211 results about "Epidemic diarrhea" patented technology

The invention discloses a porcine epidemic diarrhea virus antigen fusion protein, a coding gene and a porcine epidemic diarrhea vaccine prepared from the porcine epidemic diarrhea virus antigen fusion protein. The method comprises the following steps: screening to obtain a homosensory sequence of the spike protein of the porcine epidemic diarrhea virus, and fusing the homosensory sequence with a monomeric porcine ferritin heavy chain subunit to obtain an antigen fusion protein; in order to improve the expression quantity or the titer of the antigen fusion protein, the obtained antigen fusion protein is subjected to sequence optimization and then subjected to single-site or multi-site mutation, and the expression quantity and the titer of the obtained mutant are remarkably improved. According to the invention, a recombinant protein is expressed by using a silkworm or AcMNPV-insect cell and other eukaryotic expression systems to obtain a self-assembled ferritin nanoparticle antigen for displaying an S protein antigen protein on the surface of a cage structure of porcine ferritin, and a vaccine prepared from the prepared nanoparticle antigen can induce a widely neutralized anti-porcine epidemic diarrhea virus antibody; the porcine epidemic diarrhea virus vaccine has the potential of becoming a universal porcine epidemic diarrhea virus vaccine with cross immune efficacy.

The invention discloses an application of phloretin in preparation of a medicine or a feed additive for treating or preventing swine envelope virus infection, the medicine is phloretin or oral liquid, granules, tablets and powder prepared from phloretin as an effective component and a solvent or an auxiliary material, and the use dosage is 2.5-10mg / kg of the body weight of a pig; or the phloretin is directly added into the feed, and the addition amount of the phloretin is 0.01-0.03% of the total weight of the pig feed. The phloretin or the composition containing the phloretin is used as a medicine or a feed additive, is high in safety and free of toxic and side effects, and has the effects of remarkably inhibiting replication of envelope viruses such as porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus and African swine fever virus and reducing the toxicity of the porcine envelope viruses; the defects of immune failure possibly caused by vaccination, virus variation caused by use of antiviral drugs and the like are overcome, the morbidity and mortality of the piglets infected with the swine envelope virus are effectively reduced, and the effect of preventing and treating or preventing the swine envelope virus infection diseases is achieved.

The invention discloses an indirect ELISA kit for rapidly detecting a porcine epidemic diarrhea virus IgA antibody and application of the indirect ELISA kit, and belongs to the technical field of immunology. The invention provides a recombinant PEDV-RBD protein. The amino acid sequence of the recombinant PEDV-RBD protein is shown as SEQ ID NO. 3. The indirect ELISA kit for detecting the porcine epidemic diarrhea virus IgA antibody is constructed based on the recombinant PEDV-RBD protein, the PEDV IgA antibody in pig breast milk or serum can be accurately captured for detection, the kit has the advantages of high sensitivity and strong specificity, and a technical means is provided for prevention and control of porcine epidemic diarrhea.

The invention relates to a method for inhibiting in-vitro infection of a porcine epidemic diarrhea virus by using JPH203 and application. The JPH203 can be used for preparing an antiviral drug for inhibiting infection of the porcine epidemic diarrhea virus. JPH203 with the concentration of 5 [mu] M is added in the process that Vero cells are infected with PEDV, compared with a control group without JPH203, the virus infection condition, the protein expression quantity, the RNA content and the virus titer of the PEDV in a treatment group with JPH203 are all remarkably reduced, and it is indicated that JPH203 can be used for inhibiting in-vitro infection of the porcine epidemic diarrhea virus.

The invention discloses an application of a porcine GDPD2 gene in preparation of a medicine for inhibiting porcine epidemic diarrhea virus replication. It is found for the first time that overexpression of the porcine GDPD2 gene can effectively inhibit replication of the porcine epidemic diarrhea virus, and the porcine GDPD2 gene can serve as a target spot for inhibiting porcine epidemic diarrhea virus infection to be used for preparing related drugs.

The invention belongs to the technical field of biology, and relates to a latex immunochromatography test strip for rapidly detecting porcine epidemic diarrhea virus (PEDV) and a preparation method of the latex immunochromatography test strip. The test strip comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, a quality control line C and a detection line T are arranged on the nitrocellulose membrane. A latex microsphere labeled PEDV N protein monoclonal capture antibody is sprayed on the combination pad, a detection line T is coated with a PEDV N protein monoclonal detection antibody 1E1, and a quality control line C is coated with goat anti-mouse IgG. The test strip can specifically recognize PEDV, and does not react with other porcine intestinal coronaviruses with similar clinical symptoms. The test strip is good in specificity, relatively high in sensitivity and simple to operate, a result is interpreted within 10 minutes, and compared with a traditional colloidal gold detection technology, the stability and the color development effect are obviously improved.

The invention relates to the technical field of biological detection, relates to a multiple ddPCR detection method for porcine intestinal coronavirus, and in particular relates to a monitoring and quantitative detection method for samples with relatively low porcine intestinal coronavirus content or complex matrix, such as feeds, animal tissues and animal-derived products. The droplet digital PCR detection method for the porcine intestinal coronavirus established in the invention has the advantages of strong specificity, high sensitivity, good repeatability and strong anti-interference capability, can be used for absolute quantification of the copy concentration of the porcine acute diarrhea syndrome coronavirus (SADS-CoV), the porcine epidemic diarrhea virus (PEDV), the porcine delta coronavirus (PDCoV) and the porcine transmissible gastroenteritis virus (TGEV) in an actual sample, and can be used for the detection of the porcine intestinal coronavirus. The invention provides a novel and reliable technical method for trace early warning and monitoring of early-stage infection of four porcine intestinal coronavirus, differential diagnosis of other common viruses, research of propagation rules of the viruses, epidemiological risk assessment and other systematic applications.

The invention belongs to the technical field of biology, and particularly relates to a porcine epidemic diarrhea virus broad-spectrum neutralizing antibody and application thereof. A porcine epidemic diarrhea virus broad-spectrum neutralizing antibody 6G2 is screened, the virus neutralizing activity of the antibody is evaluated through a cell model, and it is found that the antibody can effectively block infection of G1a, G1b, G2a, G2b, G2c and S-INDEL (porcine epidemic diarrhea virus) gene subtype strains with high epidemic rate on cells. Further, animal infection experiments of pigs prove that the neutralizing antibody provided by the invention has good prevention and treatment effects on the porcine epidemic diarrhea virus, and has important significance on improving the capability of preventing and controlling porcine epidemic diarrhea in a pig farm.

Provided are an RNA vaccine for porcine epidemic diarrhea and a preparation method therefor. The RNA vaccine is obtained by means of gene synthesis, in-vitro transcription, and lipid nanoparticle encapsulation, which can rapidly respond to the currently prevalent new PEDV strains, reduces the research and production cycle of new vaccine development, and provides an effective technical approach for the prevention and control of newly mutated highly virulent strains. The prepared RNA vaccine for porcine epidemic diarrhea has high production efficiency, can induce relatively high levels of porcine epidemic diarrhea virus neutralizing antibodies and binding antibodies in vivo, effectively reducing the occurrence of diarrhea in piglets, and thus holds great promotional value for the prevention and control of porcine epidemic diarrhea.

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection method of a porcine epidemic diarrhea virus antibody SIgA, and belongs to the technical field of animal antibody detection. In order to solve the problems of limited detection range and single immune evaluation dimension of a porcine epidemic diarrhea virus detection method in the prior art, the invention provides an indirect ELISA detection method of a porcine epidemic diarrhea virus antibody SIgA, and the detection method adopts a pichia pastoris eukaryotic secretion platform to prepare a high-activity PEDV S1 antigen; meanwhile, a secreting type IgA antibody is used as a detection target; the method has the advantages of high detection sensitivity, low background noise, better variant compatibility and practical popularization value, and provides a clinical and industrial-level solution for maternal immunity monitoring, piglet passive protection evaluation and vaccine immunity strategy optimization.

The invention relates to the technical field of veterinary biological products, and particularly provides a porcine epidemic diarrhea virus S protein and related products. The amino acid sequence of the porcine epidemic diarrhea virus S protein fragment is as shown in SEQ ID NO.4. The trimer S protein is constructed by fusing a trimer tag at the C terminal of a protective antigen in a truncated form of the porcine epidemic diarrhea virus S protein. After expression and purification, immunogenicity is judged through animal evaluation, and it is verified that the vaccine prepared from the trimer S protein has the advantages of being high in safety, good in immunogenicity, stable in batches, capable of inducing piglets to generate good immunoreaction and the like, and porcine epidemic diarrhea can be effectively treated or prevented.

The present invention relates to a kit for detecting porcine epidemic diarrhea and a preparation method thereof, belonging to the technical field of virus detection, and specifically comprising the following steps: after extracting the antigen, treating it with a virus purification agent containing polyethylene glycol, nuclease and concanavalin, and then obtaining a virus antigen suspension with a composite dissociation solution. The suspension is coated on a microporous reaction plate to form a solid-phase antigen. Subsequently, the porcine epidemic diarrhea virus solid-phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution are aseptically stored to prepare a kit for detecting porcine epidemic diarrhea. The present invention provides a novel kit and a preparation method thereof, which can not only effectively purify porcine epidemic diarrhea virus, but also significantly reduce the risk of protein precipitation, while overcoming nucleic acid interference and improving purification efficiency.

The invention provides a preparation method of a novel PEDV (porcine epidemic diarrhea virus) subunit vaccine, and belongs to the technical field of biology. The novel PEDV subunit vaccine is prepared by adopting a method comprising the following steps: (1) treating lactococcus lactis by using a high-pressure damp-heat method to obtain peptidoglycan particles; (2) treating the peptidoglycan particles by adopting an ultrahigh-pressure microjet technology to obtain a peptidoglycan carrier with the particle size of 280-850nm; and (3) mixing the peptidoglycan carrier with a PEDV subunit antigen of which the amino acid sequence is SEQ ID NO: 1 to obtain the novel PEDV subunit vaccine. The novel PEDV subunit vaccine comprises a peptidoglycan carrier with a specific size, can improve the in-vivo presentation efficiency of an antigen and rapidly induce efficient body fluid and mucous membrane immune response, and is of great significance to research and development of vaccines for porcine epidemic diarrhea diseases and the like.

The invention discloses application of sodium tanshinone IIA sulfonate in preparation of anti-PEDV (porcine epidemic diarrhea virus) products, and belongs to the technical field of biology. According to the application disclosed by the invention, 2320 compounds are screened, and the tanshinone IIA sodium sulfonate is found to have a good inhibition effect on PEDV (Porcine Epidemic Diarrhea Virus). Tanshinone IIA sodium sulfonate with the concentration of 200 [mu] M is added in the process that African green monkey kidney cells are infected with PEDV in vitro, compared with a control group, the protein expression level, the mRNA content of N genes and the virus titer of PEDVN of an experimental group with the tanshinone IIA sodium sulfonate are obviously reduced, and it is indicated that the tanshinone IIA sodium sulfonate can be used for inhibiting infection and proliferation of the porcine epidemic diarrhea virus. The invention verifies that the tanshinone IIA sodium sulfonate can effectively inhibit the PEDV proliferation and the action mechanism thereof in an in-vitro environment, and the discovery not only widens the biological characteristics of the tanshinone IIA sodium sulfonate, but also provides a powerful experimental basis for the development of the tanshinone IIA sodium sulfonate as a novel potential anti-PEDV drug.

The invention discloses a porcine epidemic diarrhea virus S protein monoclonal antibody and application thereof. Specifically, two monoclonal antibodies GC42G2-1 and GC33D8-1 which have broad-spectrum binding characteristics with the porcine epidemic diarrhea virus S protein are obtained firstly, the GC42G2-1 antibody has good universality and can have strong affinity with different strains, and the GC33D8-1 antibody can be used for binding the porcine epidemic diarrhea virus S protein in a broad-spectrum manner. The GC42G2-1 can be independently used, and also can be matched with a polyclonal antibody or GC33D8-1 to be used for sandwich ELISA (enzyme-linked immunosorbent assay) so as to detect and quantify PEDV (porcine epidemic diarrhea virus). Secondly, the two monoclonal antibodies are respectively used as a coating antibody and a detection antibody, a double-antibody sandwich ELISA method is established, and the double-antibody sandwich ELISA method is applied to PEDV vaccine S protein quantification, has better parallelism compared with other antibodies, and can be widely applied to detection of different strains. The method disclosed by the invention has the advantages of high sensitivity, high accuracy, high repeatability and high flux, and can be used for the development of blocking ELISA (Enzyme-Linked Immunosorbent Assay), capturing ELISA, indirect ELISA, competitive ELISA and detection of porcine epidemic diarrhea viruses by colloidal gold test strips.

According to the RNA vaccine for porcine epidemic diarrhea and the preparation method of the RNA vaccine, the RNA vaccine is obtained through gene synthesis, in-vitro transcription and lipid nanoparticle wrapping, the current epidemic new PEDV strain can be rapidly dealt with, the research and development and production cycle of new vaccine development is shortened, and an effective technical means is provided for prevention and control of new variant virulent strains. The RNA vaccine for porcine epidemic diarrhea prepared by the invention is high in production efficiency, can generate a high-level porcine epidemic diarrhea virus neutralizing antibody and binding antibody in vivo, can effectively reduce the occurrence of piglet diarrhea, and has great popularization value for prevention and control of porcine epidemic diarrhea.

The invention provides a porcine epidemic diarrhea virus G2c subtype strain and application thereof. According to the invention, the porcine epidemic diarrhea virus G2c subtype HN2501 strain is separated from porcine intestinal tissues and is subjected to passage purification, and the microbial preservation number of the porcine epidemic diarrhea virus G2c subtype HN2501 strain is CGMCC (China General Microbiological Culture Collection Center) No.46417. The isolated strain can stably proliferate on passage cells to generate typical cytopathy. The G2c subtype isolated strain of the porcine epidemic diarrhea virus has excellent immunogenicity; the vaccine prepared from the isolated strain can induce piglets to generate high-level neutralizing antibodies; meanwhile, the ELISA kit prepared from the isolated strain can be used for well detecting the porcine epidemic diarrhea virus antibody in serum.

The invention provides a subunit vaccine composition for porcine epidemic diarrhea, porcine delta coronal and porcine rotavirus as well as a preparation method and application of the subunit vaccine composition, and is characterized in that the subunit vaccine composition comprises prokaryotically expressed swine erysipelas filamentous bacillus SpaA protein, viral subunit protein and pharmaceutically acceptable adjuvants; wherein the amino acid sequence of the prokaryotically expressed swine erysipelas filamentous bacillus SpaA protein is as shown in SEQ No.1, and the viral subunit protein is selected from one or more of porcine epidemic diarrhea virus S protein, porcine delta coronavirus S protein and porcine rotavirus VP8 protein. The vaccine composition disclosed by the invention has the advantages of strong immunogenicity, good safety, no immune interference, high neutralizing antibody titer and long antibody duration.

The present invention relates to a method and application of TBB in inhibiting the in vitro infection of porcine epidemic diarrhea virus, and the TBB can be used to prepare antiviral drugs for inhibiting the infection of porcine epidemic diarrhea virus. In the process of PEDV infecting Vero cells in the present invention, TBB with a concentration of 10 μmol / L is added. Compared with the group without TBB treatment, the RNA content, protein expression level and virus titer of PEDV in the group treated with TBB are all significantly reduced, indicating that TBB can be used to inhibit the in vitro infection of porcine epidemic diarrhea virus.

The application belongs to the technical field of biotechnology, and particularly relates to a porcine epidemic diarrhea virus recombinant strain, a construction method and application. The S gene sequence of the existing epidemic porcine epidemic diarrhea virus PEDV CH / SX / 2016 strain is replaced by the S2015 gene sequence shown in SEQ ID NO. 1 to construct a PEDV CH / SX / 2016 strain infectious clone. A porcine epidemic diarrhea virus recombinant strain rCH / SX / 2016-S 2015 is successfully rescued by transfecting cells with a plasmid containing the infectious cDNA shown in SEQ ID NO. 4. The problem that the original strain cannot be successfully rescued by reverse genetics engineering is solved, and the recombinant virus strain rCH / SX / 2016-S 2015 has an activity comparable to that of the original strain, and can be used for preparing a PEDV diagnostic reagent and / or vaccine.

The invention provides an application of brucein A in preparation of an anti-porcine epidemic diarrhea virus drug, and belongs to the technical field of chemical medicines. Experimental results show that the brucein A has remarkable inhibiting and blocking effects on a-1 ribosomal Frameshift process of a porcine epidemic diarrhea virus (PEDV), can effectively inhibit replication and proliferation of the PEDV, and has an inhibiting effect on a plurality of common strains of the PEDV; the brucein A can be used for preparing a medicine for resisting PEDV (porcine epidemic diarrhea virus) and a medicine for preventing and treating porcine epidemic diarrhea caused by PEDV, and the medicine is a wide-spectrum antiviral medicine, is insensitive to variation of PEDV and has a wide application prospect.

The invention discloses a primer probe combination for simultaneously detecting African swine fever virus, porcine epidemic diarrhea virus and porcine reproductive and respiratory syndrome virus, a kit and application thereof. The primer probe combination consists of a first primer probe group for detecting African swine fever virus, a second primer probe group for detecting porcine epidemic diarrhea virus and a third primer probe group for detecting porcine reproductive and respiratory syndrome virus. The primer probe combination for simultaneously detecting the African swine fever virus, the porcine epidemic diarrhea virus and the porcine reproductive and respiratory syndrome virus, provided by the invention, is high in sensitivity and good in specificity, and can be effectively used for detecting the African swine fever virus, the porcine epidemic diarrhea virus and the porcine reproductive and respiratory syndrome virus.

The invention discloses a vaccine freeze-dried heat-resistant protective agent and a preparation method and application thereof. The freeze-dried heat-resistant protective agent consists of the following components in weight percentage: 0.6-1.0% of gelatin, 4.5-6.5% of brain heart infusion, 3.5-5.5% of tryptone, 6-10% of trehalose, 2.0-3.0% of sucrose, 1.5-2.5% of sodium thiosulfate, 0.6-1.2% of polyvinyl pyrrolidone (PVPK30), 0.4-0.6% of dextran, 0.3-0.5% of mannitol, 0.2-0.4% of glycine, 0.04-0.06% of sodium glutamate, 0.2-0.3% of L-arginine, and the balance is water for injection. The freeze-dried heat-resistant protective agent of the vaccine disclosed by the invention has components that interact with each other, thereby improving the temperature tolerance of porcine infectious gastroenteritis, porcine epidemic diarrhea and porcine rotavirus triple live vaccines during freeze-drying, reducing the loss of efficacy, and the virus loss rate before and after freeze-drying does not exceed 0.2 titers.

The application discloses a RT-MIRA-LFD kit for detecting PEDV and belongs to the technical field of molecular biology. The application firstly provides a RT-MIRA-LFD primer probe combination for rapidly detecting porcine epidemic diarrhea virus, which comprises an upstream primer shown as SEQ ID NO. 5, a downstream primer shown as SEQ ID NO. 7 and a probe shown as SEQ ID NO. 9; secondly, the application provides a kit and a detection method for rapidly detecting porcine epidemic diarrhea virus. The application designs the primer and the probe for detecting porcine epidemic diarrhea virus based on the RT-MIRA-LFD technology, and designs the RT-MIRA-LFD detection method. The detection method has the advantages of high sensitivity and high specificity, short detection time and visual results, and provides a new experience method for rapid detection of porcine epidemic diarrhea virus.

This invention belongs to the field of biotechnology, specifically relating to a broad-spectrum neutralizing antibody against porcine epidemic diarrhea virus (PEDV) and its applications. This invention screened a broad-spectrum neutralizing antibody against PEDV, 6G2. Cell model evaluation revealed that the antibody effectively blocked infection of cells by several highly prevalent PEDV genotypes: G1a, G1b, G2a, G2b, G2c, and S-INDEL. Further animal infection experiments in pigs demonstrated that the neutralizing antibody provided by this invention has good preventive and therapeutic effects against PEDV, which is of great significance for improving the ability of pig farms to control PEDV.

The invention discloses a porcine epidemic diarrhea and porcine rotavirus (G9 type) bigeminy attenuated vaccine and a preparation method thereof, and relates to the technical field of veterinary biological products. According to the present invention, dominant genotype strains such as PEDV G2 type KQ01 strain (the preservation number is CCTCC NO: V202005) and PoRV G9 type CH09 strain (the preservation number is CCTCC NO: V202191) are adopted as parent strains, a cell continuous passage method is adopted to carry out passage attenuation, and the virus content, the purity, the specificity, the hereditary stability, the safety and the immunogenicity are systematically inspected during the passage process; and then, carrying out proliferation process research on passage attenuated strains PEDV HB17-R and PoRV JS01-R in a bioreactor, harvesting a cell culture, mixing the two antigens according to a proper ratio, adding a proper amount of a stabilizer, and carrying out a freeze vacuum drying process to obtain the high-purity PoRV-HB17-R / PoRV-HB17-R / PoRV-HB17-R / PoRV-HB17-R / PoRV-JS01-R composite antigen. The bigeminy attenuated vaccine is highly matched with current dominant prevalent strains (PEDV G2 type and PoRV G9 type), is based on a suspension culture technology and has both safety and high efficiency.

The invention discloses an RT-MIRA-LFD kit for detecting PEDV (Porcine Epidemic Diarrhea Virus), and belongs to the technical field of molecular biology. The invention firstly provides an RT-MIRA-LFD primer probe combination for rapidly detecting the porcine epidemic diarrhea virus. The RT-MIRA-LFD primer probe combination comprises an upstream primer as shown in SEQ ID NO.5, a downstream primer as shown in SEQ ID NO.7 and a probe as shown in SEQ ID NO.9; the invention further provides a kit and a detection method for rapidly detecting the porcine epidemic diarrhea virus. The primer and the probe for detecting the porcine epidemic diarrhea virus and the RT-MIRA-LFD detection method are designed based on the RT-MIRA-LFD technology, the detection method has the advantages of high sensitivity and high specificity, the detection time is short, the result is visual, and a new experience method is provided for rapid detection of the porcine epidemic diarrhea virus.

Use of MMPD for the preparation of a medicament for inhibiting porcine epidemic diarrhea virus (PEDV) infection in vitro.

The invention relates to the technical field related to immunological detection, in particular to a mouse anti-porcine epidemic diarrhea virus monoclonal antibody, an immunogen and application thereof. The amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively as follows: GFSLSTSGVV, RIDPSDSYT and ARDREKSDNSGYFDY, and the amino acid sequences of complementary determining regions CDR1, CDR2 and CDR3 of a light chain variable region of the monoclonal antibody or the antigen binding fragment of the monoclonal antibody are respectively as follows: RASKSISKYLA, EASSLES and LQHTPKKNK. The monoclonal antibody can be specifically combined with nucleocapsid proteins (N proteins) of all subtypes of the porcine epidemic diarrhea virus, and a porcine epidemic diarrhea virus detection kit prepared from the monoclonal antibody has the advantages of high sensitivity, strong specificity, wide detection range, short detection time and the like.

A composition comprising Bidens pilosa or an active compound isolated from the Bidens pilosa for use in treating, inhibiting and / or decreasing the presence of pathogenic gut microbiota in an animal in need thereof is disclosed. The pathogenic gut microbiota are at least one selected from the group consisting of Brachyspira, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis coronavirus (TGEV) and Rotavirus (RV). The composition is also for use in treating, inhibiting and / or decreasing occurrence of swine diarrhea, swine dysentery, swine viral infection and increasing the percentage of swine carcass and bone weight. In one embodiment, the composition is for use in treating, inhibiting and / or decreasing occurrence of swine dysentery associated with Brachyspira.