A Monoclonal Antibody Specific to Small Ruminant Disease Virus and Its Application

Abstract

This invention discloses a monoclonal antibody specific to peste des petits ruminants (PPR) virus and its application. The monoclonal antibody is obtained by screening PPR N protein, the amino acid sequence of which is shown in SEQ ID NO:1. The PPR N protein is transcribed from the PPR N gene, and the codon-optimized nucleic acid sequence of the PPR N gene is shown in SEQ ID NO:2. The monoclonal antibody provided by this invention can specifically bind to the PPR N protein, exhibiting high titer and good affinity. Furthermore, the detection kit prepared based on this antibody can effectively detect PPR, providing technical support for the epidemiological monitoring and diagnosis of PPR infection.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a small ruminant plague virus-specific monoclonal antibody and its application. Background Technology

[0002] Peste-des-petits-ruminants virus (PPRV) is an RNA virus belonging to the Paramyxoviridae family and the Measlesvirus genus. The PPRV genome is approximately 16 kb in length, encoding six structural proteins and two non-structural proteins. The structural proteins include N, M, H, F, P, and L proteins. The non-structural proteins include C and V proteins. The N protein is the nucleocapsid protein of PPRV, with a nucleotide length of 1674 bp, composed of 525 amino acids, and a molecular weight of 128 kDa. PPRV has only one serotype, divided into four genotypes: I, II, III, and IV. Most strains in China belong to genotype IV. The N protein shares approximately 95% homology with domestically prevalent strains and exhibits high immunogenicity and good conservation, often serving as a target protein for serological diagnosis, vaccine development, and antiviral drug design.

[0003] The most susceptible animals to peste des petits ruminants (PPR) virus are sheep and goats. There are reports that buffalo, camels, and antelopes can also be infected. Currently, there are no reported cases of human infection with PPR. PPR is primarily transmitted through the respiratory system. It has a high morbidity and mortality rate, causing significant economic losses to local communities.

[0004] my country is a major sheep-raising country, with an average annual sheep population exceeding 550 million since 2005, accounting for more than 20% of the global annual sheep population. Therefore, routine monitoring is crucial for the sustainable and healthy development of the livestock industry and for increasing the income of farmers and herdsmen. It is of great significance for promoting the development of the sheep industry. However, the current detection methods for this disease are relatively simple and the detection rate is not high. Therefore, it is urgent to propose a specific monoclonal antibody for peste des petits ruminants virus and its application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a specific monoclonal antibody against peste des petits ruminants (PPR) virus and its application. This antibody can specifically bind to the PPR N protein, exhibiting high titer and good affinity. Furthermore, the detection kit prepared based on this antibody can effectively detect PPR, providing technical support for the epidemiological monitoring and diagnosis of PPR infection.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a monoclonal antibody specific to peste des petits ruminants (PPR), wherein the monoclonal antibody is obtained by screening PPR N protein, the amino acid sequence of which is shown in SEQ ID NO:1, the PPR N protein is transcribed from the PPR N gene, and the codon-optimized nucleic acid sequence of the PPR N gene is shown in SEQ ID NO:2.

[0008] Preferably, the monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3.

[0009] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-37 of SEQ ID No. 3;

[0010] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 55-57 of SEQ ID No. 3;

[0011] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 94-102 of SEQ ID No. 3;

[0012] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 4;

[0013] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 4;

[0014] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-108 of SEQ ID No. 4.

[0015] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4.

[0016] Preferably, the monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of both the heavy chain variable region and the light chain variable region are composed of CDR1, CDR2 and CDR3.

[0017] The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-31 of SEQ ID No. 5;

[0018] The amino acid sequence of CDR2 in the light chain variable region is shown as positions 49-51 of SEQ ID No. 5;

[0019] The amino acid sequence of CDR3 in the light chain variable region is shown as positions 88-96 of SEQ ID No. 5;

[0020] The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 6;

[0021] The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-57 of SEQ ID No. 6;

[0022] The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 96-110 of SEQ ID No. 6.

[0023] Preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 6.

[0024] The second objective of this invention is to provide a chemiluminescent detection kit containing a specific monoclonal antibody against peste des petits ruminants virus.

[0025] Preferably, the kit contains immunomagnetic beads conjugated with the monoclonal antibody, a small ruminant virus N protein calibration quality control sample, the monoclonal antibody labeled with alkaline phosphatase, and a chemiluminescent substrate.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The monoclonal antibody provided by this invention can specifically bind to the N protein of peste des petits ruminants virus (PPR), exhibiting high titer and good affinity. Furthermore, the detection kit prepared based on this antibody can effectively detect PPR, providing technical support for the epidemiological monitoring and diagnosis of PPR infection. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the recognition of the N protein of peste des petits ruminants virus by the 2D5 and 3G7 antibodies in Embodiment 4 of the present invention.

[0029] Figure 2 This is a schematic diagram of SDS-PAGE analysis of antibodies 1E10, 2D5, 2E3 and 3G7 in Example 5 of the present invention.

[0030] Figure 3 This is the calibration curve of the peste des petits ruminants virus detection kit in Example 8 of the present invention. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0032] Example 1: Codon optimization of the N gene of small ruminant virus.

[0033] Based on the sequence of the N gene vaccine strain (HQ197753) reported in NCBI, the N gene sequence of peste des petits ruminants (PPR) was optimized using E. coli codon bias. The optimized sequence had a CAI of 0.79 and a GC content of 54.29%. The optimized N gene nucleic acid sequence is shown in SEQ ID NO: 2.

[0034] Example 2: Optimized expression and purification of small ruminant virus N protein.

[0035] Construction and expression of the truncated PPR N protein: Based on the amino acid sequence of the PPR N protein as shown in SEQ ID NO: 1, N-terminal and C-terminal sequences were designed and ligated into the expression vector pET28a, which was then inserted into the expression strain BL21(DE3). Soluble expression of the truncated PPR N protein was induced overnight in LB medium at 37°C with 1 mmol / L IPTG. Prokaryotic recombinant expression was purified by 6×His tag affinity chromatography. The purified proteins were subjected to SDS-PAGE electrophoresis, and the purity was above 90%. The nucleic acid sequence of the PPR N protein-N fragment is shown in SEQ ID NO: 7. The nucleic acid sequence of the PPR N protein-C fragment is shown in SEQ ID NO: 8.

[0036] Example 3: Preparation of hybridoma cell line for monoclonal antibody against small ruminant virus N protein.

[0037] BALB / c mice were immunized with inactivated peste des petits ruminants (PPR) virus solution as an immunogen. For the initial immunization, the inactivated virus was mixed with Freund's complete adjuvant in equal proportions and administered subcutaneously and intraperitoneally, with 100 μL per mouse, for a total of 5 mice. A second and third immunization were performed every 14 days using Freund's incomplete adjuvant, administered subcutaneously, with 100 μL per mouse. Ten days after the third immunization, blood was collected by tail amputation, and serum was extracted by centrifugation. The serum was serially diluted seven times, and the serum titer of the immunized mice was determined using PPR N protein as a detectant. The results are shown in Table 1.

[0038] Table 1: Serum titer detection in mice.

[0039] Dilution ratio Mouse number 1 Mouse number 2 Mouse number 3 1：1000 2.43 2.357 2.46 1：2000 2.338 2.161 2.268 1：4000 2.225 2.172 2.22 1：8000 1.89 1.921 2.027 1：16000 1.61 1.803 1.798 1：32000 1.412 1.811 1.635 1：64000 1.013 1.444 1.439 1：128000 0.989 0.654 0.750 negative control 0.111 0.171 0.125

[0040] Mice with the highest serum titer were boosted with an intraperitoneal injection of 50 μL of immunogen. Three days later, spleen cells were harvested under aseptic conditions. SP2 / 0 and spleen cells were mixed in centrifuge tubes according to the specified ratio. 1 mL of pre-warmed PEG was added to a 50 mL centrifuge tube and incubated for 1 min. Then, pre-warmed 37°C serum-free DMEM medium was added to a final volume of 35 mL. The mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. 50 mL of pre-warmed HAT medium was added, and the mixture was gently pipetted to mix. The mixture was then transferred to 100 μL per well of a 96-well plate containing feeder cells and incubated. The cell supernatant was analyzed using small ruminant virus N protein detection. Positive hybridoma clones were screened using a limiting dilution method, yielding four hybridoma cell lines secreting specific monoclonal antibodies: 1E10, 2D5, 2E3, and 3G7. Antibody subtypes were identified, with 1E10 showing IgG. 2a 2D5 is IgG 2b 2E3 and 3G7 are IgG1.

[0041] Example 4: Identification of monoclonal antibody epitopes of the N protein of peste des petits ruminants virus.

[0042] Western blotting (WB) was used to confirm antibody recognition epitopes: 5 μg of each of the three proteins (N, C, and full-length) of the PPR N protein were loaded and subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to 0.45 μm PVDF under constant voltage of 150 V. After transfer, the membrane was blocked at 37°C for two hours with PBST solution containing 5% BSA. The membrane was then incubated at 37°C for one hour with 1 μg / mL of 2D5 and 3G7 monoclonal antibody solutions, respectively. After incubation, 10 ng / mL of goat anti-mouse IgG-HRP was added and incubated for one hour. After washing, ECL chemiluminescence was performed. The full-length PPR N protein was used as a positive control. The appearance of a band at the N / C fragment of the PPR N protein indicated that the antibody recognized the target fragment. The results showed that the 3G7 antibody recognized the N fragment of the PPR N protein, and the 2D5 antibody recognized the C fragment of the PPR N protein. Figure 1 As shown.

[0043] Example 5: Preparation, purification, and titer detection of monoclonal antibodies against peste des petits ruminants virus.

[0044] Liquid paraffin was injected into mice via intraperitoneal injection. Seven days later, the four hybridoma cell lines were inoculated into mice. Ascites fluid was collected after the mice's abdomens swelled.

[0045] The collected ascites fluid was centrifuged at 4°C, and the precipitate was discarded, retaining the supernatant. 10-20 volumes of Protein A equilibration buffer were added to the supernatant for Protein A affinity chromatography purification. The antibody was eluted with 0.1M glycine-hydrochloric acid solution (pH 2.7) and neutralized with 1M pH 9.0 Tris buffer. The purified antibody was dialyzed twice with 1×PBS solution. SDS-PAGE was used to determine antibody purity. The results showed that all four antibody strains exhibited bands at 50KD and 25KD, and the purity of all strains was greater than 90% according to grayscale analysis. Figure 2 As shown.

[0046] The N protein of peste des petits ruminants virus (PPR) was diluted to 2 μg / ml with carbonate buffer (CBS, pH 9.6) and coated overnight at 4°C. The coating solution was discarded, and the plate was blocked with 10% skim milk powder at 37°C for 2 h. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST. The antibody was serially diluted and added at 100 μl / well, with a negative control included. The plate was incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-HRP was added, and the plate was incubated at 37°C for 1 h. After incubation, the plate was washed again, and TMB was added for color development. OD450 was read after termination. The titers of monoclonal antibodies 1E10, 2D5, 2E3, and 3G7 were all 8 × 10⁻⁶. 6 1.6×10 7 6.4×10 7 3.2×10 7 .

[0047] Example 6: Pairing verification of monoclonal antibodies against peste des petits ruminants virus.

[0048] Purified monoclonal antibodies 1E10, 2D5, 2E3, and 3G7 were used as capture and detection antibodies, respectively, for antibody pairing verification. Antibodies were coated overnight at 4°C at 5 μg / ml (100 μl / well). Blocking with 10% skim milk powder at 37°C for 2 h. Serial dilutions of PPR antigen were added at 100 μl / well and incubated at 37°C for 1 h. Serial dilutions of PPR N protein were added at 100 μl / well and incubated at 37°C for 1 h. After washing, HRP-labeled 1E10, 2D5, 2E3, and 3G7 were added at 100 μl / well and incubated at 37°C for 1 h. After washing, substrate was added and the reaction was allowed to proceed for 15 min. The reaction was terminated with stop solution, and the OD450 absorbance was measured using a microplate reader. The results are shown in Table 2. Ultimately, 2D5 was selected as the capture antibody for the kit, and 3G7 as the detection antibody for the detection of PPR.

[0049] Table 2: Pairing verification of monoclonal antibodies against peste des petits ruminants virus.

[0050]

[0051] Example 7: Specificity analysis of monoclonal antibody against the N protein of peste des petits ruminants virus.

[0052] ELISA plates were coated with 2 μg / ml (100 μl / well) of PPR virus N protein, bovine respiratory syncytial virus (BRSV) N protein, canine distemper virus (CDV) N protein, and Nipah virus (NiV) N protein, respectively. The plates were incubated with 10% skim milk powder at 37°C for 2 h. After serial dilution of the antibodies, 100 μl was added to each well, and the plates were incubated at 37°C for 1 h. Diluted goat anti-mouse IgG-HRP (100 μl / well) was added, and the plates were incubated at 37°C for 1 h. After washing the plates, the substrate was added, and the reaction was allowed to proceed for 15 min. The reaction was terminated with stop solution, and the OD450 absorbance was measured using a microplate reader. The results showed that PPR monoclonal antibodies 2D5 and 3G7 specifically recognized PPR, but showed no immune response against BRSV, CDV, or Nipah virus. The results are shown in Table 3.

[0053] Table 3: Specificity of monoclonal antibodies against the N protein of peste des petits ruminants virus.

[0054]

[0055] Example 8: Chemiluminescent detection kit for peste des petits ruminants virus.

[0056] The chemiluminescent assay kit for peste des petits ruminants virus (PPR) includes: immunomagnetic beads labeled with monoclonal antibodies against PPR N protein, monoclonal antibodies against PPR N protein labeled with alkaline phosphatase, and PPR N protein calibrators. The kit components are shown in Table 4, and the standard curve is shown in Table 5. Figure 3 As shown.

[0057] Table 4: Chemiluminescence detection kit for peste des petits ruminants virus.

[0058]

[0059] Example 9: Sensitivity and specificity of the chemiluminescent detection kit for peste des petits ruminants virus.

[0060] 9.1 Sensitivity of the kit: Positive control samples were serially diluted and detected by chemiluminescence immunoassay (CLIA) and ELISA, respectively. The results are shown in Table 5. The analysis showed that the linear range of the chemiluminescence immunoassay was 50-16000 pg / ml, while the linear range of the ELISA method was 500-8000 pg / ml. The chemiluminescence immunoassay method has a wider linear range and higher detection sensitivity than the ELISA method.

[0061] Table 5: Sensitivity test results.

[0062]

[0063]

[0064] 9.2 Positive sample detection rate: 10 positive reference samples and 10 negative samples were tested using the kit, and the detection results and the concordance rate were 100%; the results are shown in Table 6.

[0065] Table 6: Detection results of positive samples.

[0066] Sample number Concentration value (pg / ml) determination 1 1.84 - 2 8.53 - 3 12.37 - 4 10.24 - 5 19.43 - 6 9.19 - 7 11.35 - 8 21.15 - 9 9.53 - 10 18.85 - 11 315.76 + 12 103.42 + 13 465.53 + 14 319.33 + 15 187.59 + 16 213.45 + 17 406.38 + 18 218.43 + 19 178.92 + 20 215.66 +

[0067] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody specific to peste des petits ruminants virus, characterized in that, The monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-37 of SEQ ID No. 3; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 55-57 of SEQ ID No. 3; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 94-102 of SEQ ID No. 3; The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 4; The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-58 of SEQ ID No. 4; The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 97-108 of SEQ ID No.

4.

2. The small ruminant virus-specific monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

4.

3. A monoclonal antibody specific to peste des petits ruminants virus, characterized in that, The monoclonal antibody contains a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the light chain variable region is shown as positions 27-31 of SEQ ID No. 5; The amino acid sequence of CDR2 in the light chain variable region is shown as positions 49-51 of SEQ ID No. 5; The amino acid sequence of CDR3 in the light chain variable region is shown as positions 88-96 of SEQ ID No. 5; The amino acid sequence of CDR1 in the heavy chain variable region is shown as positions 26-33 of SEQ ID No. 6; The amino acid sequence of CDR2 in the heavy chain variable region is shown as positions 51-57 of SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region is shown as positions 96-110 of SEQ ID No.

6.

4. The peste des petits ruminants virus-specific monoclonal antibody according to claim 3, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID No. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

6.

5. A chemiluminescent detection kit containing a monoclonal antibody specific to peste des petits ruminants virus as described in any one of claims 1-4.

6. The chemiluminescence detection kit according to claim 5, characterized in that, The kit contains immunomagnetic beads conjugated with the monoclonal antibody as described in claim 1 or 2, a small ruminant virus N protein calibration quality control, an alkaline phosphatase-labeled monoclonal antibody as described in claim 3 or 4, and a chemiluminescent substrate.

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