescence intensity of EGFP and RFP at 24h.pi, 48h.pi, and 72h.pi. Figure 8 (A and 8D). Simultaneously, the TCID values ​​of the cell culture supernatant collected at the corresponding time points... 50 The results show V H +V L (1C1-4D11) and V H +V L (3C4-2E9) significantly inhibited extracellular EGFP-LSDV and RFP-LSDV viral titers at 24h.pi, 48h.pi, and 72h.pi. Figure 8 Protein samples were collected at the corresponding time points (C and 8F). Western blotting results showed that V was co-transfected. H +V L (1C1-4D11) and V H +V L(3C4-2E9) can significantly inhibit the replication of intracellular EGFP-LSDV and RFP-LSDV viruses. Figure 8 B and 8E). The above fluorescence observation results, Western blot and TCID50 analysis results all indicate that V of exogenously transfected 1C1-4D11 and 3C4-2E9 hybridoma cell lines H +V L Both strains effectively inhibited the replication of EGFP-LSDV and RFP-LSDV fluorescently labeled strains, indicating that the antibody genes V expressed exogenously in hybridoma cell lines 1C1-4D11 and 3C4-2E9 through genetic engineering were effective. H and V L It has neutralizing antibody activity.

[0064] The specific experimental procedure is as follows: Vero cells were seeded into 12-well plates. When the cell density reached 70-80%, 1C1-4D11 and 3C4-2E9 cells were co-transfected with Vero. H and V L 1 μg of pCDNA and 3.41 μg of empty pCDNA were added, along with a positive control (untransfected plasmid). Exogenous co-transfection with V... H and V L The virus strain used in the plasmid experiment was EGFP-LSDV (EGFP-LSDV viral load 10). -5.5 / 0.1mL) and RFP-LSDV (RFP-LSDV viral load 10 -6.25 / 0.1mL), transfected with plasmid 18-24h before inoculation. Positive controls for the two groups were EGFP-LSDV and RFP-LSDV, respectively. Fluorescence intensity was observed and recorded at 24h, 48h, and 72h post-inoculation. It was found that compared with the positive control group, co-transfected V H and V L The fluorescence intensity was significantly reduced after plasmidization. Figure 8 A and 8D). Simultaneously, corresponding protein samples were collected and subjected to Western blotting experiments to further assess the proliferation of intracellular fluorescently labeled strains. Figure 8 B and 8E). In parallel experiments, 2.5 μL of EGFP-LSDV virus (containing 10 μL of virus) was inoculated into each well 18 h after 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 To assess the extracellular LSDV viral load and plot the viral growth curve ( Figure 8 C and 8F).

[0065] 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. Two LSDV ORF29 monoclonal antibodies, comprising heavy and light chains, characterized in that: The nucleotide sequences of the three complementarity-determining regions (CDRs) of the light chain variable region are as follows: 1C1-4D11's CDR1: CAGAATGTGGGTACAAAT CDR2 of 1C1-4D11: TCGGCATCC CDR3 of 1C1-4D11: CAACAATATAACAGCTTTCCGCTCACG CDR1 of 3C4-2E9: CAGAATGTGGATACTAAT CDR2 of 3C4-2E9: TCGGCATCC CDR3 of 3C4-2E9: CAGCAATATAACAGCTATCCGCTCACG The nucleotide sequences of the three complementarity-determining regions (CDRs) of the heavy chain variable region are as follows: 1C1-4D11's CDR1: GGCTACACCTTCACAAGCTACTAT CDR2 of 1C1-4D11: ATTATCCTGGAAGTGTTAATGCT 1C1-4D11's CDR3: GCGAACTACGGTAGTAGTGACTTCGATGTC 3C4-2E9's CDR1: GGCTACACCTTCACATCCTACTAT 3C4-2E9's CDR2: ATTATCCTGGAAATGTTAATACT 3C4-2E9's CDR3: GCGAACTACGGTAGTAGGGACTTCGATATC.

2. The two LSDV ORF29 monoclonal antibodies according to claim 1, characterized in that, The amino acid sequences of the light chain variable region are as follows: 1C1-4D11 of V L : DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSRSGTDFTLTISNVQSEDLADYFCQQYNSFPLTFGAGTKLELK 3C4-2E9's V L : DIVMTPSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVKPEDLAEYFCQQYNSYPLTFGAGTKLELK The amino acid sequences of the heavy chain variable region are as follows: 1C1-4D11 of V H : QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGSVNAKYNEKFKGKATLTADKSSSTAYIHLNSLTSEDSAVYFCANYGSSDFDVWGAGTTVTVSS 3C4-2E9's V H : QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIHWLKQRPGQGLEWIGWIYPGNVNTKYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCANYGSRDFDIWGAGTTVTVSS。

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