74 results about "Circovirus" patented technology

The present application relates to the field of veterinary diagnostic technology, and specifically to a probe composition, a primer composition, and a triple fluorescence PCR kit and related methods for differential diagnosis of three genes of different strains of African swine fever composed of the above-mentioned probe composition and primer composition. The kit includes primer probes specific to the B646L gene, the CD2v gene, and the I177L gene. The present application designs fluorescent PCR primers and probes for the B646L gene, the CD2v gene, and the I177L gene, respectively, and establishes a triple fluorescence PCR detection method, which can quickly identify African swine fever virus strains and gene-deficient strains. At the same time, it has no cross-reaction with swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine circovirus, porcine parvovirus, healthy whole blood, oropharyngeal swabs, nasal swabs, anal swabs, environmental swabs, pig tissues, etc., and has good specificity.

The invention relates to the technical field of avian virus detection, and particularly discloses a primer probe combination, a kit and a method for simultaneously detecting chicken infectious anemia virus and chicken circle virus type 3 based on fluorescent RAA and application. The method can be used for simultaneously detecting the chicken infectious anemia virus and the chicken circle virus type 3, and has the advantages of simplicity in operation, short detection time, good specificity, high sensitivity, no need of expensive instruments and equipment, support of on-site rapid detection, suitability for large-scale popularization and application and the like; and effective technical support can be provided for rapid detection and screening of the chicken infectious anemia virus and the chicken circle virus type 3.

The invention provides a triple inactivated vaccine for porcine circovirus type 2, porcine mycoplasmal pneumonia and porcine reproductive and respiratory syndrome as well as a preparation method and application of the triple inactivated vaccine, and belongs to the technical field of biology. The triple inactivated vaccine is prepared on the basis of a porcine circovirus type 2 protective antigen Cap protein, a mycoplasma hyopneumoniae protective antigen recombinant fusion protein F-P46-P65 and a porcine reproductive and respiratory syndrome virus PRRSV-NADC30-like LF strain. According to the triple inactivated vaccine, three major diseases caused by the porcine circovirus type 2, the mycoplasma hyopneumoniae and the porcine reproductive and respiratory syndrome virus can be prevented at the same time through one-time inoculation, the immunization procedure is greatly simplified, the stress reaction caused by multiple immunization to pigs is effectively reduced, and the healthy growth of the pigs is guaranteed. The immune cost of a farm is remarkably reduced, the effect of'immune burden reduction 'is really achieved, and the efficiency and economic benefits of breeding management are improved.

The present invention discloses a recombinant porcine circovirus type 3 trimer protein, a preparation method and an application thereof. The present invention uses bioinformatics methods and resources to predict the B cell antigen epitopes and T cell antigen epitopes of the PCV3Cap protein. Taking into account the stability and immunogenicity of the antigen epitopes, a recombinant porcine circovirus type 3 trimer protein based on the PCV3Cap protein antigen epitope is designed, and its efficient soluble expression in Escherichia coli is achieved. The expressed recombinant porcine circovirus type 3 trimer protein can be mass-produced and purified by Ni-NTA affinity chromatography. The purified recombinant porcine circovirus type 3 trimer protein can assemble into a stable trimer structure. The subunit vaccine prepared using the recombinant porcine circovirus type 3 trimer protein can induce experimental pigs to produce a high level of antibodies and has a significant protective effect on the experimental pigs. The recombinant porcine circovirus type 3 trimer protein designed by the present invention provides a new idea for the development of PCV3 vaccine.

The present invention discloses a detection system for simultaneously detecting avian gyrovirus type 1 and human gyrovirus type 1, and its application, relating to the technical field of biological detection. The detection system includes an RAA amplification primer pair for GyVg1, an RAA amplification primer pair for GyH1, 12-crRNA-3, 13-crRNA-2, an ssDNA probe, an ssRNA probe, LbCas12a nuclease, LbuCas13a nuclease and T7 transcriptase. Based on the conserved genes of GyVg1 and GyH1, the present invention respectively designs specific crRNAs and RAA primers, and by combining the RAA amplification technology with the CRISPR-Cas fluorescence detection system, a detection system for simultaneously detecting GyVg1 and GyH1 is established. The detection system can simultaneously detect two different subtypes of gyrovirus, improves the detection efficiency, and provides a new technical means for the dynamic study of virus infection.

The invention discloses a recombinant antigen protein, a recombinant vector, a recombinant host cell, a virus-like particle and application thereof and a vaccine, and relates to the technical field of antigen preparation, the recombinant antigen protein comprises an icosahedral porcine circovirus capsid protein and an A104R protein loaded on the surface of the icosahedral porcine circovirus capsid protein, the A104R protein comprises a protein coded by an African swine fever virus A104R gene. The recombinant antigen protein capable of simultaneously reacting with positive serum of the African swine fever virus and positive serum of the porcine circovirus is prepared, and the vaccine prepared by mixing the recombinant antigen protein with an adjuvant can effectively stimulate a pig body to generate specific antibodies of the African swine fever virus and the porcine circovirus; therefore, reference is provided for joint defense of the two viruses. The recombinant antigen protein provided by the invention has the characteristics of stable expression, high yield and high solubility.

The invention belongs to the field of biological agriculture, and discloses a recombinant vector for preparing a swine triple vaccine, a recombinant virus and application of the recombinant vector and the recombinant virus. The technical problem to be solved by the invention is how to prepare the triple vaccine of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and hog cholera virus. The recombinant vector for preparing the swine triple vaccine disclosed by the invention is a vector which is obtained by inserting an encoding gene of E2-Cap2 fusion protein into a recombinant vector capable of rescuing porcine reproductive and respiratory syndrome virus and enabling the encoding gene of the E2-Cap2 fusion protein to be expressed, wherein the E2-Cap2 fusion protein is a protein with an amino acid sequence of SEQ ID No.3. Experiments prove that the porcine triple vaccine obtained by using the recombinant vector has good immunogenicity, and can be used for simultaneously preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and hog cholera virus.

The present invention discloses a method for preparing a metal organic framework-based Pickering emulsion vaccine and its application, comprising the following steps: using Al(NO3)3·9H2O and 4,4'-diphenyldicarboxylic acid as raw materials, obtaining white DUT-5 powder by a solvothermal method; adding the prepared DUT-5 to ultrapure water, and mixing by simple ultrasound to obtain a Pickering emulsion; mixing the obtained Pickering emulsion with PCV2Cap protein and placing it in a shaking table overnight; ultrafiltration and centrifugation of the obtained mixed emulsion to obtain DUT-5-PE@CAP. The vaccine of the present invention can effectively deliver antibodies or proteins to specific locations and stimulate a strong immune response, providing new possibilities for the development of new vaccines, and providing a new method for the prevention and treatment of porcine circovirus type II, which is of great significance for controlling the disease.

The invention relates to crRNA for detecting an African swine fever virus gene type I or type II and a kit. The kit comprises crRNA-1 for detecting the African swine fever virus gene type I and crRNA-2 for detecting the African swine fever virus gene type II. And the like. The kit has high specificity on African swine fever virus gene type I and gene type II, has no cross reaction with porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine circovirus and porcine Delta coronavirus, has high detection sensitivity, and can detect 4 copies / [mu] L of ASFV gene type I plasmid and 2 copies / [mu] L of ASFV gene type II plasmid. The kit provided by the invention is simple to operate, does not need large-scale instruments and equipment, and can be used for detecting the African swine fever virus in a field environment; the whole detection process is completed within 40 minutes.

The invention relates to a fermentation medium of porcine circovirus type 3 (PCV3) Cap protein virus-like particles and a fermentation method thereof. The fermentation culture medium comprises tryptone, a yeast extract, casein hydrolysate, sorbitol, glycerol, polyoxyethylene mono-tert-octyl phenyl ether and the like. Methanol is supplemented for three times in the fermentation process, so that the expression efficiency of the Cap protein of PCV3 is remarkably improved, and correct folding and self-assembly of the Cap protein into the virus-like particles are promoted. According to the culture medium and the method, generation of impure protein is reduced, the purification cost is reduced, and key technical support is provided for industrial production of PCV3 subunit vaccines.

The present application belongs to the field of molecular biology detection, and particularly relates to a composition, method and use for simultaneous detection and differentiation of swine fever virus, psittacosis chlamydia, porcine reproductive and respiratory syndrome virus and porcine circovirus type II. The composition for simultaneous detection provided by the present application can realize rapid detection of four pathogen target genes in one tube of reaction solution by using multiplex fluorescence RPA detection technology, and has the advantages of high throughput, short time and low cost. On one hand, the present application can accurately detect pathogens and analyze mixed infection of pathogens, so as to achieve the effect of precise medication, reduce the use of antibiotics, save costs, and reduce the cost and increase the benefit of enterprises, and on the other hand, compared with fluorescence PCR, the detection time is greatly shortened, and the purpose of rapid detection is achieved.

The present application relates to the technical field of veterinary biological products, and particularly provides a porcine circovirus type 3 (PCV3) Cap protein and a related product thereof. The PCV3 Cap protein is an NLS region 8-16aa truncated Cap protein. The amino acid sequence of the Cap protein is as shown in SEQ ID NO: 2. The present application further provides a PCV3 virus-like particle antigen, which is the PCV3 Cap protein. The Cap protein is expressed at a higher level. A vaccine prepared from the antigen exhibits strong immunogenicity and provides excellent protective efficacy.

The invention discloses a porcine circovirus type 4 Cap protein antigen epitope peptide and a monoclonal antibody thereof. The amino acid sequence of the antigen epitope peptide is as shown in SEQ ID No. 11; the heavy chain variable region of the monoclonal antibody comprises CDR1-3 as shown in SEQ ID No.1 to SEQ ID No.3; the light chain variable region comprises CDR1-3 as shown in SEQ ID No. 4 to 6; the monoclonal antibody does not react with a plurality of other swine viruses such as PCV2, PCV3, PEDV, PRRSV and the like, and has good specificity; the antibody can be obtained by biological means such as conventional genetic engineering, and the problem that the genetic sequence of the antibody is easy to lose due to long-term cryopreservation of traditional hybridoma cells is avoided; optimization of the antibody on the gene or protein level is facilitated, and the specificity and affinity of the antibody are further improved.

The present application relates to the technical field of protein purification, and particularly relates to a porcine circovirus vaccine protein purification device, which comprises a protein purification instrument, a tray is arranged on the top of the protein purification instrument, and a connecting mechanism is arranged at the connecting position of the tray and the protein purification instrument. The tray is installed and fixed through the mutual insertion of the convex block and the pin rod, so that the tray is more convenient to take down and install and is easy to clean. The internal inclined surface of the tray achieves the effect of flow guiding, liquid is stored in the water collecting box, dripping on the experiment table is avoided, the safety of the experiment is improved, the height adjustment of the bracket is more convenient through the cooperation of the arc-shaped clamping block and the arc-shaped groove, the positioning effect is achieved through the cooperation of the spherical convex and the spherical groove, the adjustment of the installation position of the pre-packed column is more convenient, and the operation is more simple and convenient during the protein purification operation.

The invention discloses construction and application of a recombinant PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) strain for expressing porcine circovirus Cap protein. The recombinant PRRSV capable of expressing the Cap protein of the porcine circovirus type 2 is successfully constructed and saved by adopting a reverse genetic manipulation technology. The modified virus is inoculated to Marc-145 cells for subculture, and the result shows that the modified virus can trigger cytopathy and stably express Cap protein. The obtained recombinant strain shows a stable passage capability in a Marc-145 cell, and has an extremely high virus load. The strain can be used as a vaccine candidate strain, provides multiple protection for PRRSV and porcine circovirus of different pedigree, and provides a brand new solution for healthy breeding of pigs.

The application provides a construction and application of a recombinant pseudorabies virus strain stably and efficiently expressing green fluorescent protein, in the research, a green fluorescent protein gene (EGFP) is inserted into different positions of a double gene deletion vaccine strain (JS-2012-△gE / gI) of a pseudorabies virus mutant strain by using a homologous recombination method. Finally, it is proved that the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP has good genetic stability, and the EGFP gene can also be stably and efficiently expressed, and the growth rate on cells is also comparable to that of the parent virus, so that the VP1 / 2 gene is the best exogenous gene insertion site. Subsequently, the classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be replaced or inserted into the EGFP expression frame in a single or combined manner to construct different types of multi-vaccines, and the multi-vaccines have extremely high research value and platform significance.

The invention provides a porcine circovirus type 3 Cap recombinant protein gene, a recombinant rod grain, a protective antigen and a purification method, and belongs to the technical field of biology. The invention utilizes an insect baculovirus eukaryotic expression system to prepare the porcine circovirus type 3 Cap protein protective antigen, and provides a chromatographic purification process of the porcine circovirus type 3 Cap protein protective antigen, which adopts a one-step column chromatography purification method and adopts splitting, filtering and composite affinity chromatography processes to obtain the porcine circovirus type 3 Cap protein protective antigen. According to the method, the porcine circovirus type 3 Cap protein protective antigen is specifically purified from the complex cell lysis solution rapidly and efficiently, the recovery rate in the purification process is high, the purity reaches 90% or above, the treatment capacity is large, the assembly rate of the purified product is high, and the method is simple and suitable for large-scale production.

The invention discloses an mRNA vaccine composition for combined treatment of porcine circovirus and application of the mRNA vaccine composition. The combination comprises a nucleotide combination, the nucleotide combination comprises a sequence for coding the Cap protein of the porcine circovirus and a CpG adjuvant, and the sequence for coding the Cap protein of the porcine circovirus and the CpG adjuvant are respectively assembled into a porcine circovirus mRNA vaccine and a CpG adjuvant mRNA vaccine. In order to solve the problem that the existing mRNA vaccine generally depends on inherent immune stimulation of mRNA and LNP, and generally, an exogenous adjuvant is not additionally added, so that the immune response intensity or persistence is insufficient possibly, the CpG adjuvant and the PCV2 Cap mRNA-LNP vaccine are creatively combined for use; experiments show that the combination can significantly improve the neutralizing antibody level and enhance the immune response of Th1 type cells compared with mRNA independent immunization, has good safety, and can be used for preventing and / or treating PCV2 infection.

The present invention relates to a porcine circovirus type 2 antibody detection kit. The porcine circovirus type 2 antibody detection kit of the present invention comprises an antibody detection chip, wherein the antibody detection chip comprises a solid phase carrier and polypeptides represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively immobilized on the solid phase carrier. The porcine circovirus type 2 antibody detection kit of the present invention can be used to distinguish pigs immunized with porcine circovirus type 2 inactivated vaccine from pigs infected with porcine circovirus type 2 wild-type virus.

The application discloses a kind of anti-swine circovirus type 3 Cap protein monoclonal antibodies, the anti-swine circovirus type 3 Cap protein monoclonal antibodies is monoclonal antibody P3, wherein the amino acid sequence of the heavy chain variable region and light chain variable region of monoclonal antibody P3 is respectively shown as SEQ ID NO.3 and SEQ ID NO.4.The application further discloses a kind of swine circovirus type 3 antibody detection kit, the reagent kit includes monoclonal antibody P3 and swine circovirus type 3 Cap protein and matched detection reagent.The monoclonal antibody P3 has the advantages of good specificity and good affinity, and can be used for the detection of swine circovirus type 3 antibody and the development of related detection products.Meanwhile, the reagent kit of the application has good specificity and sensitivity, and is suitable for wide range detection of swine circovirus type 3 antibody.

The invention discloses a preparation method and application of a Pickering emulsion vaccine based on a metal organic framework, and the preparation method comprises the following steps: taking Al (NO3) 3.9 H2O and 4, 4 '-diphenyldicarboxylic acid as raw materials, and obtaining white DUT-5 powder through a solvothermal method; adding the prepared DUT-5 into ultrapure water, and carrying out simple ultrasonic uniform mixing, so as to prepare a Pickering emulsion; mixing the obtained Pickering emulsion with PCV2Cap protein, putting the mixture into a shaking table and staying overnight; and carrying out ultrafiltration and centrifugation on the obtained mixed emulsion to obtain DUT-5-PE (at) CAP. The vaccine provided by the invention can effectively transfer an antibody or protein to a specific position and stimulate a strong immune response, provides a new possibility for developing a novel vaccine, provides a new method for preventing and treating the porcine circovirus type II, and has a significant meaning for controlling the disease.

The invention discloses a porcine circovirus type 2 neutralizing antibody and a hybridoma cell strain secreting the same. The invention also discloses sequence information of heavy chain and light chain variable regions of the neutralizing antibody and fully verifies a key site for binding the neutralizing antibody with Cap protein. The porcine circovirus type 2 neutralizing antibody can specifically recognize porcine circovirus type 2 and porcine circovirus type 2 Cap protein, has high neutralizing activity on PCV2b and PCV2d, and is of great significance in guiding PCV2 vaccine design and developing therapeutic antibodies.

The present invention provides a fluorescent PCR amplification reaction reagent, a kit and a detection method for African swine fever virus that are resistant to interference from multiple disinfectants. DTT, BSA, Brij35 and betaine anti-interference components are added to the fluorescent PCR amplification reaction reagent. The detection limit of the fluorescent PCR detection kit reaches 1 copies / μL, and there is no positive cross reaction with swine fever virus, epidemic diarrhea virus, blue ear disease virus, circovirus, and pseudorabies virus. Therefore, the fluorescent PCR detection kit has the advantages of strong specificity, high sensitivity, good result interpretation accuracy, and especially can avoid false negative interpretation. In addition, the fluorescent PCR detection kit can be used to detect African swine fever virus in pig blood, saliva, tissue grinding fluid, environmental swabs, and vehicle swabs, and can effectively resist the interference of unknown disinfectants in farms.

The invention provides a porcine circovirus type 5 Cap protein, a virus-like particle, an elisa plate, an ELISA kit and application thereof, and belongs to the technical field of animal viruses. The invention provides a porcine circovirus type 5 Cap protein with an amino acid sequence as shown in SEQ ID NO. 1. The porcine circovirus type 5 Cap protein provided by the invention has the advantages of stable structure and good antigenicity, can be specifically combined with a circovirus type 5 antibody in a serum sample, and can be self-assembled into 60 virus-like particles. The ELISA plate and the ELISA kit for detecting the porcine circovirus type 5, provided by the invention, have the advantages of strong specificity and high sensitivity, and can accurately detect whether a blood sample contains the porcine circovirus type 5 or not. The porcine circovirus type 5 virus-like particle provided by the invention has the advantages of high yield, good uniformity and strong stability, and can be subsequently prepared into a porcine circovirus type 5 subunit vaccine.

The application belongs to the technical field of biotechnology, and particularly discloses a synthetic peptide antigen of porcine circovirus type 2 Cap protein, a polyclonal antibody and application thereof. In order to solve the technical defects of low specificity and inaccurate detection of the amino acid sequence of the synthetic peptide antigen in the prior art, the application provides a synthetic peptide antigen of PCV2 Cap protein, a polyclonal antibody and application thereof. The application obtains an amino acid sequence of an antigen peptide by performing antigen peptide prediction on PCV2 Cap protein, and the applicant synthesizes the antigen peptide by using an Fmoc solid phase method, and immunizes a New Zealand white rabbit with the obtained antigen peptide to prepare a polyclonal antibody of Cap protein, the antibody can effectively react with a transiently expressed Cap protein and a Cap protein in PCV2b-infected PK15 cells, and provides a powerful tool for detection and biological function research of the PCV2 Cap protein.

This invention relates to the field of cell culture technology, specifically to a cell culture tank for producing porcine circovirus vaccine. The tank includes a main body with a feeding port at the top, a fixing box at the top, and a heat dissipation vent at the top of the fixing box. A stirring mechanism is located inside the main body. The first cone design achieves a shearing and flow-diverting effect, the arc surface guides the flow, reducing cell damage, and the spherical protrusions and through-holes further enhance the flow disturbance effect. The row of staggered spherical protrusions further improves the mixing effect. The two flow-dispersing blocks are arranged in opposite directions, achieving mixing in both forward and reverse rotation. The second cone design reduces resistance during reverse rotation without affecting mixing.

The present invention in particular relates to a method for reducing virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen. In one example, an immunogenic composition is administered via the oral route to a pig, wherein the immunogenic composition comprises a recombinantly produced and purified PCV2 ORF2 protein. This allows to simply reduce virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by associated with a PCV2 infection in the pigs, while simultaneously reducing the propensity for adverse reactions, eliminating the risk of injection site reactions, avoiding pain, and putting less stress on the animals.

The invention discloses an application of a porcine ZC3HAV1 gene and a regulatory region SNP (Single Nucleotide Polymorphism) marker of the porcine ZC3HAV1 gene in resisting porcine circovirus, and belongs to the technical field of molecular genetics. According to the present invention, the first research discovers that the overexpression of the ZC3HAV1 gene can inhibit the replication of PCV2 in the porcine macrophage line, such that the new path is provided for the prevention and treatment of the porcine circovirus disease; a further research discovers that T / C nucleotide polymorphism exists at the site 1859 of the 5'regulatory region of the ZC3HAV1 gene, and the transcriptional activity of the ZC3HAV1 gene can be enhanced by mutating the nucleotide at the site 1859 of the 5 'regulatory region of the ZC3HAV1 gene from T to C; due to the fact that stable expression of the ZC3HAV1 is consistent with that of porcine circovirus disease resistance, by detecting the molecular marker, pigs with high expression of the ZC3HAV1 are selected to establish a base group for breeding, and the molecular marker has important significance on improving the circovirus disease resistance of pig groups.

The application discloses a bovine viral diarrhea virus chimeric virus expressing porcine circovirus type 3 capsid protein and a preparation method thereof. In the experiment, a gene sequence for encoding a porcine circovirus type 3 capsid protein and a His tag fused with the C terminal of the protein is inserted into different sites of a bovine viral diarrhea virus genome through a circular polymerase extension reaction, and the chimeric virus is prepared through virus rescue of the obtained circular DNA. The His tag sequence in the gene sequence and the bovine viral diarrhea virus genome are connected through a 2A peptide sequence, so as to realize the target of effectively releasing the antigen through the co-expression of the heterologous protein. The chimeric virus prepared by the application retains the infectivity and replication capacity of the parent virus, can stably replicate in cells and efficiently express the target antigen, and provides an efficient and flexible virus carrier platform for the research and development of a porcine circovirus type 3 vaccine.

The application belongs to the field of biological detection, and particularly relates to an isothermal amplification kit for detecting porcine circovirus type 3. The application provides an isothermal amplification detection primer group for porcine circovirus type 3 (PCV3), which comprises a pair of outer primers (LOF and LOR) and a pair of inner primers (LIF and LIR) and is designed according to the conserved sequence of the porcine circovirus type 3. The primer group has low design difficulty, and maintains similar sensitivity and specificity to other isothermal amplification reactions such as a loop-mediated isothermal amplification method, can be suitable for target sequences with higher GC content and below 200 bp, has no denaturation and annealing process, is suitable for on-site detection, and has great application potential.