269 results about "Porcine epidemic diarrhoea" patented technology

The invention belongs to the technical field of biological vaccine preparation, and particularly relates to a porcine epidemic diarrhea (PED) recombinant baculovirus gene engineering subunit vaccine, a preparation method and an application thereof. According to the present invention, S1 gene and M gene of the current new PEDV epidemic strain are selected as reference sequences, a baculovirus expression system is adopted to express S1 protein or partial S1 protein and M protein, and the obtained recombinant protein is prepared into a subunit vaccine for effectively controlling PED occurrence; with the PED recombinant baculovirus gene engineering subunit vaccine produced by using the method, the defect of the current PEDV traditional vaccine is solved; and the PED recombinant baculovirus gene engineering subunit vaccine can be used for prevention and treatment of PEDV infections and related diseases caused by PEDV, and can further be used for preparation of coating antigen of PEDV detection antibody ELISA kits.

The invention relates to a porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine and a preparation method of the porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine. The porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine is prepared by performing virus amplification on a swine testicular cell line (ST cells) or an African green monkey kidney cell line (Vero cells) by using a self-attenuated and preserved transmissible gastroenteritis virus SD/L strain and a self-attenuated and preserved porcine epidemic diarrhea virus LW/L strain, and carrying out the steps of harvesting, uniformly mixing, freeze-drying and the like. The porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine can effectively prevent two diseases namely swine transmissible gastroenteritis and epidemic diarrhea.

The invention provides a fusion protein, a porcine epidemic diarrhea vaccine composition containing the fusion protein and application thereof. The fusion protein contains porcine epidemic diarrhea virus antigenic protein and immunoglobulin Fc segment, wherein the porcine epidemic diarrhea virus antigenic protein contains a protein formed by series combination of porcine epidemic diarrhea virus S protein segments. The invention also provides a porcine epidemic virus vaccine composition which contains the fusion protein and a carrier. The invention also provides a preparation method of the vaccine composition and application of the vaccine composition in preparing drugs for preventing and/or treating diseases initiated by porcine epidemic diarrhea virus. The vaccine composition prepared from the fusion protein avoids the technical problem that the porcine epidemic diarrhea virus totivirus can not be easily separated and cultured in the traditional vaccine inactivation process. The fusion protein can utilize the gene engineering technique to perform abundant recombinant expressions, has the advantage of short time consumption, and is convenient for large-scale production.

The invention discloses a porcine epidemic diarrhea virus low-virulent stain ZJ08 and application thereof. The microbial collection number of the low-virulent stain is CGMCC No.7806. The PEDV low-virulent stain has high safety, and is safe for pregnant sows, baby pigs and pigs in all ages. The active protection ratio of the low-virulent stain for 3-day baby pigs reaches 100%, and the passive protective ratio reaches higher than 94.7%, which indicates that the low-virulent stain has favorable immunoprotection effect on porcine epidemic diarrhea.

The invention provides a method for preparing triple vaccine for preventing porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus. The method comprises the following steps: inoculating a host-cell line with a 90 percent grown monostratum against a porcine transmissible gastroenteritis virus, a porcine epidemic diarrhea virus and a porcine rotavirus respectively, and adding a cell maintenance media into the host-cell lines respectively to be cultured at 37 DEG C; after cytopathic effect reaches over 75 percent, collecting viruses to be stored at 20 DEG C below zero for standby; mixing the viruses according to 10 TCID50 in 1:1:1, and simultaneously adding Freund's complete adjuvant and immunopotentiator into the mixture to inactivate the mixture by formaldehyde at 37 DEG C for 24 hours; and adding an oil adjuvant into the mixture to prepare a vaccine of water-oil-water preparation. The method can be used for preparing the triple vaccine for preventing the porcine transmissible gastroenteritis, the porcine epidemic diarrhea and the porcine rotavirus so as to solve the problem that the diseases do not have an effective medicine to treat currently.

The invention discloses a porcine epizootic diarrhea virus strain with good immunogenicity and an inactivated vaccine prepared through the porcine epizootic diarrhea virus strain. The porcine epizootic diarrhea virus strain is a current epidemic strain, and good immunogenicity and stability are achieved; compared with a commercially available vaccine strain, the vaccine prepared through the strain has the advantages of being good in safety, high in immune protective capability, high in immune efficacy and the like, and porcine epizootic diarrhea can be comprehensively and effectively prevented and treated.

The invention provides a porcine epidemic diarrhea vaccine composition. The vaccine composition contains a porcine epidemic diarrhea virus subunit antigen and a vector. The invention also provides a porcine epidemic diarrhea vaccine composition, and the vaccine composition comprises a porcine epidemic diarrhea virus subunit antigen, a porcine epidemic diarrhea totivirus inactivated antigen and a vector. The invention also discloses a preparation method of the vaccine composition and application thereof to preparation of drugs for preventing and / or treating porcine epidemic diarrhea. An S1 protein in the vaccine composition can conduct large amount of recombinant expression of components in the vaccine composition by means of genetic engineering, so as to realize short time consumption as well as facilitate large-scale production. When the vaccine composition contains the S1 protein and porcine epidemic diarrhea totivirus inactivated antigen, the vaccine composition has better immune effect than single usage of totivirus inactivated antigen, and can effectively prevent diseases caused by variants of porcine epidemic diarrhea and common strains.

The invention provides a triple live vaccine for a swine transmissible gastroenteritis virus, a swine epidemic diarrhea virus and a swine rotavirus and a preparation method thereof. The content of the three viruses is not less than 107.5 TCID50 (Tissue Culture Infectious Dose 50)/mL, and the volume ratio is 1:1:1. The triple live vaccine provided by the invention solves the problem that a multiple vaccine for effectively preventing and treating such three diseases as swine transmissible gastroenteritis, swine epidemic diarrhea and the swine rotavirus is not available on the current market, and especially realizes the prevention and control on the swine rotavirus. Compared with the existing method of inoculating with three simplex vaccines to prevent such three transmissible diseases, the triple live vaccine provided by the invention is economical to use, simplifies the immunization procedure and lowers the epidemic prevention cost, thereby providing a new simple and convenient immunization way for farms in China.

The invention provides feed which comprises a puffing feed carrier, a micro-organism bacterial agent, a trace element additive, an amino acid additive and a traditional Chinese medicine additive, wherein the traditional Chinese medicine additive further comprises galangal, cumin, root of chinese barberry, herba lagotis, partrinia, phellodendron, astragalus, atractylodes, myrobalan, limonitum, dendrobium, liriope, hairy euphorbia, aristolochia debilis, coriolus versicolor, Kapok and schisandra chinensis. According to the invention, the traditional Chinese medicine composition has the efficacies that the treatment of epidemic diarrhea of pigling pays an attention to treating both principal and secondary aspect of epidemic diarrhea; the treatment effect is good; drug residue does not exist, functions of an organism can be aroused, the resistance of the organism is improved.

The invention discloses a traditional Chinese medical composition for treating porcine epizootic diarrhea. The composition comprises the following components in parts by weight as follows; 80+/-5 parts of honeysuckle, 150+/-10 parts of Agastache rugosus, 80+/-5 parts of Codonopsis pilosula, 80+/-5 parts of Rhizoma Atractylodis Macrocephalae, 10+/-5 parts of Mangnolia officinalis, 100+/-5 parts of Pinellia ternate, 80+/-5 parts of Poria cocos, 50+/-5 parts of Poria cocos and 80+/-5 parts of liquorice. The traditional Chinese medical composition disclosed by the invention has the advantages of high integral curative effect, difficulty in generation of tolerance, low residue, small toxic and side effects and the like, has the remarkable inhibiting effect to growth and propagation of many virus pathogenies, even has the pharmacological effect of killing pathogenies, and has very important economic and social meanings for promoting healthy development of pig industry.

The invention relates to an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for a porcine epidemic diarrhea virus (PEDV) and a preparation method of the ELISA detection kit. The ELISA detection kit for the porcine epidemic diarrhea virus comprises an ELISA plate, a detection antibody and an ELISA second antibody, wherein the ELISA plate is coated with a specific egg yolk antibody of the porcine epidemic diarrhea virus; the detection antibody is a polyclonal antibody of the porcine epidemic diarrhea virus; the ELISA second antibody is an antibody which is labeled by an enzyme and resists the detection antibody. The ELISA detection kit for the porcine epidemic diarrhea virus takes the PEDV of vomitus and excrement of diarrhea pigs as a detection object and can find PEDV infected pigs as soon as possible; meanwhile, the operation difficulty of the detection is reduced.

The invention provides a PED (Porcine Epedemic Diarrhea) inactivated vaccine and a preparation method thereof and relates to the field of biopharmacy. The PED inactivated vaccine comprises inactivated PEDV (Porcine Epedemic Diarrhea Virus), and is characterized in that the PED inactivated vaccine comprises 0.05-10 mg/mL Beta-glucosylceramide, 0.1-21 mg/mL monophosphoryl lipid A, 1.5-125 mg/mL muramyl dipeptide and 0.7-4.5 mg/mL Beta-glucan. According to the ingredients of the PED inactivated vaccine, Beta-glucosylceramide, monophosphoryl phosphoryl lipid A, muramyl dipeptide and Beta-glucan have the synergistic effect, the immune response of animals to antigens in the vaccine is significantly improved, the immune window phase is shortened, the antibody production duration of the animal body is obviously prolonged, the serum antibody level is improved, and the level of total intestinal mucosa secretory antibodies (the total SIgA) is improved.

The invention discloses a combined live vaccine of transmissible gastroenteritis virus of swine (TGEV) and porcine epidemic diarrhea virus (PEDV) and a preparation method thereof. An attenuated swine transmissible gastroenteritis virus HB08 and an attenuated porcine epidemic diarrhea virus ZJ08 which are self-separated, attenuated and stored respectively undergo viral multiplication on ST cells and VeroE6 cells, and seedling and freeze drying are then carried out by adding a freeze-drying protective additive into a virus solution which is qualified after inspected. The two diseases, transmissible gastroenteritis of swine and porcine epidemic diarrhea virus, which are epidemic in clinic at present, can be effectively prevented by the use of the combined live vaccine.

The invention discloses hyper-immune serum prepared by immunizing a hybridized pig with the age of 50-60 days by taking an inactivated vaccine of porcine epidemic diarrhea virus ZJ08 strain CGMCC No.7806 as an immunogen due to immunization for three times. The prepared hyper-immune serum is high in properties, high in specificity and high in safety and is not polluted by bacteria, moulds, mycoplasma and exogenous viruses, and the artificial infection cure rate is 100 percent. The neutralization titer of the serum is over 26, the serum is separately filled at the temperature of minus 70 DEG C, and the retention period is 12 months.

The invention discloses a Chinese herb extract feed additive capable of effectively preventing and treating porcine epizootic diarrhea, and belongs to the field of feed additives. The feed additive is prepared from the main materials of snow grass meal, curcuma powder, semen cassiae powder, burdock powder, shallot white powder, Chinese herbal medicine preparation and curcuma extract, and toadstool and bacillus subtilis are scientifically compounded, so that the application amount of antibiotics medicine in cultivation of pigs is effectively reduced, the use amount of the antibiotic is reduced by above 60%, the drug property of the Chinese herbal medicine is improved, the porcine epizootic diarrhea caused by porcine epidemic diarrhea virus can be effectively prevented and treated, and the Chinese herb extract feed additive is safe and non-toxic. Besides, the amount of the additive in the feed is low, the quality is stable, an acting effect is improved by combining different Chinese herb extracts, the porcine epizootic diarrhea is reduced by 60-80%, and the taste and quality of animal products are obviously improved. A sense experimental evaluation proves that the evaluation score on the taste and quality of the animal products using the additive is improved by above 65%.

The invention discloses a porcine epidemic diarrhea S1 protein fusion gene, recombinant bacillus megaterium strain and application. An antigen fusion gene shown as SEQ ID No. 1. is obtained by connecting an antigen locus of porcine epidemic diarrhea virus (PEDV) S glycoprotein and a cell wall anchoring sequence. The invention further constructs a secretion expression vector containing the antigen fusion gene, and secretion expression vector is converted into the bacillus megaterium to express recombinant protein on the cell wall or surface of the bacillus megaterium. Immunoblotting experiments indicate that the expressed recombinant protein can react with PEDV immune serum and has the same antigenicity as PEDV natural antigens. Immunofluorescent tests of live bacteria which are subjected to induced expression indicate that the expressed recombinant protein is positioned to the surfaces of the bacteria. Experiment results indicate that the recombinant protein can be prepared into safe and effective mucous immune live vaccines for preventing and treating porcine epidemic diarrhea.

The invention discloses a method for preparing a double yolk antibody of a porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The method comprises the following steps of: performing porcine transmissible gastroenteritis virus multiplication on porcine kidney cells (PK15); performing porcine epidemic diarrhea virus multiplication on African green monkey kidney cells (Vero); emulsifying the two cell cultures used as antigen with an oil emulsion adjuvant to prepare immunogen, namely, mixing the two kinds of viruses in a ratio of (1-3):(1-3) to prepare the immunogen; immunizing non-immunologic laying hens; and obtaining the double yolk antibody which can prevent and treat porcine transmissible gastroenteritis and porcine epidemic diarrhea based on the collection and purification of the yolk. When the double yolk antibody is used for curing experimental pigs, the clinical symptoms in the experiment are obviously reduced compared with a control group, and the death rate of the experimental group is obviously lower than that of the control group. The double yolk antibody has obvious preventing and treating functions when applied in a pig farm with high incidence rate of the porcine transmissible gastroenteritis and the porcine epidemic diarrhea.

The invention provides a method for detecting swine epidemic diarrhea by reverse transcription-loop-mediated isothermal amplification. According to the conserved domain gene of PEDV (Porcine Epidemic Diarrhea Virus) N protein, three pairs of primers aiming at six areas on a target gene are designed, two RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primers are combined, and PED (Porcine Epidemic Diarrhea) viruses are identified by the RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) technology; RT-LAMP reacts; reverse transcription is performed on obtained RNA(Ribose Nucleic Acid); and the obtained cDNA is used for PCR reaction. The method provided by the invention ensures amplification specificity, has high amplification speed and can obtain a result within 30-60 minutes, and people can observe amplification effect with eyes without electrophoresis. Reverse transcription and nucleic acid amplification can be realized in 1 hour at the constant temperature of 65 DEG C by using enzyme mixture; RT-LAMP detection is carried out on the basis of LAMD amplification DNA; a reverse transcription enzyme is added to realize amplified detection of RNA; and reverse transcription and amplification are finished in one step so as to omit the reverse transcription step which is firstly carried out by the traditional RT-PCR.

The invention relates to a porcine epidemic diarrhea virus strain MY01 and application thereof and belongs to the field of porcine epidemic diarrhea virus vaccine reagents. The porcine epidemic diarrhea virus strain MY01 is collected in China General Microbiological Culture Collection Center under CGMCC No. 11495. The porcine epidemic diarrhea virus strain MY01 is higher in pathogenicity for pigs and good in immunogenicity, a deactivated oil emulsion vaccine prepared using the strain is safe and reliable, homologous attacking protection can be provided, protection is also provided for PEDV (porcine epidemic diarrhea virus) epidemic strains, higher immunity can be generated after immunization, inoculated pigs are significantly lower in morbidity and mortality, the immunization effect is superior than that of existing commercial vaccines in the market, and the strain has the advantage of competing with like products home and abroad, can effectively prevent the prevalence and spread of porcine epidemic diarrhea virus and reduce economic cost caused by the disease and has a promising application prospect.

The invention provides fusion protein, which comprises monomeric ferritin subunit protein connected to porcine epidemic diarrhea virus antigenic protein, which includes porcine epidemic diarrhea virus S protein and/or fragments thereof, and further includes S1 protein, M protein, N protein and/or fragments thereof. The invention further provides nanoparticles containing the fusion protein. The invention further provides a vaccine composition containing the nanoparticles and/or a carrier. The invention further discloses a preparation method of the vaccine composition and an application of the vaccine composition to prepare a medicine for preventing and/or treating porcine epidemic diarrhea virus. The vaccine composition made from the nanoparticles overcomes the technical problem that whole viruses are hard to separate and culture in a process of manufacturing a conventional inactivation vaccine from porcine epidemic diarrhea whole viruses. The nanoparticles can be subjected to massive recombinant expression via genetic engineering technology. The time consumption is short, and the fusion protein is convenient for massive production.

The invention provides a double-antibody sandwich ELISA quantitative determination kit of a porcine epidemic diarrhea virus and an application and relates to the field of biodetection. A preservation number of a hybridoma cell strain PEDV of an anti-PEDV N protein monoclonal antibody is CCTCC NO: C201410. The double-antibody sandwich ELISA quantitative determination kit of the porcine epidemic diarrhea virus comprises a pre-coated elisa plate and an enzyme labeled antibody. The pre-coated elisa plate is the elisa plate coated by the anti-PEDV N protein monoclonal antibody, and the enzyme labeled antibody is a horse radish peroxidase labeled anti PEDV N protein polyclonal antibody; the anti PEDV N protein polyclonal antibody is obtained by immunizing a rabbit through the protein N of the porcine epidemic diarrhea virus to obtain serum of the rabbit. The kit provided by the invention is good in specificity, high in sensitivity and good in stability, can quickly, simply and efficiently detect the porcine epidemic diarrhea virus quantitatively, and is low in cost and suitable for high throughput detection.

The invention discloses an egg yolk antibody for preventing and treating porcine epidemic diarrhea and a preparation method of the egg yolk antibody. The preparation method comprises the following steps: using an inactivated vaccine of a porcine epidemic diarrhea virus as immunogen for immunizing a laying hen; harvesting egg yolk liquid and purifying from the egg yolk liquid to obtain the egg yolk antibody, wherein a porcine epidemic diarrhea virus strain is PEDV/CH/2014, with a preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 10111. The egg yolk antibody prepared by the preparation method disclosed by the invention has the advantages of good characters, easiness in storage, high safety and good preventing and treating effects; by an in vitro neutralization test, the neutralizing titer of the egg yolk antibody is measured to reach 1:128; by means of a clinical prevention test, the diarrhea incidence of tested pigs can be reduced by 20 percent, and the cure rate of artificial infection reaches 100 percent.