45 results about "Porcine circovirus Type II" patented technology

The present invention belong to veterinary new biological medicine technology field, relates to porcine circovirus type II (PCV2) inactivated vaccine of and method for preparing same. The vaccine used seed virus is porcine circovirus type II DBN-SX07 strain, the preservation number is CGMCC No 3064, the virus strain is used as antigen preparation of inactivation by alkyl agents and emulsification by adding oil adjuvant. Using the invention provided PCV2 inactivated vaccine to immune pig can generate an uniform and effective protection force-shorting PCV2 infection windows period obviously, and prolong immune duration-reducing times of booster immunization.

The invention provides a method for producing porcine circovirus type II capsid protein by utilizing a silkworm bioreactor, and belongs to the field of biotechnology. In the method, by taking a bombyx nuclear polyhedrosis virus as a vector, porcine circovirus type II capsid protein genes are integrated into a polyhedrosis promoter of the bombyx nuclear polyhedrosis virus so as to express target protein by a mode of the homologous recombination of a homologous arm of the nuclear polyhedrosis virus in a transfer vector and bombyx nuclear polyhedrosis virus (BmNPV) genes; a recombinant bombyx nuclear polyhedrosis virus containing target protein genes is obtained by a plaque sieving method, and the target protein is expressed in large scale by using a bombyx bioreactor so as to prepare a subunit vaccine containing recombinant porcine circovirus type II capsid protein; and piglet infection experiments verify that the subunit vaccine has the excellent immune protective effect. The method has the characteristics of high expression efficiency, good activity of the target protein, low production cost and the like, and is suitable for large-scale production.

The invention provides a kluyveromyces marxianus engineering bacterium obtained from recombinant expression of porcine circovirus type II capsid protein; and porcine circovirus type II virus-like particles are obtained by cloning porcine circovirus type II capsid protein genes into an expression vector and conducting recombinant expression in kluyveromyces marxianus. The invention also provides a preparation method of a porcine circovirus type II virus-like particle vaccine, wherein the preparation method comprises the following steps: preparing the porcine circovirus type II virus-like particles through high-density fermentation of the recombinant kluyveromyces marxianus, and then conducting separation and purification so as to prepare the vaccine. The porcine circovirus type II virus-like particle vaccine provided by the invention, which can generate a high-level serum IgG antibody just by implementing injection immunization once, has the advantages of being good in immunizing effect, high in safety, simple in culture operation, high in yield, low in production cost and the like, and the porcine circovirus type II virus-like particle vaccine can achieve large-scale enlarged production; and the porcine circovirus type II virus-like particle vaccine can be used for relieving and preventing related clinical symptoms caused by the infection of porcine circovirus type II (PCV2).

The invention relates to construction and application of porcine circovirus type II-porcine mycoplasma pneumoniae expressing strains and belongs to the technical field of biology. By a method provided by the invention, prokaryotic expression engineering strains of expressing porcine circovirus type II-porcine mycoplasma pneumoniae major antigenic protein are constructed. The strains can simultaneously express the major antigenic protein R1 of porcine mycoplasma pneumoniae and a major antigenic protein catabolite activator protein (CAP) of porcine circovirus, purified recombinant protein can stimulate organisms to produce a protective immune response which resists attacks of porcine circovirus type II and the porcine mycoplasma pneumoniae, and the infection of the porcine circovirus and the porcine mycoplasma pneumoniae can be effectively prevented.

The present invention discloses a porcine circovirus type II strain, wherein the latin name is Circoviridae, and the preservation number is CCTCC NO:V201312. According to the invention, in vitro PCV2 culture proliferation conditions are optimized, and a PK15 cell clone strain with high PCV2 sensitivity and named L8 is screened so as to establish a foundation for further PCV2 researches.

The invention belongs to the biological field and particularly relates to preparation and application of recombinant porcine circovirus PCV2-Cap/Phage display particle. A preparation method of the capsid protein phage display particle of recombinant II porcine circovirus specifically comprises the following four steps: (1), optimizing and coding nucleotide of the capsid protein of the recombinant II porcine circovirus; (2), constructing a recombinant plasmid; (3), constructing a recombinant phage genome; and (4), preparing the capsid protein phage display particle of recombinant II porcine circovirus. An animal experiment proves that the recombinan display plasmid has good antigenicity, can stimulate a pig body to generate good humoral immunity and cellular immunity reaction, and can prevent and control II porcine circovirus infection; time needed for copying the process is short, production cost of the recombinant display particle is low, and product is convenient to use, simple and convenient to store and high in cloning yield.

The invention discloses a recombinant porcine pseudorabies virus (Herpesviridae) strain used for expression of porcine circovirus type II ORF2 gene. Preservation number of the recombinant porcine pseudorabies virus strain is CCTCC No.V201315. According to the preparation method, PCV2ORF2 gene is inserted into common carrier PG so as to construct transferring plasmid PGO; monolayer ST cells are inoculated with PRVTK<->/gG<->/gE<-> virus by 2h of adsorption; ST cells are transfected with plasmid PGO, wherein fusion degree of the monolayer ST cells is 80 to 90%; the recombinant porcine pseudorabies virus PGO strain is obtained by plaque purification, and is used for immunization of mouse. It is shown by results that commercial PCV2 inactivated vaccine and a group immunized by the recombinant porcine pseudorabies virus PGO strain are both capable of inducing specific humoral immune response of mouse, antibody titers of both the commercial PCV2 inactivated vaccine and the group are obviously higher than that of a group immunized by DMEM medium, and difference is significant (p<0.05). It is shown by the results of mouse lymphocyte proliferation test that specific ceullar immune response caused by the group immunized by the recombinant porcine pseudorabies virus PGO strain is more obvious than that caused by the commercial PCV2 inactivated vaccine and the group immunized by DMEM medium, and difference is obvious (p<0.05). In addition, the group immunized by the recombinant porcine pseudorabies virus PGO strain is capable of resisting severe attack by PCV2. Therefore application of the recombinant porcine pseudorabies virus strain in development of a novel PCV2 vaccine is possible.

The invention discloses a system for simultaneously detecting six pig viruses and a special LAMP primer for the system. The system comprises an LAMP complete primer for detecting the six pig viruses and/or a reagent required for LAMP and/or an instrument and/or an amplified product data processor, wherein the amplified product data processor is used for distinguishing whether a to-be-detected sample amplification product obtained through loop-mediated isothermal amplification on a to-be-detected sample through the LAMP complete primer for detecting the six pig viruses comprises a specific amplified product or not. The system for detecting the six pig viruses and the special LAMP primer for the system can meet simultaneous and rapid detection of a pig foot-and-mouth disease virus O/A/Asia type I, a hog cholera virus, a porcine respiratory and reproductive syndrome virus American type, a porcine circovirus type II, a porcine parvovirus and a porcine pseudorabies virus, the detection system is simple in operation, the result is convenient to observe and a strong technical support is provided for epidemic disease monitoring of a pig farm.

The invention discloses a porcine circovirus type-2 recombinant CAP protein and a subunit vaccine of the recombinet CAP protein. The invention provides the porcine circovirus type-2 recombinant CAP protein, 8 histidine tags are fused at the C-end of the Cap protein, and the amino acid sequence of the protein is as shown in SEQ ID No.1. Compared with a conventional used PCV2 inactivated vaccine, the porcine circovirus type-2 Cap protein expressed by recombinant escherichia coli, which is disclosed by the invention, is relatively simple in vaccine preparation process, relatively low in cost, and therefore has relatively wide application prospects in preventing and treating PCV2.

The invention relates to a veterinary antiviral and antifebrile drug composition and belongs to the field of traditional Chinese veterinary medicine. The drug composition provided by the invention comprises the components including Bupleurum, cassia twig, radix isatidis, kudzu, honeysuckle and analgin. The composition is Chinese and western compound, has a synergistic effect, and is used for preventing and treating hyperpyrexia caused by viral infection of livestock and poultry; the antiviral and the antifebrile effects are better than those of prescribed preparations of chemical drug and TCM (Traditional Chinese Medicine) preparations; and the drug composition has a very good effect on preventing and treating infection of various virus such as porcine circovirus type II, swine influenza virus, Newcastle Disease Virus (NDV) and the like, and has the advantages of less toxic side effects and low cost of drug material.

The invention aims to provide a quantitative detection method for porcine circovirus type II virus-like particles simple to operate, and high in sensitivity, repeatability and applicability. The method comprises the following steps: S1, diluting to-be-tested samples and filtering and clarifying or centrifugalizing and clarifying the samples; S2, serially diluting the porcine circovirus type II virus-like particle standard substances with determined protein contents, and carrying out HPLC detection on the diluted serial standard substances to establish a regression equation which takes concentration as a horizontal ordinate and peak height as a vertical ordinate; S3, carrying out HPLC detection on the diluted to-be-tested samples to obtain corresponding peak heights of the samples; and S4,calculating the concentrations of the porcine circovirus type II virus-like particles in the to-be-tested samples according to the regression equation and the peak heights of the samples, wherein chromatographic columns used in HPLC in the S2 and S3 are gel chromatographic columns, and detectors are ultraviolet detectors, the detection wavelengths of which are 280 nm.

The invention relates to a PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof. Porcine circovirus type II PCV2d VLPs immunizing mice are used for screening hybridoma cells; the prepared antibody can specifically recognize PCV2, wherein the PCV2b and PCV2d can be recognized; and the antibody can be applied to PCV2 related detection of WB, IFA and the like.When the antibody is used for coating in ELISA detection, the antibody does not react with Cap protein and can be used for distinguishing VLPs and Cap. The sandwich ELISA is established through optimization of a series of reaction conditions according to the monoclonal antibody and can be used for specific quantification and qualitative determination of PCV2 VLPs; the lowest detection concentration can reach 62.5 ng/ml, the sensitivity is high, the correlation coefficient R2 is 0.992, and the quantification accuracy is high. The PCV2 virus-like particle sandwich quantitative ELISA detection method is suitable for quantification of PCV2b and PCV2d VLPs and can be applied to quantification and assembly efficiency identification of different genotypes of VLPs of PCV2 and PCV2 virus infectionidentification.

The invention discloses a visible gene chip for a pig pseudorabies virus, porcine parvovirus and porcine circovirus type-II. The visible gene chip comprises a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe comprises gene segments as shown in SEQ ID NO:1, 3, and 4; and the gene segments are used for respectively detecting the pig pseudorabies virus, the porcine parvovirus and the porcine circovirus type-II. The invention further discloses a kit for detecting the pig pseudorabies virus, the porcine parvovirus and the porcine circovirus type-II. By adopting the gene chip and the kit disclosed by the invention, pathogens of the pig pseudorabies virus, the porcine parvovirus and the porcine circovirus type-II can be effectively detected, infection caused by vaccine strains and wild strains of the pig pseudorabies virus can be distinguished, the gene chip and the kit have the characteristics of good specificity, high sensitivity and long preservation period, are short in time, rapid in detection, visible in detection result observation and good in clinical application prospect, no expensive equipment is needed, and the infection situation of livestock diseases can be rapidly handled.

The invention relates to a preparation method of a porcine circovirus type II gene engineering subunit oral vaccine. The method comprises the following steps: 1, sequence synthesis; 2, construction of a recombinant vector in E. coli; 3, construction of a recombinant vector in Lactococcus lactis MG1363; and 4, protein expression, recombinant bacteria preservation experiment, and vaccine preparation. The invention has the following beneficial effects: no special treatment for the antigen avoids antigen purification and reduces antigen loss; optimization of the gene sequence improves the antigen expression; optimization of expression conditions solves the problems of toxicity to animals caused by addition of inducing agent for other prokaryotic expression and high costs, and avoids the increase of the chance of contamination and increase of the workload in the expression process. The method provided by the invention has low cost and significant effect; the invention employs oral vaccine to avoid the problems of stress and absorption caused by routine vaccination.

The invention provides a loop-mediated isothermal amplification primer for detecting type 2 porcine circovirus. The loop-mediated isothermal amplification primer comprises a first outer primer, a second outer primer, a first inner primer and a second inner primer. The invention further provides a method for detecting the type 2 porcine circovirus by the loop-mediated isothermal amplification primer. The loop-mediated isothermal amplification primer can rapidly detect the type 2 porcine circovirus and is high in detection sensitivity, good in specificity, high in detection rate, short in reaction time and simple to operate, and results are conveniently observed.

The invention discloses a gene chip for detecting pathogenic microorganisms of the porcine epidemic disease. The gene chip comprises an oligonucleotide probe and a positive quality controller, which are fixed on a solid phase carrier and correspond to the pathogenic microorganisms to be detected. The invention further discloses a method for detecting porcine actinobacillus pleuropneumonia, hog cholera virus, haemophilus parasuis, swine influenza virus, mycoplasma hyopneumoniae, porcine circovirus type II, porcine parvovirus, porcine reproductie and respiratousyndrome virus, porcine pseudorabies virus and streptococcus suis by adopting the gene chip. The gene chip realizes efficient detection, aims at 10 kinds of swine pathogenic microorganisms at a time, and greatly improves the detection efficiency. Besides, the gene chip is high in sensibility and strong in specificity.

The invention discloses a microcarrier biological reaction tank and a method for culturing porcine circovirus type II by the microcarrier biological reaction tank. An interlayer is arranged on the side wall of a tank body; heating tubes are arranged in the interlayer for heating jacket water in the interlayer so as to heat liquid in the tank body; a telescopic pipe in the tank body is used for sampling; the sampling position can be adjusted according to the amount of culture carriers, and the convenience in operation is realized; an observation pipe equipped with a transparent observation areais connected with the inner part of the tank body; a filtering gas head and a switch valve body which correspond to each other are matched for enabling carrier cells to flow into a transparent tube;cell states are observed at any time under a microscope; in addition, residual carriers are returned into the tank after observation so as to avoid waste; a carrier filter screen is arranged on the bottom side face of the reaction tank and is matched with a ventilation pipe, so that the blockage of the filter screen by the carriers when a final antigen removes the carriers during harvesting is avoided. The microcarrier biological reaction tank disclosed by the invention has the advantages that a culture device of the microcarrier biological reaction tank is improved; real-time observation of cells in the tank is facilitated; a waste solution is conveniently discharged; the carriers are favorably filtered; the efficiency is improved and 24-hour cell density is improved; cells with better states are cultured.

The invention discloses a preparation process and application of sulfated saccharosan. The sulfated saccharosan is prepared by the following steps: (1) preparing saccharosan solution, lauroyl chloride solution and chlorosulfonic acid solution; (2) mixing the lauroyl chloride solution and the saccharosan solution, reacting at the temperature of 60-80 DEG C, and incubating at room temperature for 18 hours; (3) mixing the chlorosulfonic acid solution with the solution obtained in the step (2), reacting at the temperature of 60-80 DEG C, and incubating at room temperature for 18 hours; (4) regulating the pH value of the reaction solution in the step (3) to be neutral by using NaOH solution, distilling to remove the solvent, washing and dialyzing to remove impurities, and drying to obtain the sulfated saccharosan, wherein a molecular ratio of the lauroyl chloride to saccharosan is (1.2-3.6):1, and a molecular ratio of the chlorosulfonic acid to saccharosan is (0.5-2.0):1. When an oil-in-water adjuvant is prepared by using the sulfated saccharosan, the concentration of porcine circovirus type II specific antibodies IgG and antibody subclasses IgG1 and IgG2a can be obviously increased, and the activity is equivalent to that of an oil adjuvant. The sulfated saccharosan can effectively improve the antigen immunocompetence and is expected to serve as a novel oil-in-water adjuvant for development and application.

The invention relates to a method for rapidly detecting a porcine circovirus type II, in particular to a method for rapidly detecting the porcine circovirus type II by using a primer pair based on a closed neck loop-mediated isothermal nucleic acid amplification (CDA) technology. The invention discloses a CDA primer pair for detecting porcine circovirus type II, a kit and application of the CDA primer pair. The CDA primer pair is a primer for amplifying a conserved region fragment of the porcine circovirus type II, and is PCV2-MF/PCV2-MR (porcine circovirus type II); wherein the nucleotide sequence of the PCV2-MF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the PCV2-MR is as shown in SEQ ID NO. 2. According to the method provided by the invention, rapid and accurate detection of the porcine circovirus type II can be completed without expensive instruments or complicated operation, so that the method is suitable for on-site rapid detection of places such as customs and farms with low professional degree.