Monoclonal antibody of nairobi sheep disease virus and use thereof

Abstract

This invention proposes a monoclonal antibody against Nairobi sheep disease virus and its application, belonging to the field of monoclonal antibody technology. It solves the problem of accurate detection of Nairobi sheep disease virus. This invention provides a monoclonal antibody against Nairobi sheep disease virus. The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; the amino acid sequence of CDR-H3 is shown in SEQ ID NO.5; the amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of CDR-L2 is LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.7. The monoclonal antibody against Nairobi sheep disease virus provided by this invention is mainly used to detect whether sheep are infected with Nairobi sheep disease virus.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, and in particular relates to a monoclonal antibody against Nairobi sheep disease virus and its application. Background Technology

[0002] Nairobi sheep disease virus (NSDV) is an important tick-borne virus belonging to the genus Orthonairovirus in the family Nairoviridae. It causes Nairobi sheep disease (NSD), resulting in a mortality rate exceeding 90% in sheep and goats. This virus primarily infects small ruminants such as sheep and goats, causing severe economic losses to livestock production. NSDV infection in sheep can cause symptoms such as depression, high fever, progressive weight loss, severe diarrhea (some cases presenting with characteristic bloody watery stools), and respiratory symptoms. The acute course and high mortality of the disease make it one of the most important infectious diseases affecting the health of ruminants. However, currently, there is a lack of both commercially available effective preventative vaccines and specific treatments for NSD, highlighting the urgent need for relevant research.

[0003] Nairobi sheep disease virus (NSDV) is a tick-borne hemorrhagic fever virus that causes high mortality in sheep and goats. The virus was first reported in an outbreak in Kenya in 1917, causing a 90% mortality rate in sheep populations. It is currently listed as a notifiable animal disease by the World Organisation for Animal Health (WOAH). NSDV belongs to the Nairovirus genus, the same as Crimean-Congo hemorrhagic fever virus (CCHFV). Its genome encodes polymerases and glycoproteins with similar immune evasion mechanisms, suggesting a potential zoonotic risk. Large-scale outbreaks often occur when susceptible animals are introduced from non-endemic areas into endemic areas.

[0004] Nairobi sheep disease (NSD) is a vector-borne disease primarily caused by the ticks *Dermacentor appendiculatus* (East Africa) and *Haemaphysalis intermedia* (Asia), posing a widespread threat to public health. The virus enters the host through tick bites, targeting vascular endothelial cells and triggering a systemic inflammatory response, clinically manifesting as hemorrhagic gastroenteritis, fever, and abortion. Traditionally, this virus has been prevalent in East Africa and South Asia. In recent years, NSDV has been detected in tick serum collected from Liaoning, Jilin, Heilongjiang, and Hubei provinces in Northeast China. This suggests that climate change may be driving the spread of vector ticks to temperate zones. Research indicates that the widespread transmission of NSDV in China may pose serious potential risks, not only significantly threatening livestock production in China but also potentially having a profound negative impact on the economic value of related industries. Detection methods for this virus are urgently needed. Summary of the Invention

[0005] In view of this, the present invention aims to provide a monoclonal antibody against Nairobi sheep disease virus and its application, in order to solve the problem of accurately detecting whether sheep are infected with Nairobi sheep disease virus.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a monoclonal antibody against Nairobi sheep disease virus, wherein the amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.5.

[0007] The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of CDR-H2 is LVS; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.7.

[0008] Further specifying, the heavy chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO.8.

[0009] Further specifying, the light chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO.9.

[0010] This invention provides a gene that encodes the above-mentioned monoclonal antibody.

[0011] Further specifying, the gene sequence encoding the heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.10.

[0012] Further specifying, the gene sequence encoding the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 11. This invention provides a recombinant vector comprising the above-described gene.

[0013] Further specifying, the starting carrier is the pCold carrier.

[0014] The present invention provides a recombinant host cell containing the above-described recombinant vector.

[0015] Further specifying, the recombinant host cell is a microbial cell or an animal cell.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This study uses the NSDV nucleocapsid protein (N protein) N gene sequence indexed in the NCBI database as a template, designs specific amplification primers, amplifies the NSDV N gene using PCR technology, purifies it, and then clones it into the expression vector pCold-Gst to construct the recombinant plasmid pCold-Gst-NSDV-N. The truncated fragment of the NSDV nucleocapsid protein (N protein) is recombinantly expressed using a prokaryotic expression system. SDS-PAGE electrophoresis analysis confirmed that the recombinant protein exists mainly in a soluble form in the expression cells. Western blot results showed that the NSDV-N protein can significantly bind to specific antibodies, confirming that the protein has ideal immunoreactivity. The purified recombinant protein was used to immunize four mice. Serum titers were determined using an established indirect ELISA method. The mouse with the highest serum titer was selected for booster immunization. Following immunization, cell fusion experiments were performed. Positive hybridoma cells were then screened using indirect ELISA and IFA methods. After three subcloning processes, a monoclonal antibody, named 6D12, was obtained. Western blot and IFA experiments showed that this monoclonal antibody specifically binds to NSDV, indicating it is a high-titer, high-specificity monoclonal antibody. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 This is a diagram showing the PCR amplification results of the N gene; Note: M: Marker, 1: Negative control, 2: PCR product;

[0019] Figure 2The image shows the results of bacterial culture PCR identification; Note: M: Marker, 1: Positive control, 2: Negative control, 3, 4: Selected different strains;

[0020] Figure 3 Figure showing the enzyme digestion identification results of recombinant plasmid pCold-Gst-NP; Note: M: Marker, 1: Enzyme digestion vector;

[0021] Figure 4 The image shows the SDS-PAGE results of protein expression identification; Note: M: Marker, 1: Before induction, 2: After induction, 3: Ultrasonic supernatant, 4: Ultrasonic precipitation;

[0022] Figure 5 The image shows the SDS-PAGE results of protein purification. Note: M: Marker, 1: Before column chromatography, 2: After column chromatography, 3: Washing, 4: Elution buffer.

[0023] Figure 6 Figure showing the results of Western blot identification of NSDV N protein; Note: M: Marker, 1: Negative control, 2: Target protein;

[0024] Figure 7 Image showing the SDS-PAGE identification results of the 6D12 strain monoclonal antibody purified from mouse ascites fluid; Note: M: protein marker, 1: unpurified monoclonal antibody, 2: purified monoclonal antibody;

[0025] Figure 8 Figure 150x shows the IFA (in vitro analysis) results of monoclonal antibodies; A: IFA test performed with 12D6 as the primary antibody after NSDV inoculation; B: Negative control;

[0026] Figure 9 Figure showing the Western blot results of the anti-NSDV 6D12 monoclonal antibody; Note: M: Protein Marker; 1: pCold-Gst empty vector; 2: Target protein;

[0027] Figure 10 This is a graph showing the results of competitive detection using monoclonal antibodies. Detailed Implementation

[0028] Example 1. Preparation of monoclonal antibodies

[0029] 1. N Protein Primer Design: A primer pair for amplifying the N gene was designed by comparing and analyzing the whole genome sequence of Nairobi sheep disease virus (NSDV) already included in the NCBI database. This primer pair can amplify a 1449 bp fragment of the N gene. The primer design and synthesis were completed by Ruiboxingke Biotechnology Co., Ltd., and the specific primer sequence information is as follows: NSDV-F: (XhoI) CTCGAG ATGCAGAATCAGATT(SEQ ID NO.1);

[0030] NSDV-R: (ECORI) GAATTC TTAAAATAATCTGAATGCTGGTCGC (SEQ ID NO. 2).

[0031] 2. Construction and identification of recombinant plasmids:

[0032] PCR amplification of the NSDV N gene: Using the cDNA of the NSDV strain (SEQ ID NO.13) as a template, PCR amplification was performed. The reaction program was as follows: pre-denaturation 95℃, 2 min; denaturation 95℃, 15 s; annealing 55℃, 15 s; extension 72℃, 1 min, 35 cycles; final extension 72℃, 5 min. The PCR system was as follows: NSDV-F, 1 μL; NSDV-R, 1 μL; template, 2 μL; 2xRapid Taq Master Mix, 12.5 μL; RNase-free water to 20 μL. The PCR amplification products were subjected to 1% agarose gel electrophoresis. The target band size was normal. The gel was cut, labeled, and used for later use.

[0033] Nucleic acid from the NSD virus strain was extracted for RT-PCR. The N gene of NSDV was amplified using specific primers, such as... Figure 1 As shown, the agarose gel electrophoresis results indicate that the amplification product size is approximately 1500 bp, which is consistent with the expected size, indicating that the N gene amplification was successful.

[0034] 3. Gene and vector ligation and transformation: The target gene fragment and the enzyme-digested linearized vector were purified by gel electrophoresis and directionally ligated using the In-Fusion high-efficiency ligase system to obtain the pCold-Gst-NSDV-N recombinant plasmid. The specific reaction system is as follows: 5× In-Fusion HD Enzyme Premix, 2 μL; pClod-GST, 1 μL; NSDV-N, 1 μL; RNase-free water to a final volume of 10 μL. The reaction conditions were set at 50℃ for 15 min. Immediately after the ligation reaction, 5 μL of the ligation product was transformed into DH5α chemocompetent cells.

[0035] 4. Identification of the pCold-Gst-NSDV-N recombinant plasmid: 1% agarose gel electrophoresis analysis showed that one colony from two randomly selected bacterial cultures exhibited a specific amplification band at approximately 1500 bp. The results are as follows: Figure 2 The size shown matches the expected N gene fragment size (1457 bp), preliminarily identifying it as a positive clone. Double enzyme digestion identification of the recombinant plasmid: The recombinant plasmid pCold-Gst-NSDV-N was digested with Kpn I and EcoRI restriction endonucleases. Nucleic acid electrophoresis showed two specific bands with molecular weights of approximately 5050 bp and 1457 bp, respectively, consistent with the expected size. The results indicate that the N gene of NSDV has been accurately inserted between the Kpn I and EcoRI restriction sites of the pCold-Gst prokaryotic expression vector. Figure 3 ).

[0036] 5. NSDV N protein induction expression: The pCold-Gst-NSDV-N recombinant plasmid was introduced into BL21 competent cells using a heat shock conversion method, with no control group samples induced. Pre-prepared IPTG solution (100 μM stock solution) was added to the remaining bacterial culture to achieve a final concentration of 1 μM. The induced culture was lysed using an ultrasonic cell disruptor. The supernatant was purified by column chromatography, followed by protein electrophoresis on a 12% protein gel. After SDS-PAGE electrophoresis, the protein was efficiently transferred from the gel to a PVDF membrane. After blocking, the membrane was incubated with NSDV positive serum primary antibody diluted 1:10 with 5% skim milk powder. After primary antibody incubation and secondary antibody incubation, signals were acquired using a chemiluminescence imaging system to obtain clear protein detection results.

[0037] Results: SDS-PAGE analysis of the bacterial supernatant and bacterial pellet before and after induction confirmed the successful production of the target protein, which was mainly present in the lysate supernatant in a soluble form. Figure 4 ).

[0038] The purified NSDV N protein was identified using SDS-PAGE electrophoresis. The results are as follows: Figure 5 As shown, the purified product exhibited a single, clear band at approximately 79 kDa, consistent with the expected size. The band showed no obvious contamination from other proteins, indicating that the protein purification was effective and met the requirements for subsequent experiments.

[0039] Identification of NSDV N protein (SEQ ID NO.12): Western blot results are as follows Figure 6As shown, after SDS-PAGE, the recombinant N protein was transferred to a PVDF membrane and incubated with NSDV positive serum as the primary antibody. A clear band appeared at 79 kDa, which was consistent with the expected size, confirming that the recombinant N protein has a natural antigenic epitope and reacts specifically with the N protein.

[0040] 6. Preparation of NSDV monoclonal antibody

[0041] (1) Animal immunization: Six-week-old male mice were immunized three times with NSDV-N protein as the immunogen, with each immunization occurring 14 days apart. The immunization was performed via subcutaneous injection at multiple sites. For the first immunization, 100 μg of NSDV N protein was emulsified with Freund's complete adjuvant at a 1:1 ratio. For the second and third immunizations, 100 μg of NSDV N protein was emulsified with Freund's incomplete adjuvant at a 1:1 ratio. Seven days after the third immunization, mouse serum was collected for serum titer testing. Mice with the highest serum titer were selected for booster immunization via indirect ELISA. The booster immunization was performed via intraperitoneal injection of NSDV N protein. Three days after the booster immunization, cell fusion experiments were conducted.

[0042] (2) Preparation of positive and negative serum: Blood was collected from the cheeks of unimmunized healthy mice and mice immunized with NSDV N protein. The mice were centrifuged at 4000×g for 10 min at 4℃. After centrifugation, the upper serum was collected to obtain NSDV negative serum and NSDV positive serum.

[0043] (3) Serum titer determination: After immunization three times, blood was collected from the cheeks of mice to collect serum. The serum titer was determined by indirect ELISA. NSDV N protein was coated onto an ELISA plate at a concentration of 1:800. The ELISA plate was incubated at 4°C overnight to ensure that the protein was fully adsorbed to the bottom of the plate. The coating solution was discarded, and the plate was blocked with 5% skim milk and incubated at 37°C for 1 h. The plate was washed three times with PBST by rapid shaking for 5 min on a shaker. The serum was serially diluted in eight ways: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:640000. The diluted solution was then added to each well of the ELISA plate at 100 μL and incubated at 37°C for 1 h. The serum was discarded, and the plate was washed three times with PBST by rapid shaking for 5 min on a shaker. Add 100 μL of HRP goat anti-mouse IgG secondary antibody (1:5000 dilution) and incubate at 37°C for 1 h. Discard the secondary antibody and wash the plate three times with PBST using rapid shaking for 5 min on a shaker. Finally, add 100 μL of TMB chromogenic solution and incubate at room temperature in the dark for 15 min. Immediately afterward, add 100 μL of 2 M H2SO4 to stop the reaction and immediately measure the OD of each well. 450nm .

[0044] Following the third immunization, blood was collected from four immunized mice, and serum was separated. Serum was serially diluted from 1:500 to 1:640,000, and serum titer was determined using an indirect ELISA method. The positive value was defined as the OD value of negative serum. 450 The titer was twice that of mice. According to the results, the serum titers of all four mice reached positive values. Since the serum titer of mouse number 4 was the highest, it was given a booster immunization, and the cell fusion experiment was carried out 3 days later.

[0045] Table 1. Determination of serum titer in mice

[0046]

[0047] (4) Preparation of SP2 / 0 cells: Five days before cell fusion, SP2 / 0 myeloma cells need to be revived in advance, and the culture medium is changed to 20% FBS 1640 medium for culturing SP2 / 0 myeloma cells. When the cell density reaches 80-90% confluence, passage is performed at a ratio of 1:3.

[0048] (5) Spleen cell preparation: BALB / c mice with satisfactory serum titers were selected, and their spleens were transferred to new culture dishes containing 10 mL of serum-free 1640 medium. Spleen cells were flushed out using a hydraulic shock method. Cell counting was performed using a hemocytometer under an inverted microscope, requiring a viable cell percentage ≥90% and a cell density adjusted to 1×10⁻⁶. 7 Units / mL are available for use.

[0049] (6) Cell fusion: Mix spleen cells and SP2 / 0 cells at the optimal ratio of 1:8 in a new 50 mL centrifuge tube, and gently invert 10 times to ensure full cell contact. Add 1 mL of preheated 45% PEG fusion agent (containing 5% DMSO) and incubate at 37°C for 2 min. The fusion reaction is terminated by gradient dilution.

[0050] (7) Five days after fusion, observe whether the cells have fused successfully under an inverted microscope. When the cells in the plate have grown to about 1 / 3, aspirate the supernatant and use the indirect ELISA method. Based on the results of the indirect ELISA test, determine whether the following is true: (1) Antigen coating: Coat the NSDVN protein on the ELISA plate at a ratio of 1:800, 100 μL / well, overnight at 4°C to ensure that the protein is fully adsorbed on the bottom of the plate. (2) Blocking: Block with 5% skim milk and incubate at 37°C for 1 h to block non-specific binding sites. (3) Primary antibody incubation: Wash 3 times with PBST for 5 min each time, add 100 μL of monoclonal antibody supernatant to each well, and incubate at 37℃ for 1 h. (4) Secondary antibody incubation: Wash 3 times with PBST for 5 min each time, then incubate with HRP goat anti-mouse IgG1 diluted at 5000 at 37℃ for 1 h. (5) Color development: Wash 3 times with PBST for 5 min each time, add 100 μL of Beyotime TMB color development solution to each well, and incubate at room temperature in the dark for 15 min. (6) Stop color development and reading: Finally, add 100 μL of 2 M H2SO4 to the microplate to stop the reaction, and immediately measure the OD of each well. 450nmPreliminary screening was conducted. Based on the results of indirect ELISA, strongly positive wells with high antibody secretion levels were selected and labeled for subsequent subcloning experiments. Subcloning was performed using a cell counting method. Cells from the original wells were resuspended in 100 μL of HT medium. 20 μL of the medium was then mixed with 20 μL of trypan blue at a 1:1 ratio, and cell counts were performed. Cells were seeded into plates containing 300 cells per 200 μL for culture. (This process was repeated twice in the original text.)

[0051] After each subcloning, a single cell was selected for testing. The cell supernatant was tested using an indirect ELISA method. When the positive rate reached 100%, a second screening was performed using an indirect immunofluorescence assay. Positive wells were then screened using indirect ELISA and IFA assays. Finally, the monoclonal antibody strain was selected and named 6D12.

[0052] (8) Ascites preparation: Seven days before the ascites preparation experiment, 1 mL of sterile liquid paraffin was injected into the peritoneal cavity of 12-week-old BALB / c female mice via intraperitoneal injection. Seven days later, hybridoma cells were blown off with serum-free 1640 medium, and 10 cells were counted and collected. 5 The mice were injected intraperitoneally with cells, and the changes in the peritoneum were observed daily. One week after the intraperitoneal injection, the mice showed obvious abdominal distension and fluctuation. Ascites was collected using an ascites needle. The collected ascites was centrifuged at 2000 r / min at 4℃ for 30 min, and the supernatant was collected and stored at -20℃.

[0053] (9) Ascites purification: The pretreated ascites samples were purified using Protein G Resin.

[0054] Example 2.

[0055] 1. Monoclonal antibody titer assay: Purified ascites fluid was diluted 1:200 to 1:25600 and the titer was determined by indirect ELISA. A negative control was prepared. HRP goat anti-mouse IgG secondary antibody was diluted 1:5000, and 100 μL was added to each well. The mixture was incubated at 37°C for 1 h, followed by the addition of 100 μL of TMB chromogenic buffer and incubation at room temperature in the dark for 15 min. The reaction was immediately terminated by adding 100 μL of 2 M H2SO4. The OD values ​​for each dilution were read using a microplate reader. 450nm value.

[0056] 2. Monoclonal Antibody Subclass Identification: Using the prepared ascites fluid as the primary antibody, the following procedures were followed according to the Beyotime reagent kit instructions: 100 ng of the corresponding Ag specifically bound by the monoclonal antibody was coated onto the ELISA plate with CBS buffer. The plate was incubated at 4°C on a shaker for 12 h. The coating solution was discarded, and the plate was washed once with PBST for 5 min each. 200 μL of ELISA blocking buffer was added to each well, and the plate was incubated at 37°C for 1 h. The plate was then washed three times with PBST for 5 min each. 100 μL of the prepared ascites fluid was diluted 1:1000 with PBS and added to each well. The plate was incubated at 37°C for 1 h. The plate was washed three times with PBST for 5 min each. 100 μL of each of the eight enzyme markers was added to two wells, and the plate was incubated at 37°C for 1 h. The plate was washed five times with PBST for 5 min each. TMB chromogenic buffer was added and the plate was incubated at 37°C for 30 min. The reaction was immediately terminated by adding 100 μL of 2 M H2SO4. The OD values ​​were read using a microplate reader. 450nm The value is used to determine the result.

[0057] 3. Indirect immunofluorescence identification: Trypsin-digested BSR cells were seeded into 6-well plates. When the cells reached approximately 90% confluence, they were incubated in 2% FBS medium containing 1 μg / mL NSDV medium for 2 h. After 2 h, the medium was discarded, and 100 μL of 2% FBS medium was added for further incubation. The medium was discarded after 72 h. Cell fixation: 500 μL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 20 min. The fixative was then discarded. Permeabilization: Permeabilize with 0.02% Trion X-100 at room temperature for 20 min. Blocking: Block with 1% BSA at room temperature for 1 h, then wash 3 times with PBS. Primary antibody incubation: Add 100 μL of monoclonal antibody to each well and incubate overnight at 4°C, then wash 3 times with PBS the next day. Secondary antibody incubation: Dilute 488 goat anti-mouse secondary antibody 1:1000 with PBS, add 100 μL to each well and incubate at room temperature for 1 h, then wash 3 times with PBS under dark conditions. DAPI staining: Dilute DAPI 1:1000 and incubate at room temperature for 10 min, then wash 3 times with PBS. Observe the experimental results using an indirect immunofluorescence instrument.

[0058] 4. Monoclonal antibody reactivity identification and sample preparation: Add 100 μL of the prepared NSDV N protein to an EP tube containing 25 μL of 5× loading buffer and mix well. After mixing, boil the sample in a metal bath at 100℃ for 10 min to denature the protein. Perform Western blot experiments on the prepared protein samples. The specific operation is as follows: Place the prepared 12% SDS-PAGE gel into the electrophoresis tank, add SDS electrophoresis buffer, and spot the prepared protein marker and protein sample into the gel wells. The sample loading volume is 20 μL per well. Perform electrophoresis experiments with a program of 80V for 30 min and 120V for 1 h. Soak the transfer sponge and filter paper in the transfer buffer for later use. Cut the PVDF membrane to a suitable size and soak it in anhydrous methanol for 1-2 min to activate the PVDF membrane. Remove the protein gel and cut off the excess gel. Transfer was performed using a rapid wet transfer apparatus. After transfer, the membrane was blocked with 5% skim milk at room temperature for 1 h, followed by washing three times with PBST on a shaker for 5 min each time. The monoclonal antibody was diluted 1:1000 with 5% skim milk as the primary antibody and incubated overnight at 4°C. The next day, it was washed three times with PBST on a shaker for 5 min each time. Secondary antibody (HRP goat anti-mouse IgG diluted 1:5000) was used and incubated at room temperature for 1 h, followed by washing three times with PBST on a shaker for 5 min each time. Western blotting was then performed.

[0059] Results: Hybridoma monoclonal antibody cells were injected intraperitoneally into mice, and the induced ascites fluid was then purified using Protein G affinity chromatography. The purified product was analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 7 As shown, the purified sample exhibited specific bands at the heavy chain (50 kDa) and light chain (20 kDa), respectively, and no obvious impurities were observed, indicating that the purification effect was good.

[0060] Monoclonal antibody titer assay: The purified monoclonal antibody was tested for titer. The results showed that the ascites produced by each hybridoma cell line had a titer of over 1:12800 (negative control OD). 450nm The value was 0.275, and the judgment criterion was: twice the negative control was considered positive.

[0061] Table 2. Determination of serum titer of monoclonal antibodies

[0062]

[0063] Monoclonal antibody subtype identification was performed using the Beyotime IgG class / subclass identification kit to analyze the subtypes of monoclonal antibodies secreted by hybridoma cell line 6D12. The results are as follows: the heavy chain of monoclonal antibody from strain 6D121 was identified as IgG I subtype, and the light chain type was IgGκ.

[0064] Monoclonal antibody IFA identification: After NSDV was inoculated into BSR cells, the purified monoclonal antibody was identified by IFA. Mouse negative serum was used as a negative control. All monoclonal antibodies showed specific green fluorescence, while the negative control showed no fluorescence, indicating that the antibody specifically binds to NSDV. The results are as follows: Figure 8 As shown.

[0065] Western blot results of the reactivity of the monoclonal antibody with the protein showed that when the prepared 6D12 monoclonal antibody was used as the primary antibody for incubation, the target band of NSDV N protein was visible at 79 kDa. Figure 9 As shown, no bands were found in the pCold-Gst empty vector, verifying the specificity of the monoclonal antibodies and indicating that the two monoclonal antibodies can recognize NSDV N protein and have good specificity.

[0066] Example 3.612D Monoclonal Antibody Sequencing

[0067] 1. Amino acid sequence:

[0068] (1) The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is shown in SEQ ID NO.3: GYTFTRYW;

[0069] (2) The amino acid sequence of CDR-H2 is shown in SEQ ID NO.4: INPTNGHI;

[0070] (3) The amino acid sequence of CDR-H3 is as shown in SEQ ID NO.5: SANIY;

[0071] (4) The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6: QSLLYSNGKTY;

[0072] (5) The amino acid sequence of CDR-L2 is shown below: LVS;

[0073] (6) The amino acid sequence of CDR-L3 is shown in SEQ ID NO.7: VQGTHFPQT;

[0074] (7) The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.8: QVQLQQPGAELVKPGAAVKLSCKASGYTFTRYWMHWVKQRPGQGLEWIGEINPTNGHINYNEKFKSKAILTVDKSSSTAYMQLSSLTSEDSAVYYCSANIYWGQGTLVTVSA;

[0075] The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.9: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK.

[0076] 2. Nucleic acid sequence:

[0077] (1) Heavy chain coding gene: SEQ ID NO.10

[0078] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTGCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGGTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTACCAACGGT CATATTAATTACAATGAGAAGTTCAAGAGCAAGGCCATCCTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAGCCAATATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA;

[0079] (2) Light chain coding gene: SEQ ID NO.11

[0080] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTG TCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTCCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0081] Example 4. Preliminary Establishment of a Competitive ELISA Method

[0082] I. Antigen Coating: Dilute NSDV N protein 1:800 with CBS coating buffer, mix thoroughly, and add 100 μL / well to the microplate. Incubate overnight (12-16 h) at 4°C. Washing: Discard the liquid in the wells, add 200 μL PBST to each well, and wash rapidly on a shaker for 5 min. Wash 3 times, and blot dry after washing. Blocking: Block with 5% skim milk, 200 μL / well, cover with plastic film, and block at 37°C for 1 h. Washing: Discard the liquid in the wells, add 200 μL PBST to each well, and wash rapidly on a shaker for 5 min. Wash 3 times, and blot dry after washing. Incubation with Primary Antibody: Add 50 μL NSDV positive serum to each well, followed by an equal volume of monoclonal antibody supernatant. Mix gently, cover with plastic film, and incubate at 37°C for 1 h. Prepare a negative serum control at the same time. Washing: Discard the liquid in each well. Add 200 μL of PBST to each well and wash rapidly on a shaker for 5 min. Wash 3 times, then blot dry. Secondary antibody incubation: Use HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution), add 100 μL to each well. Incubate at 37℃ for 1 h. Washing: Discard the liquid in each well. Add 200 μL of PBST to each well and wash rapidly on a shaker for 5 min. Wash 3 times, then blot dry. Color development: Add 100 μL of Beyotime TMB color development solution to each well and incubate at room temperature in the dark for 15 min. Stop color development: 100 μL / well, 2 M H2SO4 stop solution. Microplate reader reading: Read the OD at 450 nm using a microplate reader. 450nm Value. Calculate the P / N value and the PI value (inhibition rate (PI) = [(negative control well OD)]).450nm -Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Select the group with the smallest P / N value and the largest PI value as the optimal condition (P represents the sample well, and N represents the negative control well).

[0083] II. Optimization of Competitive ELISA Detection Methods

[0084] 1. Competitive Detection: Five different NSDV positive and NSDV negative sera were selected for competitive ELISA experiments. The steps for the competitive ELISA experiment are as described in Example 4. The primary antibody was incubated with 6D12 monoclonal antibody, the P / N value was calculated, the PI value was calculated, and the monoclonal antibody with the largest difference in PI value between the positive and negative sera was selected.

[0085] 2. Determination of antigen coating conditions and optimal serum dilution: The optimal antigen coating conditions and optimal serum dilution were determined using the checkerboard method. The procedure was as follows: NSDV N protein was diluted with coating buffer at ratios of 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000 to coat ELISA plates and incubate overnight at 4°C. The next day, NSDV-infected positive and negative sera were diluted at ratios of 1:1, 1:2, 1:4, 1:8, and 1:16 to perform competitive ELISA experiments. The coating conditions were determined based on the P / N and PI values, and the group with the lowest P / N and highest PI value was selected.

[0086] 3. Determination of the optimal blocking solution: NSDV N protein was subjected to a competitive ELISA experiment according to the determined coating conditions and optimal serum dilution. Blocking solutions were prepared using ELISA stabilizer I, ELISA stabilizer II, 5% skim milk, and 5% whey protein, respectively, and incubated at 37°C for 1 h. The optimal blocking solution was determined based on the P / N and PI values, and the solution with the lowest P / N and highest PI value was selected.

[0087] 4. Determination of optimal blocking time: Determine the antigen coating conditions, optimal serum dilution factor, and blocking solution. Change the blocking time to 5% skim milk and block at 37℃ for 0.5 h, 1 h, 1.5 h, and 2 h respectively, while keeping other conditions unchanged. Determine the optimal blocking time based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0088] 5. Determination of optimal blocking concentration: Determine the antigen coating conditions, optimal serum dilution factor, blocking solution, and blocking time. Change the blocking conditions and use 1%, 3%, 5%, and 10% skim milk for blocking at 37°C. Keep other conditions unchanged. Determine the optimal blocking solution concentration based on P / N and PI, and select the group with the lowest P / N and highest PI value.

[0089] 6. Determination of the optimal working concentration of monoclonal antibody: Determine the antigen coating conditions, optimal serum dilution factor, blocking solution, and optimal blocking time. Change the monoclonal antibody dilution factor by diluting the monoclonal antibody with PBS at 1:50, 1:100, 1:150, and 1:200 and then perform the assay. Keep other conditions unchanged. Determine the optimal working concentration of the monoclonal antibody based on the P / N and PI values, and select the group with the lowest P / N and the highest PI value.

[0090] 7. Determination of Optimal Monoclonal Antibody Incubation Time: With the antigen coating conditions, optimal serum dilution, blocking buffer conditions, and monoclonal antibody concentration unchanged, the monoclonal antibody incubation time was varied. Primary antibody incubation was performed at 37℃ for 30 min, 60 min, 90 min, and 120 min, respectively. The optimal incubation time was determined based on the P / N and PI values, and the group with the lowest P / N and highest PI value was selected.

[0091] 8. Determination of enzyme-labeled secondary antibody dilution: Determine the antigen coating conditions, optimal serum dilution factor, blocking buffer, optimal blocking time, monoclonal antibody concentration, and optimal monoclonal antibody incubation time. Adjust the dilution factor of goat anti-mouse HRP-IgG enzyme-labeled secondary antibody by diluting it with PBS at 1:2000, 1:4000, 1:8000, and 1:16000, and then perform the assay. Determine the enzyme-labeled secondary antibody dilution based on P / N and PI, and select the group with the lowest P / N and highest PI value.

[0092] 9. Determination of enzyme-labeled secondary antibody incubation time: Determine the antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, optimal monoclonal antibody conditions, and enzyme-labeled secondary antibody dilution. Change the enzyme-labeled secondary antibody incubation time. Incubate goat anti-mouse HRP-IgG enzyme-labeled secondary antibody at 37℃ for 30 min, 45 min, 60 min, and 75 min. Determine the enzyme-labeled secondary antibody incubation time based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0093] 10. Determination of Chromogenic Conditions: Determine the antigen coating conditions, optimal serum dilution, blocking buffer, optimal blocking time, monoclonal antibody concentration, optimal monoclonal antibody incubation time, enzyme-labeled secondary antibody dilution, and enzyme-labeled secondary antibody incubation time. Incubate the TMB chromogenic solution at room temperature and 37°C for 5 min, 10 min, 12 min, 15 min, and 20 min, respectively. Select the group with the lowest P / N ratio and the highest PI value.

[0094] 11. Criteria for Determining Positive and Negative Results of the Competitive ELISA Method: Forty-one sheep negative serum samples stored in the laboratory were tested using the optimized competitive ELISA method described above. A positive control was also included. The absorbance (OD) values ​​of the samples at 450 nm were recorded. 450nm According to the formula for calculating the blocking rate (PI): (PI) = [(OD of negative control wells)] 450nm -Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Calculate the mean (xˉ) and standard deviation (SD) of the blocking rate for negative samples. The positive threshold is set as the mean blocking rate of negative serum plus 3 times the standard deviation, and the negative threshold is set as the mean blocking rate of negative serum plus 2 times the standard deviation. When the blocking rate of the sample is ≥ the positive threshold, it is judged as positive; if the blocking rate is ≤ the negative threshold, it is judged as negative. For samples with blocking rates between the two, repeated testing is required. If the repeated test results are still within this range, it is finally judged as negative.

[0095] 12. Sensitivity Evaluation of Competitive ELISA Method: A random sample of NSDV-positive serum was diluted 1:1, 1:2, 1:4, 1:8, and 1:16 for neutralization and competitive ELISA experiments. The neutralization experiment was performed as follows: Diluted sheep serum was inactivated in a water bath at 56°C for 30 min. A sterile 96-well cell culture plate was prepared, with 50 μL of serum-free DMEM medium added to each well. In the first row of wells, 50 μL of serum was added to each well, with four replicates per serum sample. 50 μL of the mixture was drawn from the first row and diluted downwards row by row. The last row was mixed thoroughly and 50 μL of liquid was discarded. NSDV virus strains with known titers were diluted with serum-free DMEM medium to a concentration of 1:1, 1:2, 1:4, 1:8, and 1:16. Working concentration of virus particles / mL. Gently vortex to mix, incubate at 37°C for 5%. Incubate for 2 hours in an incubator to promote virus-antibody neutralization. Digest healthy BSR cells at appropriate density, centrifuge them, resuspend them in 1 mL of 2% FBS DMEM medium, and perform cell counting. Dilute to 4.5 × 10⁻⁶. 5Cells were seeded at a density of 100 μL per well in a 96-well plate. IFA assay was performed after 4 days. The concordance rate between the two methods for detecting clinical samples was calculated using a competitive ELISA method.

[0096] 13. Specificity evaluation of the competitive ELISA method: Positive sera for foot-and-mouth disease, small ruminant virus, sheep pox virus, sheep oral thrush virus, and akaban virus stored in the laboratory were serially diluted 2-fold and used as primary antibodies for incubation in a competitive ELISA experiment. NSDV positive serum was used as a positive control, and negative serum as a negative control. Specificity was evaluated based on OD... 450nm Value determination of specific results.

[0097] 14. Repeatability evaluation of the competitive ELISA detection method: Five NSDV positive serum samples and five negative serum samples were selected and subjected to competitive ELISA detection using ELISA plates coated from the same batch under standard experimental conditions. The intra-assay repeatability of the method was evaluated by calculating the coefficient of variation of the serum inhibition rate (CV = S / X × 100%, where S is the standard deviation and X is the arithmetic mean).

[0098] Five NSDV-positive serum samples and five NSDV-negative serum samples were collected and tested using different batches of ELISA plates under the same experimental conditions. The coefficient of variation (CV = S / X × 100%) of the serum inhibition rate was calculated to evaluate the reproducibility of this method among different batches of ELISA plates.

[0099] 15. Clinical application of competitive ELISA method: 179 local sheep serum samples were tested using competitive ELISA and neutralization test, and the concordance rate of the two methods in detecting clinical samples was calculated.

[0100] Results: The results of the competitive testing are as follows Figure 10 As shown: The ability of monoclonal antibodies to distinguish between positive and negative sera was determined by cELISA. The shape of each symbol indicates the identity inhibition of the serum against the corresponding monoclonal antibody. Five positive NSDV sera (red) and five negative sera (blue) were tested, and the mean PI for positive and negative sera for each monoclonal antibody was recorded. According to the competitive detection results, the 6D12 monoclonal antibody performed very well.

[0101] Determination of antigen coating conditions and optimal serum dilution: Using the checkerboard method, under otherwise identical conditions, the optimal antigen coating concentration and optimal serum dilution were determined. N protein coating buffer was diluted to 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000. Positive and negative controls were diluted to 1:1, 1:2, 1:4, 1:8, and 1:16, respectively. Checkerboard titration was performed using positive and negative controls at different protein concentrations and dilutions, and OD was measured using a competitive ELISA method. 450nm The optimal conditions were determined by the PI value, and the results are shown in Table 3. When the antigen coating concentration was 300 μg / mL and the serum dilution ratio was 1:2, the highest PI value and the highest blocking rate were detected.

[0102] Table 3 Screening results based on antigen coating concentration and serum dilution factor

[0103]

[0104] Determination of the optimal blocking solution: Based on the optimized antigen coating conditions and optimal serum dilution factor, competitive ELISA experiments were conducted using different blocking solutions, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 4. The optimal blocking solution is 5% skim milk.

[0105] Table 4 Determination of the Optimal Sealing Fluid

[0106]

[0107] Determination of optimal blocking time: Based on the optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution type, competitive ELISA experiments were conducted using different blocking times, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 5. The optimal closure time is 1.5 h.

[0108] Table 5 Determination of Optimal Closure Time

[0109]

[0110] Determination of optimal blocking concentration: Based on the optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution and blocking time, competitive ELISA experiments were conducted using different blocking solution concentrations, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 6. The optimal blocking solution concentration is 5% skim milk blocking.

[0111] Table 6 Determination of Optimal Blocking Concentration

[0112]

[0113] Determination of optimal working concentration of monoclonal antibody: Based on optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution conditions, competitive ELISA experiments were conducted using different monoclonal antibody dilution factors, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 7. The optimal dilution factor for the monoclonal antibody is 1:320.

[0114] Table 7 Optimal dilution factor for antibodies

[0115]

[0116] Determination of optimal antibody incubation time: Based on optimized antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, and antibody dilution, competitive ELISA experiments were conducted using different antibody incubation times, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 8. The optimal antibody action time is 1.5 h.

[0117] Table 8 Optimal Antibody Inaction Time

[0118]

[0119] The determination of enzyme-labeled secondary antibody dilution was based on optimized antigen coating conditions, optimal serum dilution factor, optimal blocking buffer conditions, and optimal antibody conditions. Different secondary antibody dilution factors were used in competitive ELISA experiments to measure OD. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 9. The optimal dilution factor for the second antibody is 1:8000.

[0120] Table 9 Optimal Dilution Factors for Secondary Antibodies

[0121]

[0122] The determination of enzyme-labeled secondary antibody incubation time was based on optimized antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, and optimal antibody conditions. Different incubation times were used in competitive ELISA experiments to measure OD. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 10: the optimal incubation time for the two antibodies is 1 hour.

[0123] Table 10 Optimal Action Time of Secondary Antibody

[0124]

[0125] Color development conditions determined: Based on the above conditions, the development times for the color development solution were 5 min, 10 min, 12 min, 15 min, and 20 min, respectively. OD was measured after the color development was completed. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 11. The optimal color development condition is to develop the color at room temperature for 15 minutes.

[0126] Table 11 Determination of Optimal Color Development Time

[0127]

[0128] The determination of the critical value: According to the formula for calculating the blocking rate (PI): (PI) = [(OD of negative control wells)] 450nm -Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Calculate the OD of 41 sheep negative serum samples. 450nm The results showed that the average blocking rate of negative serum was 21.275%, with a standard deviation of 4.59%. The cutoff values ​​for positive and negative sera were determined as follows: Positive threshold = Average blocking rate of negative serum + 3 times the standard deviation of the negative serum blocking rate = 21.275% + 3 × 4.59% = 35.06%; Negative threshold = Average blocking rate of negative serum + 2 times the standard deviation of the negative serum blocking rate = 21.275% + 2 × 4.59% = 30.47%. When the PI ≥ 35.06%, it is considered positive; when the PI ≤ 30.47%, it is considered negative; when the PI is between 30.47% and 35.06%, it is considered suspicious and requires repeat testing. If it is suspicious again, it is considered negative.

[0129] Table 12 Results of competitive ELISA detection of negative serum samples

[0130]

[0131] The sensitivity evaluation of the competitive ELISA method was based on the results of the competitive ELISA and neutralization assays (Table 13). The competitive ELISA result was determined by the PI value; a PI value > 35.06% was considered positive. The neutralization assay criteria were: a neutralizing antibody titer ≥ 4 was considered positive, and < 4 was considered negative. The results of the competitive ELISA and neutralization assays were consistent; positive serum could be diluted to 1:4, demonstrating the good sensitivity of this method.

[0132] Table 13 Sensitivity test results

[0133]

[0134] Specificity evaluation of the competitive ELISA method: Positive sera for peste des petits ruminants virus (PPR), foot-and-mouth disease virus (FMD), sheep pox virus (EPV), sheep stomatitis virus (SPV), and akaban virus (AKAV), as well as NSDV positive sera, were subjected to a competitive ELISA experiment, and the specificity was determined by measuring OD. 450nm The specificity of the competing ELISA was assessed, and the results are shown in Table 14. Only NSDV-positive serum showed a blocking rate of ≥35.06% (68.50%), and the result was considered positive. The blocking rates of other pathogen-positive serum samples were all ≤30.47%, and the results were considered negative. The experimental data indicate that this method can effectively distinguish NSDV from other related viral antibodies and has high detection specificity.

[0135] Table 14 Specificity Test Results

[0136]

[0137] Repeatability evaluation of the competitive ELISA detection method: Five NSDV positive serum samples were randomly selected for competitive ELISA testing. Inter-batch and intra-batch replicates were performed, and the competitive inhibition rate, standard deviation, and coefficient of variation were measured. According to the results in Table 15, the coefficients of variation for different batches were all low (maximum 6.01%). A coefficient of variation ≤10% is generally considered to indicate good repeatability, suggesting that the test results have good reproducibility.

[0138] Table 15 Repeatability Test Results

[0139]

[0140] 179 local sheep serum samples were tested using a competitive ELISA method and a neutralization assay. Using the established competitive ELISA method, 35 samples were positive, with a positive rate of 19.5%. Using the neutralization assay, 42 samples were positive, with a positive rate of 23.4%. The positive concordance rate between the two methods was 83%, and the negative concordance rate was 95.8%. Kappa test results showed a Kappa value of 0.78 > 0.610, indicating a high degree of consistency between the two methods. The calculated concordance rate was 93%, which is relatively high.

[0141] Table 16 Comparison of detection results between competitive ELISA method and neutralization method

[0142]

Claims

1. A monoclonal antibody against Nairobi sheep disease virus, characterized in that, The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

5. The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of CDR-L2 is LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

7.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.

8.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

9.

4. A gene characterized in that, Encoding the monoclonal antibody according to any one of claims 1-3.

5. The gene according to claim 4, characterized in that, The gene sequence encoding the amino acid sequence of the heavy chain variable region of a monoclonal antibody is shown in SEQ ID NO.

10.

6. The gene according to claim 4, characterized in that, The gene sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

11.

7. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 4.

8. The recombinant vector according to claim 7, characterized in that, The departure carrier is the pCold carrier.

9. A recombinant host cell, characterized in that, The host cell contains the recombinant vector as described in claim 7.

10. The recombinant host cell according to claim 9, characterized in that, The recombinant host cell is a microbial cell or an animal cell.

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