Preparation and application of colloidal gold strip for detecting porcine epidemic diarrhea virus based on nanobodies

Abstract

The application discloses a kind of based on nanobody detection swine epidemic diarrhea virus (PEDV) colloidal gold test strip preparation and application, belong to animal pathogen detection technical field.The application is based on the specific nanobody of 2 strains of anti-PEDV fiber projection glycoprotein screening as basic material, by gene engineering modification to nanobody, they are respectively with the Fc region of pig IgG and ferritin fusion expression, successfully prepared two fusion proteins.Then, with two fusion proteins as matched antibody, wherein the protein labeled with colloidal gold as detection antibody, another protein fused with ferritin is capture antibody, the colloidal gold detection test strip of PEDV is established.The test strip has the advantages of strong specificity, high sensitivity, simple operation, low production cost, etc., and can be applied to the rapid detection of PEDV in clinical pig diarrhea samples.

Description

TECHNICAL FIELD

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

[0002] Porcine epidemic diarrhea (PED) is a highly contagious diarrhea disease in pigs caused by porcine epidemic diarrhea virus (PEDV) infection. After the virus invades the body, it mainly destroys the epithelial cells and intestinal villi of the pig intestinal tissue, damaging the intestinal mucosa of the pig. PEDV infection in pigs under one week old will cause severe diarrhea and vomiting, resulting in 80% to 100% mortality. Currently, PEDV mainly has two subgroups of GI and GII, and the classic strain (CV777) belongs to GI-b subgroup. However, since 2010, a global outbreak of suspected highly pathogenic PEDV variant infection has occurred, 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, PEDV infection has caused diarrhea in piglets, which has become one of the infectious diseases that need to be prevented and controlled in pig farms in China. Therefore, a simple, rapid and specific PEDV detection method in the clinic 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, which is a type I membrane glycoprotein composed of 1383 amino acids, with a size of about 150-220 kDa. PEDV S protein is involved in recognizing host cell surface receptors and mediating fusion of the virus envelope with the host cell membrane. PEDV-S protein has strong antigenicity, contains important antigenic epitopes and neutralizing epitopes of the virus, and is an important target for disease detection and diagnosis technology and vaccine development.

[0004] The clinical symptoms of diarrhea disease caused by PEDV are similar to those caused by other pathogens such as porcine transmissible gastroenteritis virus, porcine rotavirus, porcine delta coronavirus, and Escherichia coli in piglets. Therefore, rapid and accurate differential diagnosis is of great significance for the accurate prevention and control of the disease. Currently, the commonly used detection methods for PEDV in the laboratory include virus isolation and culture, PCR and qPCR, ELISA, etc. However, the above methods require sample processing, complex experimental operation steps, and the presence of professional operators and appropriate equipment, which are not suitable for rapid diagnosis on the spot. Gold immunochromatography assay (GICA), also known as immunogold labeling technique, is a new type of solid-phase immunolabeling rapid detection technology based on antigen-antibody reaction. This method uses nitrocellulose membrane as a solid-phase carrier for qualitative or semi-quantitative detection of antigens, antibodies, and half antibodies through labeled colloidal gold particles. It has been widely used in the rapid detection of pathogenic microorganisms, hormones, tumors, markers, agricultural and veterinary drug residues, illegal drugs, biological toxins, and drugs. Colloidal gold immunochromatography technology does not require expensive equipment and professional technicians, and the results can be obtained quickly and interpreted clearly. Therefore, it can be used for rapid detection of diseases on the spot, facilitating early diagnosis and timely isolation of sick animals. Currently, colloidal gold immunochromatography is widely used for on-site detection of animal infectious diseases such as porcine reproductive and respiratory syndrome (PRRS), avian influenza (AI), canine parvovirus (CPV), and feline distemper (FDV). However, most traditional colloidal gold test strips are based on monoclonal antibodies, which have complex preparation processes and long production cycles, resulting in high production costs and difficulties in practical application. Therefore, developing a colloidal gold paper strip for PEDV with low production cost and high detection sensitivity is of great significance for early clinical diagnosis and detection of the disease.

[0005] Nanobody (Nb) as the third generation of genetically engineered antibody is the smallest antibody with antigen binding ability known at present. Compared with traditional monoclonal antibody, Nb has small molecular weight, but the loop structure produced by its long CDR3 region can bind to the groove site of antigen. In addition, Nb is composed of only 130 amino acids, is easy to be genetically edited, can be expressed in large quantities in vitro by using prokaryotic or yeast expression system, and has low production cost in vitro. Nb can also be expressed by fusion with different proteins or enzymes, and can simultaneously play the biological functions of Nb binding to antigen and fusion protein or enzyme. For example, Nb is expressed by fusion with ferritin, Nb is displayed on the surface of ferritin nanoparticle, and the affinity of Nb to antigen is improved. The fusion protein is called Fenobody. In addition, Fenobody can be prepared by prokaryotic expression of E. coli, and has the advantages of simple production process, easy scale production and low cost. Nb is expressed by fusion with IgG Fc fragment, and the characteristics of traditional antibody can be well simulated by nanobody. Since nanobody is only a single-chain antibody, the production cost of the fusion protein is also low. However, there is no related report on the use of nanobody technology for PEDV detection. SUMMARY

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

[0007] To achieve the above purpose, the present application provides the following solutions.

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

[0009] In the second technical solution of the present application, the DNA molecule encoding the fusion protein Nb61-pFc has the nucleotide sequence shown in SEQ ID NO. 6.

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

[0011] In the fourth technical solution of the present application, the DNA molecule encoding the fusion protein Fenobody 86 has the nucleotide sequence 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 conjugation pad, a detection pad and a water-absorbing pad stacked in sequence on the base plate, wherein the conjugation pad comprises the 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 Protein A.

[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 Protein A on a detection membrane, and evenly 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.

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

[0016] The present invention uses two screened, specific nanobodies against the PEDV S protein as the base material. Through genetic engineering, these nanobodies were fused to the Fc region of porcine IgG and ferritin, respectively, to successfully produce two fusion proteins. Subsequently, a colloidal gold test strip for PEDV was developed using these two fusion proteins as paired antibodies: one fused to the porcine IgG Fc region was labeled with colloidal gold and used as the detection antibody, while the other fused to ferritin served as the capture antibody. This test strip offers strong specificity, high sensitivity, ease of operation, and low production cost, and can be used clinically for the rapid detection of PEDV in porcine diarrhea samples. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 Lymphocytes from the 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, where M is a nucleic acid marker, 1 is the double enzyme digestion pMECS vector, and 2 is the double enzyme digestion VHH fragment.

[0022] Figure 6 The positive rate of the constructed phage library was analyzed by agarose gel electrophoresis. Forty-eight randomly selected monoclonal colonies were used to PCR amplify the VHH gene inserted into the constructed phage library. M is a marker; 1-48 represents the 48 selected monoclonal colonies.

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

[0024] Figure 8 In order to compare the amino acid sequences of nanobodies specific for PEDV-S protein, 36 positive clones of crude extracts of nanobodies were sent for sequencing and classified according to the amino acid sequences of the CDR regions. A total of 6 specific nanobody sequences against PEDV-S protein were screened.

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

[0026] Figure 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 the pCMV-PEDV-S-Nbs-HRP fusion protein in HEK293T cells was analyzed by immunofluorescence, where Negative was the pCMV-N1-HRP empty vector.

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

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

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

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

[0031] Figure 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; C is the electron microscope scan of the colloidal gold particles.

[0032] Figure 16 The optimal colloidal gold-labeled pH values ​​for the fusion proteins Fenobody61, Fenobody86, Nb61-pFc, and Nb86-pFc are determined. A represents the optimal pH for the gold-labeled antibodies, B represents the 400-600 nm wavelength scan results for gold-labeled Fenobody61 at different pH values, C represents the 400-600 nm wavelength scan results for gold-labeled Fenobody86 at different pH values, D represents the 400-600 nm wavelength scan results for gold-labeled antibody Nb61-pFc at different pH values, and E represents the 400-600 nm wavelength scan results for gold-labeled antibody Nb86-pFc at different pH values.

[0033] Figure 17 The optimal colloidal gold-labeled protein amount determination for the fusion proteins Fenobody61, Fenobody86, Nb61-pFc, and Nb86-pFc is shown in Figure A. The optimal colloidal gold-labeled protein amount determination for the gold-labeled antibody is shown in Figure B. The 400-600 nm wavelength scan results for gold-labeled Fenobody61 at different protein concentrations are shown in Figure C. The 400-600 nm wavelength scan results for gold-labeled Fenobody86 at different protein concentrations are shown in Figure D. The 400-600 nm wavelength scan results for gold-labeled Nb61-pFc at different protein concentrations are shown in Figure E. The 400-600 nm wavelength scan results for gold-labeled Nb86-pFc at different protein concentrations are shown in Figure 1.

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

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

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

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

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

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

[0040] Figure 24 To evaluate the consistency of porcine epidemic diarrhea antigen detection 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0043] The present invention provides a fusion protein Nb61-pFc, the amino acid sequence of which is shown in SEQ ID NO. 5. The fusion protein pFc-Nb61 is prepared by linking the nanobody Nb61 with the Fc fragment of porcine IgG via GS Linker and secretory expression in HEK-293F cells.

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

[0045] The present invention also provides a fusion protein Fenobody 86, the amino acid sequence of which is shown in SEQ ID NO. 7. Fenobody 86 is obtained by linking ferritin and nanobody Nb86 via GS Linker and expressing 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 conjugation pad, a detection pad and a water-absorbing pad stacked in sequence on the base plate, wherein the conjugation pad comprises the 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 Protein A.

[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 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 Protein A on a detection membrane, and evenly spraying the colloidal gold-labeled fusion protein Nb61-pFc on a conjugate pad; then, affixing the detection membrane to the center of a base plate, affixing a water-absorbing pad and a conjugate pad to both sides of the detection membrane, and affixing a sample pad to the other end of the conjugate 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 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, two nanoantibodies targeting the PEDV-S protein are provided. Through genetic engineering modification, the sensitivity and specificity of the nanoantibodies are enhanced, and they have good biological activity, reducing the production cost of the antibodies, 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] 编码纳米抗体Nb86的核苷酸序列为(SEQ ID NO.4):CAGGTGCAGCTGCAG GAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTAGGTTTCTCATGAAATGGGTCCGCCAAATTCCAGGGAAGGGACTCGAGTGGGTCGCAAGTATTGGCCGGAGTGATGATTACACATCGTATGGAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAATTGAACAGTCTGAAAACTGAGGACGAGCAGAAACTCATCTCTGAAGAGGATCTGTCTGTCGATGGTACTAGACCTCTTTCGGGGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCG;

[0058] Nb61-pFc的氨基酸序列为(SEQ ID NO.5):QVQLQESGGGSVQTGGSLRLSCA ASGHTYTICGMAWYRQAPGKGRELVSNILRGGDSYYADSVKGRFTISQDNAKNTVYLQMNSLRPEDTEQKLISEEDLDRGSCVWHNDWGQGTQVTVSSTKTKPPCPICPGCEVAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVSKEHAEVQFSWYVDGVEVHTAETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAIGQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYRTTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK;

[0059]

[0060] Fenobody86的氨基酸序列为(SEQ ID NO.7):MLSERMLKALNDQLNRELYSA YLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDGGGGSGGGGSGGGGSESGGGLVQPGGSLRLSCVASGFTFSRFLMKWVRQIPGKGLEWVASIGRSDDYTSYGDSVEGRFTISRDNDKNTLYLQLNSLKTEDEQKLISEEDLSVDGTRPLSGRGQGTQVTVSS;

[0061] Fenobody86的核苷酸序列为(SEQ ID NO.8):ATGCTGAGCGAACGCATGCTG AAGGCTTTAAACGACCAGCTGAATCGTGAACTGTACAGCGCCTATTTATACTTCGCCATGGCCGCCTATTTTGAAGATCTGGGTTTAGAGGGTTTCGCCAATTGGATGAAAGCCCAAGCTGAAGAAGAGATCGGTCATGCTTTACGCTTCTACAACTATATCTATGATCGCAACGGCCGCGTGGAACTGGACGAAATTCCGAAACCGCCGAAAGAGTGGGAAAGCCCGCTGAAAGCCTTTGAGGCCGCCTACGAGCACGAAAAATTCATCAGCAAAAGCATTTATGAACTGGCCGCTTTAGCCGAGGAAGAAAAAGACTATAGCACCCGCGCCTTTCTGGAGTGGTTTATCAACGAGCAAGTTGAAGAAGAAGCCAGCGTGAAGAAAATTTTAGACAAACTGAAATTTGCCAAGGACGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTGGCCTCTGGATTCACCTTCAGTAGGTTTCTCATGAAATGGGTCCGCCAAATTCCAGGGAAGGGACTCGAGTGGGTCGCAAGTATTGGCCGGAGTGATGATTACACATCGTATGGAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACGCTGTATCTGCAATTGAACAGTCTGAAAACTGAGGACGAGCAGAAACTCATCTCTGAAGAGGATCTGTCTGTCGATGGTACTAGACCTCTTTCGGGGCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0062] 实施例1

[0063] 抗PEDV-S蛋白特异性纳米抗体的筛选与制备

[0064] (1)动物免疫

[0065] 1 mL of concentrated, purified PEDV (isolated from clinical specimens from a porcine epidemic diarrhea outbreak at a pig farm in Yangling, Shaanxi Province, amplified 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 four-year-old Alxa Bactrian camels. For the remaining four immunizations, 1 mL of purified virus was emulsified with an equal volume of incomplete Freund's adjuvant, administered every two weeks for a total of five immunizations. Seven days after the final immunization, 150 mL of peripheral blood was collected from the immunized Bactrian camels.

[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 ), demonstrating 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 medium (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 medium was the lymphocyte ( Figure 2 The 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 segments in peripheral blood lymphocytes

[0071] according to The total RNA (larger than 200 bp) of isolated lymphocytes was extracted using the Plus Mini RNA Extraction Kit (QIAGEN). The extracted total RNA was used as a template to synthesize first-strand cDNA using PCR-III reverse transcriptase (Life Technologies). The VHH gene was then amplified using nested PCR. First, an RNA / primer mixture was prepared, as 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, the system of which 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 well, incubated at 50°C for 50 min, and terminated by 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 products were identified by 1.2% agarose gel electrophoresis, and the results showed that there was a band at the 700 bp and 900 bp positions ( Figure 3 A 700-bp PCR product was recovered using the EasyPure Quick Gel Extraction Kit according to the manufacturer's instructions. This recovered product was then used as a template for a second round of PCR amplification using the aforementioned 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 as follows: pre-denaturation at 94°C for 5 min; 18 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and extension at 72°C for 5 min.

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

[0091] 3) Construction of VHH phage display vector

[0092] The recovered PCR product and the pMECS phage display vector were simultaneously digested with Pst I and Not I. The specific enzyme digestion systems are shown in Tables 6 and 7.

[0093] Table 6 pMECS plasmid double enzyme digestion system

[0094]

[0095] Table 7 PCR product double enzyme digestion system

[0096]

[0097]

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

[0099] The recovered PCR digestion product was ligated into the digested phage display vector pMECS using T4 DNA ligase 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 nanobody library

[0105] The ligation product was added to freshly prepared E. coli TG1 competent cells, mixed thoroughly, and added to an electroporation cuvette. The competent cells were electroporated using an Eppendorf electroporator (1.8 kV, 25 μF, 200 Ω, 1 mm). Immediately after 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 and autoclaved at 121°C for 30 min) was added to the culture medium. After the culture medium cooled to room temperature, 2 mL of 20% glucose solution, 1 mL of 1 M MgCl2 solution, and 1 mL of 1 M 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). After 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 prepare the 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 libraries

[0110] The obtained phage library was inoculated into 2×YT / AMP-GLU medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl dissolved in 900 mL ultrapure water and autoclaved at 121°C for 30 min. After the medium cooled to room temperature, 100 mL of 20% glucose solution and 1 mL of ampicillin stock solution were added). The culture was cultured at 37°C at 200 rpm until logarithmic phase. M13KO7 helper phage was then added and the culture was allowed to stand at 37°C for 30 min. The culture was then centrifuged at 2800 rpm for 10 min, the supernatant discarded, and the pelleted cells resuspended in 200 mL of 2×YT / AMP-KAN medium (16 g tryptone, 10 g yeast extract, and 5 g NaCl dissolved in 1000 mL ultrapure water, 1 mL of kanamycin stock solution and 1 mL of ampicillin stock solution added). The culture was then cultured at 37°C at 200 rpm for 14 h. After centrifugation at 3800 rpm for 30 minutes at 4°C, collect the supernatant and add 1 / 5 volume of pre-chilled PEG / NaCl solution. Centrifuge again at 3800 rpm for 30 minutes at 4°C, and resuspend the phage pellet in 1 mL of PBS to fully dissolve it. This is the rescued phage library.

[0111] Take a small amount of phage library solution and dilute it 10 times, then take a dilution of 10 -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] PEDV-S protein was coated onto an ELISA plate, with PBS used as a control. The rescued phage library was added to the ELISA plate and incubated at room temperature for 1 hour. The phage sample was discarded. Freshly prepared 0.1 M triethylamine was then added to each well and allowed to stand at room temperature for 10 minutes. The eluate was then quickly neutralized with an equal volume of 1 M Tris-HCl (pH 7.4).

[0114] Take the eluate and infect 4 mL of logarithmic phase TG1 cells, let it stand at 37°C for 30 min, then add 2×YT / AMP-GLU medium and culture at 37°C at 200 rpm until OD 600nmto 0.6-0.8. The above operation was repeated to rescue the phage library, and then the second and third rounds of screening were performed, and the phage titers were determined each round. The results showed that after three rounds of selection, the P / N value was increased from 0.42 to 8.3 x 10 3 , proving that the anti-PEDV-S protein phage was effectively enriched (Table 9).

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

[0116]

[0117] 3) Preparation of recombinant nanobody crude extract

[0118] 96 single colonies were randomly picked from the plate for determining the third round of eluted phage titers, and were sequentially labeled 1-96, inoculated into a 96-well plate, and 37°C 200 r / min cultured for 8 h with LB / AMP-GLU medium added to each well.

[0119] 10 μL of culture solution was taken from each clone, and inoculated into 1 mL of TB medium, and cultured in a 24-well culture plate at 37°C 200 r / min until the logarithmic phase OD 600nm was 0.6-0.8, and 0.1 mM IPTG was added to each well to induce expression. The expressed bacteria were centrifuged and precipitated, repeatedly frozen and thawed 3 times, centrifuged at 3500 r / min for 15 min at 4°C, and the supernatant was collected as the soluble recombinant nanobody crude extract.

[0120] 4) Indirect ELISA detection of specific binding of recombinant nanobody crude extract to PEDV-S protein

[0121] The ELISA plate was coated with PEDV-S protein, and irrelevant E. coli was used as a negative control. The soluble recombinant nanobody crude extract was taken, 2.5% skimmed milk powder was added to the enzyme-labeled plate for blocking, and incubated at room temperature for 1 h. After washing the plate with PBS'T (8.5 g NaCl, 3.352 g Na2HPO4·12H2O, 0.286 g NaH2PO4·2H2O dissolved in deionized water and made up to 1000 mL, pH value is 7.2-7.4, and 0.5 mL Tween-20 is added), a commercial mouse anti-HA-tag antibody (Beijing Quanshi Gold Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 h. Then a commercial HRP-labeled goat anti-mouse IgG (Beijing Boaosen Biotechnology Co., Ltd.) was added and incubated at room temperature for 1 h. After adding TMB color developing solution (Solarbio Company) and developing at room temperature for 15 min in the dark, 3M concentrated sulfuric acid was added to stop the reaction, and the OD 450nm value was read by an automatic enzyme-labeled instrument. The results showed that 36 of the 96 crude extracts could specifically bind to 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 for 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, 2019, Vol. 17, No. 1) and the PCR-encoded nanobody product were double-digested using Pst I and Not I endonucleases. The agarose gel electrophoresis results showed that the target bands of about 5000 bp and 400 bp were successfully obtained, respectively ( Figure 9 ). The VHH gene obtained by enzyme digestion was then 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 nanobody 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 and incubated at room temperature with a commercial His monoclonal antibody (Quanshijin Biotechnology Co., Ltd.), followed by incubation with FITC-labeled goat anti-mouse IgG. Immunofluorescence was used to detect whether the recombinant fusion protein was expressed in the HEK293T cells; the results showed that the HEK293T cells transfected with the positive plasmid emitted green fluorescence, indicating that the fusion protein was successfully expressed ( Figure 10 ).

[0128] The supernatant collected after transfection of HEK293T cells was diluted proportionally (1:1, 1:10, 1:100, 1:1000) and directly added to the ELISA plate coated with PEDV-S protein (100 ng / well). After TMB color development, the results showed that the OD values ​​of the six nanoantibodies reacting with PEDV-S protein after 100-fold dilution were 0. 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 ( Figure 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 fenobides 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 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 800bp and 4800bp were successfully obtained respectively. The gene obtained by enzyme digestion was connected to the vector using T4 ligase, and the vector construction was successfully confirmed by bacterial liquid PCR ( Figure 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 rpm until the logarithmic phase (OD 600nm The expression was induced by adding IPTG at a final concentration of 0.1 mM and culturing was continued at 37 ° C for 8 h. The bacterial solution was collected and purified using a Ni column after ultrasonic disruption. The expected target band of 30 kDa was obtained by SDS-PAGE and Western blot analysis ( Figure 13 ).

[0138] (4) Expression of nanoantibody and porcine IgG Fc fragment fusion protein

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

[0140] The successfully constructed pCDNA3.1-PEDV-S-Nbs-pFc positive plasmid was mixed evenly with culture medium and PEI transfection reagent, and then added to HEK293F cells. After incubation at 37°C, 140 rpm, 5% CO2 for 120 hours, 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 instructions. After SDS-PAGE and Western blot analysis, the expected target band of 38 kDa was obtained ( Figure 14 ).

[0141] (5) Screening of optimal paired nanoantibodies

[0142] The sandwich ELISA method was used to screen the nanoantibody pairing combination with the best capture effect. Fenobody 41, 61 and 86 were coated on 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 determined according to the OD value. 450nm Calculate the P / N value, and the one with the highest ratio is the best pair. The specific steps are as follows:

[0143] 1) Purified Fenobody 41, 61, and 86 were coated onto ELISA plates (200 ng / well) at 4°C overnight.

[0144] 2) After washing the plate 4 times with PBS, block it with 2.5% skim milk powder in a 37°C incubator for 1 hour.

[0145] 3) Add 100 μL of PEDV and PRRSV samples to each well of the ELISA plate, incubate in a 37°C incubator for 1 hour, and then wash the plate four times with PBS.

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

[0147] 5) After washing the plate four times with PBS, 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 minutes in the dark, stop with 3M H2SO4, and read the OD 450nm The P / N ratio was calculated using the PEDV-S-Fenobody 41 / 61 / 86 protein as the capture antibody and the Nb41 / 61 / 86-pFc protein as the detection antibody. Sandwich ELISA results showed that the PEDV-S-Fenobody 41 / 61 / 86 proteins were able to capture PEDV. The sandwich ELISA with the paired Fenobody 61 and Nb86-pFc antibody had the highest P / N of 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] Colloidal gold nanoparticles were prepared using the sodium citrate reduction method. The specific preparation steps are as follows:

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

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

[0157] 3) The shape of the colloidal gold particles was observed using a transmission electron microscope, and the uniformity of the 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, transparent, and free of precipitation. By full-band scanning, the colloidal gold particles showed a single peak with a sharp peak shape, indicating that the particle size was uniform ( Figure 15 Middle B). Scanning by transmission electron microscopy shows that the colloidal gold particles are regular in shape and about 20 nm in size ( Figure 15 Middle C).

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

[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 of different pH values.

[0160] 2) Transfer 125 μL of colloidal gold solution with different pH values ​​to the corresponding wells of a microtiter plate, add 20 μL of Fenobody 61, Fenobody 86, Nb61-pFc, and Nb86-pFc, mix thoroughly, and react at room temperature for 15 minutes.

[0161] 3) Add 125 μL of 10% NaCl solution to each well and let it stand at room temperature for 5 minutes. 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 to add 2 μL / mL of K2CO3; Fenobody86 was to add 2 μL / mL of K2CO3; Nb61-pFc was to add 8 μL / mL; Nb86-pFc was to add 2 μL / mL ( Figure 16 ).

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

[0164] Add 10 μL of PBS to wells 1 through 7 of the ELISA sample wells. Serially dilute 10 μL of purified Fenobody 61, Fenobody 86, Nb61-pFc, and Nb86-pFc proteins. Add 125 μL of colloidal gold solution at the optimal pH to each well, mix thoroughly, and let stand at room temperature for 15 minutes. Then, add 125 μL of 10% NaCl solution to each well, stir thoroughly, and let stand at room temperature for 10 minutes. Observe the color change of each well. The protein concentration corresponding to the dilution at which the color of the solution remains unchanged is the optimal labeling concentration.

[0165] OD analysis 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 ( Figure 17 ).

[0166] 5) Colloidal gold labeling of proteins

[0167] Adjust 1 mL of colloidal gold solution to the optimal pH for protein labeling. Mix thoroughly, then add the optimal amount of labeled protein and allow to react at room temperature for 60 minutes. Add 1 mL of 20 mmol / L sodium borate solution containing 10% BSA, mix rapidly, and allow to react at room temperature for 60 minutes. Centrifuge at 10,000 rpm for 30 minutes at 4°C, carefully remove the supernatant, and resuspend the pellet in 20 mmol / L sodium borate solution containing 1% BSA. Store the resulting gold-labeled antibody at 4°C until ready for use.

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

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

[0170] Sample pad treatment: Soak the glass wool strips in 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 them in a 37°C drying oven for 12 hours. They can be used to assemble trial test strips.

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

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

[0173] On the NC membrane, a colloidal gold three-dimensional spraying device was used to label the same concentration of proteins Fenobody 61, Fenobody 86, Nb61-pFc and Nb86-pFc on the test line (T line). The same concentration of Protein A (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 strip was assembled. The positive sample was tested and the color development of the test strip was observed to determine the best T-line labeled protein. 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 ( Figure 18 ).

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

[0175] Fenobody86 was diluted to 1.0, 1.5 and 2.0 mg / mL using PBS, and the NC membrane was placed in an incubator for equilibrium for 10 minutes. Then, a colloidal gold three-dimensional sprayer was used to mark three concentrations of protein on the detection line (T line). The C line was then separated by 6 mm above the T line, and the C line was marked with Protein A at a concentration of 1.0 mg / mL. The marked NC membrane was placed in a 37°C drying oven and dried 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 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 tested, 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 ( Figure 19 ).

[0176] The NC membrane was placed in an incubator for equilibrium for 10 minutes, and a colloidal gold three-dimensional sprayer was used to mark the detection line (T line) with a concentration of 1.5 mg / mL of Fenobody 86. 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 line 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 C line concentration was determined to be 0.8 mg / mL ( Figure 20 ).

[0177] ④Assembly of test strips

[0178] First, attach the NC test membrane with T lines (Fenobody 861.5 mg / mL) and C lines (Protein A 0.8 mg / mL) to the center of the support plate. Then, attach the gold label pad and sample pad, in sequence, to the sample end of the test membrane. Finally, attach the absorbent pad to the other end of the test membrane, overlapping the NC membrane by 2 mm. Then, use a strip cutter to cut the assembled test paper into 4 mm strips. These strips are placed in plastic card holders to create test paper cards. Add desiccant, seal, and store at 4°C.

[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 as described 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 sample is PEDV positive, and the virus content of the sample is proportional to the depth of the T line color; when only one red line appears at the C line and the T line is colorless, it indicates that the 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 repeated ( Figure 21 ).

[0182] ②Sensitivity evaluation

[0183] The PEDV stored in the laboratory was titered, and then the virus was serially diluted twofold. The sample was added to the sample well and reacted for 15 minutes to obtain the experimental results. The samples were then subjected to real-time fluorescence RT-PCR analysis to compare the test results. 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 ( Figure 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] ③Specificity evaluation

[0185] The specificity of the colloidal gold test strip was evaluated using laboratory samples of PEDV, PRRSV, swine fever virus, and porcine parvovirus. The sample to be tested was added dropwise to the sample well and the reaction was allowed to proceed for 15 minutes to obtain the experimental results. The results showed that the colloidal gold test strip assembled in this case only showed red bands on both the T and C lines when testing PEDV; the other viruses only showed red bands on the C line ( Figure 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, 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 ( Figure 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 a commercial fluorescent RT-PCR kit were used to simultaneously test 50 clinical samples. The test results of the fluorescent RT-PCR 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 method was calculated and evaluated.

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

[0191] Table 1 Test results of 350 clinical samples

[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 and clinical samples, had no cross-reaction with other major porcine viruses, and had a lower production cost than test strips assembled using traditional monoclonal antibodies.

[0194] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection 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 in 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 according to claim 1 and the fusion protein Fenobody 86 according to claim 3 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 conjugation pad, a detection pad, and a water-absorbing pad stacked on the base plate in sequence, characterized in that: The conjugate pad comprises the fusion protein Nb61-pFc of claim 1 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 of claim 3, and the quality control line comprises Protein A.

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

8. The method for preparing a colloidal gold test strip according to claim 6 or 7, wherein: The method comprises the following steps: first, labeling the fusion protein Fenobody 86 and Protein A 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.

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 holder.

10. The preparation method according to claim 9, characterized in that The position of the test paper cartridge corresponding to the sample pad is provided with a sample addition hole, and the position of the detection membrane is provided with an observation window.

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