108 results about "Porcine serum" patented technology

The invention discloses a hybridoma cell line of a monoclonal antibody against African swine fever virus and the secreted monoclonal antibody thereof. The preparation method of the invention comprises the following steps: preparing a recombined P30 soluble antigen by prokaryotic expression; immunizing a BALB/c mouse; and finally fusing, screening and cloning by a hybridoma technology to obtain the hybridoma cell line which can stably secrete the monoclonal antibody against African swine fever virus P30 protein. The invention further discloses a method for preparing the monoclonal antibody with the cell line, an antibody purification method and a labeling method for horseradish peroxidase of the antibody. The monoclonal antibody can be used in detecting the African swine fever viral antibody in pig serum.

The invention discloses an indirect ELISA kit for detecting an African swine fever virus antibody and an application thereof, and belongs to the technical field of biology. The kit adopts prokaryotic expression recombinant P30 protein as a coating antigen, and detects the antibody of African swine fever virus in porcine serum based on the indirect ELISA principle. The coating antigen in a 96-well plate of the kit is prokaryotic expression recombinant P30 protein which has good antigenicity. The enzyme-linked immunoassay kit provided by the invention comprises a 96-well plate coated with P30 protein, a positive control, a negative control, a horseradish peroxidase-labeled rabbit anti-porcine IgG polyclonal antibody, a concentrated washing liquid, a serum diluent, a TMB substrate, and a terminating liquid. The kit of the invention is applicable to the screening of large quantities of samples, and main reagents in the kit are provided in a form of operating fluid which is convenient for use.

The invention discloses a preparation method of pig serum protein antioxidant peptide by adopting microbial fermentation and a product thereof. The preparation method comprises steps of: (1) carrying out micro-filtering on pig serum protein; (2) inoculating the filtered pig serum protein with bacillus subtilis bacterial suspension and carrying out fermentation; and (3) decentralizing fermentation liquor, collecting liquid supernatant, and carrying out concentrating, freezing and drying to obtain the finished product. The pig serum protein antioxidant peptide prepared by the method has higher antioxidation activity, capability of eliminating free radical and better antioxidation effect both in vitro and in vivo. The preparation method is simple for operation, and is safe and reliable compared with the method of synthesizing antioxidant; and the preparation method is applicable to industrial production and can greatly enhance the utilization rate of pig serum protein resource and raise the comprehensive utilization level of pig blood so as to effectively increase additional value of pig blood resource.

The invention discloses an indirect ELISA kit for detecting African swine fever virus (ASFV) antibody, and a use thereof, and belongs to the technical field of biology. The kit is characterized by: adopting prokaryotic expressed recombined P54 protein as coating antigen; detecting antibody against to the ASFV in swine serum according to an indirect ELISA principle. The coating antigen in 96-well plates of the kit is the prokaryotic expressed recombined P54 protein which has good antigenicity. The enzyme-linked immunospecific assay kit provided by the present invention comprises the 96-well plates coated by the P54 protein, positive control, negative control, horseradish peroxidase-labeled rabbit anti-porcine IgG polyclonal antibody, a concentrated cleaning solution, a serum dilution, a TMB substrate indicator and a stop buffer. The kit provided by the present invention can be provided for screening mass samples. In addition, the main reagents in the kit are provided in a working fluid manner such that the reagents are used conveniently.

The invention relates to an indirect ELISA (enzyme-linked immunosorbent assay) method for detecting a porcine reproductive and respiratory syndrome antibody, which comprises the following steps: by using a pGEX-6p-1 prokaryotic expression vector, performing tandem repeat on two epitopes to improve the antigen activity of an expressed protein, thus constructing a gene engineering bacterium BLpGEX-6p-GP5 capable of realizing secretory expression of the GP5 protein dominant antigen epitopes, wherein one epitope is a linear conservative neutralizing epitope (epitope B) of a screened PRRSV (porcine reproductive and respiratory syndrome virus) GP5 protein, which can be identified by a monoclonal antibody and can also be identified by porcine anti-PRRSV neutralizing serum, and the other epitope is a high-variability immunodominant epitope (A); and purifying and renaturing the expressed recombinant protein, and coating an ELISA plate, thus establishing the indirect ELISA method for detecting a PRRSV antibody to detect the PRRSV antibody level in porcine serum. Results show that the method has the characteristics of favorable repetitiveness and high specificity and can be used for PRRSV serological search.

The invention discloses application of a microRNA (micro ribonucleic acid)-miR-23a molecular marker miR-23a to quickly detecting the quality of oocytes of sows. The miRNA molecular marker miR-23a in the cumulus oocytes with low potentiality development is abnormally high in expression and can be secreted in serum via active secretion procedures of tissues, and serum miR-23a is the molecular marker with a function of quickly detecting the quality of the oocytes of the sows as discovered by large-sample detection on the expression level of the serum miR-23a of the sows with low production performance. The application has the advantages that the reproductive potential of the sows can be predicted by means of detecting the quality of the oocytes, so that the sows with low production potential can be eliminated as early as possible, the feed and labor costs can be greatly reduced, and the application is beneficial to vigorous development of the pig industry in China.

The invention belongs to the technical field of cell biology, and more specifically discloses a porcine islet cell frozen stock solution and a cryopreservation method. The porcine islet cell frozen stock solution is composed of a basic medium, porcine serum, DMSO, catalase, tocopherol, trehalose, and Z-VAD-FMK. The porcine islet cell frozen stock solution is capable of reducing apoptosis of porcine islet cells in freeze preservation process, improving resistance of porcine islet cells on severe environment, and increasing cell survival rate. According to the freeze preservation method, a programmable cooler is used for cooling, wherein when the temperature is higher than -25 DEG C, the cooling speed is controlled to be 1 DEG C/min, when the temperature is -40 DEG C or higher, and -25 DEG C or lower, the cooling speed is controlled to be 0.25 DEG C/min, and when the temperature is -80 DEG C or higher and is lower than -40 DEG C, the cooling speed is controlled to be 5 DEG C/min. The cryopreservation method is capable of reducing generation of ice crystals, and protecting porcine islet cells more conveniently.

The invention provides a monoclonal antibody for detecting porcine C-reactive protein (CRP), and preparation and application thereof. A gene engineering process is performed to obtain high-purity CRP recombinant protein; and the recombinant protein is utilized to screen out the hybridoma cell strain with the highest stability and highest antibody activity for secreting CRP protein antibody, wherein the collection number is CGMCC NO.9345. The monoclonal antibody generated by the hybridoma cell strain has the advantages of high specificity, high affinity and simple and efficient preparation method, and can monitor the CRP content in porcine serum, perform differential diagnosis on bacterial and virus diseases and perform auxiliary observation on treatment effects, thereby promoting the rational use of antibiotics, reducing the drug residues and ensuring the safety of animal food.

The invention discloses a test paper card for detecting a pseudorabies virus gE protein antibody in porcine serum as well as a preparation method and application thereof. The test paper card is formed by an immuno-colloidal gold test strip and a card body; a nitrocellulose membrane is adhered to a bottom plate of the immuno-colloidal gold test strip; a gold-marking binding pad and a sample pad are adhered to one end of the nitrocellulose membrane, and a water absorbing pad is adhered to the other end of the nitrocellulose membrane; the nitrocellulose membrane is provided with a porcine pseudorabies virus EA strain coated detection line and a goat anti-mouse IgG coated quality control line, wherein the detection line and the quality control line are parallel to each other, the detection line is arranged near to one end of the gold-marking binding pad, and the quality control line is arranged near to one end of the water absorbing pad; the immuno-colloidal gold test strip is arranged in the test paper card body; and the detection line and the quality control line are arranged in positions which correspond to detection holes of the test paper card. The test paper card disclosed by the invention is used for detecting the pseudorabies virus gE protein antibody in the porcine serum rapidly and has the advantages of simple structure, convenience of operation, high sensitivity and clear result.

The invention provides a porcine source single B cell antibody of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (N) and a competitive ELISA antibody detection kit, and belongs to the technical field of virus detection. The invention provides a porcine source single B cell antibody C8 of PRRSV protein. Tests show that 3A epitope monoclonal antibody 3A24 of FMDV is taken as a coating antibody, 3AN fusion protein is captured to form a coating ELISA plate, a C8 monoclonal antibody is taken as a detection antibody, a competitive ELISA reaction mode is employed for detecting a porcine serum PRRSV antibody, the prepared PRRSV antibody detection kit has good sensitivity and specificity, and a new tool is provided for PRRSV serum epidemiological investigation.

The present invention discloses a technique for extracting high-purity porcine serum albumin. The technique includes the following steps: sodium citrate and sodium chloride are added into porcine blood to separate plasma and erythrocytes; after being centrifugated, filtered by a membrane and extracted, the plasma is put into an interlayered reactor; alcohol, sodium caprylate and sodium chloride are added into the plasma and stirred slowly after the interlayered reactor is heated; hydrochloric acid is added to adjust the pH value for two times; after centrifugation, supernatant is added into precipitating reagent, ground into paste and stirred; after being refrigerated overnight, the mixed solution is centrifugated and left for precipitation; the precipitate is resolved in water and, after the pH is adjusted by sodium hydroxide solution, kept still; supernatant is separated out by centrifugation; and after membrane filtration and virus inactivation, the filtrate is frozen out into the finished product. The technique has the advantages of little material usage, short process flow, high production efficiency, easy operation, low production cost and high product quality and is suitable for mass production. The product can be used as an auxiliary material for the preparation of fibrin glue sealant, a biochemical material and a substitute for other albumins.

The invention relates to an indirect ELISA kit for detecting a porcine transfusion transmitted virus 2 (TTV2) antibody and belongs to the field of biotechnology. The invention comprises antigen recombinant protein preparation, indirect ELISA establishing, and use of determination standard and clinical serological test. Through pcoldI prokaryotic expression vectors, a gene engineering bacterium pcoldI-ORF1 for expression of a porcine TTV2ORF1 truncated protein is constructed and the expressed antigen recombinant protein is purified and is used as an antigen so that an indirect ELISA detection method is established. The indirect ELISA detection method is used for detecting a TTV2 antibody level of porcine serum, has good repeatability and high singularity, can be used for porcine TTV2 serology investigation and is a fast and simple serological test method for prevention, treatment and prevalence state control of porcine transfusion transmitted diseases. The indirect ELISA detection method utilizes the porcine TTV2ORF1 recombinant protein to detect the porcine TTV2 antibody first in China.

The invention relates to a blocking ELISA antibody detection kit for porcinecircovirus type 2 and a preparation method of the blocking ELISA antibody detection kit. The blocking ELISA antibody detection kit for the porcinecircovirus type 2 comprises a coated plate, a PVC2 enzyme labelled antibody, sample diluent, a concentrated washing solution (10*), a positive serum contrast, a negative serum contrast, TMB substrate diluent and a stop solution, wherein the coated plate is prepared from a purified porcine circovirus type 2 nucleocapsid protein CapC protein serving as a non-antigen, and the PVC2 enzyme labelled antibody is prepared from monoclonal antibody hybridoma which is labeled with horse radish peroxidase and secretes PCV2. According to the preparation method, the CapC protein is expressed by virtue of a prokaryotic expression system, and the blocking ELISA is established by taking a monoclonal antibody of PCV2 as the enzyme labelled antibody; and the blocking ELISA antibody detection kit has the characteristics of high specificity and sensitivity and can be applied to the detection of PCV2 antibodies of a porcine serum sample.

The invention discloses a prokaryotic soluble expression method of porcine circovirus 2b subtype Cap protein. The method comprises the following steps: taking a PCV2 strain genome as a reference sequence, coupling an SUMO solubility-promoting expression tag coding gene at the upstream of a Cap protein coding gene of PCV2b, introducing 6 His tag encoding genes at the upstream of SUMO, performing gene optimization, recombining the gene to a PUC57 plasmid, taking the recombinant plasmid as a template, designing a specific primer, after PCR amplification, cloning the product into a pMAL-C4x prokaryotic expression vector, constructing a recombinant plasmid, transferring the recombinant plasmid into escherichia coli, and performing induction and purification. The method successfully constructs the pMAL-MBP-HisSUMO-Cap recombinant expression vector, MBP-HisSUMO-Cap fusion protein can realize soluble expression in BL21(DE3), after purification, the MBP-HisSUMO-Cap fusion protein can be used asimmunogen and can have a specific reaction with PCV2b positive porcine serum, a rabbit anti-MBP-tag polyclonal antibody and immunized mouse antiserum, and results indicate that the protein has good immunogenicity, and lays a theoretical foundation for development of a PCV2b diagnostic kit.

The invention discloses a porcine circovirus type 2 (PCV2) competitive ELISA antibody detection kit. The kit includes an enzyme-labeled monoclonal antibody secreted by a hybridoma cell strain having the preservation number of CGMCC NO.10205. The invention also discloses a competitive ELISA method established by using the monoclonal antibody and used for detecting a porcine serum PCV2 antibody. The method comprises that firstly an ELISA plate is wrapped with a PCV2 positive serum to capture a PCV2 antigen, then the PCV2 antigen undergoes a reaction with a to-be-detected porcine serum antibody and an HRP-labeled mono-antibody 3A5, and a competitive ELISA detection result is showed through measuring the enzyme-labeled mono-antibody. The method and an IPMA method perform parallel tests on 237 porcine serum samples, the two methods have the detecting coincidence rate being 94.1%, and the method provided by the invention has the sensitivity of 92.6% and the specificity of 98.4%, and has no cross reaction with other porcine virus reference positive serums. The kit has the characteristics of simple operation, good specificity, high sensitivity and the like, and provides an effective technical means for PCV2 epidemiological investigation and serum antibody detection.

The invention provides a fusion protein 3AN for capturing a PRRSV nucleocapsid protein antibody and application thereof, and belongs to the technical field of virus detection. The invention provides a fusion protein 3AN for capturing a porcine reproductive and respiratory syndrome virus nucleocapsid protein antibody. The amino acid sequence of the fusion protein 3AN is shown as SEQ ID NO: 3. Tests show that a 3A epitope monoclonal antibody 3A24 of FMDV serves as a coating antibody, a coating ELISA plate is formed after 3AN fusion protein is captured, a C8 monoclonal antibody serves as a detection antibody, a porcine serum PRRSV antibody is detected in a competitive ELISA reaction mode, the prepared PRRSV antibody detection kit has good sensitivity and specificity, and a new tool is provided for PRRSV serum epidemiological investigation.

The invention discloses a composite interferon inducer for pigs. The composite interferon inducer consists of the following components in percentage by weight: 5 to 50 percent of echinacea extract, 10 to 50 percent of hypericin and 5 to 50 percent of yeast nucleotide. The composite interferon inducer for the pigs can greatly improve the interferon level in serum of piglets, so the composite interferon inducer can well induce the piglets to generate I-type interferon.

The invention discloses a traditional Chinese medicine feed additive for improving immune function of pigs as well as a preparation method and application of the additive. The additive is prepared from the following components in parts by weight: astragalus membranaceus, fructus alpiniae oxyphyllae, Chinese yam, endothelium corneum gigeriae galli, poria cocos, hawthorn, pinellia ternate, rhizoma atractylodis, ginger, fructus amomi and liquorice. The preparation method comprises the steps of drying, smashing, screening and uniformly mixing the raw materials by adopting a traditional Chinese medicine production device to prepare, adding a prepared traditional Chinese medicine preparation into basic ration of a small healthy Meishan pig of 60 days, uniformly blending to feed, and carrying out free choice feeding and free water drinking. Upon verification of tests, the method can obviously improve the concentration of total protein and albumin in porcine serum; can significantly improve the content of immune globulin IgG and the content of a complement C3 in the porcine serum; obviously improves the antibody level of post-inoculation swine fever; increases the pig spleen indexes. Moreover, the defects of various chemicals and antibiotics feed additives are overcome, toxic and side effects and drug residues do not exist, and the body immune function is improved while the production of green and high-quality animal products is guaranteed.

The invention discloses a recombinant porcine IL-29 fusion protein and a preparation method and application thereof. The fusion protein is formed by directly or indirectly connecting porcine IL-29 with an immunoglobulin Fc fragment or porcine serum albumin (PSA) through a connecting element. The porcine IL-29 fusion protein can be prepared by utilizing a mammalian cell expression system based on agene engineering technology. Compared with natural porcine IL-29, the recombinant porcine IL-29 fusion protein is longer in half-life period and higher in antiviral activity, and can be used for preparing medicines for preventing and treating porcine viral diseases.

The invention relates to a mycoplasma synoviae culture medium and a preparation method thereof. The culture medium comprises a basal culture medium and auxiliary components. The basal culture medium comprises a phosphate buffer solution, glucose, lactoalbumin hydrolysate, coenzyme I, arginine hydrochloride, L-cysteine hydrochloride, MEM, yeast extract powder, 1% phenol red, and trehalose. The auxiliary components comprise pig serum and penicillin. According to the culture medium, the antigen content of mycoplasma synoviae bacterial liquid is remarkably increased, the bacterial liquid titer reaches up to 1012.0-13.0 CCU/ml. Meanwhile, the separation rate of the MS wild strains is remarkably improved and is as high as 93%. According to the preparation method of the culture medium, the antigen content of mycoplasma synoviae is increased, the production process is simplified, and the production cost is reduced.

The invention relates to a vaccine production technology in the field of biotechnology, in particular to a CHO cell strain expressing recombinant protein Pigpsa-PCV2cap2 and constructed with a genetic engineering method, and a preparation method and application of the CHO cell strain. The recombinant fusion protein Pigpsa-PCV2cap2 provided by the invention is shown as A1) or A2) as follows: A1) the amino acid sequence is as shown as SEQ ID No. 2; and A2) superseding/missing/adding one or more amino acid residues in the amino acid sequence of the protein of A1) to obtain a protein having Pigpsa-PCV2cap2 activity. The monoclonal cell strain capable of secreting and expressing Pigpsa-PCV2cap2, obtained with the method, has the relatively high expression level of the fusion protein; the fusion protein obtained after His affinity, separation and purification can be bonded with a monoclonal antibody, so as to be better than other current marketing products in the aspects of immunizing animals and generating a neutralizing antibody; the fusion protein can be used for preparing porcine circovirus vaccine, so as to reduce the production cost, and avoid death and immune failure loss caused by endotoxin residues.