Porcine epidemic diarrhea recombinant rS1 antigen and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a porcine epidemic diarrhea recombinant rS1 antigen as well as a preparation method and application thereof. A coding sequence of the recombinant rS1 antigen sequentially comprises the following elements from an N terminal to a C terminal: a signal peptide, PEDV S1 protein and a trimer tag, and the elements are connected with one another through linkers. According to the recombinant rS1 antigen, a signal peptide capable of assisting S1 protein secretion is added to the upstream of an S gene to assist soluble expression of S1 protein in periplasmic space, so that the protein solubility is enhanced; a trimer tag GCN4 is added to the downstream to assist correct folding of S1 in the expression process, so that the protein solubility is further enhanced, and correct expression of conformation is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a porcine epidemic diarrhea recombinant rS1 antigen and a preparation method and application thereof. Background Art

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enteric disease affecting pigs of all ages, caused by the porcine epidemic diarrhea virus (PEDV). It primarily infects newborn piglets, with a 100% mortality rate. Currently, there is no effective vaccine. Immunity for newborn piglets primarily comes from passive immunity acquired through the colostrum of lactating sows. In enterovirus-infected sows, secretory IgA antibodies (sIgA) are produced through immune cells that migrate to the mammary gland (MG), where they produce high titers of secretory IgA (sIgA) in the colostrum (via the gut-mammary-sIgA axis), providing passive immunity to newborn piglets. Monitoring sIgA levels in pig herds is crucial for the prevention and control of PEDV.

[0003] PEDV is a positive-sense, single-stranded, enveloped RNA virus of the Coronaviridae family. Its genome consists of seven open reading frames (ORFs), encoding 16 nonstructural proteins (Nsp1-16) and four structural proteins: spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E). Among these viral proteins, the S glycoprotein on the surface of the virus particle plays a crucial role in interacting with the host. The PEDV S protein is subdivided into the S1 globular domain (amino acids 1-789) and the S2 domain (amino acids 790-1383). S1 contains the main neutralizing and cell receptor binding residues, interacts with the host, and possesses neutralizing antibody activity, making it a diagnostic protein for diagnosing PEDV infection.

[0004] Currently, PEDV-S1 is expressed using eukaryotic expression systems such as baculovirus and mammalian cells. Due to high process requirements and high costs, some people choose to express it using prokaryotic systems, but they are all expressed in the form of inclusion bodies and cannot fully demonstrate the reactivity of the protein.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant rS1 antigen for porcine epidemic diarrhea, a preparation method and an application thereof; the recombinant rS1 antigen assists the soluble expression of the S1 protein in the periplasmic space by adding a signal peptide that can assist the secretion of the S1 protein upstream of the S gene, thereby enhancing the protein solubility; the trimer tag GCN4 is added downstream to assist the correct folding of S1 during the expression process, further enhancing the protein solubility and achieving the correct expression of the conformation.

[0007] In order to overcome the deficiencies of the prior art, the present invention provides the following technical solutions:

[0008] A porcine epidemic diarrhea recombinant rS1 antigen, wherein the coding sequence of the recombinant rS1 antigen comprises the following elements in sequence from N-terminus to C-terminus: a signal peptide, a PEDV S1 protein and a trimer tag; the elements are connected by a linker.

[0009] Furthermore, the nucleotide sequence of the recombinant rS1 antigen is shown as SEQ ID NO.1; the amino acid sequence of the recombinant rS1 antigen is shown as SEQ ID NO.2.

[0010] Furthermore, the signal peptide is selected from any one of the PeIB signal peptide, the OmpA signal peptide, and the OmpF signal peptide;

[0011] And / or, the trimer tag is selected from any one of GCN4, T4, and CMP;

[0012] And / or, the linker sequence is GGGGSGGGGSGGGGS.

[0013] In addition, the present invention also provides a method for preparing the porcine epidemic diarrhea recombinant rS1 antigen as described above, comprising the following steps:

[0014] S1. Clone the nucleotide sequence shown in SEQ ID NO. 1 into an expression vector, and design amplification primers rS1-F-Nde I and rS1-R-Xho I;

[0015] S2. Amplify the rS1 gene fragment using the primer set rS1-F-Nde I and rS1-R-Xho I. Double-digest the expression vector and the rS1 gene fragment using Nde I and Xho I restriction endonucleases, and then ligate them using T4 DNA ligase to obtain a ligation product.

[0016] S3. Transform the obtained ligation product into competent Escherichia coli cells, culture them, and extract the plasmid; double-digest the plasmid with Nde I and Xho I restriction endonucleases to obtain a recombinant plasmid;

[0017] S4. The recombinant plasmid was transformed into competent Escherichia coli cells, amplified and cultured, and then induced for expression. The bacterial cells were collected by centrifugation, resuspended with PBS, ultrasonically disrupted, the supernatant was collected, filtered, and the protein was purified to obtain the porcine epidemic diarrhea recombinant rS1 antigen.

[0018] Further, in step S1, the vector is selected from any one of pET28a, pET30a, and pET32a;

[0019] And / or, the nucleotide sequence of the amplification primer set rS1-F-Nde I is shown as SEQ ID NO.3; the nucleotide sequence of the rS1-R-Xho I is shown as SEQ ID NO.4.

[0020] Furthermore, in step S3, the specific steps of transforming the obtained ligation product into Escherichia coli competent cells and extracting the plasmid after culturing include:

[0021] The obtained ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB resistance plates, and cultured in a constant temperature incubator at 37°C until single colonies were clear;

[0022] A single colony was inoculated into LB liquid culture medium, cultured at 37°C and 220 rpm for 6 h, and then the plasmid was extracted using a plasmid extraction kit.

[0023] And / or, in step S4, the specific steps of transforming the recombinant plasmid into competent E. coli cells and inducing expression after amplification and culture include:

[0024] The recombinant plasmid was transformed into Escherichia coli BL21 competent cells and inoculated into LB liquid medium. After constant temperature shaking culture at 37°C and 220 rpm for 10 h, the culture was inoculated into LB shake flasks at a 1% inoculation ratio and cultured at 37°C and 220 rpm until OD600 reached 0.6. The culture was then cooled to 25°C, IPTG inducer was added, and the culture was continued with shaking for 12 h before the fermentation was terminated.

[0025] And / or, after protein purification, the purity of the porcine epidemic diarrhea recombinant rS1 antigen is ≥90%.

[0026] In addition, the present invention also provides a porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit, the diagnostic antigen of which is the porcine epidemic diarrhea recombinant rS1 antigen mentioned above.

[0027] Furthermore, it includes magnetic particle suspension, enzyme-labeled antibody, calibration product, quality control product, sample diluent, washing solution and luminescent liquid.

[0028] Furthermore, the preparation method of the magnetic particle suspension comprises the following steps:

[0029] (1) Mix the magnetic beads and EDC at a ratio of 100 μl of 4 mg / ml EDC solution to 50 mg of magnetic beads. After activation at 40°C for 5 min, wash the activated magnetic beads with magnetic particle washing solution, centrifuge, and discard the supernatant. Repeat this three times to obtain activated magnetic beads.

[0030] (2) The activated microspheres were mixed with recombinant rS1 antigen at a ratio of 15 μg recombinant antigen / mg magnetic beads, coated with coating buffer at 25°C, centrifuged, and the supernatant discarded. The microspheres were then washed with magnetic microparticle washing solution, centrifuged, and the supernatant discarded. This was repeated three times to obtain coated magnetic beads.

[0031] (3) Add 4 mL of magnetic particle blocking solution to the obtained coated magnetic beads, mix at 100 r / min for 15 minutes, then centrifuge and discard the supernatant; then add 100 mL of magnetic particle preservation solution, mix at 100 r / min for 5 minutes, and store at 2°C to 8°C to obtain the magnetic particle suspension.

[0032] Furthermore, in step (2), the coating buffer is prepared as follows: 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate are weighed, the volume is adjusted to 1000 mL, the pH is measured to be 9.7±0.2, and the coating buffer is stored at 2°C to 8°C to obtain the coating buffer;

[0033] And / or, in step (3), the magnetic particle blocking solution is prepared as follows: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride are added to purified water; then 1 g of fish gelatin and 1 mL of Proclin-300 are added in sequence, stirred to dissolve, and the volume is adjusted to 1000 mL, and stored at 2°C to 8°C to obtain the magnetic particle blocking solution;

[0034] And / or, in step (3), the preparation method of the magnetic particle preservation solution is as follows: weigh 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride, dilute to 1000 mL, measure the pH to 7.4±0.2, and obtain a phosphate buffer solution for use; then, add 1 g of fish gelatin and 0.1 g of multivitamins to the phosphate buffer solution in sequence, stir evenly, dilute to 1000 mL, and store at 2°C to 8°C to obtain the magnetic particle preservation solution.

[0035] Compared with the prior art, the technical solution of the present invention has at least the following technical effects:

[0036] The present invention utilizes an Escherichia coli prokaryotic expression system to solublely express a recombinant rS1 antigen, thereby ensuring the stability of the protein conformation and the reactivity of the protein; by adding a signal peptide upstream of the S gene that can assist in the secretion of the S1 protein, the soluble expression of the S1 protein in the periplasmic space is assisted, thereby enhancing the protein solubility; and by adding a trimer tag downstream, the correct folding of S1 during the expression process is assisted, thereby further enhancing the protein solubility and achieving the correct expression of the conformation. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The process parameters for which specific conditions are not specified in the following examples are generally in accordance with conventional conditions.

[0038] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0039] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0040] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0041] The professional terms involved in the present invention are explained as follows:

[0042] The term "N-terminus to C-terminus": In proteins, the N-terminus is the amino terminus, and the C-terminus is the carboxyl terminus. Amino acids are linked by peptide bonds to form peptide chains, and the amino acids at each end of the peptide chain have unique structures. The N-terminus corresponds to the beginning of the peptide chain, and the C-terminus is the end of the peptide chain. They determine the orientation of the protein, and the sequence from N-terminus to C-terminus is the basic sequence of proteins. The amino acid sequence and chemical modifications at the N-terminus and C-terminus can have a significant impact on the protein's activity, localization, folding, and interactions with other molecules.

[0043] Term "signal peptide": Signal peptide is a short peptide chain that guides the transfer of newly synthesized proteins to the secretory pathway. It is generally located at the N-terminus of the newly synthesized protein and is usually 15-30 amino acids in length.

[0044] The term "trimer tag" refers to a tool used in biochemistry and biotechnology; it is a specific tag that can induce proteins to form trimers. A trimer is a structure composed of three identical or different subunits bound together by specific interactions. This tag is typically an amino acid sequence derived from a protein with a natural tendency to form trimers.

[0045] The term "flexible linker" is a short peptide sequence used to connect two different functional domains, protein subunits or other biological molecules; its peptide chain contains multiple amino acid residues with a certain degree of flexibility. For example, when constructing a fusion protein, a flexible linker can connect two different protein domains.

[0046] The term "PelB signal peptide" refers to a short peptide chain that directs protein translocation across membranes, typically located at the N-terminus of a newly synthesized polypeptide chain. It has a specific sequence composition and function, primarily used to direct proteins to different membrane structures and subcellular organelles within the cell.

[0047] The term "OmpA signal peptide" refers to the signal peptide sequence of Escherichia coli Outer Membrane Protein A (OmpA). A signal peptide is a short peptide chain located at the N-terminus of a newly synthesized protein, typically consisting of 5-30 amino acids. Its primary function is to guide protein translocation and localization across the membrane.

[0048] The term "OmpF signal peptide" refers to the signal peptide of the Escherichia coli outer membrane protein F (OmpF). It is an amino acid sequence located at the N-terminus of the OmpF protein, generally consisting of 21 amino acids. During protein synthesis, nascent peptide chains are synthesized on the ribosome. When the OmpF signal peptide emerges from the ribosome, it is recognized and bound by the signal recognition particle, temporarily halting peptide chain extension. Subsequently, the ribosome binds to the translocon on the endoplasmic reticulum membrane, and the signal peptide guides the peptide chain across the endoplasmic reticulum membrane and into the endoplasmic reticulum lumen. Once in the endoplasmic reticulum lumen, the signal peptide is removed by a signal peptidase, allowing the protein to continue folding and processing, ultimately being transported to its specific location to function.

[0049] The term "GCN4" refers to a transcription factor that plays an important role in yeast and belongs to the basic region-leucine zipper (bZIP) domain model. GCN4 is a key transcriptional activator of amino acid biosynthesis genes in yeast, and its expression is primarily upregulated under stresses such as amino acid starvation and ribosomal protein (RP) gene deletion.

[0050] The term "T4" refers to a tag that can induce the formation of a trimer structure in host cells. T4 fibritin is a protein derived from the T4 bacteriophage that can be used as a tag for protein trimerization. In molecular biology research, scientists use this property of T4 fibritin to add it as a tag to the C-terminus of the target protein, allowing the target protein to exist in the form of a trimer. This trimerization is crucial for the function of some proteins. For example, the surface proteins of certain viruses need to be in the form of trimers to exert their biological activity.

[0051] Term "CMP": Chicken cartilage matrix protein (CMP) is an abundant non-collagenous extracellular matrix protein with a trimer structure. Cloning the target protein to the N-terminus can achieve the maintenance of its target protein structure.

[0052] According to a first aspect of the present invention, a recombinant porcine epidemic diarrhea (PED) rS1 antigen is provided. The coding sequence of the recombinant rS1 antigen comprises, from N-terminus to C-terminus, the following elements: a signal peptide, a PEDV S1 protein (full-length S1 sequence), and a trimer tag. These elements are connected by a flexible linker (GGGGSGGGGSGGGGS). The nucleotide sequence of the recombinant rS1 antigen is shown in SEQ ID NO. 1, and the amino acid sequence of the recombinant rS1 antigen is shown in SEQ ID NO. 2.

[0053] The coating antigen, rS1, is a highly immunogenic recombinant protein that provides reactivity with the sample being tested, indicating the antibody level in the sample. The signal peptide directs periplasmic expression of the S1 protein, enabling soluble expression. The linker connects the signal peptide to S1 and the trimer tag. The trimer tag ensures that the S1 protein is expressed as a trimer, ensuring correct conformational expression. The PEDV S protein, PelB signal peptide, and linker are all codon-optimized for E. coli BL21 (DE3).

[0054] SEQ ID NO.1

[0055]

[0056] SEQ ID NO.2

[0057] MKYLLPTAAAGLLLLAAQPAMAGGGGSGGGGSGGGGSMRSLIYFWLLLPVLPTLSLPQDVTRCQSTTNFRRFFSKFNVQAPAVVVLGGYLPSMNSSSWYCGTGIETASGVHGIFLSYIDSG

[0058] QGFEIGISQEPFDPSGYQLYLHKATNGNTNAIARLRICQFPDNKTLGPTVNDVTTGRNCLFNKAIPAYMRDGKDIVVGITW

[0059] DNDRVTVFADKIYHFYLKNDWSRVATRCYNRRSCAMQYVYTPTYYMLNVTSAGEDGIYYEPCTANCTGYAANVFATDSNG

[0060] HIPEGFSFNNWFLLSNDSTLLHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNHTMDGVCNGAAVDRAPEALRFNINDTSVI

[0061] LAEGSIVLHTALGTNLSFVCSNSSDPHLAIFAIPLGATEVPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYL

[0062] HLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTSIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHE

[0063] QPISFVTLPSFNDHSFVNITVSAAFGGLSSANLVASDTTINGFSSFCVDTRQFTITLFYNVTNSYGYVSKSQDSNCPFTLQSV

[0064] NDYLSFSKFCVSTSLLAGACTIDLFGYPAFGSGVKLTSLYFQFTKGELITGTPKPLEGITDVSFMTLDVCTKYTIYGFKGEGIITL

[0065] TNSSILAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVNDDIVGVISSLSNSTFNNTRELPGFFYHS

[0066] GGGGSGGGGSGGGGSRMKQLEDKVEELLSKNYHLENEVARLKKLVGER

[0067] In the aforementioned porcine epidemic diarrhea recombinant rS1 antigen, as a preferred embodiment, the signal peptide includes, but is not limited to, any one of the PelB signal peptide, OmpA signal peptide, and OmpF signal peptide; preferably, the PelB signal peptide is secreted into the periplasmic space of the large intestine for expression, achieving soluble expression. The trimer tag includes, but is not limited to, any one of GCN4, T4, and CMP; preferably, when the trimer tag sequence is GCN4, the trimer structure of the recombinant antigen can be correctly expressed. Preferably, when the flexible linker sequence is GGGGSGGGGSGGGGS, the epitope of the recombinant antigen can be better displayed.

[0068] According to a second aspect of the present invention, there is provided a method for preparing the porcine epidemic diarrhea recombinant rS1 antigen as described above, comprising the following steps:

[0069] Step 1: Clone the nucleotide sequence shown in SEQ ID NO. 1 into an expression vector, and design amplification primers rS1-F-Nde I and rS1-R-Xho I;

[0070] Step 2: Use the primer set rS1-F-Nde I and rS1-R-Xho I to amplify the rS1 gene fragment, and then double-digest the expression vector and rS1 gene fragment using Nde I and Xho I restriction endonucleases, and then ligate them with T4 DNA ligase to obtain a ligation product;

[0071] Step 3: Transform the obtained ligation product into competent Escherichia coli cells, culture them, and extract the plasmid; double-digest the plasmid with Nde I and Xho I restriction endonucleases to obtain a recombinant plasmid;

[0072] Step 4: Transform the recombinant plasmid into competent Escherichia coli cells, induce expression after amplification and culture, collect the bacterial cells by centrifugation, resuspend with PBS, ultrasonically disrupt, collect the supernatant, filter, and purify the protein to obtain the porcine epidemic diarrhea recombinant rS1 antigen.

[0073] In the above preparation method, as a preferred embodiment, in step S1, the vector is selected from any one of pET28a, pET30a, and pET32a; preferably pET28a, so that the recombinant antigen can be expressed in a soluble form.

[0074] Optionally, the nucleotide sequence of the amplification primer set rS1-F-Nde I is shown as SEQ ID NO.3; the nucleotide sequence of the rS1-R-Xho I is shown as SEQ ID NO.4.

[0075] SEQ ID NO.3

[0076] CCCATATGAAATATTTACTACC

[0077] SEQ ID NO.4

[0078] ATCGAGTTAGCGCTCACCAACC

[0079] In the above preparation method, as a preferred embodiment, in step S3, the specific steps of transforming the obtained ligation product into Escherichia coli competent cells and extracting the plasmid after culturing include:

[0080] The obtained ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB resistance plates, and cultured in a constant temperature incubator at 37°C until single colonies were clear;

[0081] A single colony was inoculated into LB liquid culture medium, cultured at 37°C and 220 rpm for 6 h, and then the plasmid was extracted using a plasmid extraction kit.

[0082] In the above preparation method, as a preferred embodiment, in step S4, the specific steps of transforming the recombinant plasmid into Escherichia coli competent cells and inducing expression after amplification and culture include:

[0083] The recombinant plasmid was transformed into Escherichia coli BL21 competent cells and inoculated into LB liquid medium. After constant temperature shaking culture at 37°C and 220 rpm for 10 h, the culture was inoculated into LB shake flasks at a 1% inoculation ratio and cultured at 37°C and 220 rpm until OD600 reached 0.6. The culture was then cooled to 25°C, IPTG inducer was added, and the culture was continued with shaking for 12 h before the fermentation was terminated.

[0084] Optionally, after protein purification, the purity of the porcine epidemic diarrhea recombinant rS1 antigen is ≥90%.

[0085] According to a third aspect of the present invention, a porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit is provided, wherein the diagnostic antigen thereof is the porcine epidemic diarrhea recombinant rS1 antigen.

[0086] The components of the porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit include: magnetic particle suspension, enzyme-labeled antibody, calibration product, quality control product, sample diluent, washing solution and luminescent liquid.

[0087] The preparation method of the magnetic particle suspension comprises the following steps:

[0088] Mix magnetic beads and EDC at a ratio of 100 μl of 4 mg / ml EDC solution to 50 mg of magnetic beads and activate at 40°C for 5 minutes. Accurately pipette 4 mL of magnetic microparticle wash buffer (1x concentrated wash buffer) into the reaction vessel. Wash at 100 rpm for 2 minutes, then perform magnetic separation until the supernatant is clear and discard. Repeat three times. Combine the two at a ratio of 15 μg recombinant protein / mg of magnetic beads and coat at 25°C.

[0089] Magnetic separation was performed on the reaction vessel until the supernatant was clear and the supernatant was discarded. Accurately pipette 4 mL of magnetic particle wash solution into the reaction vessel. Wash at 100 rpm for 2 minutes, then perform magnetic separation until the supernatant was clear and discard the supernatant. Repeat three times. Accurately pipette 4 mL of magnetic particle blocking solution into the reaction vessel. Mix at 100 rpm for 15 minutes, and finally perform magnetic separation until the supernatant was clear and discard the supernatant.

[0090] Accurately measure 100 mL of magnetic microparticle storage solution and add it to the reaction vessel. Mix at 100 rpm for 5 minutes. Securely cap the bottle / container, label it, and store at 2°C to 8°C.

[0091] Among them, the configuration of relevant reagents is as follows:

[0092] Preparation of EDC solution: Weigh 0.42 g of EDC, dissolve in 100 mL of purified water, and store at 2°C to 8°C.

[0093] Coating buffer: Weigh 1.59g of sodium carbonate and 2.93g of sodium bicarbonate, add purified water to 1000mL, measure the pH to 9.7±0.2, and store at 2°C to 8°C. The preferred coating buffer formulation of the present invention, when combined with the raw materials of the present invention, can achieve the optimal signal-to-noise ratio for the coated magnetic beads of this product.

[0094] Magnetic particle blocking solution: Weigh 2.9 g of disodium hydrogen phosphate (dodecyl sulfate), 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride, add 800 mL of purified water, weigh 1 g of fish gelatin, add 1 mL of Proclin-300, stir to dissolve, then add distilled water to make up to 1000 mL, and store at 2°C to 8°C.

[0095] Magnetic Microparticle Preservative Solution: Weigh 2.9g of disodium hydrogen phosphate (dodecyl sulfate), 0.2g of potassium dihydrogen phosphate, 8g of sodium chloride, and 0.2g of potassium chloride. Add purified water to a volume of 1000mL and adjust the pH to 7.4±0.2. Set aside. The preferred magnetic microparticle preservative solution of the present invention provides long-lasting preservation, extending the shelf life of the coated magnetic beads to 24 months. Furthermore, the preferred preservative solution of the present invention can reduce the background value of the coated magnetic beads to less than 2 orders of magnitude.

[0096] Take 800 mL of prepared phosphate buffer (PH7.4±0.2), add 1 g of fish gelatin and 0.1 g of multivitamin, stir evenly and adjust the volume to 1000 mL, and store at 2°C~8°C.

[0097] Sample diluent: Weigh 2.9 g of disodium hydrogen phosphate (dodecanoate), 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride, add 800 mL of purified water and stir to dissolve. Then weigh 15 g of bovine serum albumin and add it, then add purified water to make up to 1000 mL. Filter and sterilize, aliquot aseptically and store at 2°C to 8°C.

[0098] 25x concentrated washing solution: Weigh 72.5 g of disodium hydrogen phosphate (dodecyl), 5 g of potassium dihydrogen phosphate, 200 g of sodium chloride, and 5 g of potassium chloride, add 800 mL of purified water, add 25 mL of Tween-20, and then add purified water to make up to 1000 mL. Aliquot quantitatively and store at 2°C–8°C.

[0099] Magnetic particle washing solution (1x concentrated washing solution): dilute the 25x concentrated washing solution with distilled water or purified water at a ratio of 1:25 for later use.

[0100] Enzyme conjugate diluent: Weigh 2.9 g of disodium hydrogen phosphate (dodecanoate), 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride, add 800 mL of purified water and stir to dissolve, then weigh 15 g of bovine serum albumin and add it, then add purified water to make up to 1000 mL, filter and sterilize, aseptically aliquot and store at 2°C to 8°C.

[0101] Enzyme-labeled antibody: The enzyme-labeled antibody described in the present invention is a goat anti-pig IgG labeled with a chemiluminescent marker. The specific process is to prepare an enzyme-labeled secondary antibody by diluting HRP-labeled goat anti-pig IgG with an enzyme conjugate diluent at a ratio of 1:1500, adding 0.01% carmine pigment, stirring evenly, filtering through a 0.22μm filter membrane for sterilization, aseptically quantitatively packaging, and storing at 2°C to 8°C.

[0102] The present invention will be described in detail below with reference to embodiments of the present invention. Each example is provided by way of explanation of the present invention and is not intended to limit the present invention. In fact, it will be apparent to those skilled in the art that modifications and variations may be made in the present invention without departing from the scope or spirit of the present invention. For example, a feature shown or described as part of one embodiment may be used in another embodiment to produce yet another embodiment. Therefore, it is intended that the present invention encompasses such modifications and variations within the scope of the appended claims and their equivalents.

[0103] In the examples of the present invention, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0104] Example 1

[0105] 1.1 Construction of rS1 recombinant antigen expression vector

[0106] 1.1.1 Design of synthetic primers

[0107] The SEQ ID NO: 1 sequence was synthesized into the pUC57 vector by Suzhou Jinweizhi Biotechnology Co., Ltd. and named pUC57::rS1. A primer set for amplifying the SEQ ID NO. 1 gene sequence was designed using Primer Premier 6.0 and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The specific sequence of the primer set is as follows:

[0108] rS1-F-Nde I:

[0109] CCCATATGAAATATTTACTACC

[0110] rS1-R-Xho I:

[0111] ATCGAGTTAGCGCTCACCAACC

[0112] 1.1.2 Construction of recombinant expression vector

[0113] Using the synthetic pUC57::rS1 vector as a template, the rS1 gene fragment was amplified using the primer set rS1-F-Nde I and rS1-R-Xho I. The pET28a vector and rS1 gene fragment were then double-digested with Nde I and Xho I restriction endonucleases. The digested products were recovered and ligated using T4 DNA ligase. The resulting ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB resistance plates (containing 50 μg / mL kanamycin), and cultured in a 37°C incubator until single colonies were clearly visible.

[0114] A single colony was picked from an LB resistance plate and inoculated into LB liquid medium (containing 50 μg / mL kanamycin). After constant temperature shaking at 37°C and 220 rpm for 6 hours, the plasmid was extracted using a plasmid extraction kit. Double digestion with Nde I and Xho I restriction endonucleases yielded fragments of approximately 1908 bp and 5020 bp, respectively. The plasmids were sequenced and verified by Suzhou Jinweizhi Biotechnology Co., Ltd. The correct recombinant plasmid was named pET28a::rS1.

[0115] 1.2 Expression of rS1 fusion protein

[0116] The recombinant plasmid pET28a::rS1 was transformed into competent Escherichia coli BL21(DE3) cells, and the resulting recombinant strain was named BL21(DE3) / pET28a::rS1. The recombinant strain BL21(DE3) / pET28a::rS1 was inoculated into LB liquid medium (supplemented with kanamycin at a final concentration of 50 μg / mL) and cultured with constant shaking at 37°C and 220 rpm for approximately 10 hours. The culture was then inoculated into LB shake flasks (500 mL shake flasks, LB liquid medium volume: 200 mL) at a 1% inoculation ratio. Culture was continued at 37°C and 220 rpm until the OD600 reached approximately 0.6. The culture was then cooled to 25°C and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L. The culture was then shaken for another 12 hours to terminate the fermentation.

[0117] The bacterial cells were collected by centrifugation at 8000r / min for 10min, and the cells were resuspended in PBS (8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, fixed to 1L). The cells were broken using an ultrasonic disruptor (800w, ultrasonic 2s, stop 5s, ultrasonic time 20min), and then centrifuged at 13000r / min for 30min to separate the supernatant and precipitate. The precipitate was resuspended in an equal proportion of the above PBS buffer and subjected to SDS-PAGE analysis. The results showed that a target protein band of about 70KDa could be detected in the supernatant, and the protein expression level was about 0.5mg / mL.

[0118] 1.3 Purification of rS1 recombinant protein

[0119] The supernatant containing the target protein described in 1.2 was filtered using a 0.22 μm filter, and the treated sample was purified using a protein purifier (Taidu Biotechnology Co., Ltd.). Ni NTA Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) was used as filler, with a column volume of about 10 mL and a sample loading flow rate of 2 mL / min. Contaminants were eluted with a buffer containing 50 mmol / L imidazole, and the target protein was eluted with a buffer containing 500 mmol / L imidazole. After elution, the target protein was identified by SDS-PAGE. The results showed that the purity of the purified rS1 recombinant antigen was about 90%, which met the requirements for the use of diagnostic reagent raw materials.

[0120] Test Example 1

[0121] Reactivity identification of recombinant protein: Western blot was used to verify the reactivity and specificity of the purified rS1 recombinant antigen described in 1.3. First, the rS1 recombinant antigen was subjected to SDS-PAGE. After electrophoresis, it was transferred to an NC membrane by wet transfer. After blocking with 5% skim milk (prepared with PBST, where PBST is PBS supplemented with 0.05% Tween 20) at room temperature for 1 hour, PEDV positive and negative sera obtained from clinical isolation were used as primary antibodies. The sera were diluted 1:100 with 5% skim milk. After incubation with the primary antibody at room temperature for 1 hour, the cells were washed three times with PBST. HRP (horseradish peroxidase)-labeled goat anti-pig antibody was used as a secondary antibody. The secondary antibody was diluted 1:50,000 with 5% skim milk and incubated at room temperature for 1 hour. After washing three times with PBST, the cells were developed using DAB colorimetric solution (Kangwei Century Biotechnology Co., Ltd.). Results The rS1 recombinant antigen showed a specific color reaction with PEDV-positive serum, but had no reaction with PEDV-negative serum, which proved that the rS1 recombinant antigen could specifically bind to the antibodies produced after PEDV immunization or infection.

[0122] Test Example 2

[0123] Performance index test of porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit:

[0124] 1. Test methods

[0125] 1.1 Sensitivity test:

[0126] Positive standard serum (purchased from the China Veterinary Drug Administration) was diluted with sample diluent at a ratio of 1:2, 1:8, 1:32, 1:64, 1:128, and 1:256, respectively, and named SQC1, SQC2, SQC3, SQC4, SQC5, and SQC6, respectively, as sensitivity quality control products. The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit of the present invention was used for performance index testing. The S / P of SQC1 was required to be greater than 5.0, the S / P of SQC2 was between 3-5, the S / P of SQC3 was between 2-3, the S / P of SQC4 was between 1-2, the S / P of SQC5 was between 0.5-1, and the S / P of SQC6 was less than 0.5. (The S / P value refers to the ratio of the sample test signal value (S) to the positive control signal value (P). It is an important indicator for measuring the test results. The larger the S / P value, the higher the relative content of the target substance (antibodies related to porcine epidemic diarrhea virus) in the sample.)

[0127] 1.2 Specificity test

[0128] The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit of the present invention was used to detect the antibody-positive serum (20 copies) of this project; the porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit was used to detect the antibody-negative serum (20 copies); and the remaining sera (1 copy each); as shown in Table 1, the S / P ratio was calculated based on the measured luminescence value to determine the positive or negative.

[0129] Table 1

[0130] Antibody-positive serum Magnetic microparticle chemiluminescence detection kit of the present application Porcine epidemic diarrhea virus S / P values are all greater than or equal to 0.8 (all judged as positive) Porcine circovirus type II S / P values were all less than 0.8 (all judged as negative) Pseudorabies virus S / P values were all less than 0.8 (all judged as negative) African swine fever virus S / P values were all less than 0.8 (all judged as negative) Porcine reproductive and respiratory syndrome virus S / P values were all less than 0.8 (all judged as negative) Swine foot-and-mouth disease virus S / P values were all less than 0.8 (all judged as negative)

[0131] 1.3 Repeatability test

[0132] The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit of the present invention was used to perform 20 repeated tests on one strongly positive serum and one weakly positive serum, respectively. The mean and standard deviation of the luminescence value were calculated, and the coefficient of variation CV was calculated; and the CV% of a total of 60 tests on the same serum was calculated.

[0133] 1.4 Accuracy test

[0134] Twenty portions of standard serum were collected and tested using the porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit of the present invention, and the serum compliance rate was calculated.

[0135] 1.5 Stability test

[0136] The test kit was stored at 37°C for 3 days, 7 days, and 10 days. Three standard sera (strongly positive, weakly positive, and negative) were taken out and compared with the test kit stored at 4°C. The luminescence value ratio of the three standard sera was calculated.

[0137] 2. Results

[0138] 2.1 Sensitivity test

[0139] The six serum samples from SQC1 to SQC6 described in 1.1 were subjected to sensitivity testing. The results showed that SQC1 to SQC5 were all positive, while SQC6 was negative. The results are shown in Table 2 below:

[0140] Table 2

[0141]

[0142]

[0143] 2.2 Specificity test

[0144] The 46 sera described in 1.2 were tested, and the positive and negative results were determined based on the measured S / P ratios. The results showed that all 20 PED-positive sera tested by the three batches of kits were positive; all 20 PED-negative sera tested were negative; and the other six sera were all negative, indicating no cross-reaction. The results are shown in Tables 3 and 4 below:

[0145] Table 3

[0146]

[0147]

[0148] Table 4

[0149]

[0150] 2.3 Repeatability test

[0151] As described in 1.3, the porcine epidemic diarrhea virus magnetic microparticle chemiluminescent antibody detection kit was used to perform 20 repeated tests on one strongly positive serum and one weakly positive serum, respectively. The mean and standard deviation of the luminescence values were calculated, and the coefficient of variation (CV) was less than 4%. The CV of 60 tests on the same serum was less than 5%, indicating good repeatability, as shown in Table 5 below:

[0152] Table 5

[0153]

[0154]

[0155]

[0156] 2.4 Stability test

[0157] The kit was stored at 37°C for 3, 7, and 10 days. Three standard sera (strongly positive, weakly positive, and negative) were taken out and compared with the kit stored at 4°C. The luminescence value ratios of the three standard sera were all below 20%, indicating good stability of the kit, as shown in Table 5 below:

[0158] Table 6

[0159]

[0160]

[0161] The above describes and evaluates the efficacy of some embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments. Any person skilled in the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments with equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A porcine epidemic diarrhea recombinant rS1 antigen, characterized in that: The coding sequence of the recombinant rS1 antigen comprises the following elements in order from N-terminus to C-terminus: a signal peptide, a PEDV S1 protein and a trimer tag; the elements are connected by a linker.

2. The porcine epidemic diarrhea recombinant rS1 antigen according to claim 1, characterized in that The nucleotide sequence of the recombinant rS1 antigen is shown in SEQ ID NO.1; the amino acid sequence of the recombinant rS1 antigen is shown in SEQ ID NO.

2.

3. The porcine epidemic diarrhea recombinant rS1 antigen according to claim 1, characterized in that The signal peptide is selected from any one of the following: PeIB signal peptide, OmpA signal peptide, and OmpF signal peptide; And / or, the trimer tag is selected from any one of GCN4, T4, and CMP; And / or, the linker sequence is GGGGSGGGGSGGGGS.

4. A method for preparing the porcine epidemic diarrhea recombinant rS1 antigen according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Clone the nucleotide sequence shown in SEQ ID NO. 1 into an expression vector, and design amplification primers rS1-F-Nde I and rS1-R-Xho I; S2. Amplify the rS1 gene fragment using the primer set rS1-F-Nde I and rS1-R-Xho I. Double-digest the expression vector and the rS1 gene fragment using Nde I and Xho I restriction endonucleases, and then ligate them using T4 DNA ligase to obtain a ligation product. S3. Transform the obtained ligation product into competent Escherichia coli cells, culture them, and extract the plasmid; double-digest the plasmid with Nde I and Xho I restriction endonucleases to obtain a recombinant plasmid; S4. The recombinant plasmid was transformed into competent Escherichia coli cells, amplified and cultured, and then induced for expression. The bacterial cells were collected by centrifugation, resuspended with PBS, ultrasonically disrupted, the supernatant was collected, filtered, and the protein was purified to obtain the porcine epidemic diarrhea recombinant rS1 antigen.

5. The preparation method according to claim 4, characterized in that In step S1, the vector is selected from any one of pET28a, pET30a, and pET32a; And / or, the nucleotide sequence of the amplification primer set rS1-F-Nde I is shown as SEQ ID NO.3; the nucleotide sequence of the rS1-R-Xho I is shown as SEQ ID NO.

4.

6. The preparation method according to claim 4, characterized in that In step S3, the steps of transforming the obtained ligation product into competent E. coli cells and extracting the plasmid after culturing include: The obtained ligation product was transformed into Escherichia coli DH5α competent cells, coated on LB resistance plates, and cultured in a constant temperature incubator at 37°C until single colonies were clear; A single colony was inoculated into LB liquid culture medium, cultured at 37°C and 220 rpm for 6 h, and then the plasmid was extracted using a plasmid extraction kit. And / or, in step S4, the specific steps of transforming the recombinant plasmid into competent E. coli cells and inducing expression after amplification and culture include: The recombinant plasmid was transformed into Escherichia coli BL21 competent cells and inoculated into LB liquid medium. After constant temperature shaking culture at 37°C and 220 rpm for 10 h, the culture was inoculated into LB shake flasks at a 1% inoculation ratio and cultured at 37°C and 220 rpm until OD600 reached 0.

6. The culture was then cooled to 25°C, IPTG inducer was added, and the culture was continued with shaking for 12 h before the fermentation was terminated. And / or, after protein purification, the purity of the porcine epidemic diarrhea recombinant rS1 antigen is ≥90%.

7. A porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit, characterized in that: The diagnostic antigen is the porcine epidemic diarrhea recombinant rS1 antigen according to any one of claims 1 to 3.

8. The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit according to claim 7, characterized in that: It includes magnetic particle suspension, enzyme-labeled antibody, calibration product, quality control product, sample diluent, washing solution and luminescent liquid.

9. The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit according to claim 8, characterized in that: The preparation method of the magnetic particle suspension comprises the following steps: (1) Mix the magnetic beads and EDC at a ratio of 100 μl of 4 mg / ml EDC solution to 50 mg of magnetic beads. After activation at 40°C for 5 min, wash the activated magnetic beads with magnetic particle washing solution, centrifuge, and discard the supernatant. Repeat this three times to obtain activated magnetic beads. (2) The activated microspheres were mixed with recombinant rS1 antigen at a ratio of 15 μg recombinant antigen / mg magnetic beads, coated with coating buffer at 25°C, centrifuged, and the supernatant discarded. The microspheres were then washed with magnetic microparticle washing solution, centrifuged, and the supernatant discarded. This was repeated three times to obtain coated magnetic beads. (3) Add 4 mL of magnetic particle blocking solution to the obtained coated magnetic beads, mix at 100 r / min for 15 minutes, then centrifuge and discard the supernatant; then add 100 mL of magnetic particle preservation solution, mix at 100 r / min for 5 minutes, and store at 2°C to 8°C to obtain the magnetic particle suspension.

10. The porcine epidemic diarrhea virus magnetic particle chemiluminescent antibody detection kit according to claim 9, characterized in that: In step (2), the coating buffer is prepared as follows: 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate are weighed, the volume is adjusted to 1000 mL, the pH is measured to be 9.7±0.2, and the coating buffer is stored at 2°C to 8°C to obtain the coating buffer; And / or, in step (3), the magnetic particle blocking solution is prepared as follows: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride are added to purified water; then 1 g of fish gelatin and 1 mL of Proclin-300 are added in sequence, stirred to dissolve, and the volume is adjusted to 1000 mL, and stored at 2°C to 8°C to obtain the magnetic particle blocking solution; And / or, in step (3), the preparation method of the magnetic particle preservation solution is as follows: weigh 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride, dilute to 1000 mL, measure the pH to 7.4±0.2, and obtain a phosphate buffer solution for use; then, add 1 g of fish gelatin and 0.1 g of multivitamins to the phosphate buffer solution in sequence, stir evenly, dilute to 1000 mL, and store at 2°C to 8°C to obtain the magnetic particle preservation solution.

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