A portable immunochromatographic device for detecting neutralizing antibodies of bovine nodular dermatosis virus and its application

Abstract

The present invention discloses a portable immunochromatographic device for detecting neutralizing antibodies of bovine nodular dermatosis virus and its application. The device comprises a sampling body and a detection body connected in an integrated manner from front to back. A spring sampler and a siphon are arranged in the sampling body. A sample pretreatment chamber, a filter membrane, an antibody enrichment chamber and an immunochromatographic detection chamber connected in sequence from front to back are arranged in the detection body. The siphon is communicated with the sample pretreatment chamber. A diluent storage chamber and a plastic needle are also arranged outside the sample pretreatment chamber. The diluent storage chamber is communicated with the sample pretreatment chamber, and the two are separated by a diaphragm seal. A liquid absorption pad and a chromatography pad are arranged in the antibody enrichment chamber. The liquid absorption pad is connected to the filter membrane and the chromatography pad respectively. An immunochromatographic test strip is arranged in the immunochromatographic detection chamber. The chromatography pad is connected to the liquid absorption pad and the immunochromatographic test strip respectively. The device is integrated with sampling and testing, saves time and is accurate, reduces non-specific reactions, is easy to operate, and is suitable for large-scale detection and monitoring of LSDV neutralizing antibodies.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and more specifically, to a portable immunochromatographic device for detecting bovine lumpy dermatosis virus neutralizing antibodies and an application thereof. Background Art

[0002] Lumpy skin disease (LSD) is also called bovine nodular dermatitis, bovine pimple skin disease, bovine nodular rash, etc. It is an acute, subacute or chronic infectious disease caused by the infection of cattle by the Lumpy skin disease virus (LSDV). At present, my country has adjusted bovine nodular skin disease from a Class I animal epidemic disease to a Class II animal epidemic disease.

[0003] LSDV belongs to the Poxviridae family, the Chordopoxvirinae subfamily, and the Capripoxvirus (CPV) genus. It is a linear double-stranded enveloped DNA virus that presents a typical "brick-shaped" or "large oval-shaped" shape. It has only one serotype, and the representative strain is the South African Neethling strain. Due to the cross-protection between CaPVs, isolates from sheep or goats can cross-protect cattle from LSD infection, and LSDV isolates can protect sheep and goats from infection with sheep pox virus (SPPV) and goat pox virus (GTPV) to varying degrees. Therefore, vaccination is currently the main preventive measure for bovine nodular dermatitis. Common vaccine types include homologous vaccines (LSDV attenuated live vaccine, inactivated vaccine) and heterologous vaccines (goat pox vaccine, sheep pox vaccine). For most epidemic diseases, the level of immune antibodies is a reference indicator for evaluating the effectiveness of immunity. The most representative antibody in the immune effect is neutralizing antibodies. However, there are large differences in antibody levels after vaccination with this disease vaccine, and most farms are located in remote areas from urban areas, without laboratory testing conditions. After sample collection, it needs to be stored and transported under special conditions before it can be tested at the testing agency. Even if there are portable testing equipment, samples still need to be pre-processed by centrifugation to separate serum, which is cumbersome and can easily lead to hemolysis of samples, affecting the accuracy of the test results. Therefore, it is urgent to develop a portable detection device for detecting LSDV neutralizing antibodies. Summary of the invention

[0004] The purpose of the present invention is to provide a portable immunochromatographic device that integrates sampling and detection and is suitable for detecting LSDV neutralizing antibodies in bovine serum and plasma. The device can evaluate the neutralizing antibody level of cattle after immunization with LSDV vaccine, and can also perform auxiliary diagnosis of LSDV infection. It is particularly suitable for use in areas with simple conditions such as farms.

[0005] According to one aspect of the present invention, a portable immunochromatographic device for detecting neutralizing antibodies of bovine nodular dermatitis virus is provided, comprising a sampling body and a detection body connected in an integral manner front and back, a spring sampler and a siphon tube being arranged in the sampling body, the spring sampler comprising a blood collection needle, the blood collection needle being tightly fitted with the siphon tube, a sample pretreatment chamber, a filter membrane, an antibody enrichment chamber and an immunochromatographic detection chamber being sequentially connected front and back in sequence being arranged in the detection body, the siphon tube being in communication with the sample pretreatment chamber, a diluent storage chamber and a plastic needle being further arranged on the outside of the sample pretreatment chamber, the diluent storage chamber being in communication with the sample pretreatment chamber, the two being sealed and separated by a diaphragm, a sample diluent being stored in the diluent storage chamber, the plastic needle being located in the diluent storage chamber and being located above the diaphragm, a liquid-absorbing pad and a chromatography pad being arranged in the antibody enrichment chamber, the front and rear ends of the liquid-absorbing pad being respectively connected to the filter membrane and the chromatography pad, an immunochromatographic test strip being arranged in the immunochromatographic detection chamber, the front and rear ends of the chromatography pad being respectively connected to the liquid-absorbing pad and the immunochromatographic test strip.

[0006] In some embodiments, the spring sampler also includes a button and a spring, one end of the spring is connected to the blood collection needle, and the other end is connected to the button, the button is located on the outside of the sampling body, the sampling body is also provided with an openable protective cover, one end of the sampling body is provided with a sampling hole, the protective cover is covered on the sampling hole, one end of the siphon tube extends out of the sampling hole and is located inside the protective cover.

[0007] In some embodiments, the immunochromatographic detection cavity is further provided with two annular clips and an observation window, the two annular clips are fixedly installed in the immunochromatographic detection cavity, the front and rear ends of the immunochromatographic test strip are respectively fixed by the two annular clips, and the observation window is provided in the outer shell of the immunochromatographic detection cavity and is located above the immunochromatographic test strip, so as to facilitate the observation of the test results of the immunochromatographic test strip.

[0008] In some embodiments, the immunochromatographic test strip includes an NC membrane, a gold label pad, a sample pad, an absorbent pad and a PVC bottom plate. The sample pad, the gold label pad, the NC membrane and the absorbent pad are overlapped and adhered to the PVC bottom plate in sequence. The NC membrane includes a detection line and a quality control line arranged in sequence along the sample flow direction. The gold label pad contains colloidal gold-labeled LSDV-A27L-063-1 recombinant protein, the detection line is coated with anti-bovine IgG antibody, and the quality control line is coated with a polyclonal antibody against the LSDV-A27L-063-1 recombinant protein. The sample pad extends into the antibody enrichment cavity and overlaps with the chromatography pad.

[0009] In some embodiments, the sample diluent is 0.01 mM PBS buffer containing 1 wt % BSA, 0.25 wt % Tween, 0.1 wt % trehalose, and 1 wt % glycerol, and the filter membrane is any one of a sterile microporous filter membrane, a PTFE composite membrane, or a hemagglutinin-treated filter membrane.

[0010] In some embodiments, the chromatography pad is soaked with a polypeptide that can bind to bovine immunoglobulin and fully wrap the non-specific binding site of the antibody Fc segment, the amino acid sequence of the polypeptide is WPIGRSTETAVL, and the chromatography pad is subjected to low-temperature drying after adsorbing the polypeptide.

[0011] In some embodiments, the amino acid sequence of the LSDV-A27L-063-1 recombinant protein is shown in SEQ ID NO:10.

[0012] In some embodiments, the method for preparing the LSDV-A27L-063-1 recombinant protein comprises the following steps:

[0013] S1. The amino acid sequence shown in SEQ ID NO: 8 is sequentially connected in series with the amino acid sequences shown in SEQ ID NO: 2-3 through a flexible linker, and then sequentially connected with the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 6 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 10, which is the LSDV-A27L-063-1 recombinant protein;

[0014] S2. The amino acid sequence shown in SEQ ID NO: 10 was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO: 11, which was inserted into the pFastbac1 vector to construct a recombinant plasmid pFastBac-LSDV-A27L-063-1 with correct sequencing;

[0015] S3. The recombinant plasmid pFastBac-LSDV-A27L-063-1 was transferred into Escherichia coli DH10bac competent cells, cultured, extracted and identified to obtain the recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1;

[0016] S4. The recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1 is transfected into sf9 cells, cultured, and inoculated to obtain the recombinant baculovirus, and the recombinant baculovirus is induced to express, and the LSDV-A27L-063-1 recombinant protein with the amino acid sequence shown in SEQ ID NO: 10 is obtained after separation and purification, i.e., the recombinant protein for detecting bovine lumpy dermatitis virus neutralizing antibodies.

[0017] In some embodiments, the polyclonal antibody is prepared by immunizing animals with LSDV-A27L-063-1 recombinant protein.

[0018] According to another aspect of the present invention, there is provided a portable immunochromatographic device for detecting neutralizing antibodies to bovine lumpy skin disease virus, and its use in immunological detection of neutralizing antibodies to bovine lumpy skin disease virus for non-disease diagnosis purposes.

[0019] Beneficial effects of the present invention: 1. The portable immunochromatographic device of the present invention integrates sampling and detection, eliminating the links of sample pretreatment, storage and transportation, saving time and cost; and the detection speed is fast, the operation is simple, and no professional personnel are required to operate. It can be applied to areas with simple conditions such as farms, and can also be applied to disease detection agencies, third-party detection agencies, etc. to conduct large-scale detection and monitoring of LSDV neutralizing antibodies, providing a simpler and faster on-site detection device for LSDV epidemic prevention and control;

[0020] 2. The sampling wound of the portable immunochromatographic device of the present invention is small, which has little stress on the cattle herd. The sample diluent isolation setting ensures the effective processing of the sample. The setting of the antibody enrichment chamber improves the sensitivity and accuracy of the detection and reduces non-specific reactions.

[0021] 3. The test strip in the portable immunochromatographic device of the present invention uses the LSDV-A27L-063-1 recombinant protein of bovine nodular dermatosis virus, and the polyclonal antibody titer of the recombinant protein is as high as 1:2.048×10 7 It has good immunogenicity and antigenicity, and has high sensitivity and good specificity for the detection of bovine nodular skin disease virus neutralizing antibodies in serum. It can be used to detect bovine nodular skin disease virus antibodies in bovine serum, and plays an auxiliary role in the diagnosis of bovine nodular skin disease. It can also be used to monitor and evaluate the level of neutralizing antibodies in cattle herds after immunization with bovine nodular skin disease vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the portable immunochromatography device in Example 1 of the present invention.

[0023] Figure 2 This is the mass spectrum of polypeptide A FIRWTCLDSVAG synthesized in Example 2 of the present invention.

[0024] Figure 3 This is the mass spectrum of the polypeptide B WPIGRSTETAVL synthesized in Example 2 of the present invention. Figure 4 is the mass spectrum of the polypeptide C QANAFDYETIEL synthesized in Example 2 of the present invention, Figure 5 This is the mass spectrum of the polypeptide DRHWRGSWTQVEI synthesized in Example 2 of the present invention.

[0025] Figure 6This is an SDS-PAGE detection and identification diagram of the recombinant protein in Example 3 of the present invention, wherein 1 is the LSDV-A27L-063-2 recombinant protein, and 2 is the LSDV-A27L-063-1 recombinant protein.

[0026] Figure 7 Figure A is a graph showing the results of Western-blot analysis of the LSDV-A27L-063-1 recombinant protein in Example 3 of the present invention, wherein 1 is a protein Marker; 2 is an inactivated LSDV-positive serum; 3 is an inactivated GPTV-positive serum; 4 is an inactivated SPPV-positive serum; 5 is an inactivated PPRV vaccine virus-positive serum; 6 is an inactivated Akabane disease (AKAV)-positive serum; 7 is a bovine negative serum, and Figure B is a graph showing the results of Western-blot analysis of the LSDV-A27L-063-2 recombinant protein in Example 3 of the present invention, wherein 1 is a protein Marker; 2 is an inactivated LSDV-positive serum; 3 is an inactivated GPTV-positive serum; 4 is an inactivated SPPV-positive serum; 5 is an inactivated PPRV vaccine virus-positive serum; 6 is an inactivated Akabane disease (AKAV)-positive serum; 7 is a bovine negative serum.

[0027] Figure 8 A is a graph showing the titer detection result of the polyclonal antibody serum used to measure the LSDV-A27L-063-1 recombinant protein in Example 3 of the present invention, and B is a graph showing the titer detection result of the polyclonal antibody serum used to measure the LSDV-A27L-063-2 recombinant protein. DETAILED DESCRIPTION

[0028] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0029] According to the technical solution of the present disclosure, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see Table 1.

[0030] Table 1 Conservatively substituted amino acids

[0031]

[0032] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be replaced without changing the activity or function of the polynucleotide sequence. The recombinant protein for detecting the neutralizing antibody of bovine nodular dermatitis virus provided by the present invention is soluble, and can form a correctly folded functional conformation during the expression of eukaryotic cells, which is closer to the structure and function of the real protein, avoiding the biosafety risks brought by the use of real viruses.

[0033] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0034] Example 1 A portable immunochromatographic device for detecting neutralizing antibodies to bovine nodular skin virus

[0035] A portable immunochromatographic device for detecting neutralizing antibodies of bovine nodular dermatosis virus comprises a sampling body 1 and a detection body 2 connected in an integral manner from front to back. The sampling body 1 is provided with a spring sampler 11, a siphon 12 and a protective cover 13. The spring sampler 11 comprises a blood collection needle 14, a spring 15 and a button 16, the button 16 is located outside the sampling body 1, and the blood collection needle 14, the spring 15 and the siphon 12 are all located inside the sampling body 1. One end of the spring 15 is connected to the blood collection needle 14, and the other end is connected to the button 16. A sampling hole 17 is provided at one end of the sampling body 1, and a protective cover 13 is provided on the sampling hole 17, and one end of the siphon 12 extends out of the sampling hole 17 and is located inside the protective cover 13. The blood collection needle 14 is tightly fitted with the siphon 12 and is located below the siphon 12. When not in use, the needle tip of the blood collection needle 14 is located inside the sampling body 1 and does not extend out of the sampling hole 17.

[0036] The detection body 2 is provided with a sample pretreatment chamber 21, a filter membrane 22, an antibody enrichment chamber 23 and an immunochromatographic detection chamber 24 which are connected in sequence. The other end of the siphon 12 is in communication with the sample pretreatment chamber 21, and a diluent storage chamber 25 and a plastic needle 26 are also provided outside the sample pretreatment chamber 21. The diluent storage chamber 25 is in communication with the sample pretreatment chamber 21, and the two are sealed and separated by a diaphragm 27. The sample diluent is stored in the diluent storage chamber 25, and the plastic needle 26 is located in the diluent storage chamber 25 and above the diaphragm 27. When the plastic needle 26 is pressed, the plastic needle 26 penetrates the diaphragm 27, so that the sample diluent flows into the sample pretreatment chamber 21. The sample diluent is a 0.01mM PBS buffer containing 1wt% BSA, 0.25wt% Tween, 0.1wt% trehalose, and 1wt% glycerol.

[0037] A filter membrane 22 is provided between the sample pretreatment chamber 21 and the antibody enrichment chamber 23. The filter membrane 22 is any one of a sterile microporous filter membrane, a PTFE composite membrane or a hemagglutinin-treated filter membrane, and is used to filter the sample treated by the sample pretreatment chamber to remove red blood cells in the sample.

[0038] The liquid-absorbing pad 29 and the chromatography pad 28 are both located in the antibody enrichment chamber 23, one end of the liquid-absorbing pad 29 is in contact with the filter membrane 22, and the other end is connected to the chromatography pad 28. The chromatography pad 28 is a chromatography pad soaked and adsorbed with a polypeptide that binds to bovine immunoglobulin and fully wraps the non-specific binding site of the antibody Fc segment, and the amino acid sequence of the polypeptide is WPIGRSTETAVL. After the chromatography pad 28 adsorbs the polypeptide WPIGRSTETAVL, it is subjected to a low-temperature drying treatment. The front and rear ends of the chromatography pad 28 are respectively connected to the liquid-absorbing pad 29 and the immunochromatographic test strip 241 in the immunochromatographic detection chamber 24.

[0039] The immunochromatographic detection chamber 24 is provided with an immunochromatographic test strip 241, two annular buckles 242 and an observation window 243. The two annular buckles 242 are fixedly installed in the immunochromatographic detection chamber 24. The front and rear ends of the immunochromatographic test strip 241 are respectively fixed by the two annular buckles 242. The observation window 243 is provided in the outer shell of the immunochromatographic detection chamber 24 and is located above the immunochromatographic test strip 241, so as to facilitate observation of the test results of the immunochromatographic test strip 241.

[0040] The immunochromatographic test strip 241 includes an NC membrane, a gold label pad, a sample pad, a water-absorbing pad and a PVC bottom plate. The sample pad, the gold label pad, the NC membrane and the water-absorbing pad are overlapped and adhered to the PVC bottom plate in sequence. The NC membrane includes a detection line and a quality control line arranged in sequence along the sample flow direction. The gold label pad contains colloidal gold-labeled LSDV-A27L-063-1 recombinant protein, the detection line is coated with anti-bovine IgG antibodies, and the quality control line is coated with polyclonal antibodies against the LSDV-A27L-063-1 recombinant protein. The sample pad extends into the antibody enrichment cavity 23 and overlaps with the chromatography pad 28.

[0041] When the device is in use, the protective cover 13 is opened, and one end of the siphon tube 12 extending out of the sampling hole 17 is placed against the venous skin of the sampled cattle, and the button 16 is pressed, the blood collection needle 14 pops out, and its needle tip extends out of the sampling hole 17. After piercing the skin, the blood collection needle 14 immediately retracts, and the blood flows out and is siphoned upward to the sample pretreatment chamber 21 by the tightly fitted siphon tube 12. The plastic needle 26 is pressed to pierce the film 27, so that the sample diluent enters the sample pretreatment chamber 21 and mixes with the collected blood sample. Subsequently, the mixed sample reaches the antibody enrichment chamber 23 through the filter membrane 22 through chromatography, and the filter membrane 22 filters out red blood cells. The liquid absorption pad 29 can effectively help the sample flow through the filter membrane 22 to the antibody enrichment chamber 23. The chromatography pad 28 in the antibody enrichment cavity 23 is pretreated with a polypeptide WPIGRSTETAVL that can bind to bovine immunoglobulin and fully wrap the non-specific binding site of the antibody Fc segment. When the sample flows through this cavity, the antibodies in the serum sample bind to the polypeptide WPIGRSTETAVL with a high affinity, which can compete with other large proteins or impurities that are easy to non-specifically bind to the antibodies in the serum, reduce their resistance to serum chromatography, and thus speed up the chromatography speed of the sample. It can not only play a role in enriching the antibodies in the serum, but also promote the chromatography of the sample toward the immunochromatographic test strip 241 in the immunochromatographic detection cavity 24, color development, and thus achieve the purpose of detection. Through the observation window 243 on the immunochromatographic detection cavity 24, the test results of the immunochromatographic test strip 241 can be directly observed.

[0042] Example 2 Screening of polypeptides with high affinity binding to bovine serum antibody Fc segment and preparation of chromatography pad

[0043] 2.1 Molecular simulation software and equipment

[0044] All molecular simulations and calculations were performed using the molecular simulation software CHARMM and SYBYL 6.92 (Tripos).

[0045] 2.2 Construction of peptide library

[0046] The initial conformation of the peptide was drawn using CHARMM software combined with the CHARMM27 force field, and then the initial conformation was optimized using the energy minimization method. Steepest Descent and adopted basis Newton-Raphson algorithms were used for minimization, with a maximum number of optimizations of 500 times, an energy convergence of 0.05 kcal / (mol•Å), and the rest were set to default values.

[0047] 2.3 Amino Acid Residues of Bovine Immunoglobulin Fc Region

[0048] The residues of the Fc fragment of bovine immunoglobulin G1 (GenBank No. AAB37381.2) were obtained by searching the NCBI database, which consisted of 108 amino acids from 222 to 329, as shown in SEQ ID NO: 14, and consisted of the following amino acids:

[0049] PAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK

[0050] 2.4 Molecular docking

[0051] Molecular docking uses the Flex X2 module in the SYBYL software. Flex X2 is a fast semi-flexible docking program that uses a fragment growth algorithm to fully consider the flexibility of the ligand and treat the receptor cavity as rigid. The software automatically adds formal charge during docking, and the maximum number of dockings for each peptide is set to 200. The drugscore scoring function is used to evaluate the affinity of the peptide and the Fc fragment. The peptide length is set to 10-15 amino acids, and the rest of the parameters use the default values ​​of FlexX2.

[0052] Affinity peptides used for purification are generally selected with an affinity of 10 4 ~10 8 M -1 Because if the affinity is too weak, the target protein cannot be adsorbed, and if it is too strong, elution will be difficult. In the present invention, the affinity range is set to 10 in order to utilize the high affinity of the affinity peptide to the Fc segment of the immunoglobulin to reduce non-specific reactions and improve the detection sensitivity without elution. 5 ~10 10 M -1 After software screening and scoring, 4 peptides were finally selected for subsequent experimental research. The amino acid sequences of the 4 peptides are:

[0053] Polypeptide A (SEQ ID NO: 15): FIRWTCLDSVAG

[0054] Polypeptide B (SEQ ID NO: 16): WPIGRSTETAVL

[0055] Polypeptide C (SEQ ID NO: 17): QANAFDYETIEL

[0056] Polypeptide D (SEQ ID NO: 18): RHWRGSWTQVEI

[0057] The above peptide AD was synthesized by Hangzhou Dangang Biotechnology Co., Ltd., and the mass spectrum is attached. Figure 2-5 The above polypeptide AD has a high affinity with the bovine immunoglobulin Fc segment and can encapsulate the non-specific binding site of the Fc segment, thereby reducing the non-specific reaction caused by the non-specific binding site of the Fc segment during the detection process.

[0058] 2.5 Preparation of chromatography pad

[0059] Dissolve the four polypeptides in PBS at 1 mg / mL respectively, then place the chromatography pad in the corresponding polypeptide solution, soak it at 4°C overnight, dry it at low temperature the next day, cut it into appropriate sizes, seal it and store it for later use.

[0060] Example 3 Preparation of Bovine Lumpy Dermatosis Virus Neutralizing Antibody Immunochromatographic Test Strip

[0061] Materials, reagents and instruments used in the examples:

[0062] Material

[0063] 1. Bacteria

[0064] Escherichia coli competent cells DH5α and DH10Bac were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and pFastbac1 vector and sf9 cells were preserved by the Animal Inspection and Quarantine Laboratory of the Chinese Academy of Inspection and Quarantine;

[0065] 2. Serum

[0066] The LSDV-positive serum inactivated by 0.3% TNBP and 1% triton X-100 was kindly donated by China Animal Husbandry Research Institute, the GPTV-positive serum, SPPV-positive serum, PPRV vaccine virus-positive serum, and Akabane disease (AKAV)-positive serum inactivated by 0.3% TNBP and 1% triton X-100 were stored by China Animal Disease Prevention and Control Center, and the bovine negative control serum was purchased from Gibco.

[0067] Reagents

[0068] SIM SF serum-free medium was purchased from Beijing Sino Biotech Co., Ltd., Ni-NTA agarose gel resin and plasmid extraction kit were purchased from QIAGEN, BCA protein concentration assay kit was purchased from Beijing Solebow Technology Co., Ltd., fetal bovine serum was purchased from Gibico, BamH I / Xho I restriction endonuclease, T4 ligase, TaKaRa LA PCR™Kit were purchased from TaKaRa, DNA extraction kit DNeasy Blood and Tissue Kit was purchased from Qiagen, gel recovery kit Agarose Gel DNA Purification Kit, IPTG, X-gal, and ampicillin were purchased from Beijing Quanshijin Biotechnology Co., Ltd., HRP-rabbit anti-bovine IgG and HRP-rabbit anti-goat IgG were purchased from Bio-Aolong, and rabbit anti-bovine IgG secondary antibody was purchased from Beijing Bio-Ao Lab Technology Co., Ltd.;

[0069] instrument

[0070] ABI's ordinary PCR instrument (AB Applied Biosystems), Sigma's desktop centrifuge (3-18k), constant temperature shaking incubator (HZQ-F100), fluorescence microscope (ZEISS, AXIO), 6-1 electrophoresis instrument, dual infrared laser scanning imaging system (Proteinsimple), and microplate reader.

[0071] 3.1 Preparation and identification of A27L-063 recombinant protein for detection of neutralizing antibodies against bovine lumpy skin disease virus

[0072] 3.1.1 Design and construction of A27L-063 recombinant protein

[0073] Based on the full-length amino acid sequence of the LSDV A27L protein of the Xinjiang strain of bovine nodular skin disease virus (OP508345.1) published in GenBank, as shown in SEQ ID NO: 1, after bioinformatics analysis, the epitope prediction of the LSDV A27L protein was performed, and two dominant epitope regions were obtained, and their amino acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3. Based on the full-length amino acid sequence of the LSDV063 protein of the Xinjiang strain of bovine nodular skin disease virus (OP508345.1) published in GenBank, as shown in SEQ ID NO: 4, after bioinformatics analysis, the epitope prediction of the LSDV063 protein was performed, and three dominant epitope regions were obtained, and their amino acid sequences are shown in SEQ ID NO: 5-SEQ ID NO: 7.

[0074] Two designs were made for the LSDV-A27L-063 recombinant protein. The first design was to sequentially connect the N-terminal amino acid sequence of the LSDV A27L protein containing the dominant epitope region 1 and the dominant epitope region 2 of the A27L protein (SEQ ID NO: 8) with the dominant epitope region 1 (SEQ ID NO: 2) and the dominant epitope region 2 (SEQ ID NO: 3) of the LSDV A27L protein through a flexible linker (GGGGGGGG), and then continue to couple the N-terminal amino acid sequence of the LSDV063 protein containing the dominant epitope region 1 and the dominant epitope region 2 of the LSDV-063 protein (SEQ ID NO: 9) through a flexible linker (GGGGGGGG), and finally sequentially couple the amino acid sequence of the dominant epitope region 1 (SEQ ID NO: 5) and the amino acid sequence of the dominant epitope region 2 (SEQ ID NO: 6) of LSDV-063 to form the final tandem recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 10, denoted as LSDV-A27L-063-1 protein, and the nucleotide sequence of the tandem recombinant protein after codon optimization is shown in SEQ ID NO: 11.

[0075] The second design is to couple the N-terminal amino acid sequence of the A27L protein containing the dominant epitope region 1 and the dominant epitope region 2 of A27L (SEQ ID NO: 8) with the N-terminal amino acid sequence of the LSDV-063 protein containing the dominant epitope region 1 and the dominant epitope region 2 of the LSDV-063 protein (SEQ ID NO: 9) and the dominant epitope region 3 of the LSDV-063 protein (SEQ ID NO: 7) through a flexible linker (GGGGGGGG) to form a recombinant tandem protein, whose amino acid sequence is shown in SEQ ID NO: 12, recorded as LSDV-A27L-063-2 protein, and the nucleotide sequence of the tandem recombinant protein after codon optimization is shown in SEQ ID NO: 13.

[0076] The nucleotide sequences of the above two recombinant tandem proteins were artificially synthesized by Beijing Qingke Biotechnology Co., Ltd. and inserted into the pfastbac1 vector. BamH I and Xho I restriction sites were introduced at both ends of the sequence, and 10 His and 3 Flags were introduced at the N-terminus of the sequence to construct recombinant plasmids pFastBac-LSDV-A27L-063-1 and pFastBac-LSDV-A27L-063-2, respectively. Escherichia coli DH5α competent cells were transformed, plated and cultured overnight, and positive clones were picked for sequencing the next day. The positive bacterial solution with correct sequencing was extracted to obtain the recombinant plasmids pFastBac-LSDV-A27L-063-1 and pFastBac-LSDV-A27L-063-2 with correct sequencing for use.

[0077] 3.1.2 Construction and identification of recombinant baculovirus shuttle plasmid

[0078] 3.1.2.1 Construction of recombinant baculovirus shuttle plasmid

[0079] Take 1 μL of the correctly sequenced recombinant plasmid pFastBac-LSDV-A27L-063-1 and add it to 100 μL of E. coli DH10bac competent cells, mix gently, place on ice for 30 min, heat shock in a 42°C water bath for 45 s, and immediately place on ice for 2 min; add 900 μL of preheated LB medium and culture on a shaker at 37°C and 225 r / min for 4 h to allow the bacteria to recover; take the cultured bacterial liquid and use LB medium for 10 -1 , 10 -2 and 10 -3 Three dilutions were prepared, and 100 μL of each was added with x-gal and IPTG, and then spread on triple antibody (gentamicin, kanamycin and tetracycline) screening plates, cultured at 37°C in the dark for 48 h, and then white colonies were selected for identification.

[0080] The recombinant plasmid pFastBac-LSDV-A27L-063-2 was transformed and identified using the same steps as above.

[0081] 3.1.2.2 Extraction and identification of recombinant baculovirus shuttle plasmid

[0082] A single white colony containing the recombinant plasmid pFastBac-LSDV-A27L-063-1 was picked from the plate and inoculated into 100 ml LB liquid medium containing gentamicin (7 µg / mL), kanamycin (50 µg / mL) and tetracycline (10 µg / mL), and cultured at 37°C and 180 r / min for 12 h; the cells were collected by centrifugation at room temperature at 12,000 r / min for 1 min, the supernatant was discarded, and the recombinant baculovirus shuttle plasmid was extracted using Qiagen's plasmid mini kit (containing solutions I, II and III), 300 μL of pre-cooled solution I was added to resuspend the cells, and vortexed to completely disperse them; 300 μL of solution II was added, gently inverted to mix, and allowed to stand on ice for 5 min, and slowly Add 300 μL of precooled solution III, gently invert to mix, let stand on ice for 10 min, centrifuge at 4°C and 12,000 r / min for 15 min; aspirate the supernatant to another centrifuge tube, centrifuge at 4°C and 12,000 r / min for 15 min, aspirate the supernatant to another centrifuge tube, add an equal amount of -20°C precooled isopropanol, gently invert to mix, let stand at -20°C for 30 min, centrifuge at 4°C and 12,000 r / min for 15 min; discard the supernatant, gently add 1 mL of -20°C precooled 75% ethanol to wash the precipitate, discard the liquid, wash again in the same way, absorb as much ethanol as possible, place the precipitate in a ventilated place to dry for 15 min, add 50 μL of RNAse water to dissolve the precipitate at 37°C for 30 min, and then store the extracted plasmid at 4°C to obtain the recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1.

[0083] Because the bacmid DNA genome of baculovirus is very large (>135kb), it is difficult to identify whether the foreign gene is successfully transposed by double enzyme digestion reaction, and the PCR method is generally used for identification. In the present invention, by analyzing the structure of bacmid DNA, M13 upstream primer and M13 downstream primer are used to carry out PCR amplification with the extracted recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1 as a template. After the plasmid with successful gene insertion is amplified by M13 primers, it can be seen that the size is a target band of about 3200bp, while the plasmid with unsuccessful gene insertion has only a target band of about 300bp after amplification by M13 primers.

[0084] The same steps as above were used to extract and identify the white single colony containing the recombinant plasmid pFastBac-LSDV-A27L-063-2 to obtain the recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-2.

[0085] 3.1.3 Preparation and identification of recombinant baculovirus

[0086] 3.1.3.1 Preparation of recombinant baculovirus

[0087] The recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1 was transfected into sf9 cells. The specific steps are as follows: one day before transfection, sf9 cells were inoculated in a 6-well cell culture plate. When the cell monolayer grew to 70%-80%, transfection was prepared. Two 1.5mL sterilized centrifuge tubes were taken, 6μL Cellfectin Reagent and 94μL Grace basal medium were added to one tube, and gently mixed. In the other tube, 50μL of the bacmid DNA to be transfected and 50μL Grace basal medium were added, and gently mixed. The mixture was allowed to stand at room temperature for 5min. The two tubes of liquid were mixed, gently mixed, and allowed to stand at room temperature for 20min. During this period, the medium in the wells of the cell culture plate was aspirated and discarded, and the cells were washed twice with Grace basal medium, and the liquid was aspirated and discarded. 800μL Grace basal medium was added to the 1.5mL centrifuge tube and gently added to the cell surface. The cell culture plate was placed in a 27°C incubator. After culturing for 5h, the supernatant was discarded and 2mL The culture was continued in Grace's complete medium for 3-5 days. When obvious cytopathic effects were observed, the supernatant was collected, centrifuged to remove cell debris, and appropriate amounts were aliquoted and stored at 4°C to obtain the P1 generation recombinant baculovirus of LSDV-A27L-063-1;

[0088] Inoculate serum-free sf9 cells in a T75 cell culture flask to make the monolayer spread to 70%-80%. After 2 hours, inoculate the cells with the P1 generation recombinant baculovirus of LSDV-A27L-063-1 at a volume of 1:50, and place it in a 27°C incubator for about 3 days. Obvious cytopathic effects can be seen. At this time, collect the cell supernatant and centrifuge at 5000r / min for 5 minutes to remove the cell precipitate, and store it in appropriate amounts at 4°C to obtain the P2 generation recombinant baculovirus. In the same way, inoculate the cells with the P2 generation recombinant baculovirus at a volume of 1:50, then culture, collect the supernatant, and centrifuge to remove the precipitate to prepare the P3 generation recombinant baculovirus of LSDV-A27L-063-1.

[0089] The recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-2 was used to prepare the P1-P3 recombinant baculovirus of LSDV-A27L-063-2 by the same steps as above.

[0090] 3.1.3.2 Identification of recombinant baculovirus

[0091] 3.1.3.2.1 Virus identification and sequence analysis

[0092] The nucleic acid of each passage of virus culture fluid was extracted respectively, and common PCR primers were designed according to the optimized nucleotide sequence fragments shown in SEQ ID NO: 11 and SEQ ID NO: 13 to amplify the extracted nucleic acid samples to detect whether the recombinant baculovirus was successfully packaged.

[0093] It was determined that the P1, P2, and P3 recombinant baculoviruses all amplified the target bands by PCR, and the sizes were consistent with expectations, indicating that the P1, P2, and P3 recombinant baculoviruses containing LSDV-A27L-063-1 and the P1, P2, and P3 recombinant baculoviruses containing LSDV-A27L-063-2 of this example were successfully packaged.

[0094] 3.1.3.2.2 Expression characteristics identification

[0095] The corresponding P2 generation recombinant baculovirus was expressed in a small amount to determine the expression characteristics of the virus. The corresponding P2 generation recombinant baculovirus was infected with sf9 generation cells at a ratio of 1:50, and the cells were collected, then resuspended with 3mL buffer, and placed in a 10mL culture bottle for ultrasound, ultrasound for 3s, interval 5s, ultrasound duration 7min, then 15000 rpm, 4℃ centrifugation for 1h, the supernatant was combined with the treated beads for 2h, and then the impurities were washed with 5ml each time, washed 3 times, and finally resuspended with 500μL buffer for SDS-GEL identification.

[0096] The identification results are as follows Figure 6 As shown, the recombinant protein LSDV-A27L-063-1 has a band at about 33KD, and the recombinant protein LSDV-A27L-063-2 has a band at about 24KD, both of which are in line with the expected target band size. Both the recombinant protein LSDV-A27L-063-1 and the recombinant protein LSDV-A27L-063-2 are soluble, and can form a correctly folded functional conformation during the expression of eukaryotic cells, which is closer to the structure and function of the real protein, avoiding the biosafety risks brought by the use of real viruses.

[0097] 3.1.4 Large-scale expression and purification of recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2

[0098] The corresponding P3 generation recombinant baculovirus was used to infect sf9 suspension cells in the logarithmic growth phase in a culture flask at a volume ratio of 1:50, and the cells were placed in a 28°C incubator and cultured at 120 rpm. After induction of expression for 48-72 hours, the transfected cells were collected by centrifugation at 1000×g for 20 minutes, and the cell pellet was resuspended with a resuspension buffer containing 20 mM hepes buffer, pH 7.4, 300 mM NaCl and PMSF protease inhibitor, sonicated on ice for 3 minutes, and centrifuged at 160000 rpm at 4°C for 20 minutes. The supernatant was removed from the impurities with a 0.22μm filter, and anti-flag tag affinity magnetic beads were added to the supernatant for binding. The bound protein was washed twice with resuspension buffer, and then the target protein was eluted with elution buffer (0.1 mg / mL 3×flag peptide, 20 mM HEPES and 300 mM NaCl, pH 7.4). The eluate was further purified using a Superdex 200 10 / 300GL prepacked column, and the impurities were eluted with a 20mM imidazole buffer (20mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH7.5), and the target protein was eluted with 200mM imidazole (20mM Tris-HCl, 300mM NaCl, 200mM imidazole, pH7.5). The eluate of the recombinant protein was collected, and the target protein was concentrated to a certain volume using a 10KD ultrafiltration concentrator. The protein concentration was determined using the BCA method, and the concentration was adjusted to 1 mg / mL with PBS. After aliquoting, it was stored at -80°C for future use. The recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2 were obtained according to the above steps.

[0099] 3.1.5 Identification of recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2

[0100] Take appropriate amount of recombinant protein LSDV-A27L-063-1 and LSDV-A27L-063-2 samples for SDS-PAGE, take out the gel, prepare polyacrylamide gel-membrane "sandwich" in the order of filter paper-gel-PVDF membrane-filter paper for transfer, remove the PVDF membrane for incubation and washing of primary and secondary antibodies, and identify the specificity of recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2. The primary antibodies are inactivated LSDV positive serum, GTPV positive serum, SPPV positive serum, PPRV vaccine virus positive serum, Akabane disease (AKAV) positive serum, and bovine negative serum, and the secondary antibodies are HRP-labeled rabbit anti-bovine IgG and rabbit anti-goat IgG.

[0101] The identification results are as follows Figure 7 As shown, from Figure 7It can be seen that the recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2 only showed obvious positive reactions with inactivated LSDV positive serum, GTPV positive serum, and SPPV positive serum, but did not cross-react with PPRV vaccine virus positive serum, Akabane disease (AKAV) positive serum, and cattle negative serum, proving that the recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2 have good specificity and can simultaneously detect LSDV and sheeppox virus.

[0102] 3.1.6 Immunogenicity of recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2

[0103] 100 μg of recombinant protein LSDV-A27L-063-1 was mixed with an equal volume of Freund's complete adjuvant and then immunized with two 8-week-old BALB / c mice by intraperitoneal injection. At the same time, two negative mice were immunized with Freund's complete adjuvant emulsified with PBS. The second immunization was performed 14 days later. The positive group of mice was immunized with an equal volume of Freund's incomplete adjuvant and 100 μg of recombinant protein LSDV-A27L-063-1, and the negative group of mice was treated in the same way. The third immunization was performed 28 days later. 100 μg of recombinant protein LSDV-A27L-063-1 was used to immunize the positive group of mice, and the negative group of mice was immunized with PBS. 14 days after the three immunizations, the blood of mice was collected and the serum was collected to obtain the polyclonal antibody of the recombinant protein LSDV-A27L-063-1. The antibody was diluted at 1:10000, 1:20000, 1:40000~1:40960000 and then detected by indirect ELISA to determine the specific antibody titer in the serum of the immunized mice after the three immunizations.

[0104] The recombinant protein LSDV-A27L-063-2 was prepared using the same steps as above to obtain a polyclonal antibody to the recombinant protein LSDV-A27L-063-2, and the polyclonal antibody was diluted at 1:10000, 1:20000, 1:40000~1:40960000, and then detected by indirect ELISA to determine the specific antibody titer in the serum of immunized mice after three immunizations.

[0105] Test results such as Figure 8 As shown, the titer of the mouse serum after three immunizations of the recombinant protein LSDV-A27L-063-1 was 1:20480000, and the titer of the mouse serum after three immunizations of the recombinant protein LSDV-A27L-063-2 was 1:2560000, which proved that the immunogenicity of the recombinant protein LSDV-A27L-063-1 was better than that of LSDV-A27L-063-2, and it had better immunogenicity and antigenicity, and was more conducive to the binding and detection of LSDV neutralizing antibodies in bovine serum.

[0106] The polyclonal antibody serum samples of the recombinant proteins LSDV-A27L-063-1 and LSDV-A27L-063-2 were purified by protein purifier, and the target elution bands were collected, concentrated, and the protein concentration was determined. The protein concentration was adjusted to 1 mg / mL with PBS and stored in aliquots for later use.

[0107] 3.2 Preparation of immunochromatographic test strips for detecting neutralizing antibodies against LSDV

[0108] 3.2.1 Preparation of colloidal gold solution

[0109] Dilute 1% chloroauric acid solution to 0.01% (mass fraction) with double distilled deionized water, take 100 mL of 0.01% chloroauric acid solution and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, quickly add 1.6 mL of 1% trisodium citrate solution at one time, continue stirring at a uniform speed, stop stirring when the solution turns translucent red, continue heating for 5 minutes after the color stabilizes, cool to room temperature, and restore to the original volume with deionized water to obtain a colloidal gold solution, which is stored at 4°C;

[0110] 3.2.2 Preparation of recombinant protein colloidal gold label

[0111] Take 100 mL of the prepared 0.01% colloidal gold solution, adjust the pH value of the colloidal gold solution to about 8.5 with 0.1 mol / L potassium carbonate under magnetic stirring, add 30-40 μg of recombinant protein per milliliter of colloidal gold solution, add the prepared recombinant protein to the colloidal gold solution, stir evenly and let stand at room temperature 25°C for 20 min. 1 mL of 10% bovine serum albumin (BSA) solution was added, and the mixture was stirred with a magnetic stirrer (speed 150 r / min, 30 min). After standing for 20 min, the mixture was centrifuged at 12000 rpm for 40 min at 4°C, and the supernatant was discarded. The remaining precipitate was resuspended with 20 ml of colloidal gold resuspension solution (2% sucrose, 1% BSA, 0.3% Tween 20, prepared with 0.01 mol / L phosphate buffer solution with a pH of 7.4) to prepare the colloidal gold label of the recombinant protein LSDV-A27L-063-1 and the colloidal gold label of LSDV-A27L-063-2, and stored at 4°C for future use;

[0112] 3.3 Preparation of colloidal gold detection device

[0113] The above two recombinant protein colloidal gold labels were evenly sprayed on the gold label pad at an amount of 5 μL / cm by using a film sprayer, and dried at 37°C for 2 hours. A detection area (T line) and a quality control area (C line) were provided on the NC membrane. The detection area (T line) was coated with commercial rabbit anti-bovine antibodies, and the quality control area (C line) was coated with the polyclonal antibody against the recombinant protein LSDV-A27L-063-1 or the polyclonal antibody against the recombinant protein LSDV-A27L-063-2 prepared in 3.1.6 of Example 3, respectively. The coating concentration of the detection area (T line) and the quality control area (C line) was 1:1, ranging from 0.5 mg / mL to 0.8 mg / mL. The NC membrane, gold label pad, sample pad, and absorbent pad were sequentially pasted on the PVC base plate, and cut into 4 mm wide test strips after pasting to prepare immunochromatographic test strips for detecting LSDV neutralizing antibodies, which were stored at room temperature.

[0114] 3.3 Preparation of full-length immunochromatographic test strips of LSDV-A27L and LSDV-063 proteins

[0115] 3.3.1 Preparation of full-length recombinant proteins of LSDV-A27L and LSDV-063

[0116] The recombinant proteins LSDV-A27L and LSDV-063 full-length proteins were prepared according to the method in 3.1 of Example 3 and labeled as LSDV-A27L-F and LSDV-063-F, respectively.

[0117] 3.3.2 Preparation of polyclonal antibodies against LSDV-A27L-F and LSDV-063-F

[0118] Polyclonal antibodies to LSDV-A27L-F and LSDV-063-F were prepared according to the method in 3.1.6 of Example 3.

[0119] 3.3 Preparation of LSDV-A27L-F and LSDV-063-F immunochromatographic test strips

[0120] The immunochromatographic test strips of LSDV-A27L-F and LSDV-063-F were prepared by referring to the method in 3.2 of Example 3.

[0121] 3.4 Comparison of test results of four immunochromatographic test strips

[0122] 3.4.1 Test result determination

[0123] The results are determined by comparing the color depth of the quality control area (C line) and the test area (T line). Determination criteria:

[0124] Positive: When both the quality control area (C line) and the test area (T line) show bands, it is judged as positive, that is, the sample contains LSDV neutralizing antibodies, which is indicated by “+”;

[0125] Negative: When the quality control area (C line) shows a band and the detection area (T line) does not show color, it is judged as negative, that is, there is no LSDV neutralizing antibody in the sample or the content of LSDV neutralizing antibody is lower than the detection limit, which is indicated by "-";

[0126] Invalid: When no band is displayed in the quality control area (C line), the test strip is invalid.

[0127] 3.4.2 Comparison of test results of four immunochromatographic test strips

[0128] Four types of immunochromatographic test strips were used to simultaneously detect LSDV-positive serum and control serum (GPTV-positive serum, SPPV-positive serum, PPRV vaccine virus-positive serum, Akabane disease (AKAV)-positive serum) in multiple dilution ratios. The results are shown in Table 2.

[0129] Table 2 Comparison of sensitivity and specificity of four immunochromatographic test strips

[0130]

[0131] As shown in Table 2, the LSDV-A27L-063-1 immunochromatographic test strip prepared in Example 3 of the present invention has better detection sensitivity and specificity. The LSDV-A27L-063-1 immunochromatographic test strip can detect LSDV positive serum diluted 1:500, and can simultaneously detect LSDV, GTPV, and SPPV, and has no cross-reaction with the control viruses PPRV and AKAV. The sensitivity of the LSDV-A27L-063-2 immunochromatographic test strip is also high, but lower than that of the LSDV-A27L-063-1 immunochromatographic test strip. Due to the use of the full-length antigen sequence, the LSDV-A27L-F immunochromatographic test strip and the LSDV-063-F immunochromatographic test strip not only have low detection sensitivity, but also have non-specific reactions, cross-react with PPRV positive serum, and have poor specificity. Therefore, the LSDV-A27L-063-1 immunochromatographic test strip was selected for the assembly of the portable immunochromatographic device.

[0132] Example 4 Assembly and application of a portable immunochromatographic device for detecting neutralizing antibodies to bovine lumpy dermatosis virus

[0133] 4.1 Test animals: 4-month-old calves immunized with the recombinant protein LSDV-A27L-063-1 and healthy 4-month-old calves.

[0134] 4.2 Assembly of portable immunochromatographic device:

[0135] The LSDV-A27L-063-1 immunochromatographic test strip is placed in the immunochromatographic detection cavity of the portable immunochromatographic device of Example 1, so that one end of the sample pad of the LSDV-A27L-063-1 immunochromatographic test strip overlaps the chromatography pad, and the portable immunochromatographic device is placed in a sealed bag, desiccant is added, and sealed to prepare a portable immunochromatographic detection device for detecting bovine nodular dermatitis virus neutralizing antibodies, which is stored at room temperature.

[0136] According to the different polypeptides adsorbed by the chromatographic pad of the antibody enrichment cavity, four types of portable immunochromatographic devices were prepared, which were named Type A, Type B, Type C and Type D portable immunochromatographic devices respectively. At the same time, a control device with a blank chromatographic pad that had not undergone any treatment was set up, which was named Type E portable immunochromatographic device.

[0137] 4.3 Detection: Place the above-mentioned portable immunochromatographic devices of type A, type B, type C, type D and type E close to the ear vein of the calf, press the button, collect blood samples and perform the test. After the sample to be tested is siphoned into the sample pretreatment chamber, press the plastic needle to puncture the diaphragm, release the sample diluent into the sample pretreatment chamber, and place the device horizontally to facilitate the mixing of samples for chromatography. After the sample enters the LSDV-A27L-063-1 immunochromatographic test strip, start the timing and interpret the results.

[0138] After testing and comparison, it was found that within 5 minutes, the blood sample of the type B portable immunochromatographic device can be chromatographed to the immunochromatographic test strip, and after another 5 minutes, a darker band can appear on the T line. Through the observation window on the top of the device, it can be clearly seen that the 4-month-old calves with immune recombinant protein LSDV-A27L-063-1 showed obvious red bands in the test area (T line) and the quality control area (C line), while the healthy 4-month-old calves only showed obvious red bands in the quality control area (C line); while for the three types of portable immunochromatographic devices A, C, and D, it takes 5 to 10 minutes for the blood sample to be chromatographed to the immunochromatographic test strip, and after another 5 minutes, the T line begins to show color, but the final color is lighter than the former; and for the type E portable immunochromatographic device, it takes at least 15 minutes for the blood sample to be chromatographed to the immunochromatographic test strip, and after another 10 minutes, the T line begins to slowly show color. This indicates that when blood flows through the chromatography pad treated with polypeptide B, polypeptide B will bind to the Fc segment of immunoglobulin in bovine blood, wrap the non-specific binding site of the Fc segment, and reduce the binding of other macromolecules and impurities in the blood to the immunoglobulin Fc segment, thereby accelerating the chromatography speed of the blood and improving the detection sensitivity.

[0139] In summary, the LSDV-A27L-063-1 portable immunochromatographic device prepared by the present invention and equipped with a chromatography pad treated with polypeptide B has high sensitivity and specificity, can be used for the detection of bovine nodular dermatitis virus neutralizing antibodies, and can also be used for the detection of bovine nodular dermatitis antibodies, playing an auxiliary diagnosis role in LSDV infection.

[0140] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatitis virus, characterized in that: The invention comprises a sampling body and a detection body connected in an integral manner front and back, wherein a spring sampler and a siphon are arranged in the sampling body, wherein the spring sampler comprises a blood collection needle, wherein the blood collection needle is tightly fitted with the siphon, wherein a sample pretreatment chamber, a filter membrane, an antibody enrichment chamber and an immunochromatographic detection chamber which are sequentially connected front and back are arranged in the detection body, wherein the siphon is communicated with the sample pretreatment chamber, wherein a diluent storage chamber and a plastic needle are arranged on the outside of the sample pretreatment chamber, wherein the diluent storage chamber is communicated with the sample pretreatment chamber, and the two are sealed and separated by a diaphragm, wherein a sample diluent is stored in the diluent storage chamber, wherein the plastic needle is located in the diluent storage chamber and above the diaphragm, wherein a liquid absorption pad and a chromatography pad are arranged in the antibody enrichment chamber, wherein the front and rear ends of the liquid absorption pad are respectively connected with the filter membrane and the chromatography pad, wherein the immunochromatographic detection chamber is provided with a liquid absorption pad. An immunochromatographic test strip is provided, wherein the front and rear ends of the chromatography pad are respectively connected to the absorbent pad and the immunochromatographic test strip, the immunochromatographic test strip comprises an NC membrane, a gold-labeled pad, a sample pad, a water-absorbing pad and a PVC bottom plate, the sample pad, the gold-labeled pad, the NC membrane and the water-absorbing pad are overlapped and adhered to the PVC bottom plate in sequence, the NC membrane comprises a detection line and a quality control line arranged in sequence along the sample flow direction, the gold-labeled pad comprises a colloidal gold-labeled LSDV-A27L-063-1 recombinant protein, the detection line is coated with an anti-bovine IgG antibody, the quality control line is coated with a polyclonal antibody against the LSDV-A27L-063-1 recombinant protein, the sample pad extends into the antibody enrichment cavity and overlaps with the chromatography pad, and the amino acid sequence of the LSDV-A27L-063-1 recombinant protein is as shown in SEQ As shown in ID NO: 10, the chromatography pad is soaked with a polypeptide that can bind to bovine immunoglobulin and fully wrap the non-specific binding site of the antibody Fc segment, the sequence of the polypeptide is WPIGRSTETAVL, and the chromatography pad is subjected to low-temperature drying after adsorbing the polypeptide.

2. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatosis virus according to claim 1, characterized in that: The spring sampler also includes a button and a spring, one end of the spring is connected to the blood collection needle, and the other end is connected to the button, the button is located on the outside of the sampling body, the sampling body is also provided with an openable protective cover, one end of the sampling body is provided with a sampling hole, the protective cover is covered on the sampling hole, one end of the siphon tube extends out of the sampling hole and is located inside the protective cover.

3. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatosis virus according to claim 1, characterized in that: The immunochromatographic detection cavity is also provided with two annular buckles and an observation window. The two annular buckles are fixedly installed in the immunochromatographic detection cavity. The front and rear ends of the immunochromatographic test strip are respectively fixed by the two annular buckles. The observation window is provided in the outer shell of the immunochromatographic detection cavity and is located above the immunochromatographic test strip, so as to facilitate the observation of the test results of the immunochromatographic test strip.

4. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatosis virus according to claim 1, characterized in that: The sample diluent is a 0.01 mM PBS buffer solution containing 1 wt% BSA, 0.25 wt% Tween, 0.1 wt% trehalose, and 1 wt% glycerol. The filter membrane is any one of a sterile microporous filter membrane, a PTFE composite membrane, or a filter membrane treated with hemagglutinin.

5. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatosis virus according to claim 1, characterized in that: The method for preparing the LSDV-A27L-063-1 recombinant protein according to claim 1 comprises the following steps: S1. The amino acid sequence shown in SEQ ID NO: 8 is sequentially connected in series with the amino acid sequences shown in SEQ ID NO: 2-3 through a flexible linker, and then connected in series with the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence shown in SEQ ID NO: 5, and the amino acid sequence shown in SEQ ID NO: 6 through a flexible linker to obtain the amino acid sequence shown in SEQ ID NO: 10, which is the LSDV-A27L-063-1 recombinant protein; S2. The amino acid sequence shown in SEQ ID NO: 10 was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO: 11, which was inserted into the pFastbac1 vector to construct a recombinant plasmid pFastBac-LSDV-A27L-063-1 with correct sequencing; S3. The recombinant plasmid pFastBac-LSDV-A27L-063-1 was transferred into Escherichia coli DH10bac competent cells, cultured, extracted and identified to obtain a recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1; S4. The recombinant baculovirus shuttle plasmid bacmid-LSDV-A27L-063-1 is transfected into sf9 cells, cultured, and inoculated to obtain recombinant baculovirus, and the recombinant baculovirus is induced to express, and the LSDV-A27L-063-1 recombinant protein with the amino acid sequence shown in SEQ ID NO: 10 is obtained after separation and purification, i.e., the recombinant protein for detecting bovine lumpy dermatitis virus neutralizing antibodies.

6. A portable immunochromatographic device for detecting neutralizing antibodies against bovine nodular dermatitis virus according to claim 1, characterized in that: The polyclonal antibody is prepared by immunizing animals with the LSDV-A27L-063-1 recombinant protein according to claim 1.

7. Use of a portable immunochromatographic device for detecting neutralizing antibodies to bovine lumpy skin disease virus according to claims 1-6 in immunological detection of neutralizing antibodies to bovine lumpy skin disease virus for non-disease diagnosis purposes.

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