A canine distemper virus protein NFH truncated tandem antigen and its application

Abstract

The embodiment of the present invention discloses a canine distemper virus protein N‑F‑H truncated tandem antigen and its application, which belongs to the field of pet disease detection. The amino acid sequence of the truncated tandem antigen is shown in SEQ ID No. 7; the nucleotide sequence of the truncated tandem antigen is shown in SEQ ID No. 8. According to the existing and predicted three-dimensional structure of the protein, the present invention compares and analyzes the structures of the three antigenic proteins N, F and H of the canine distemper virus, thereby selecting fragments with better stability, and expressing them in series using a eukaryotic insect cell expression system. The canine distemper virus tandem antigen protein has higher sensitivity, better specificity and stronger stability. The Elisa antibody detection kit and antibody fluorescence detection test strip of the canine distemper virus protein N‑F‑H truncated tandem antigen provided by the present invention are used to detect the antibody level of canine distemper virus in canine serum, and have the characteristics of high specificity and good sensitivity.

Description

Technical Field

[0001] The invention relates to the field of pet disease detection, and in particular to a canine distemper virus protein NFH truncated body tandem antigen and an application thereof. Background Art

[0002] Canine distemper (CD) is an ancient infectious disease of canines, caused by canine distemper virus (CDV) of the Morbillivirus genus of the Paramyxoviridae family. It is an acute, highly contagious disease. Canine distemper occurs widely throughout the world, and confirmed hosts include Canidae, Mustelidae, Felidae, Procyonidae, Civetidae, and Ursidae. It can be transmitted through direct contact, air, and food, and has obvious epidemic characteristics of age (puppies are susceptible to infection), seasonality (winter and spring are susceptible to infection), and breed (purebred dogs are more susceptible to infection). The mortality rate of canines can be as high as 30%-80%, and the mortality rate of ferrets can reach 100%. Canine distemper virus CDV is highly contagious and has a high mortality rate, which poses great difficulties to the prevention and control of CDV and seriously threatens the related breeding industry and the health of pet dogs.

[0003] Canine distemper virus (CDV) is a single-stranded negative-sense RNA virus. Its particles are polymorphic, mostly spherical, and have a capsule. They are very sensitive to the outside world, and the symptoms they cause include fever, rash, respiratory inflammation, gastrointestinal inflammation, and neurological symptoms. Prevention and control of canine distemper virus (CDV) is mainly based on prevention, mainly through vaccination. The single-stranded negative-sense RNA genome of canine distemper virus (CDV) contains approximately 15,690 nucleotides, encoding 6 structural proteins: nucleocapsid protein N, phosphoprotein P, matrix membrane protein M, fusion protein F, hemagglutinin protein H, and large protein L. At present, there have been many studies on canine distemper virus (CDV), mainly focusing on hemagglutinin protein H, which has large differences among different strains. This type of protein determines host specificity and is usually used as the basis for virus typing to study the variation and evolution of the virus. Fusion protein F can mediate the membrane fusion reaction between the viral envelope protein and the host cell, completing the process of canine distemper virus invading the host. Nucleocapsid protein N is often used for antibody detection due to its high conservatism.

[0004] The serological detection methods of canine distemper virus CDV mainly include enzyme-linked immunosorbent assay (ELISA) and fluorescent microsphere immunochromatography technology. ELISA has the advantages of high specificity, strong sensitivity, wide application range, and fast detection speed. Fluorescent microsphere immunochromatography technology has strong specificity, simple and fast operation, and is suitable for single sample detection. It has been widely used in veterinary clinics and pet hospitals and is an important auxiliary means of diagnosis and treatment. The establishment of both methods requires the provision of specific antigens or antibodies to obtain diagnostic products with high sensitivity and strong specificity.

[0005] Therefore, the present invention prepares canine distemper virus-specific antigen protein as a raw material for antibody detection methods such as the indirect Elisa method, which is of great significance for accurate clinical detection of antibody levels in the future. Summary of the invention

[0006] The purpose of the present invention is to provide a canine distemper virus tandem antigen protein with higher sensitivity, better specificity, better stability, higher expression level and lower cost, and provide the application of the tandem antigen.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] In a first aspect, the present invention provides a canine distemper virus protein NFH truncated tandem antigen, characterized in that the truncated tandem antigen is formed by connecting the amino acid residues 28-373 of the canine distemper virus N protein from the N-terminus, the amino acid residues 136-565 of the canine distemper virus F protein from the N-terminus, and the amino acid residues 150-602 of the canine distemper virus H protein from the N-terminus through flexible random curling to form a new truncated tandem antigen CDV N 28-373 -F 136-565 -H 150-602 .

[0009] The amino acid sequence of the truncated tandem antigen is shown in SEQ ID No.7.

[0010] In a second aspect, the present invention provides a gene encoding a tandem antigen of a truncated form of canine distemper virus protein NFH, characterized in that the nucleotide sequence of the encoding gene is shown in SEQ ID No.8.

[0011] In a third aspect, the present invention provides a method for preparing the above-mentioned canine distemper virus protein NFH truncated tandem antigen, characterized in that the steps are:

[0012] (1) constructing the coding gene of the above-mentioned truncated tandem antigen into the pFastBac-HTB vector;

[0013] (2) The expression vector correctly identified in step (1) is transformed into DH10Bac engineering bacteria, and the plasmid is further extracted and then transfected into insect cells Sf9, the P1-P3 generation viruses are cultured, and the P3 generation virus is transfected into Sf9 cells for protein expression;

[0014] (3) Purify the target protein expressed in step (2) using Ni affinity chromatography.

[0015] In a fourth aspect, the present invention provides any one of the following biomaterials (a1) to (a4):

[0016] (a1) an expression cassette containing the above-mentioned coding gene;

[0017] (a2) a recombinant vector containing the above-mentioned encoding gene;

[0018] (a3) a recombinant bacterium containing the above-mentioned encoding gene;

[0019] (a4) A transgenic cell line containing the above-mentioned encoding gene.

[0020] In a fifth aspect, the present invention provides the use of the above-mentioned canine distemper virus protein NFH truncated tandem antigen in an Elisa antibody detection kit and an antibody fluorescence detection test strip.

[0021] Preferably, the kit is an indirect Elisa antibody detection kit;

[0022] Preferably, the test strip is a fluorescent microsphere detection test strip.

[0023] The embodiments of the present invention have the following advantages:

[0024] The present invention compares and analyzes the structures of three antigenic proteins N, F and H of canine distemper virus according to the existing and predicted three-dimensional protein structures, thereby selecting fragments with better stability, and expressing them in series using a eukaryotic insect cell expression system. After the three antigenic proteins are connected in series, the antigenic epitopes increase, further improving the discrimination of positive and negative samples detected, while reducing cross-reactions with other viruses. The canine distemper virus series antigenic protein has higher sensitivity, better specificity and stronger stability. The prepared Elisa antibody detection kit and antibody fluorescence detection test strip have good effects, low cost, strong specificity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0026] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0027] Figure 1 To align the predicted canine distemper N protein structure with the whale measles virus N protein structure 7OI3;

[0028] Figure 2 The published trimeric (left) and monomeric (right) structures of canine distemper virus F protein.

[0029] Figure 3 The published canine distemper virus H protein structure 7ZNY tetramer (left) and monomer (right) structures;

[0030] Figure 4 It is the published canine distemper virus H protein structure 8IAY dimeric structure;

[0031] Figure 5 The three-dimensional structure of the canine distemper virus tandem antigen protein predicted by AlphaFold;

[0032] Figure 6 These are the results of eukaryotic expression and purification of canine distemper virus tandem antigens.

[0033] Figure 1 The middle left picture shows that the N-terminal α-helix part of the canine distemper N protein (dark gray) is in a different position from 7OI3 (light gray), and the right picture shows that the C-terminal random coil of the canine distemper N protein (dark gray) occupies the RNA site in the 7OI3 (light gray) structure. DETAILED DESCRIPTION

[0034] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1: Selection and structural analysis of tandem antigens of truncated canine distemper virus protein NFH

[0036] The sequence of the canine distemper virus nucleocapsid protein N protein refers to the NP_047201 amino acid sequence of the American Center for Biotechnology Information NCBI, with a total of 523 amino acid residues, no signal peptide and transmembrane region, and the full-length corresponding nucleic acid sequence is NC_001921, 1572bp. The protein sequence was compared with the structural data in the protein structure database PDB in the NCBI Blast function, and the results showed that the structure of the whale measles virus N protein was 7OI3, covering 77% of the sequence 416 amino acid residues, and the homology was 82.80%.

[0037] The predicted structure of canine distemper N protein was compared with the structure of whale measles virus N protein 7OI3. The calculation results of Pymol software showed that the root mean square deviation of the alignment of 297 overlapping molecules was 1.27Å, and the difference in the overlapping parts was small. The parts that could not overlap were mainly concentrated in the structures of the N-terminus and C-terminus. Figure 1 As shown, the left figure shows that the first α helix at the N-terminus of the predicted structure of canine distemper N protein (dark gray) is in a completely different position from the 7OI3 structure of whale measles virus N protein (light gray), indicating that the α helix at the N-terminus and some random coils are in an unstable state, specifically 1-27aa amino acid residues. The right figure shows that in the predicted structure of canine distemper N protein, most of the C-terminus is a random coil and occupies the position of the active center RNA in the 7OI3 structure (light gray), indicating that the C-terminus of the structure (410-523aa) is in an unstable state, which is not conducive to the overall stability of the protein. The structure of the 28-337aa amino acid residues in the middle has a good degree of overlap with the published structure and can form a stable three-dimensional structure. 338-409aa is quite different from the published 7OI3 structure, among which 338-373aa is similar to the secondary structure of the relative position of the 7OI3 structure, but the position is biased, and the secondary structure and relative position of 374-409aa are quite different. From the perspective of protein stability, the middle 28-373aa truncation of the N protein should have better stability.

[0038] The sequence of the canine distemper virus fusion protein F protein refers to the AF378705_4 amino acid sequence of the American Center for Biotechnology Information NCBI, with a total of 662 amino acid residues, and the amino acid residues at positions 606-628 are transmembrane regions, without signal peptides, and the full-length corresponding nucleic acid sequence is AF378705 (genome full length 4935-6923bp), 1989bp. The protein sequence was compared with the structural data in the protein structure database PDB in the NCBI Blast function. The results showed that the B chain structure of the canine measles virus F protein structure 6XYE, the sequence covers 66% of the sequence of the C-terminus of the F protein (positions 225-662), and the homology is as high as 97.49%. The A chain structure of the canine measles virus F protein structure 6XYE, the sequence covers 13% of the sequence of the N-terminus of the F protein (positions 136-224), and the homology is as high as 98.88%. This extremely high homology confirms that the structure of the canine distemper virus fusion protein F should be the same as the published structure.

[0039] The published structure of the canine distemper virus (canine measles virus) fusion protein F 6XYE is as follows Figure 2 As shown in the figure on the left, the trimer structure formed by three proteins is in the shape of a triangular pyramid. The five α-helices near the N-terminus of the three monomers together form the top of the cone, and the single α-helices at the C-terminus of the three monomers interact with each other to form the center of the bottom of the cone. Figure 2As shown in the right figure, the single α-helix at the C-terminus is free from the two domains of the whole structure ( Figure 2 The lower left corner of the right picture) can stabilize the formation of the trimer structure, but it is not stable enough in the monomer structure due to its greater flexibility. Therefore, according to the structural information, the F protein should be more stable if the amino acids 136-565 of the C-terminal α helix (566-583aa) are removed.

[0040] The sequence of canine distemper virus hemagglutinin H protein refers to the amino acid sequence AF378705_5 of the Center for Biotechnology Information NCBI of the United States, with a total of 604 amino acid residues, 36-58 amino acid residues are transmembrane regions, no signal peptide, and the full-length corresponding nucleic acid sequence is AF378705 (genome full length 7079-8893bp), 1815bp. The protein sequence was compared with the structural data in the protein structure database PDB in the NCBI Blast function, and the results showed that the homology of the 7ZNY and 8IAY structural sequences of canine measles virus hemagglutinin H protein was 92.72% and 96.93% respectively. This extremely high homology confirms that the structure of canine distemper virus hemagglutinin protein H should be the same as the published structure.

[0041] The published structures of canine distemper virus H protein 7ZNY and 8IAY are as follows: Figure 3 and Figure 4 As shown in the figure. The overall structure of 7ZNY is 4 protein molecules, which form dimers in pairs and then form a tetramer structure. The random curl and small β sheet at the N end can stabilize the tetramer structure. 8IAY is a dimer structure formed by two protein molecules rotating 90 degrees along the X axis, showing different directions of the monomers. The interior is a barrel-shaped structure composed of multiple antiparallel β sheets, and a small amount of α helix and random curls are distributed on the periphery. The monomer structures of 7ZNY and 8IAY are basically the same. If the main chain is compared, the calculated root mean square deviation RMSD is only 0.75Å. The distribution and direction of the main chain are basically the same, indicating that the structures of the two are very similar. Only the random curl and small β sheet at the N end of the 7ZNY structure monomer that stabilizes the tetramer are different from those of the 8IAY structure. From the perspective of the monomer structure, this part is in an unstable area and can be considered for removal. Based on the two published structures, the structure composed of amino acid residues 150-602 of the canine distemper virus hemagglutinin H protein is relatively stable.

[0042] Since the N protein, F protein and H protein of canine distemper protein can all exert good antigenic activity, we connected the stable fragments of the three proteins together through flexible random curling to form a new tandem antigen CDV N 28-373 -F 136-565 -H 150-602To perform expression and purification attempts, AlphaFold software was used to calculate and predict the three-dimensional structure of the tandem antigen protein. The final predicted structure is as follows Figure 5 As shown in Figure 2, the structure is in a stable state as a whole, and there is no flexible unstable area.

[0043] Canine distemper virus N protein full-length amino acid sequence (SEQ ID No.1):

[0044] MASLLKSLTLFKRTRDQPPLASGSGGAIRGIKHVIIVLIPGDSSIVTRSRLLDRLVRLVGDPKINGPKLTGILISILSLFVESPGQLIQRIIDDPDVSIKLVEVIPSINSACGLTFASRGASLDSEADEF FKIVDEGSKAQGQLGWLENKDIVDIEVDNAEQFNILLASILAQIWILLAKAVTAPDTAADSEMRRWIKYTQQRRVVGEFRMNKIWLDIVRNRIAEDLSLRRFMVALILDIKRSPGNKPRIAEMICDIDNY IVEAGLASFILTIKFGIETMYPALGLHEFSGELTTIESLMMLYQQMGETAPYMVILENSVQNKFSAGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSALAAELGITKEE AQLVSEIASKTTEDRTIRATGPKQSQITFLHSERSEVANQQPPTINKRSENQGGDKYPIHFSDERLPGYTPDVNSSEWSESRYDTQIIQDDGNDDDRKSMEAIAKMRMLTKMLSQPGTSEDNSPVYSDKE.

[0045] Full-length nucleotide sequence of canine distemper virus N protein (SEQ ID No.2):

[0046]

[0047] Full-length amino acid sequence of canine distemper virus F protein (SEQ ID No.3):

[0048] MHKGIPKSSKTQTHTQQDRPPQPSTEPEETRTSRARHSITSAQRSTHYDPRTSDRPVSYTMDRIRSRKQTSHRLKNIPVHGNHEAIIQHIPESVSKGARSQIERRQPNAINSGSQCTWLVLWCLGIASLFLCSKAQIHWNNLSTIGIIGTDSVHYKIMTRPSHQYLVIKLMPNVSLIDNCTKAELGEYEKLLNSVLEPINQALTLMTKNVKPLQSLGSGRRQRRFAGVVLAGAALGVATAAQITAGIALHQSNLNAQAIQSLRTSLEQSNKAIEEIREATQETVIAVQGVQDYVNNELVPAMQHMSCELVGQRLGLRLLRYYTELLSIFGPSLRDPISAEISIQALSYALGGEIHKILEKLGYSGGDMIAILESRGIKTKITHVDLPGKFIILSISYPTLSEVKGVIVHRLEAVSYNIGSQEWYTTVPRYIATNGYLISNFDESSCVFVSESAICSQNSLYPMSPLLQQCIRGDTSSCARTLVSGTMGNKFILSKGNIVANCASILCKCYSTSTIINQSPDKLLTFIASDTCPLVEIDGVTIQVGGRQYPDMVYEGKVALGPAISLERLDVGTNLGNALKKLDDAKVLIDSSNQILETVRRSSFNFGSLLSVPILSCTALALLLLIYCCKRRYQQTLKQHTKVDPAFKPDLTGTSKSYVRSL。

[0049] Full-length nucleotide sequence of canine distemper virus F protein (SEQ ID No.4):

[0050]

[0051] Canine distemper virus H protein full-length amino acid sequence (SEQ ID No.5):

[0052] .

[0053] Full-length nucleotide sequence of canine distemper virus H protein (SEQ ID No.6):

[0054]

[0055] Canine distemper virus tandem antigen CDV N 28-373 -F 136-565 -H 150-602 Amino acid sequence (SEQ ID No.7), with the underlined part being the randomly coiled linker:

[0056] IRGIKHVIIVLIPGDSSIVTRSRLLDRLVRLVGDPKINGPKLTGILISILSLFVESPGQLIQRIIDDPDVSIKLVEVIPSINSACGLTFASRGASLDSEADEFFKIVDEGSKAQGQLGWLENKDIVDIEVDNAEQFNILLASILAQIWILLAKAVTAPDTAADSEMRRWIKYTQQRRVVGEFRMNKIWLDIVRNRIAEDLSLRRFMVALILDIKRSPGNKPRIAEMICDIDNYIVEAGLASFILTIKFGIETMYPALGLHEFSGELTTIESLMMLYQQMGETAPYMVILENSVQNKFSAGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSA GGGGSGGGGS QIHWNNLSTIGIIGTDSVHYKIMTRPSHQYLVIKLMPNVSLIDNCTKAELGEYEKLLNSVLEPINQALTLMTKNVKPLQSLGSGRRQRRFAGVVLAGAALGVATAAQITAGIALHQSNLNAQAIQSLRTSLEQSNKAIEEIREATQETVIAVQGVQDYVNNELVPAMQHMSCELVGQRLGLRLLRYYTELLSIFGPSLRDPISAEISIQALSYALGGEIHKILEKLGYSGGDMIAILESRGIKTKITHVDLPGKFIILSISYPTLSEVKGVIVHRLEAVSYNIGSQEWYTTVPRYIATNGYLISNFDESSCVFVSESAICSQNSLYPMSPLLQQCIRGDTSSCARTLVSGTMGNKFILSKGNIVANCASILCKCYSTSTIINQSPDKLLTFIASDTCPLVEIDGVTIQVGGRQYPDMVYEGKVALGPAIS GGGGSGGGGSFTNYCESIGIRKAIASAANPILLSALSGGRSDIFPPHRCSGATTSVGKVFPLSVSLSMSLISRTSEIINMLTAISDGVYGKTYLLVPDDIEREFDTQEIRVFEIGFIKRWLND MPLLQTTNYMVLPENSKAKVCTIAVGELTLASLCVEESTVLLYHDSSGSQDGILVVTLGIFWATPMDHIEEEVIPVAHPSMEKIHITNHRGFIKDSIATWMVPALASEKQEEQKG CLESACQRKTYPMCNQTSWEPFGGRQLPSYGRLTLPLDASVDLQLNISFTYGPVILNGDGMDYYESPLLNSGWLTIPPKNGTIVGLINKAGRGDQFTVLPHVLTFAPWESSGN CYLPIQTSQIIDRDVLIESNIVVLPTQSFRYVIATYDISRSDHAIVYYVYDPIRTISYTHPFRLTTKGRPDFLRIECFVWDDNLWCHQFYRFEADIANSTTSVENLVRIRFSC.

[0057] Canine distemper virus tandem antigen CDV N 28-373 -F 136-565 -H 150-602 Nucleotide sequence (SEQ ID No. 8), underlined are connected random curls:

[0058] GGTGGCGGTGGAAGCGGC GGTGGCGGAAGC GGTGGCGGTGGAAGCGGCGGTGGCGGAAGC

[0059] Example 2: Expression, purification and antigen activity verification of canine distemper virus protein NFH truncated tandem antigen

[0060] Based on the results of homologous structure comparison and structure prediction, canine distemper virus tandem antigen CDV N was selected. 28-373 -F 136-565 -H 150-602 Expression purification was performed. After the full-length nucleotide sequence of the tandem antigen was codon-optimized according to the insect cell expression system at the gene company, the sequence was synthesized. The synthesized sequence was constructed into the pFastBac-HTB vector and transformed into the DH10Bac engineering bacteria. The white spots after verification were expanded and cultured through blue-white screening, and the Bacmid plasmid was extracted. Bacmid was transfected into insect cells Sf9 using liposome transfection reagent to culture P1-P3 generation viruses. The P3 generation virus was transfected into Sf9 cells for protein expression. The insect cells collected by centrifugation were ultrasonically broken, and the protein was purified by Ni affinity chromatography to finally obtain the canine distemper virus tandem antigen CDV N 28-373 -F 136-565 -H 150-602 The electrophoresis results are as follows Figure 6 As shown, there is a target band at around 130kD with high purity.

[0061] The purified canine distemper virus tandem antigens were used to verify the antigen activity using the Elisa method. The purified canine distemper virus tandem antigen was coated at 1 μg / ml, 100 μl / well, coated overnight at 4°C, blocked at 37°C for 2 h with 0.1% BSA, washed 3 times with 250 μl / well PBST solution (0.1%), added 100 μl / well of diluted clinical positive serum, incubated at 37°C for 30 min (the positive control group was canine distemper virus positive serum, the negative control group was canine distemper virus negative serum, and each group was repeated three times), washed 3 times with 250 μl / well PBST solution (0.1%), patted dry, added 100 μl / well of HRP-labeled rabbit anti-dog secondary antibody (diluted 1:10000 in PBS), reacted at 37°C for 30 min, washed again 3 times, patted dry, added 100 μl / well of TMB color development solution (commercial), developed at 37°C for 10 min, finally added 0.5 M sulfuric acid, 50 μl / well, terminated the reaction, and measured OD with an enzyme reader. 450nm As shown in Table 1, the canine distemper virus tandem antigen reacted with the canine distemper monoclonal antibody, which was close to the positive control result and had good antigenic activity.

[0062] Table 1 Antigen activity verification results

[0063]

[0064] Example 3. Use and characteristics of the Elisa antibody detection kit based on the canine distemper virus tandem antigen prepared in Example 2 as the antigen raw material

[0065] The Elisa antibody detection kit prepared using the canine distemper virus tandem antigen prepared in Example 2 as an antigen is an enzyme-linked immunosorbent assay kit for detecting the level of canine distemper virus antibodies in canine serum, and its working steps are:

[0066] (1) Take out the antigen-coated plate from the kit, dilute the serum to be tested 100 times with the sample diluent on the serum dilution plate (mix 198ul sample diluent and 2ul serum to be tested), take 100ul of the mixture and add it to the ELISA plate. At the same time, add 2 wells of positive serum and 2 wells of negative serum, 100ul in each well.

[0067] (2) Gently shake the samples in each well to mix well, cover the coated plate with a sealing film, and incubate at 37°C for 30 minutes.

[0068] (3) Discard the solution in the wells and add 250ul of working washing solution to each well. Repeat 3 times. After the last wash, pat the liquid dry thoroughly.

[0069] (4) Add 100 μl of enzyme-labeled antibody to each well, cover the coated plate with sealing film, and incubate at 37°C for 30 minutes.

[0070] (5) Discard the solution in the wells and add 250ul of working washing solution to each well. Repeat 3 times. After the last wash, pat the liquid dry thoroughly.

[0071] (6) Add 100 μl of substrate solution to each well, cover the coated plate with sealing film, and incubate at 37°C for 10 minutes.

[0072] (7) Add 50 μl of stop solution to each well to terminate the reaction.

[0073] (8) Determine the absorbance of the sample and the control at 450 nm (OD 450nm ). Positive control OD 450nm Value ≥ 0.5, negative control OD 450nm Value ≤ 0.2, the test is established. Interpretation standard: S / P = (OD 450nm Mean - negative control OD 450nm Mean) / (positive control OD 450nm Mean - negative control OD 450nm S / P ≥ 0.3 is positive, and the serum antibody of the sample to be tested has reached the protective level; S / P < 0.3 is negative, and the serum antibody of the sample to be tested has not reached the protective level.

[0074] The results of the above kit characteristic identification experiment are as follows:

[0075] 1. Specificity test

[0076] The above canine distemper virus antibody level detection kit was used to detect standard antibody positive serums such as canine parvovirus CPV, rabies virus RABV and canine infectious hepatitis virus CAVI and canine distemper virus CDV antibody positive serum. The results are shown in Table 2. Except for the S / P value of canine distemper virus standard antibody positive serum significantly greater than 0.3, the S / P values ​​of the other sera were less than 0.3, which met the judgment criteria for negative serum, indicating that the specificity of the kit was good.

[0077] Table 2 Specificity test results of canine distemper virus antibody level test kit

[0078]

[0079] 2. Sensitivity test

[0080] The positive serum of CDV standard antibody was diluted 2, 4, 8, 16, 32, 64 and 128 times respectively, and the above antibody detection kit and the commercialized canine distemper virus Elisa antibody detection kit were used for simultaneous detection. The results are shown in Table 3, and the results show that both kits can detect CDV standard antibody positive serum diluted 64 times, which indicates that the above antibody detection kit has good sensitivity.

[0081] Table 3 Sensitivity test results of canine distemper virus antibody level detection kit

[0082]

[0083] 3. Comparison of compliance rate

[0084] The above antibody detection kit and the commercial canine distemper virus Elisa antibody detection kit were used to simultaneously detect 35 serum samples, and the results were compared. The conformity rate of the canine distemper virus antibody level detection kit and the commercial kit for overall sample detection was 97.1%, indicating a good correspondence with the commercial kit. The specific results are shown in Tables 4 and 5.

[0085] Table 4 Comparison of sample compliance rates

[0086]

[0087] Table 5 Analysis of compliance results

[0088]

[0089] Example 4: Application and Characterization of Antibody Fluorescence Test Strips Using the Canine Distemper Virus Tandem Antigens Prepared in Example 2 as Antigens

[0090] The antibody fluorescence detection test strip prepared using the canine distemper virus tandem antigen prepared in Example 2 as an antigen is a test strip for detecting canine distemper virus antibody levels, and its working steps are:

[0091] (1) Return the test strip and sample diluent to room temperature. Use a pipette to draw 20 μl of serum sample and add it to the sample diluent. After thorough mixing, use a pipette to draw 80 μl of the test solution and vertically add it drop by drop into the sample well of the test strip.

[0092] (2) After adding the sample, let it react at room temperature for 5 minutes, insert the card into the detection hole of the fluorescent immunoassay analyzer, and press "Quick Test" to automatically read the value.

[0093] (3) Determine the fluorescence values ​​of the T line and C line. The sample and control use an excitation light of 365nm wavelength and detect the fluorescence intensity of the emission light of 615nm. Interpretation standard: T / C = mean fluorescence value of the T line / mean fluorescence value of the C line. T / C ≥ 0.3 is positive, and the serum antibody of the sample to be tested has reached the protective level; T / C < 0.3 is negative, and the serum antibody of the sample to be tested has not reached the protective level.

[0094] The results of the above-mentioned antibody fluorescence test strip characteristic identification experiment are as follows:

[0095] 1. Specificity test

[0096] The canine distemper virus antibody fluorescent test strip was used to detect canine parvovirus CPV, rabies virus RABV and canine infectious hepatitis virus CAVI standard antibody positive serum and canine distemper virus CDV antibody positive serum. The results are shown in Table 6. Except for the canine distemper virus standard antibody positive serum with an S / P value significantly greater than 0.3, the other serum S / P values ​​were less than 0.3, which met the judgment criteria for negative serum, indicating that the fluorescent test strip had good specificity.

[0097] Table 6 Canine distemper virus antibody fluorescent test strip specific serum test results

[0098]

[0099] 2. Sensitivity test

[0100] The canine distemper virus standard antibody positive serum was diluted 2, 4, 8, 16, 32, 64, and 128 times, respectively, and the above antibody fluorescence detection test strip and the commercial canine distemper virus antibody fluorescence detection kit were used for simultaneous detection. The results are shown in Table 7. The test strip can detect canine distemper standard antibody positive serum diluted 64 times, and the purchased kit can also detect canine distemper virus standard antibody positive serum diluted 64 times, indicating that the above canine distemper virus antibody immunofluorescence detection test strip has good sensitivity.

[0101] Table 7 Canine distemper virus antibody fluorescence test strip sensitivity serum test results

[0102]

[0103] 3. Comparison of compliance rate

[0104] The above-mentioned antibody fluorescence detection test strip and the commercial canine distemper virus antibody fluorescence detection kit were used to simultaneously detect 32 serum samples, and the results were compared. The conformity rate of the antibody fluorescence detection test strip and the purchased commercial kit for overall sample detection was 100%, indicating good correspondence with the commercial kit. The specific results are shown in Tables 8 and 9.

[0105] Table 8 Comparison of conformity rates of canine distemper virus antibody fluorescent test strip samples

[0106]

[0107] Table 9 Analysis of test strip compliance results

[0108]

[0109] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A canine distemper virus protein NFH truncated tandem antigen, characterized in that: The truncated tandem antigen is formed by connecting the amino acid residues 28-373 of the canine distemper virus N protein from the N-terminus, the amino acid residues 136-565 of the canine distemper virus F protein from the N-terminus, and the amino acid residues 150-602 of the canine distemper virus H protein from the N-terminus through flexible random curling to form a new truncated tandem antigen CDV N 28-373 -F 136-565 -H 150-602 ; The amino acid sequence of the truncated tandem antigen is shown in SEQ ID No.

7.

2. The nucleic acid encoding the canine distemper virus protein NFH truncated tandem antigen according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid is shown in SEQ ID No.

8.

3. A method for preparing the canine distemper virus protein NFH truncated tandem antigen according to claim 1, characterized in that The steps are: Cloning the nucleic acid of claim 2 into the pFastBac-HTB vector; The expression vector identified correctly in step (1) is transformed into DH10Bac engineering bacteria, and the plasmid is extracted after further screening, and the plasmid is transfected into insect cells Sf9, and P1-P3 generation viruses are cultured, and the P3 generation virus is transfected into Sf9 cells for protein expression; The target protein expressed in step (2) was purified by Ni affinity chromatography.

4. Any of the following biological materials (a1) to (a4): (a1) an expression cassette containing the nucleic acid of claim 2; (a2) a recombinant vector containing the nucleic acid of claim 2; (a3) a recombinant bacterium containing the nucleic acid of claim 2; (a4) A transgenic cell line containing the nucleic acid of claim 2.

5. A canine distemper virus Elisa antibody detection kit, characterized in that, The antigen active ingredient is the canine distemper virus protein tandem antigen according to claim 1.

6. A fluorescent detection kit for canine distemper virus antibodies, characterized in that: The antigen active ingredient is the canine distemper virus protein tandem antigen according to claim 1.

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