Peste des petits ruminants virus specific monoclonal antibody and application thereof

Abstract

The invention discloses a peste des petits ruminants virus specific monoclonal antibody and application thereof.The monoclonal antibody is obtained by screening peste des petits ruminants virus N protein, the amino acid sequence of the peste des petits ruminants virus N protein is shown as SEQ ID NO: 1, the peste des petits ruminants virus N protein is obtained by transcription of a peste des petits ruminants virus N gene, and the peste des petits ruminants virus N gene is shown as SEQ ID NO: 2. The nucleotide sequence of the codon-optimized peste des petits ruminants virus N gene is shown as SEQ ID NO: 2. The monoclonal antibody provided by the invention can be specifically combined with the peste des petits ruminants virus N protein, the titer is high, the affinity is good, meanwhile, the detection kit prepared based on the antibody pair can effectively carry out peste des petits ruminants virus detection, and a technical support can be provided for epidemiological monitoring and diagnosis of peste des petits ruminants virus infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a peste des petits ruminants virus-specific monoclonal antibody and an application thereof. Background Art

[0002] Peste-des-petits-ruminants virus (PPRV) is an RNA virus belonging to the family Paramycoviridae and the genus Morbillivirus. The genome of PPR virus is about 16kb in length, encoding 6 structural proteins and 2 non-structural proteins. Structural proteins include N protein, M protein, H protein, F protein, P protein, and L protein. Non-structural proteins include C protein and V protein. Among them, N protein is the nucleocapsid protein of PPR virus, with a nucleotide length of 1674bp, composed of 525 amino acids, and a molecular weight of 128kDa. PPR virus has only one serotype, which is divided into 4 gene groups, namely I, II, III and IV gene groups. Most of the strains in China belong to the IV gene group. The homology of N protein with the domestic epidemic strain is about 95%, and N protein has high immunogenicity and good conservation. It is often used as a target protein for serological diagnosis, vaccine development, and antiviral drug design.

[0003] The susceptible animals to the PPR virus are sheep and goats. There are reports that ruminants such as buffalo, camels, and antelopes can also be infected. So far, there have been no reports of human infection with PPR. PPR is mainly transmitted through the respiratory system. The morbidity and mortality of PPR are very high, causing serious economic losses to the local area.

[0004] my country is a major sheep-raising country. Since 2005, the average annual sheep breeding volume has exceeded 550 million, which is more than 20% of the global annual breeding volume. Therefore, carrying out regular monitoring is related to the sustainable and healthy development of animal husbandry, to the increase of farmers' and herdsmen's income, and is of great significance to promoting the development of the sheep industry. However, the current detection methods of the disease are relatively simple and the detection rate is not high. Therefore, a small ruminant peste des petits ruminants virus-specific monoclonal antibody and its application are urgently needed. Summary of the invention

[0005] In order to solve the defects of the prior art, the present invention provides a peste des petits ruminants virus-specific monoclonal antibody and its application, which can specifically bind to the peste des petits ruminants virus N protein, has high titer and good affinity. At the same time, the detection kit prepared based on the antibody can effectively carry out peste des petits ruminants virus detection, and can provide technical support for the epidemiological monitoring and diagnosis of peste des petits ruminants virus infection.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first purpose of the present invention is to provide a monoclonal antibody specific for Peste des petits ruminants virus, wherein the monoclonal antibody is obtained by screening the Peste des petits ruminants virus N protein, the amino acid sequence of the Peste des petits ruminants virus N protein is shown in SEQ ID NO:1, the Peste des petits ruminants virus N protein is obtained by transcription of the Peste des petits ruminants virus N gene, and the codon-optimized Peste des petits ruminants virus N gene nucleic acid sequence is shown in SEQ ID NO:2.

[0008] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining region and a framework region; and the complementary region determining region of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3;

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

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

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

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

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

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

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

[0016] Preferably, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining region and a framework region; and the complementary region determining region of the heavy chain variable region and the light chain variable region are both composed of CDR1, CDR2 and CDR3;

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

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

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

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

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

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

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

[0024] The second purpose of the present invention is to provide a chemiluminescent detection kit containing a monoclonal antibody specific for peste des petits ruminants virus.

[0025] Preferably, the kit comprises immunomagnetic beads coupled with the monoclonal antibody, a quality control product for calibration of the N protein of peste des petits ruminants virus, 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 the present invention can specifically bind to the N protein of the peste des petits ruminants virus, has high titer and good affinity, and the detection kit prepared based on the antibody pair can effectively carry out the detection of the peste des petits ruminants virus, and can provide technical support for the epidemiological monitoring and diagnosis of the peste des petits ruminants virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0030] Figure 3 It is a calibration curve diagram of the PPR virus detection kit in Example 8 of the present invention. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] Example 1: Codon optimization of the N gene of peste des petits ruminants virus.

[0033] Based on the sequence of the N gene vaccine strain (HQ197753) reported in NCBI, the PPR N gene sequence was optimized by the codon preference of Escherichia coli. After optimization, the CAI was 0.79 and the GC content was 54.29%. The optimized N gene nucleic acid sequence is shown in SEQ ID NO: 2.

[0034] Example 2: Optimized expression and purification of peste des petits ruminants virus N protein.

[0035] Truncated PPR N protein construction and expression: According to the amino acid sequence of PPR N protein as shown in SEQ ID NO: 1, the N-terminal and C-terminal sequences were designed, connected to the expression vector pET28a, and loaded into the expression strain BL21 (DE3). In LB medium, IPTG 1mmol / L was used to induce the soluble expression of PPR N truncated protein overnight at 37 degrees. Prokaryotic recombinant expression was affinity purified by 6×His tag, and the purified protein was subjected to SDS-PAGE electrophoresis, with a purity of more than 90%. The nucleic acid sequence of PPR N protein-N fragment is shown in SEQ ID NO: 7. The nucleic acid sequence of PPR N protein-C fragment is shown in SEQ ID NO: 8.

[0036] Example 3: Preparation of hybridoma cell lines of monoclonal antibodies against the N protein of peste des petits ruminants virus.

[0037] BALB / c mice were immunized using inactivated virus solution of PPR as an immunogen. For the first immunization, the inactivated virus was mixed with Freund's complete adjuvant in equal proportions and injected subcutaneously and intraperitoneally. Each mouse was immunized with 100ul, for a total of 5 mice. Every 14 days, Freund's incomplete adjuvant was used for the second and third immunizations, subcutaneously injected, and each mouse was immunized with 100ul. Ten days after the third immunization, the tail was cut off and blood was collected. The serum was centrifuged and diluted 7 times. The N protein of PPR virus was used as the detection source to detect the titer of the serum of the immunized mice. The results are shown in Table 1.

[0038] Table 1: Mouse serum titer detection.

[0039] Dilution ratio Mouse No. 1 Mouse No. 2 Mouse No. 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] Mouse No. 1 with the highest serum titer was intraperitoneally injected with 50ul of immunogen for booster immunization, and spleen cells were removed under sterile conditions 3 days later. SP2 / 0 and spleen cells were mixed in a centrifuge tube in proportion, 1ml of preheated PEG was added to a 50ml centrifuge tube for 1min, and then 37℃ serum-free DMEM medium preheated was added to 35mL, and centrifuged at 1000rpm / min for 10min to discard the supernatant. Add 50mL of preheated HAT medium, gently blow and mix, transfer to a 96-well culture plate with feeder cells, 100μL per well, and culture in an incubator. The supernatant of the fused cells was detected using the N protein of the peste des petits ruminants virus, and the positive hybridomas were cloned and screened by the limiting dilution method to obtain 4 hybridoma cell strains that secrete specific monoclonal antibodies, namely 1E10, 2D5, 2E3 and 3G7, and the antibody subtype was identified. The results showed that 1E10 was IgG 2a , 2D5 is IgG 2b , 2E3 and 3G7 are IgG 1 .

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

[0042] Western blotting WB confirmed the antibody recognition epitope: 5μg of three proteins, N, C and full-length, of the peste des petits ruminants virus N protein were loaded and subjected to 15% SDS-PAGE electrophoresis. After the electrophoresis was completed, the protein was transferred to 0.45um PVDF under a constant voltage of 150V. After the transfer was completed, the membrane was blocked with a PBST solution containing 5% BSA at 37°C for two hours. Incubate with 1μg / mL 2D5 and 3G7 monoclonal antibody solutions at 37°C for 1 hour respectively. After the incubation was completed, 10ng / ml of goat anti-mouse IgG-HRP was added and incubated for 1 hour. After washing, ECL luminescent liquid was added for color development. The full-length protein of the peste des petits ruminants virus N protein was used as a positive control. The appearance of a band at the position of the peste des petits ruminants virus N protein-N / C fragment indicated that the antibody recognized the target fragment. The results showed that the 3G7 antibody recognized the N segment of the peste des petits ruminants virus N protein, and the 2D5 antibody recognized the C segment of the peste des petits ruminants virus N protein. Figure 1 shown.

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

[0044] Liquid paraffin was injected intraperitoneally into mice. Seven days later, the above four hybridoma cell lines were inoculated into mice respectively. After the abdomen of the mice was swollen, ascites was collected.

[0045] The collected ascites was centrifuged at 4°C, the precipitate was discarded, and the supernatant was retained. 10-20 volumes of Protein A equilibrium buffer were added to the supernatant, and Protein A affinity chromatography was performed for purification. The antibody was eluted with 0.1M glycine-hydrochloric acid solution pH 2.7, and 1M pH 9.0 Tris buffer was added for neutralization. The purified antibody was dialyzed twice with 1× PBS solution, and the antibody purity was detected by SDS-PAGE. The results showed that the 4 antibodies had bands at 50KD and 25KD, and the purity of the grayscale analysis was greater than 90%, as shown in Figure 2. Figure 2 shown.

[0046] The N protein of PPR virus was diluted to 2 μg / ml with carbonate buffer (CBS, pH 9.6), coated overnight at 4°C, the coating solution was discarded, and 10% skim milk powder was used to block at 37°C for 2h. After blocking, the blocking solution was discarded and the plate was washed three times with PBST. The antibody was diluted in a gradient manner and 100ul / well was added, and a negative control was set. The plate was incubated at 37°C for 1h, and goat anti-mouse IgG-HRP was added to the plate and incubated at 37°C for 1h. After incubation, TMB was added to the plate for color development. After termination, OD450 was read. The titers of monoclonal antibodies 1E10, 2D5, 2E3 and 3G7 were 8×10 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 1E10, 2D5, 2E3 and 3G7 monoclonal antibodies were used as capture antibodies and detection antibodies to verify antibody pairing. The antibodies were coated at 5μg / ml, 100μl / well, and overnight at 4℃. 10% skim milk powder was blocked at 37℃ for 2h. Peste des petits ruminants virus antigen was diluted in gradients, 100μl / well, and incubated at 37℃ for 1h. Peste des petits ruminants virus N protein was diluted in gradients, 100μl / well, and incubated at 37℃ for 1h. After washing the plate, HRP-labeled 1E10, 2D5, 2E3 and 3G7 were added, 100μl / well, and incubated at 37℃ for 1h. After washing the plate, the substrate was added to react for 15min, and the OD450 absorbance was measured on an ELISA instrument after the stop solution was terminated. The results are shown in Table 2. Finally, 2D5 was selected as the capture antibody of the kit, and 3G7 was selected as the detection antibody for the detection of Peste des petits ruminants virus.

[0049] Table 2: Validation of paired monoclonal antibodies against PPR virus.

[0050]

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

[0052] ELISA plates were coated with 2μg / ml, 100μl / well of N protein of Peste des Petits Ruminants virus (PPRV), N protein of bovine respiratory syncytial virus (BRSV), N protein of canine distemper virus (CDV), and N protein of Nipah virus (NiV). 10% skim milk powder was incubated at 37℃ for 2h. After gradient dilution of the antibody, 100μl was added to each well and incubated at 37℃ for 1h. Diluted goat anti-mouse IgG-HRP was added, 100μl / well, and incubated at 37℃ for 1h. After washing the plate, the substrate was added to react for 15min, and the OD450 absorbance was measured by an ELISA reader after the stop solution was terminated. The results showed that the monoclonal antibodies 2D5 and 3G7 of Peste des Petits Ruminants virus can specifically recognize Peste des Petits Ruminants virus (PPRV), and have no immune response to bovine respiratory syncytial virus (BRSV), canine distemper virus (CDV), and Nipah virus (NiV). The results are shown in Table 3.

[0053] Table 3: Specificity of monoclonal antibodies against PPR virus N protein.

[0054]

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

[0056] The chemiluminescent detection kit for peste des petits ruminants virus includes: immunomagnetic beads labeled with anti-peste des petits ruminants virus N protein monoclonal antibody, anti-peste des petits ruminants virus N protein monoclonal antibody labeled with alkaline phosphatase, and peste des petits ruminants virus N protein calibrator. The components of the kit are shown in Table 4, and the standard curve is shown in Figure 3 shown.

[0057] Table 4: Chemiluminescent 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: The positive control sample was diluted in a gradient manner and tested by chemiluminescence (CLIA) and ELISA, respectively. The results are shown in Table 5: Through analysis, it was found that the linear range of the chemiluminescence method was 50-16000pg / ml, while the linear range of the ELISA method was 500-8000pg / ml. The chemiluminescence 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: The test kit was used to test 10 positive reference samples and 10 negative samples, and the test results were consistent with the rate of 100%; the results are shown in Table 6.

[0065] Table 6: Positive sample test results.

[0066] Sample No. 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 description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody specific for peste des petits ruminants virus, characterized in that: The monoclonal antibody is obtained by screening the peste des petits ruminants virus N protein, the amino acid sequence of the peste des petits ruminants virus N protein is shown in SEQ ID NO:1, the peste des petits ruminants virus N protein is obtained by transcription of the peste des petits ruminants virus N gene, and the codon-optimized peste des petits ruminants virus N gene nucleic acid sequence is shown in SEQ ID NO:

2.

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

4.

3. The peste des petits ruminants virus-specific monoclonal antibody according to claim 2, 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.

4. The peste des petits ruminants virus-specific monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are both composed of a complementary region determining a cluster and a framework region; the complementary region determining a cluster 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 of the light chain variable region is shown in positions 27-31 of SEQ ID No. 5; The amino acid sequence of CDR2 of the light chain variable region is shown in positions 49-51 of SEQ ID No. 5; The amino acid sequence of CDR3 of the light chain variable region is shown in positions 88-96 of SEQ ID No.5; The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 26-33 of SEQ ID No. 6; The amino acid sequence of CDR2 of the heavy chain variable region is shown in positions 51-57 of SEQ ID No.6; The amino acid sequence of CDR3 of the heavy chain variable region is shown in positions 96-110 of SEQ ID No.

6.

5. The peste des petits ruminants virus-specific monoclonal antibody according to claim 4, 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.

6. A chemiluminescent detection kit containing the peste des petits ruminants virus-specific monoclonal antibody according to any one of claims 1 to 5.

7. The chemiluminescent detection kit according to claim 6, characterized in that: The kit comprises immunomagnetic beads coupled with the monoclonal antibody as claimed in claim 2 or 3, a calibration quality control product of peste des petits ruminants virus N protein, an alkaline phosphatase-labeled monoclonal antibody as claimed in claim 4 or 5, and a chemiluminescent substrate.

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