Preparation and application of colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nano antibody

Abstract

The invention discloses preparation and application of a colloidal gold test strip for detecting porcine epidemic diarrhea virus (PEDV) based on a nano antibody, and belongs to the technical field of animal pathogen detection. According to the invention, two screened specific nano antibodies resisting PEDV spike glycoprotein are taken as base materials, genetic engineering modification is carried out on the nano antibodies, and the nano antibodies are respectively subjected to fusion expression with an Fc region of porcine IgG and ferritin, so that two fusion proteins are successfully prepared. Then, the two fusion proteins serve as paired antibodies, the protein fused with porcine IgG Fc is subjected to colloidal gold labeling to serve as a detection antibody, the other protein fused with ferritin serves as a capture antibody, and a PEDV colloidal gold detection test strip is established. The test strip has the advantages of high specificity, high sensitivity, simplicity and convenience in operation, low production cost and the like, and can be applied to rapid detection of PEDV in a porcine diarrhea sample clinically.

Description

Technical Field

[0001] The invention relates to the technical field of animal pathogen detection, and in particular to the preparation and application of a colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nano antibodies. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious diarrheal disease of pigs caused by infection with porcine epidemic diarrhea virus (PEDV). After the virus invades the body, it mainly destroys the epithelial cells and intestinal villi of the pig's intestinal tissue and damages the intestinal mucosa of the pig. PEDV infection in piglets under one week old can cause severe diarrhea and vomiting, resulting in a mortality rate of 80% to 100%. At present, PEDV mainly has two subgroups, GI and GII, of which the classic strain (CV777) belongs to the GI-b subgroup. However, since 2010, an outbreak of suspected highly pathogenic PEDV variants has occurred worldwide, causing huge economic losses to the global pig industry. Genetic sequence analysis of the new PEDV variant strain found that the variant strain belongs to the GII-a type, and the existing GI type strain vaccine cannot provide complete protection. At present, piglet diarrhea caused by PEDV infection has become one of the key infectious diseases controlled in pig farms in my country. Therefore, a PEDV detection method that is simple, rapid and highly specific in clinical operation is of great significance for the prevention and control of the disease.

[0003] The spike glycoprotein (S) of PEDV is the main structural protein of the virus. It is a type I envelope glycoprotein composed of 1383 amino acids and is approximately 150-220 kDa in size. The PEDV S protein is involved in recognizing host cell surface receptors and mediating the fusion of the viral envelope with the host cell membrane. The PEDV-S protein has strong antigenicity and contains important antigenic epitopes and neutralizing epitopes of the virus. It is an important target for the detection and diagnosis of the disease and the development of vaccines.

[0004] Since the clinical symptoms of diarrhea caused by PEDV are similar to those caused by other pathogens such as porcine transmissible gastroenteritis virus, porcine rotavirus, porcine deltacoronavirus, and piglet Escherichia coli, rapid and accurate differential diagnosis is of great significance for the precise prevention and control of the disease. At present, the commonly used detection methods for PEDV in laboratories include virus isolation and culture, PCR and qPCR, ELISA, etc., but the above methods require processing of collected samples, the experimental operation steps are complicated, and certain professional operators and appropriate instruments and equipment are required, which are not suitable for rapid diagnosis of epidemic sites. Colloidal gold immunochromatography technique (Gold immunochromatography assay, GICA), also known as immunogold labeling technique (Immunogoldlabeling technique), is a new solid phase immunolabeling rapid detection technology based on antigen-antibody reaction. This type of method uses nitrocellulose membrane as a solid phase carrier, and uses labeled colloidal gold particles to perform qualitative or semi-quantitative detection of antigens, antibodies, and semi-antibodies. It has been widely used in the rapid detection of pathogenic microorganisms, hormones, tumors, markers, pesticide and veterinary drug residues, banned drugs, biological toxins, and drugs. Colloidal gold immunochromatography technology does not require expensive instruments and professional technicians, and it takes a short time to obtain results, and the results are clearly interpreted. Therefore, it can be used for rapid on-site disease detection, facilitating early diagnosis of diseases and timely isolation of sick animals. At present, colloidal gold immunochromatography is widely used in on-site detection of animal infectious diseases, such as porcine reproductive and respiratory syndrome (PRRS), avian influenza (AI), canine parvovirus (CPV), feline distemper (FDV), etc., but traditional colloidal gold test strips are mostly based on monoclonal antibodies. The preparation process of monoclonal antibodies is complicated and the production cycle is long, resulting in high production costs of colloidal gold test strips and difficulties in actual production and application. Therefore, the development of a PEDV colloidal gold paper strip with low production cost and high detection sensitivity is of great significance for the early clinical diagnosis and detection of the disease.

[0005] Nanobody (Nb), as the third generation of genetically engineered antibodies, is the smallest antibody known to have antigen binding ability. Compared with traditional monoclonal antibodies, Nb has a small molecular weight, but the ring structure generated by its longer CDR3 region can bind to the groove of the antigen. In addition, Nb is composed of only 130 amino acids, which is easy to gene edit and can be expressed in large quantities in vitro using prokaryotic or yeast expression systems, with low in vitro production costs. Nb can also be expressed by fusion with different proteins or enzymes, and can simultaneously exert the biological functions of Nb binding to antigens and fusion proteins or enzymes. For example, by fusion expression of Nb with ferritin, Nb can be displayed on the surface of ferritin nanoparticles, which increases the affinity of Nb for antigens. The fusion protein is called Fenobody. In addition, Fenobody can be prepared by prokaryotic expression of Escherichia coli, and has the advantages of simple production process, easy large-scale production, and low cost. Fusion expression of Nb with IgG Fc fragment can well simulate the characteristics of traditional antibodies with nanobodies. Since nanobodies are only single-chain antibodies, this type of fusion protein also has a lower production cost. However, there are currently no reports on the use of nanoantibody technology for PEDV detection. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation and application of a colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nano antibodies, so as to solve the problems existing in the above-mentioned prior art. The present invention first screens nano antibodies against PEDV-S protein, then prepares a fusion protein of nano antibodies with porcine IgG Fc and ferritin, pairs the two fusion proteins, and prepares an immune colloidal gold test strip for detecting PEDV in porcine diarrhea samples. The test strip has the advantages of simple operation, low production cost, intuitive results, no need for special instruments and equipment, and is suitable for rapid detection at the fence, and has a good market application prospect.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is the fusion protein Nb61-pFc, whose amino acid sequence is shown in SEQ ID NO.5.

[0009] The second technical solution of the present invention is a DNA molecule encoding the fusion protein Nb61-pFc, and its nucleotide sequence is shown in SEQ ID NO.6.

[0010] The third technical solution of the present invention is the fusion protein Fenobody 86, whose amino acid sequence is shown in SEQ ID NO.7.

[0011] The fourth technical solution of the present invention is a DNA molecule encoding the fusion protein Fenobody 86, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0012] The fifth technical solution of the present invention is the use of the fusion protein Nb61-pFc and the fusion protein Fenobody 86 in the preparation of a product for detecting porcine epidemic diarrhea virus.

[0013] The sixth technical solution of the present invention is a colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nanoantibodies, comprising a base plate and a sample pad, a binding pad, a detection pad and a water-absorbing pad stacked in sequence on the base plate, wherein the binding pad comprises a fusion protein Nb61-pFc labeled with colloidal gold; the detection pad comprises a quality control line C line and a detection line T line; the detection line comprises the fusion protein Fenobody 86, and the quality control line comprises ProteinA.

[0014] The seventh technical solution of the present invention is a method for preparing the colloidal gold test strip, comprising the following steps: first, labeling the fusion protein Fenobody 86 and ProteinA on a detection membrane, and uniformly spraying the colloidal gold-labeled fusion protein Nb61-pFc on a binding pad; then pasting the detection membrane to the center of a bottom plate, pasting a water-absorbing pad and a binding pad to both sides of the detection membrane, and pasting a sample pad to the other end of the binding pad, with each pad being stacked.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] The present invention uses two screened specific nano antibodies against PEDV S protein as the basic material, and through genetic engineering modification of the nano antibodies, they are fused with the Fc region of pig IgG and ferritin for expression, and two fusion proteins are successfully prepared. Then, the two fusion proteins are used as paired antibodies, wherein the protein fused with pig IgG Fc is colloidal gold labeled as a detection antibody, and the other protein fused with ferritin is used as a capture antibody to establish a colloidal gold detection test strip for PEDV. The test strip has the advantages of strong specificity, high sensitivity, simple operation, low production cost, etc., and can be used for rapid detection of PEDV in pig diarrhea samples in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The titer of anti-PEDV-S protein antibodies in Bactrian camel serum after the fifth immunization with PED virus particles.

[0018] Figure 2 Lymphocytes from peripheral blood of immune Bactrian camels were separated using Ficoll lymphocyte separation medium.

[0019] Figure 3 Agarose gel electrophoresis was used to detect and analyze the VHH 700bp gene product amplified by the first round of nested PCR, where M is a nucleic acid marker.

[0020] Figure 4 Agarose gel electrophoresis was used to detect and analyze the VHH 400bp gene product amplified by the second round of nested PCR, where M is a nucleic acid marker.

[0021] Figure 5 Agarose gel electrophoresis analysis of double enzyme digestion of VHH and pMECS phage display vector, wherein M is a nucleic acid marker, 1 is a double enzyme digestion pMECS vector, and 2 is a double enzyme digestion VHH fragment.

[0022] Figure 6 The positive rate of the constructed phage library was analyzed by agarose gel electrophoresis. 48 monoclonal colonies were randomly selected, and the VHH gene inserted into the constructed phage library was amplified by bacterial liquid PCR; M is the marker; 1-48 is the 48 monoclonal colonies selected.

[0023] Figure 7 Indirect ELISA was used to screen positive clones of specific nanoantibodies against PEDV-S protein.

[0024] Figure 8 For the amino acid sequence comparison of nanoantibodies specific for PEDV-S protein, 36 nanoantibody crude extracts positive clone bacterial fluids were sent for sequencing and classified according to the amino acid sequence of the CDR region. A total of 6 anti-PEDV-S protein specific nanoantibody sequences were screened.

[0025] Fig. 9 Agarose gel electrophoresis analysis of double enzyme digestion of VHH and pCMV-N1-HRP vector, wherein M is a nucleic acid marker, the target fragment is VHH, and the vector is pCMV-N1-HRP.

[0026] Fig.10 For the IFA expression identification of pCMV-PEDV-S-Nbs-HRP, the constructed pCMV-PEDV-S-Nbs-HRP recombinant expression plasmid was transfected into HEK293T cells, and the expression of pCMV-PEDV-S-Nbs-HRP fusion protein in HEK293T cells was analyzed by immunofluorescence, wherein Negative was the pCMV-N1-HRP empty vector.

[0027] Fig.11 ELISA was used to analyze the affinity of pCMV-PEDV-S-Nbs-HRP fusion protein to porcine epidemic diarrhea S protein.

[0028] Fig.12 Agarose gel electrophoresis analysis of PCR amplified Fenobody41;61;86 fragments; wherein M is a marker.

[0029] Fig.13 The expression of Fenobody-41 / 61 / 86 was identified by SDS-PAGE and Western Blot, wherein M is a protein marker.

[0030] Fig.14 The expression of Nb-41 / 61 / 86 / -pFc was identified by SDS-PAGE and Western Blot, where M is a protein marker.

[0031] Fig.15 The preparation of colloidal gold solution, wherein A is the color change during the preparation of colloidal gold solution; B is the 450-600nm band scan of the colloidal gold solution; and C is the electron microscope scan of the colloidal gold particles.

[0032] Fig.16 The optimal colloidal gold-labeled pH values ​​of fusion proteins Fenobody61, Fenobody86, Nb61-pFc, and Nb86-pFc are determined. A is the optimal pH determination of gold-labeled antibodies, B is the 400-600 nm band scanning results of gold-labeled Fenobody61 under different pH conditions, C is the 400-600 nm band scanning results of gold-labeled Fenobody86 under different pH conditions, D is the 400-600 nm band scanning results of gold-labeled antibody Nb61-pFc under different pH conditions, and E is the 400-600 nm band scanning results of gold-labeled antibody Nb86-pFc under different pH conditions.

[0033] Fig.17 The optimal colloidal gold-labeled protein amount determination of fusion proteins Fenobody61, Fenobody86, Nb61-pFc, and Nb86-pFc. Among them, A is the optimal protein amount determination of gold-labeled antibodies, B is the gold-labeled Fenobody61 400-600 nm band scanning results under different protein concentrations, C is the gold-labeled Fenobody86 400-600 nm band scanning results under different protein concentrations, D is the gold-labeled Nb61-pFc 400-600 nm band scanning results under different protein concentrations, and E is the gold-labeled Nb86-pFc 400-600 nm band scanning results under different protein concentrations.

[0034] Fig.18 To screen the best pairing combination of porcine epidemic diarrhea antigen test strip detection line and gold-labeled antibody.

[0035] Fig.19 To screen the optimal detection line concentration of porcine epidemic diarrhea antigen test strips.

[0036] Fig. 20To screen the optimal quality control line concentration for porcine epidemic diarrhea antigen test strips.

[0037] Fig.21 This is a schematic diagram of the results of the porcine epidemic diarrhea antigen test strip.

[0038] Fig. 22 To evaluate the sensitivity of porcine epidemic diarrhea antigen detection test strips.

[0039] Fig.23 To evaluate the specificity of porcine epidemic diarrhea antigen test strips.

[0040] Fig.24 To evaluate the consistency of porcine epidemic diarrhea antigen test strips. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0043] The embodiment of the present invention provides a fusion protein Nb61-pFc, whose amino acid sequence is shown in SEQ ID NO. 5. The fusion protein pFc-Nb61 is prepared by connecting the nanobody Nb61 with the Fc fragment of pig IgG through GS Linker and secreting and expressing it through HEK-293F cells.

[0044] The embodiment of the present invention also provides a DNA molecule encoding the fusion protein Nb61-pFc, and its nucleotide sequence is shown in SEQ ID NO.6.

[0045] The present invention also provides a fusion protein Fenobody 86, whose amino acid sequence is shown in SEQ ID NO. 7. The Fenobody 86 is obtained by connecting ferritin and nanobody Nb86 via GS Linker and expressing it in Escherichia coli.

[0046] The present invention also provides a DNA molecule encoding the fusion protein Fenobody 86, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0047] The embodiments of the present invention also provide the use of the fusion protein Nb61-pFc and the fusion protein Fenobody 86 in the preparation of a product for detecting porcine epidemic diarrhea virus.

[0048] An embodiment of the present invention also provides a colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nanoantibodies, comprising a base plate and a sample pad, a binding pad, a detection pad and a water absorbent pad stacked in sequence on the base plate, wherein the binding pad comprises a fusion protein Nb61-pFc labeled with colloidal gold; the detection pad comprises a quality control line C line and a detection line T line; the detection line comprises the fusion protein Fenobody 86, and the quality control line comprises ProteinA.

[0049] In some specific embodiments, the T line has a concentration of 1.5 mg / mL; the C line has a concentration of 0.8 mg / mL.

[0050] The embodiment of the present invention also provides a method for preparing the colloidal gold test strip, comprising the following steps: first, labeling the fusion protein Fenobody 86 and ProteinA on a detection membrane, and uniformly spraying the colloidal gold-labeled fusion protein Nb61-pFc on a conjugation pad; then pasting the detection membrane to the center of a bottom plate, pasting a water-absorbing pad and a conjugation pad to both sides of the detection membrane, and pasting a sample pad to the other end of the conjugation pad, with each pad being stacked.

[0051] In some specific embodiments, the method further comprises the following steps: cutting the assembled base plate into test strips and placing the test strips into the test strip cartridge.

[0052] In some specific embodiments, a sample addition hole is provided on the test paper cartridge at a position corresponding to the sample pad, and an observation window is provided at a position of the detection membrane.

[0053] The purpose of the present invention is to provide a "circle edge" colloidal gold test strip for rapid detection of PEDV, specifically providing two nano antibodies targeting PEDV-S protein, which are genetically modified to enhance the sensitivity and specificity of the nano antibodies, and have good biological activity, thereby reducing the production cost of the antibodies, and providing a new material for the development of colloidal gold test strips for PEDV.

[0054] The amino acid sequence of the nanobody Nb61 used in the present invention is (SEQ ID NO.1): QVQLQESGGG SVQTGGSLRLSCAASGHTYTICGMAWYRQAPGKGRELVSNILRGGDSYYADSVKGR FTISQDNAKNTVYLQMNSLRPEDTEQKLISEEDLDRGSCVWHNDWGQGTQVTVSSA A;

[0055] The amino acid sequence of the nanobody Nb86 used in the present invention is (SEQ ID NO.2): QVQLQESGGG LVQPGGSLRLSCVASGFTFSRFLMKWVRQIPGKGLEWVASIGRSDDYTSYGDSVEGR FTISRDNDKNTLYLQLNSLKTEDEQKLISEEDLSVDGTRPLSGRGQGTQVTVSSAA;

[0056] The nucleotide sequence encoding Nanobody Nb61 is (SEQ ID NO.3): CAGGTGCAGCTGCAG GAGTCTGGGGGAGGCTCGGTGCAGACTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGCCACACCTACACTATCTGTGGTATGGCCTGGTACCGCCAAGGCTCCAGGGAAGGGGCGCGAGTTGGTCTCAAATATTTTACGTGGCGGTGATTCATACTATGCAGACTCCGTGAAG GGCCGATTCACCATCTCCCAAGATAACGCCAAGAATACGGTATATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGAGCAGAAACTCATCTCTGAAGAGGATCTGGATCGTGGTAGTTGTGTCTGGCATAACGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGCGGCG;

[0057] The nucleotide sequence encoding nanobody Nb86 is (SEQ ID NO.4): CAGGTGCAGCTGCAG GAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTAGGTTTCTCATGAAATGGGTCCGCCAAATTCCAGGGAAGGGACTCGAGTGGGTCGCAAGTATTGGCCGGAGTGATGATTACACATCGTATGGAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAATTGAACAGTCTGAAAACTGAGGACGAGCAGAAACTCATCTCTGAAGAGGATCTGTCTGTCGATGGTACTAGACCTCTTTCGGGGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCG;

[0058] The amino acid sequence of Nb61-pFc is (SEQ ID NO.5): QVQLQESGGGSVQTGGSLRLSCA ASGHTYTICGMAWYRQAPGKGRELVSNILRGGDSYYADSVKGRFTISQDNAKNTVYLQMNSLRPEDTEQKLISEEDLDRGSCVWHNDWGQGTQVTVSSTKTKPPCPICPGCEVAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVSKEHAEVQFSWYVDGVEVHTAETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAIGQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYRTTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK;

[0059]

[0060] The amino acid sequence of Fenobody86 is (SEQ ID NO.7): MLSERMLKALNDQLNRELYSA YLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDGGGGSGGGGSGGGGSESGGGLVQPGGSLRLSCVASGFTFSRFLMKWVRQIPGKGLEWVASIGRSDDYTSYGDSVEGRFTISRDNDKNTLYLQLNSLKTEDEQKLISEEDLSVDGTRPLSGRGQGTQVTVSS;

[0061] The nucleotide sequence of Fenobody86 is (SEQ ID NO.8): ATGCTGAGCGAACGCATGCTG AAGGCTTTAAACGACCAGCTGAATCGTGAACTGTACAGCGCCTATTTATACTTCGCCATGGCCGCCTATTTTGAAGATCTGGGTTTAGAGGGTTTCGCCAATTGGATGAAAGCCCAAGCTGAAGAAGAGATCGGTCATGCTTTACGCTTCTACAACTATATCTATGATCGCAACGGCCGCGTGGAACTGGACGAAATTCCGAAACCGCCGAAAGAGTGGGAAAGCCCGCTGAAAGCCTTTGAGGCCGCCTACGAGCACGAAAAATTCATCAGCAAAAGCATTTATGAACTGGCCGCTTTAGCCGAGGAAGAAAAAGACTATAGCACCCGCGCCTTTCTGGAGTGGTTTATCAACGAGCAAGTTGAAGAAGAAGCCAGCGTGAAGAAAATTTTAGACAAACTGAAATTTGCCAAGGACGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTAGGTTTCTCATGAAATGGGTCCGCCAAATTCCAGGGAAGGGACTCGAGTGGGTCGCAAGTATTGGCCGGAGTGATGATTACACATCGTATGGAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAATTGAACAGTCTGAAAACTGAGGACGAGCAGAAACTCATCTCTGAAGAGGATCTGTCTGTCGATGGTACTAGACCTCTTTCGGGGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0062] Example 1

[0063] Screening and Preparation of Nanobodies Specific to PEDV-S Protein

[0064] (1) Animal Immunization

[0065] 1 mL of concentrated purified PEDV (the strain was isolated from clinical materials of an outbreak of porcine epidemic diarrhea in a pig farm in Yangling, Shaanxi Province, amplified and cultured in Vero cells, and purified by sucrose density gradient centrifugation) was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously into the neck of a 4-year-old Alxa Bactrian camel; the remaining 4 times, 1 mL of purified virus was emulsified with an equal volume of incomplete Freund's adjuvant, and immunized once every two weeks, for a total of 5 times. 150 mL of peripheral blood from the immune Bactrian camel was collected 7 days after the last immunization.

[0066] Using baculovirus-expressed PEDV-S protein (100 ng / well) as the coating antigen, the anti-PEDV-S antibody titer in Bactrian camel serum after 5 immunizations was 1:256000 ( Figure 1 ), proving that immunized Bactrian camels produced an immune response against PEDV.

[0067] (2) Construction and panning of VHH phage library

[0068] 1) Isolation of peripheral blood lymphocytes

[0069] The collected peripheral blood was mixed with Ficoll-Paque PLUS lymphocyte separation solution (Greiner bio-one) at a volume ratio of 2:1, and centrifuged at 1000g and 25℃ for 10min. The ring-shaped milky white substance between the plasma and the white transparent lymphocyte separation solution was lymphocytes ( Figure 2 ). Lymphocytes were aspirated, washed repeatedly with PBS, centrifuged, resuspended and counted, and the number of cells was calculated as 1×10 7 The cells were divided into aliquots and stored at -80℃ for later use.

[0070] 2) Amplification of VHH gene fragments in peripheral blood lymphocytes

[0071] according to Plus Mini RNA Extraction Kit (QIAGEN) was used to extract total RNA (RNA larger than 200 bp) from isolated lymphocytes. The extracted total RNA was used as a template to synthesize the first-strand cDNA using III reverse transcriptase (Life technology), and then the VHH gene was amplified using nested PCR. First, an RNA / primer mixture was prepared, and its system is shown in Table 1.

[0072] Table 1 RNA / primer system

[0073]

[0074] The prepared RNA / primer mixture was incubated at 65°C for 5 minutes and then immediately placed in an ice water bath for 1 minute. Subsequently, a cDNA synthesis mixture was prepared, and its system is shown in Table 2.

[0075] Table 2 cDNA mixture system

[0076]

[0077]

[0078] The cDNA synthesis mixture (10 μL) was added to the RNA / primer mixture, mixed, incubated at 50°C for 50 min, and terminated after incubation at 85°C for 5 min.

[0079] The VHH gene was amplified by nested PCR using the reverse transcribed cDNA as a template. The primer sequences used are shown in Table 3 below.

[0080] Table 3 Primers for amplifying VHH genes

[0081]

[0082] First, primers CALL001 and CALL002 were used to perform the first round of PCR amplification, and the reaction system was shown in Table 4.

[0083] Table 4 First round PCR amplification reaction system

[0084]

[0085] The reaction program was pre-denaturation at 94°C for 5 min; 28 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and extension at 72°C for 7 min.

[0086] The PCR product was identified by 1.2% agarose gel electrophoresis, and the results showed that there was a band at the 700 bp and 900 bp positions respectively ( Figure 3 ). The PCR product of 700 bp was recovered using the EasyPure Quick Gel Extraction Kit according to the instructions. The recovered product was then used as a template to perform a second round of PCR amplification using the above-mentioned VHH-FOR and VHH-REV primers. The reaction system is shown in Table 5.

[0087] Table 5 VHH PCR amplification reaction system

[0088]

[0089] The reaction program was pre-denaturation at 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 18 cycles; and extension at 72°C for 5 min.

[0090] The PCR product was subjected to 1.5% agarose gel electrophoresis, and the results showed that a 400 bp target band was obtained ( Figure 4 ). Then, the PCR product was recovered using the EasyPure Quick Gel Extraction Kit, and the PCR product was the amplified VHH gene.

[0091] 3) Construction of VHH phage display vector

[0092] The above-recovered PCR product and pMECS phage display vector were double-digested with Pst I and Not I at the same time. The specific enzyme digestion system is shown in Tables 6 and 7.

[0093] Table 6 pMECS plasmid double restriction enzyme digestion system

[0094]

[0095] Table 7 PCR product double enzyme digestion system

[0096]

[0097]

[0098] The digestion conditions were 37°C for 16 h. After digestion, the digestion products were recovered using the commercial kit EasyPure Quick GelExtraction Kit, and the digestion results were analyzed by agarose gel electrophoresis to obtain digestion fragments of about 5000 bp and 400 bp, respectively. Figure 5 ).

[0099] The above-recovered PCR digestion product was ligated into the digested phage display vector pMECS using T4 DNA ligase, and the ligation was carried out at 16° C. for 16 h. The ligation system is shown in Table 8.

[0100] Table 8 VHH and pMECS vector connection system

[0101]

[0102] 4) Preparation of E. coli TG1 competent cells

[0103] E. coli TG1 was cultured to OD 600nm After the pH value is 0.4-0.6, cool in an ice bath, centrifuge and wash three times with pre-cooled 10% glycerol, and finally resuspend the bacteria with 10% glycerol to prepare TG1 competent cells.

[0104] 5) Preparation of phage nanoantibody library

[0105] The ligation product was added to the freshly prepared E. coli TG1 competent cells, mixed and added to the electroporation cup, and the competent cells were electroporated using an Eppendorf electroporator (1.8 kV, 25 μF, 200 Ω, 1 mm). Immediately after the electroporation, SOC medium (2 g tryptone, 0.5 g yeast extract, 0.058 g NaCl and 0.0186 g KCl, dissolved in 96 mL ultrapure water, sterilized by high pressure steam at 121°C for 30 min, and the culture medium was cooled to room temperature, and 2 mL 20% glucose solution, 1 mL 1M MgCl2 solution and 1 mL 1M MgSO4 solution were added to resuspend the cells. After shaking culture at 37°C and 120 r / min for 1 h, spread on LB / AMP-GLU large plates (10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar, dissolved in 900 mL ultrapure water, sterilized by high-pressure steam at 121°C for 30 min, and when the culture medium is cooled to about 60°C, add 100 mL 20% glucose solution and 1 mL ampicillin storage solution), culture at 37°C for 6 to 8 h, collect the bacterial lawn with a cell scraper, and add 1 / 3 volume of 50% glycerol to obtain the prepared phage library.

[0106] 6) Determination of phage library diversity and capacity

[0107] The electroconversion product was diluted 10-fold to 10 -5 Then, the cells were plated on LB / AMP-GLU plates and cultured at 37°C for 12 h. The number of transformants was calculated and the final storage capacity was 3.8×10 7 48 single clones were randomly selected and identified by PCR using the VHH-FOR and VHH-REV primers described in Table 3. Positive clones were identified by agarose gel electrophoresis. The target size was about 400 bp ( Figure 6 ).

[0108] (3) Screening of anti-PEDV-S protein specific nanoantibodies

[0109] 1) Rescue of phage library

[0110] The obtained phage library was inoculated into 2×YT / AMP-GLU (16g tryptone, 10g yeast extract and 5gNaCl, dissolved in 900mL ultrapure water, sterilized by high pressure steam at 121°C for 30min, and after the culture medium was cooled to room temperature, 100mL 20% glucose solution and 1mL ampicillin stock solution were added) culture medium, cultured at 37°C and 200r / min, and after the culture medium reached the logarithmic phase, M13KO7 helper phage was added, and the culture medium was allowed to stand at 37°C for 30min. Centrifuged at 2800r / min for 10min, the supernatant was discarded, and the precipitated bacteria were resuspended in 200mL 2×YT / AMP-KAN (16g tryptone, 10g yeast extract and 5gNaCl, dissolved in 1000mL ultrapure water, 1mL kanamycin stock solution and 1mL ampicillin stock solution were added) culture medium, and cultured at 37°C and 200r / min for 14h. Then, after centrifugation at 4°C, 3800 r / min for 30 min, the supernatant was collected and 1 / 5 volume of pre-cooled PEG / NaCl solution was added. After centrifugation at 4°C, 3800 r / min for 30 min again, 1 mL of PBS was added to resuspend the phage precipitate and fully dissolve it, which is the rescued phage library.

[0111] Take a small amount of phage library solution and dilute it 10 times. -2 , 10 -4 , 10 -6 , 10 -8 , 10 -10 The sample was added to TG1 cells in the logarithmic growth phase and incubated at 37°C for 15 min. The cells were evenly spread on LB / AMP-GLU plates and cultured at 37°C for 8 h. The titer of the recombinant phage was calculated to be 9.1×10 12 PFU / mL.

[0112] 2) Selection of recombinant phages specific for PEDV-S protein

[0113] The PEDV-S protein was coated in the ELISA plate, and PBS was used as a control. The rescued phage library was added to the ELISA plate, incubated at room temperature for 1 hour, and the phage sample was discarded. Then, freshly prepared 0.1M triethylamine was added to each well, and the plate was allowed to stand at room temperature for 10 minutes. The eluate was quickly neutralized with an equal volume of 1M Tris-HCl (pH=7.4).

[0114] Take the eluate and infect 4 mL of TG1 cells in the logarithmic phase, let it stand at 37°C for 30 min, then add 2×YT / AMP-GLU medium and culture at 37°C and 200 r / min until OD 600nmThe above operation was repeated to rescue the phage library, and then the second and third rounds of panning were performed, and the phage titer was measured in each round. The results showed that after three rounds of panning, the P / N value increased from 0.42 to 8.3×10 3 , proving that anti-PEDV-S protein phages were effectively enriched (Table 9).

[0115] Table 9 Enrichment of anti-PEDV-S protein specific phage

[0116]

[0117] 3) Preparation of crude extracts of recombinant nanobodies

[0118] 96 single colonies were randomly selected from the plate for measuring the titer of the third round of eluted phages, marked 1 to 96 in sequence, inoculated into a 96-well plate, LB / AMP-GLU medium was added to each well, and cultured at 37°C, 200 r / min for 8 h.

[0119] 10 μL of culture medium was aspirated from each clone and inoculated into 1 mL of TB medium. The culture was then cultured in a 24-well culture plate at 37°C and 200 rpm until the OD value of the clone reached the logarithmic phase. 600nm The pH value was 0.6-0.8, and IPTG with a final concentration of 0.1 mM was added to each well to induce expression. The expressed bacteria were centrifuged and precipitated, and then frozen and thawed three times, centrifuged at 3500 r / min and 4°C for 15 min, and the supernatant was collected as the crude extract of soluble recombinant nanoantibodies.

[0120] 4) Indirect ELISA to detect the specific binding of recombinant nanoantibody crude extract to PEDV-S protein

[0121] The PEDV-S protein was coated on the ELISA plate, and an irrelevant E. coli was used as a negative control. The crude extract of the soluble recombinant nanoantibody was taken and blocked with 2.5% skimmed milk powder in the ELISA plate, and incubated at room temperature for 1 hour. After washing the plate with PBS'T (8.5gNaCl, 3.352gNa2HPO4·12H2O, 0.286gNaH2PO4·2H2O, deionized water, dissolved and diluted to 1000mL, pH 7.2-7.4, 0.5mL Tween-20 was added), and commercial mouse anti-HA-tag antibody (Beijing Quanshijin Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 hour. Then, commercial HRP-labeled sheep anti-mouse IgG (Beijing Boosun Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 hour. After adding TMB colorimetric solution (Solarbio Company) and coloring at room temperature in the dark for 15 minutes, the reaction was terminated with 3M concentrated sulfuric acid, and the OD was read by an automatic microplate reader. 450nm The results showed that 36 of the 96 crude extracts could specifically bind to the PEDV-S protein ( Figure 7The positive strains were sequenced and compared, and the results showed that 6 anti-PEDV-S protein nanoantibodies were successfully screened and obtained ( Figure 8 ).

[0122] Example 2

[0123] Screening of optimal paired nanoantibodies for capturing and detecting PEDV

[0124] (1) Construction of eukaryotic expression vector of nanobody and horseradish peroxidase (HRP) fusion protein

[0125] The eukaryotic expression vector pCMV-N1-HRP (Nanobody-horseradishperoxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay, Journal of Nanobiotechnology, Vol. 17, No. 1, 2019) and the PCR-encoded nanoantibody product were double-digested using Pst I and Not I endonucleases. The results of agarose gel electrophoresis showed that the target bands of about 5000 bp and 400 bp were successfully obtained, respectively ( Fig. 9 ). Then the VHH gene obtained by enzyme digestion was ligated into the pCMV-N1-HRP vector using T4 ligase to obtain a recombinant positive plasmid expressing the nanobody and HRP fusion protein, named pCMV-N1-PEDV-S-Nbs-HRP.

[0126] (2) Expression and identification of nanoantibody and HRP fusion protein

[0127] The successfully constructed pCMV-N1-PEDV-S-Nbs-HRP positive plasmid was combined with Opti- After the medium and PEI transfection reagent were mixed evenly, they were added to HEK293T cells and cultured at 37°C, 5% CO2 for 48 hours, and the supernatant was collected. The transfected HEK293T cells were fixed with 4% paraformaldehyde, incubated with commercial His monoclonal antibody (Quanshijin Biotechnology Co., Ltd.) at room temperature, and then incubated with FITC-labeled goat anti-mouse IgG. Immunofluorescence was used to detect whether the recombinant fusion protein was expressed in HEK293T cells; the results showed that the HEK293T cells transfected with the positive plasmid emitted green fluorescence, proving that the fusion protein was successfully expressed ( Fig.10 ).

[0128] The supernatant collected after transfection of HEK293T cells was diluted in proportion (1:1, 1:10, 1:100, 1:1000) and directly added to the ELISA plate coated with PEDV-S protein (100 ng / well). The results after TMB color development showed that the OD values ​​of the six nanoantibodies reacting with PEDV-S protein after dilution 100 times were 0.14, 0.23, and 0.16, respectively. 450nm The values ​​were all higher than those of the control group, indicating that the six nanoantibodies could bind to the PEDV-S protein after secretion and expression, and the affinity of PEDV-S-Nb41-HRP, PEDV-S-Nb61-HRP, and PEDV-S-Nb86-HRP was relatively high ( Fig.11 ). Subsequently, the three nanobodies were fused with ferritin and IgG Fc fragments to screen for the best matching antibodies.

[0129] (3) Expression of Nanobody and Ferritin Fusion Protein

[0130] Primers were designed based on the gene sequences of nanobodies and ferritin. The primer sequences are shown in Table 10. The three nanobody sequences screened out, PEDV-S-Nb41, PEDV-S-Nb61 and PEDV-S-Nb86, were connected to ferritin respectively using GS Linker. The obtained sequences encoding Fenobodies were then double-digested with the pET-28a vector using HindIII and BamHI. The enzyme digestion system is shown in Table 11.

[0131] Table 10 Amplification of Fenobody Primer Sequences

[0132]

[0133]

[0134] Table 11 Fenobodies and linearized pET-28a connection system

[0135]

[0136] The results of agarose gel electrophoresis showed that the target bands of about 800 bp and 4800 bp were successfully obtained. The gene obtained by enzyme digestion was connected to the vector using T4 ligase, and the vector was successfully constructed by bacterial liquid PCR ( Fig.12 ), which were named pET-28a-PEDV-S-Fenobody41, 61 and 86.

[0137] The pET-28a-PEDV-S-Fenobody 41, 61 and 86 positive plasmids were transformed into Rosetta (DE3) to obtain Rosetta (DE3)-pET-28a-PEDV-S-Fenobody 41, 61 and 86 recombinant bacteria, which were activated and expanded. The culture was carried out at 37°C and 200 r / min until the logarithmic phase (OD 600nm 0.6~0.8), add IPTG with a final concentration of 0.1mM to induce expression, continue to culture at 37℃ for 8h, collect the bacterial solution, and use Ni column to purify after ultrasonic disruption. After SDS-PAGE and Western blot analysis, the expected 30kDa target band ( Fig.13 ).

[0138] (4) Expression of Nanobody and Porcine IgG Fc Fragment Fusion Protein

[0139] According to the gene sequences of PEDV-S protein nanobody and porcine IgG Fc fragment, the fused target sequence was connected by GS Linker and handed over to Huakang Biological Company for synthesis. Then the synthesized target fragment was connected to the eukaryotic expression vector pCDNA3.1. The eukaryotic expression vector of the constructed nanobody and Fc fusion protein is pCDNA3.1-PEDV-S-Nbs-pFc.

[0140] The successfully constructed pCDNA3.1-PEDV-S-Nbs-pFc positive plasmid was mixed evenly with the culture medium and PEI transfection reagent, and then added to HEK293F cells. After culturing for 120 hours at 37°C, 140rpm, and 5% CO2, the supernatant was collected. The transfection supernatant was incubated with Protein G (Tian Di Ren He), and PEDV-S-Nbs-pFc was purified according to the protein purification method described in the manual. After SDS-PAGE and Western blot analysis, the expected target band of 38 kDa was obtained ( Fig.14 ).

[0141] (5) Screening of the best paired nanoantibodies

[0142] The sandwich ELISA method was used to screen the nanoantibody pairing combination with the best capture effect. Fenobody41, 61 and 86 were coated in the ELISA plate (200 ng / well), and then PEDV [positive (P) control] and porcine reproductive and respiratory syndrome virus (PRRSV) [negative (N) control] were added. After incubation at room temperature for 1 hour, different PEDV-S-Nbs-pFc were added, and finally sheep@pig HRP was added for color development. The color was calculated according to the OD 450nm The P / N value is calculated, and the one with the highest ratio is the best pair. The specific steps are as follows:

[0143] 1) The purified Fenobody 41, 61 and 86 were coated on ELISA plates (200 ng / well) at 4°C overnight;

[0144] 2) After washing the plate 4 times with PBS'T, block it with 2.5% skim milk powder in a 37°C incubator for 1 h;

[0145] 3) PEDV and PRRSV samples were added to the ELISA plate at 100 μL per well, incubated in a 37°C incubator for 1 h, and then washed 4 times with PBS'T;

[0146] 4) Add 100 μL of Nb41-pFc, Nb61-pFc, and Nb86-pFc fusion proteins expressed by HEK293F cells to the ELISA plate, 100 ng per well, and incubate at 37°C for 1 h;

[0147] 5) After washing the plate 4 times with PBS'T, add HRP-labeled goat anti-pig antibody (1:5000 dilution, 100 μL / blank) to the ELISA plate and incubate at 37°C for 1 h;

[0148] 6) Add TMB substrate colorimetric solution and color for 15 min in the dark, terminate with 3M H2SO4, and read OD 450nm The P / N value was calculated. The PEDV-S-Fenobody 41 / 61 / 86 protein was used as the capture antibody and the Nb41 / 61 / 86-pFc protein was used as the detection antibody. The sandwich ELISA results showed that the PEDV-S-Fenobody 41 / 61 / 86 protein could capture PEDV. Among them, the P / N of the sandwich ELISA with Fenobody 61 and Nb86-pFc paired antibodies was the highest, 20.830 (Table 12).

[0149] Table 12 Screening of optimal paired antibodies in sandwich ELISIA

[0150]

[0151] Example 3

[0152] Assembly and performance evaluation of colloidal gold test strips for detecting PEDV

[0153] (1) Preparation and identification of colloidal gold

[0154] The colloidal gold nanoparticles were prepared by sodium citrate reduction method. The specific preparation steps are as follows:

[0155] 1) Prepare a 200 mL conical flask, soak it in acid overnight, wash it with distilled water 10 times, then add 198 mL ultrapure water and 2 mL 1% chloroauric acid to boil, quickly add 6 mL of freshly prepared 1% trisodium citrate solution to the boiling water, and continue boiling;

[0156] 2) When the solution color changes from blue to black and finally gradually transitions to wine red and no longer changes, stop heating and allow to cool naturally to room temperature ( Fig.15 Middle A);

[0157] 3) The shape of colloidal gold particles was observed using a transmission electron microscope, and the uniformity of colloidal gold was analyzed using an enzyme marker. The results showed that the colloidal gold solution prepared by the trisodium citrate reduction method was wine red in color, transparent and without precipitation. Through full-band scanning, the colloidal gold particles were a single peak with a sharp peak shape, indicating that the particle size was uniform ( Fig.15 Middle B). Scanning by transmission electron microscopy shows that the colloidal gold particles are regular in shape and about 20 nm in size ( Fig.15 Middle C).

[0158] (2) Optimization of pH value of gold-labeled antibodies

[0159] 1) Add 0, 2, 4, 6, 8 and 12 μL of 0.2 mol / L K2CO3 solution to 1 mL / tube of colloidal gold solution to prepare colloidal gold solutions with different pH values;

[0160] 2) Take 125 μL of colloidal gold solution with different pH values ​​and transfer it to the corresponding wells of the microtiter plate, add 20 μL of Fenobody61, Fenobody 86, Nb61-pFc and Nb86-pFc, mix well and react at room temperature for 15 min;

[0161] 3) Add 125 μL of 10% NaCl solution to each well, let stand at room temperature for 5 minutes, and observe the color change of the solution. The well with the least amount of K2CO3 solution and the color remains unchanged corresponds to the pH value, which is the optimal pH of the protein.

[0162] OD analysis under different pH conditions by full-band scanning 400-600nm The results showed that the optimal pH value for labeling Fenobody 61 was 2 μL / mL of K2CO3; that for Fenobody86 was 2 μL / mL of K2CO3; that for Nb61-pFc was 8 μL / mL; and that for Nb86-pFc was 2 μL / mL ( Fig.16 ).

[0163] 4) Optimization of the optimal protein labeling amount of gold-labeled antibodies

[0164] Add 10μL PBS to wells 1 to 7 of the ELISA sample wells, and take 10μL purified Fenobody 61, Fenobody86, Nb61-pFc and Nb86-pFc proteins for serial dilution. Add 125μL of the optimal pH colloidal gold solution to each well, mix thoroughly, and let stand at room temperature for 15 minutes. Then add 125μL of 10% NaCl solution to each well, stir evenly, let stand at room temperature for 10 minutes, and observe the color change of the solution in each well. The protein concentration corresponding to the dilution degree when the color of the solution does not change is the optimal labeling concentration.

[0165] Analysis of OD of colloidal gold suspensions at different pH values ​​by full-band scanning 400-600nm The results showed that the optimal amount of gold-labeled protein for Fenobody 61 was 640 μg / mL, the optimal amount for Fenobody 86 was 320 μg / mL, the optimal amount for Nb61-pFc was 160 μg / mL, and the optimal amount for Nb86-pFc was 160 μg / mL ( Fig.17 ).

[0166] 5) Colloidal gold labeling of proteins

[0167] Adjust 1mL of colloidal gold solution to the optimal pH value for protein labeling, mix thoroughly, add the optimal amount of protein, and react at room temperature for 60 minutes. Add 1mL of 20mmol / L sodium borate solution containing 10% BSA, mix quickly, and react at room temperature for 60 minutes. Centrifuge at 10000r / min for 30 minutes at 4℃, and carefully remove the supernatant. Resuspend the precipitate with 20mmol / L sodium borate solution containing 1% BSA, and store the resulting gold-labeled antibody at 4℃ for later use.

[0168] 6) Assembly of colloidal gold test strips for detecting PEDV

[0169] ① Preparation of sample pad and gold label pad

[0170] Sample pad treatment: Soak the glass wool strip in a PBS (pH 8.0) solution containing 1% BSA, 2% sucrose, 0.2% Tween-20 (v / v) and 0.1% (w / v) NaN3 for 30 minutes, and dry it in a 37°C drying oven for 12 hours. It can be used to assemble trial test strips.

[0171] Gold pad treatment: Spray the gold-labeled antibody on the glass wool at 5, 10, or 15 μL / cm, place the glass wool in a drying oven, and dry it at 37°C for 2 hours. It can be used to assemble trial test strips.

[0172] ② Screening of the best detection line and gold-labeled antibody pairing combination

[0173] On the NC membrane, a colloidal gold three-dimensional spray spotter was used to label the same concentrations of proteins Fenobody 61, Fenobody 86, Nb61-pFc and Nb86-pFc on the test line (T line), and the same concentrations of ProteinA (Biyuntian, recombinant protein A) and anti-His tag monoclonal antibody were labeled 6 mm above the test line as the quality control line (C line). The labeled NC membrane was placed in a 37°C drying oven for 30 minutes, and then the test strips were assembled. The positive samples were tested, the color development of the test strips was observed, and the best T-line labeled protein was determined. The results showed that when the T-line labeling concentration was the same, the best effect of detecting positive samples was achieved by using Fenobody 86 as the detection line antibody and Nb61-pFc as the gold-labeled antibody ( Fig.18 ).

[0174] ③ Determination of the optimal detection line and quality control line concentration of colloidal gold test strips

[0175] Use PBS to dilute Fenobody86 to 1.0, 1.5 and 2.0 mg / mL, respectively. Place the NC membrane in an incubator for equilibration for 10 minutes, and then use a colloidal gold three-dimensional spray dot apparatus to mark three concentrations of protein on the test line (T line). Then, place the C line 6 mm above the T line, and mark the C line with Protein A at a concentration of 1.0 mg / mL. Place the marked NC membrane in a 37°C drying oven and dry it for 30 minutes, then assemble the test strips, detect positive samples, observe the color development of the test strips, and determine the optimal T line marking concentration. The results showed that when the T line concentration was 1.0 mg / mL, 1.5 mg / mL and 2.0 mg / mL, the same amount of sample was detected, and the detection effect of 1.5 mg / mL of Fenobody 86 was the best. Therefore, the optimal concentration of the T line was determined to be 1.5 mg / mL ( Fig.19 ).

[0176] The NC membrane was placed in an incubator for 10 minutes to equilibrate. A colloidal gold three-dimensional spray dot apparatus was used to mark the detection line (T line) with Fenobody 86 at a concentration of 1.5 mg / mL. The C line was then positioned 6 mm above the T line, and Protein A was diluted with PBS to 0.5, 0.8, and 1.0 mg / mL, respectively, and then marked on the C line. The marked NC membrane was placed in a 37°C drying oven for 30 minutes, and then the test strips were assembled. The positive samples were tested, and the color development of the test strips was observed to determine the optimal C line marking concentration. The results showed that when the same sample was tested, the color development result of the Protein A streaking concentration of 0.5 mg / mL was not obvious, and there was no significant difference between the concentrations of 0.8 and 1.0 mg / mL. Therefore, the determined C line streaking concentration was 0.8 mg / mL ( Fig. 20 ).

[0177] ④Assembly of test strips

[0178] First, paste the NC test film with T line (Fenobody 861.5mg / mL) and C line (ProteinA0.8 mg / mL) in the center of the support plate, then paste the gold label pad and sample pad to the sample end of the test film in turn, and then paste the absorbent pad to the other end of the test film, with each layer overlapping the NC film by 2mm. Then use a strip cutter to cut the assembled test paper into 4mm test strips, put them into a plastic card shell to make a test paper card, add a desiccant, seal it, and store it at 4℃.

[0179] 7) Evaluation of the detection performance of assembled colloidal gold test strips

[0180] ① Determination of judgment criteria

[0181] When the colloidal gold test strip assembled in this case is used to test the sample, when two red lines appear at both the C line and the T line, it indicates that the test sample is PEDV positive, and the virus content of the tested sample is proportional to the depth of the color of the T line; when only one red line appears at the C line and the T line is colorless, it indicates that the test sample is PEDV negative; when no red line appears at the C line, regardless of whether the T line is colored, it indicates that the test strip is invalid and needs to be tested again ( Fig.21 ).

[0182] ②Sensitivity evaluation

[0183] The PEDV stored in the laboratory was titered, and then the virus was diluted two-fold, and the sample was added to the sample well. The reaction was allowed to proceed for 15 minutes to obtain the experimental results. The samples were analyzed by real-time fluorescence RT-PCR and the test results were compared. The results showed that the titer of the stored PEDV was 1.0×10 5 TCID 50 / mL After diluting it 2 times, the colloidal gold test strip described in this case was used for detection. When the dilution was 1:32, the T line of the test strip had no color ( Fig. 22 ). The above results show that the minimum detection amount of the colloidal gold test strip for PEDV assembled in this case is 10 3.8 TCID 50 / mL The viral copy number was determined by fluorescence quantitative PCR to be 5.8×10 4 copies / μL.

[0184] ③ Specificity evaluation

[0185] PEDV, PRRSV, swine fever virus, porcine parvovirus, etc. stored in the laboratory were used to evaluate the specificity of the colloidal gold test paper. The sample to be tested was added dropwise to the sample well and reacted for 15 minutes to obtain the experimental results. The results showed that the colloidal gold test strip assembled in this case only had red bands on both the T and C lines when testing PEDV; the other viruses only had red bands on the C line ( Fig.23 ). The above results show that the colloidal gold test strip can specifically detect PEDV and does not cross-react with other porcine viruses. The test strip has good specificity.

[0186] ④ Shelf life and consistency analysis

[0187] Three different batches of colloidal gold test strips assembled in this case were used to detect PEDV, one month apart, and the test results of different batches of test strips were compared. The results showed that the positive and negative results of the test strips from different batches were consistent ( Fig.24 ). This indicates that the colloidal gold test strips have good consistency.

[0188] ⑤Compliance rate evaluation

[0189] The colloidal gold test strips assembled in this case and the commercial fluorescent RT-PCR kit were used to simultaneously test 50 clinical samples. The test results of the fluorescent RT-PCR detection method were used as the standard. A CT value ≥ 35 was considered negative, and a CT value < 35 was considered positive. The consistency rate between the colloidal gold test strips and the fluorescent RT-PCR detection method was calculated and evaluated.

[0190] The results showed that among the 50 samples, the fluorescent RT-PCR detection method detected 15 positive samples and 35 negative samples. The colloidal gold test paper described in this case detected 12 positive samples and 38 negative samples. The colloidal gold test paper and the fluorescent RT-PCR kit had a 94% consistency in detection results, and the Kappa value was 0.848, indicating that the colloidal gold test paper described in this case had good consistency with the fluorescent RT-PCR detection method.

[0191] Table 1350 clinical sample test results

[0192]

[0193] In summary, the colloidal gold test strip for PEDV assembled using nanoantibodies in this case showed good sensitivity and stability in testing both laboratory samples and clinical samples, had no cross-reaction with other major swine viruses, and had a lower production cost than the test strip assembled using traditional monoclonal antibodies.

[0194] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Fusion protein Nb61-pFc, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

5.

2. A DNA molecule encoding the fusion protein Nb61-pFc according to claim 1, characterized in that: Its nucleotide sequence is shown as SEQ ID NO.

6.

3. Fusion protein Fenobody 86, characterized in that Its amino acid sequence is shown in SEQ ID NO.

7.

4. A DNA molecule encoding the fusion protein Fenobody 86 according to claim 3, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

8.

5. Use of the fusion protein Nb61-pFc and the fusion protein Fenobody 86 in the preparation of a product for detecting porcine epidemic diarrhea virus.

6. A colloidal gold test strip for detecting porcine epidemic diarrhea virus based on nanoantibodies, comprising a base plate and a sample pad, a binding pad, a detection pad and a water-absorbing pad stacked on the base plate in sequence, characterized in that: The binding pad contains the fusion protein Nb61-pFc labeled with colloidal gold; the detection pad contains the quality control line C line and the detection line T line; the detection line contains the fusion protein Fenobody 86, and the quality control line contains ProteinA.

7. The colloidal gold test strip according to claim 6, characterized in that: The concentration of the T line is 1.5 mg / mL; the concentration of the C line is 0.8 mg / mL.

8. The method for preparing the colloidal gold test strip according to claim 6 or 7, characterized in that: The method comprises the following steps: first, labeling the fusion protein Fenobody 86 and ProteinA on the detection membrane, and uniformly spraying the fusion protein Nb61-pFc labeled with colloidal gold on the conjugate pad; then pasting the detection membrane to the center of the bottom plate, pasting the water-absorbing pad and the conjugate pad to the two sides of the detection membrane respectively, and pasting the sample pad to the other end of the conjugate pad, with each pad being stacked.

9. The preparation method according to claim 8, characterized in that: The following steps are also included: Cut the assembled base plate into test strips and put them into the test strip card housing.

10. The preparation method according to claim 9, characterized in that: A sample adding hole is arranged at a position corresponding to the sample pad on the test paper card shell, and an observation window is arranged at a position of the detection membrane.

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