30 results about "Porcine kidney" patented technology

The invention discloses a large-scale full-suspension production method of a porcine circovirus type 2. The inventors of the invention domesticate a porcine kidney cell adaptable to large-scale serum-free full-suspension culture, which is named as sPK15-YP, is collected in the China General Microbiological Culture Collection Center and has the culture collection number of CGMCC NO.13846. The sPK15-YP cell adaptable to full-suspension culture is used for achieving serum-free large-scale culture of the porcine circovirus type 2 (PCV2), so that the conventional spinner bottle culture technology is replaced, the human resource is reduced, the product quality is improved, and the bottleneck that the virus content is low during PCV2 full-virus culture is solved; by a full-suspension sPK15-YP cell technology, a high-potency PCV2 semifinished product is prepared; by a hollow fiber method, a PCV2 virus culture solution is concentrated and purified to obtain a more pure PCV2 virus concentrated antigen. By the large-scale full-suspension production method, a solid foundation is laid for studying a PCV2 multivalent vaccine, the dosage of the vaccine is reduced, the stress of a swine herd is reduced and the immune level of the swine herd is enhanced.

The invention discloses multifunctional enzyme gene HG27 and application. The multifunctional enzyme gene HG27 comprises the following steps of comparing a nucleic acid sequence spliced by rhizomys sinensis intestinal microbe macro transcriptome with a glucoside hydrolase family, screening a sequence with relatively high homology, removing original signal peptide, adding pig parotid gland secretory protein signal peptide, optimizing a pig codon, and connecting to a pcd3.1 vector; transfecting to porcine kidney epithelial cells for expression verification, collecting a cell culture solution for enzyme activity analysis, comparing the enzyme activity, selecting the optimal HG27 gene, and detecting the optimal pH value, protein tolerance and enzymatic reaction of different substrates of the HG27 gene, thereby obtaining the multifunctional enzyme gene HG27 which can be applied to the fields of livestock and poultry feed enzyme preparations, transgenic animal preparation, food and medicine industries and the like, and the problems that cellulose is difficult to degrade and absorb and excrement pollutes the environment are solved.

The invention discloses a method for improving the matrix impact resistance of anaerobic ammonium oxidation particles, which belongs to the field of wastewater treatment. During the matrix shocking period of the ANAMMOX reactor in stable operation, 1500-2500 mg / L total nitrogen shock was performed for 12-48 hours, and 0.1 mg / L-1.5 g / L porcine kidney acylase I was added to the reactor every 1 hour until the matrix The impact is over; AHLs inhibitor porcine kidney acylase I will no longer be added to the reactor after the matrix impact is stopped. The present invention suppresses the activity of the quorum sensing system of the ANAMMOX particles under the impact of the matrix by adding a lower concentration of AHLs inhibitor porcine kidney acylase I, thereby reducing the release amount of the granular AHLs to control the excessive release of LB-EPS, and improving the granule under the impact of the matrix. stability and settling capacity. At the same time, when the inhibitor concentration is low, it will not adversely affect the particle activity.

The invention provides a sgRNA targeting knockout SST gene and its CRISPR / Cas9 system and application, belonging to the technical field of gene knockout. The present invention provides a sgRNA targeted to knock out the SST gene, including SST sgRNA1 and SST sgRNA2, and also provides a CRISPR / Cas9 system comprising the sgRNA. The CRISPR / Cas9 system is transfected into porcine kidney fibroblasts, and the positive cells obtained by screening are obtained to obtain a higher gene deletion efficiency. At the same time, the CRISPR / Cas9 system can improve the efficiency of gene deletion by completely knocking out the pig SST gene. The growth rate of pigs, thereby improving the economic benefits of pig farming, and the CRISPR / Cas9 system is of great significance for further research on the function of the SST gene and its correlation with certain diseases.

The invention discloses a method for producing a swine fever live vaccine by using a porcine kidney cell line (IBRS-2). The method comprises the following steps of: (1) selecting a porcine kidney cell line (IBRS-2) as a cell for producing the vaccine; (2) carrying out sub-culturing on the cell for producing the vaccine; (3) reproducing virus cell seeds; (4) reproducing venom for producing the vaccine; and (5) preparing the vaccine, and performing split charging and freeze-drying. The method of the invention has the advantages of simple and stable production process, easy operation, high content of virus, small difference between bitches, and controllable quality, and capability of improving the quality and yield of the vaccine. The swine fever live vaccine produced by the method of the invention has high safety and high immune effect, and has a complete immune protection function on the attacks of the strong swine fever viruses.

The invention discloses a CD163 mutant and application thereof. The 534th position of the CD163 amino acid sequence is changed from glutamic acid E to lysine K. In the present invention, by comparing the amino acid sequences of CD163 of different species, the 534th amino acid is selected for site-directed mutation (glutamic acid E is mutated into lysine K, namely E534K), and a eukaryotic expression vector system is constructed; in pig kidney PK‑15 cells Recombinantly express wild type (wild type, WT) and mutant CD163; Infect cells with PRRSV classic strain BJ‑4 (GenBank number AF331831) and HP‑PRRSV strain HN07‑1 (GenBank number KX766378.1); by fluorescence Quantitative PCR, virus titer experiment (half cell infection dose, 50% tissue culture infectious dose, TCID 50 ) identification, the 534th amino acid mutation has a significant inhibitory effect on PRRSV infection of host cells, and can be applied to anti-PRRSV pig breeding, anti-virus vaccine and drug development.

The invention discloses a large-scale full-suspension production method of a porcine circovirus type 2. The inventors of the invention domesticate a porcine kidney cell adaptable to large-scale serum-free full-suspension culture, which is named as sPK15-YP, is collected in the China General Microbiological Culture Collection Center and has the culture collection number of CGMCC NO.13846. The sPK15-YP cell adaptable to full-suspension culture is used for achieving serum-free large-scale culture of the porcine circovirus type 2 (PCV2), so that the conventional spinner bottle culture technology is replaced, the human resource is reduced, the product quality is improved, and the bottleneck that the virus content is low during PCV2 full-virus culture is solved; by a full-suspension sPK15-YP cell technology, a high-potency PCV2 semifinished product is prepared; by a hollow fiber method, a PCV2 virus culture solution is concentrated and purified to obtain a more pure PCV2 virus concentrated antigen. By the large-scale full-suspension production method, a solid foundation is laid for studying a PCV2 multivalent vaccine, the dosage of the vaccine is reduced, the stress of a swine herd is reduced and the immune level of the swine herd is enhanced.

The invention discloses a method for overexpression of porcine circovirus type 2 nucleocapsid protein in cells of a mammal. According to the method disclosed by the invention, the stability is enhanced and the expression level of the virus protein in the cells of the mammal is further improved by inhibiting the ubiquitination of the porcine circovirus type 2 nucleocapsid protein. The method comprises the following steps: 1) cloning the genome sequence of a porcine circovirus type 2 SX04 strain; 2) constructing a eukaryotic expression plasmid containing the porcine circovirus type 2 (PCV2) nucleocapsid protein; 3) performing transfection on porcine kidney cells PK15 through the plasmid, and adding MG132 into a cell culture solution at 24 hours after transfection; and 4) culturing continuously for 48 hours, then collecting the cells and detecting the expression protein by immunoblotting. Compared with an ordinary eukaryotic expression method, the method has the advantage that the PCV2 nucleocapsid protein in the eukaryotic cells can be effectively stabilized. The detection by protein immunoblotting at 72 hours after transfection shows that, through the adoption of the method disclosed by the invention, the expression level of the PCV2 nucleocapsid protein can be improved by about 20 times to the greatest extent.

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 discloses a novel PCV (porcine circovirus) mutant strain and a porcine kidney cell strain co-culturing with the novel PCV mutant strain. Cells infected by the novel PCV mutant strain have visible minimal lesions, but cell death is not caused. The sieved porcine kidney cell strain carries a PCV2, so that stable passage can be realized, a large number of viruses are generated, and the prepared viruses can be used for preventing and treating the PCV.

The invention discloses a compound porcine alpha interferon gene. Compared with a yeast expressed natural porcine alpha interferon gene, the compound porcine alpha interferon gene has the advantages that the expressed protein of the compound porcine alpha interferon gene is better in antiviral activity, and the function of resisting 100TCID50VSV virus infection on a porcine kidney cell is improved by 50 times. The invention further provides a synthetic method of the compound porcine alpha interferon gene. The synthetic method adopts pichia pastoris X-33 as a template, requires no additional inducer, and is suitable for industrial production. Due to the adoption of pichia pastoris, toxins which are carried by escherichia coli expressed foreign protein and toxic to porcine cells can be avoided, that is, the toxicity is reduced. The synthetic method is suitable for synthesis of the compound porcine alpha interferon gene.

The invention discloses a large-scale cultivation method of porcine Japanese encephalitis vaccine antigen. The invention provides a large-scale culture process for porcine Japanese encephalitis vaccine antigens, using pig kidney cells PK-15 cells to culture porcine Japanese encephalitis virus, one time of virus inoculation can continuously harvest multiple batches of viruses, and the virus titer It is equivalent to or higher than the existing culture technology of BHK‑21 and VERO cells, greatly improves the virus yield and reduces the production cost, and is a porcine Japanese encephalitis vaccine antigen culture technology suitable for large-scale production.

The present invention provides an sgRNA capable of effectively editing the porcine miR-17-92 gene cluster. On the premise that the sgRNA can specifically recognize the porcine miR-17-92 gene cluster, the shRNA is successfully integrated into the porcine site using CRISPR / Cas9 and RNAi technology. Kidney cell PK‑15‑EGFP‑KI cells were screened for positive cell clones that knocked down the expression of the EGFP gene; the above sgRNA was used to site-specifically integrate shRNA into pig fetal fibroblasts, and a positive cell line for site-specific integration of shRNA was successfully screened out. The stable transcription and site-specific integration events of shRNA were detected. During the preparation of the cell line, the shRNA was stably transcribed and effectively expressed under the action of the promoter of the porcine miR‑17‑92 gene cluster without introducing any foreign promoter Subgenes and positive and negative screening marker genes increase the safety of transgenic pigs and are of great significance for removing the biological safety hazards of transgenic pigs.

The invention provides a pMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, a method for preparing the same and application of the pMKRN1 gene-knockout porcine somaticcell. The pMKRN1 gene-knockout porcine somatic cell, the method and the application have the advantages that mutant PK-15 (porcine kidney-15) cells can be obtained by the aid of gene knockout technologies, high-titer PCV2 (porcine circoviruses type 2) can be quickly replicated and obtained by the aid of the mutant PK-15 cells, and accordingly efficient virus amplification cells can be provided toresearch on PCV2 pathogenic mechanisms and preparation of PCV2 inactivated vaccine and attenuated vaccine.