109 results about "Duck plague virus" patented technology

The invention discloses an establishing method of a bacterial artificial chromosome recombinant duck plague virus rescue system platform and application of the platform. A bacterial artificial chromosome recombinant duck plague virus is obtained by inserting a recombinant duck plague virus transfer vector pUC18 / EGFP-TKAB-BAC11 in a TK domain, wherein the recombinant duck plague virus transfer vector contains a TK gene left-right homologous arm, a reporter gene EGFP and a bacterial artificial chromosome core function component. By means of the platform, the in-vitro biologics characteristics of a UL55 gene-deleted strain established through an inside-bacterium two-step RED recombination method and a back mutation strain and parent strain of the UL55 gene-deleted strain are quite close; the functions are not related to positioning of a UL26.5 gene in a cell. The method is beneficial to development of pathogenic mechanism and gene function research of DPV CHv and is beneficial to the duck plague virus prevention and the research and application of recombinant duck plague virus vaccines of other poultry infectious diseases based on the platform; in addition, due to the fact that the recombinant virus carries a TK deletion mark and an EGFP gene, a mark vaccine can be developed to clinically distinguish a wild virus and a recombinant vaccine virus.

The invention discloses a preparation method of a natural high-effective biological active substance anti-duck plague virus transfer factor. The preparation method comprises the following steps of: inoculating duck plague virus into an allantoic fluid obtained from a 10-day-aged chick embryo, and preparing duck plague virus antigen; extracting the duck plague virus antigen; immunizing a pig with the extracted duck plague virus antigen; and extracting the anti-duck plague virus transfer factor from the immunized pig. The inventive anti-duck plague virus transfer factor can prevent duck plague and protect normal body cells from being infected by the duck plague virus, so as to reduce incidence rate.

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus antibody. The method particularly comprises the steps of the preparation of solid phase antigen, primary antibody combination, secondary antibody combination, developing, detection, judgment and the like. The method is an indirect enzyme-linked immuno sorbent assay (ELISA) method established based on purified recombinant UL51 protein, which has good specificity, and when duck hepatitis virus (DHV), Riemerella anatipestifer (RA), duck Escherichia coli (E.coli) are detected, the detection results are all positive; intra batch or batch-to-batch repeated tests show that variation coefficients are all less than 10%, and duck plague virus (DPV) vaccine immunized duck positive sera diluted at ratio of 1:3200 can be detected.

The invention discloses a duck plague virus transfer vector pUC-deltagC-EGFP and a duck plague virus gC-deleted recombinant strain DPV-deltagC-EGFP. An escherichia coli DH5alpha containing the transfer vector pUC-deltagC-EGFP is collected on November 30th, 2011, in China Center for Type Culture Collection at Wuhan University of China, and has a collection number of CCTCC NO:M2011432. After the transfer vector is subjected to homologous recombination with the duck plague virus, and after plaque purification, the EGFP-labeled recombinant virus DPV-deltagC-EGFP is obtained. As a result of experiments, same as an attenuated vaccine, the recombinant virus DPV-deltagC-EGFP with three dosages can provide complete protection for ducklings attacked by virulent virus. Therefore, the recombinant strain has certain potential to be developed into a novel vaccine which can effectively prevent duck plague.

The invention aims at providing a duck plague virus gene deletion transfer vector and an application of the duck plague virus gene deletion transfer vector. The transfer vector is subjected to homologous recombination with duck plague virus to obtain duck plague virus gene deletion engineering strains which can be prepared into a high-safety and high-prevention-effect duck plague virus gene deletion vaccine. The duck plague virus gene deletion vaccine does not influence the virus immunogenicity while lowering down the virus pathogenicity; therefore, the prepared duck plague virus gene deletion vaccines are high in safety, able to remain the original virus immunogenicity, suitable for prevention and control for the current duck plague diseases in domestic, and beneficial for the cleaning treatment of duck plague in a duck farm; in addition, the recombined duck plague virus strains do not contain any exogenous genes; and compared with the similar duck plague virus gene vaccines reported in the prior art, the duck plague virus gene deletion vaccine has the advantage that no EGFP (Enhanced Green Fluorescent Protein), lacZ and other marker gene are contained, so that the bio-safety is improved.

The invention discloses a polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV). The method comprises the steps of: designing a specific primer sequence according to the gene difference between the virulent strains and the weak vaccine strains UL2 of the DPV, extracting a deoxyribonucleic acid (DNA) of a sample to be detected to be used as a template, and carrying out PCR; and analyzing the result of the PCR, wherein if a PCR amplification band is 1018bp, the sample to be detected contains the virulent strains of the DPV, and if the PCR amplification band is 490bp, the sample to be detected contains the weak vaccine strains of the DPV. The detection method for distinguishing the virulent strains and the weak vaccine strains of the DPV has the advantages of being simple, convenient, rapid, specific, sensitive and economical, and the result judgment method is simple and convenient, so that the detection method is suitable for scientific research, clinical application and a basic laboratory, and has wide application prospect.

The invention discloses a Riemerella anatipestifer OmpA / MotB truncated recombinant protein, its antibody and a preparation method and an application thereof. The recombinant protein has immunoreactivity similar to natural OmpA / MotB protein, can bind to RA antibody specifically, will not generate cross reactions with Salmonella anatum positive serum, duck colibacilosis positive serum, duck swollen head septicemia virus positive serum, avian influenza virus (H5) positive serum, duck plague virus positive serum, duck hepatitis virus positive serum and the like, and can be used as a detection antigen for detecting whole bacteria antibody. As the recombinant protein is not whole bacteria, there is no risk of spreading bacteria when the recombinant protein is applied to detection. In addition, the preparation method of the recombinant protein is simple, is suitable for large-scale industrial production, can realize standardization of products and is beneficial to result comparison between different laboratories.

The invention belongs to the technical field of duck plague virus culturing, particularly relates to a new host cell of the duck plague virus, and discloses application of a chicken liver cancer cell system as a duck plague virus host. A traditional method for culturing the duck plague virus through the duck embryo fibroblast primary cell is limited by duck embryo supply and the hatching day age, and wastes time and labor during preparation. The established method for culturing the duck plague virus chicken liver cancer cell system is good in virus growth. The method for culturing the virus through a continuous cell line is not influenced by duck embryo supply, and has the advantages of being instantly available and capable of saving time and labor. The duck plague virus can grow and reproduce on a chicken liver cancer cell system continuous cell culture medium, and by means of a newly-found virus copying host cell, a new technical approach, method and material are provided for research of related fields.

Relating to the field of biological engineering, the invention especially relates to a kit for detecting a duck plague virus antibody and a detection method thereof. The ELISA kit comprises a solid support, an antibody scavenger, an enzyme-labeled secondary antibody, a substrate and a blocking solution. The antibody scavenger is the duck plague virus gG segmented recombinant protein, which has anamino acid sequence as shown in SEQ ID NO: 1. The detection method consists of: solid phase antigen preparation, first antibody combination, secondary antibody combination, color development, detection and determination. The kit of the invention has the advantages of good specificity, sensitivity and stability. And the detection method provided in the invention is simple to operate.

The invention discloses a triple fluorogenic quantitative PCR detection primer, kit and method of the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus. The detection primer for the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus is shown in SEQ ID NO.1-6. The detection primer and kit can detect the EDSV, the H9 AIV and the DTMUV at the same time. No cross reaction with duck plague virus, goose parovovirus, muscovy duck parvovirus and escherichia coli genomic DNA and duck hepatitis A virus 1, duck hepatitis A virus 3, muscovy duck reovirus and newcastle disease virus RNA exists. The lower limits of detection for EDSV nucleic acid, H9 AIV nucleic acid and DTMUV nucleic acid are 8.0 copies, 4.8 copies and 1.3 copies respectively. The detection primer, kit and method can be used for qualitative and quantitative detection for the EDSV, the H9 AIV and the DTMUV.

The invention provides a duck virual hepatitis virus type I and duck plague virus dual-fluorescent quantitative PCR (polymerase chain reaction) method. A pair of specific detection primers, namely DHV-I-F and DHV-I-R, and a fluorescent probe, namely DHV-I-R, are designed for duck virual hepatitis virus type I; a pair of specific detection primers, namely DPV-F and DPV-R, and a fluorescent probe, namely DPV-P, are designed for duck plague virus; and concentrations of the specific primers and the fluorescent probes of the dual-fluorescent quantitative PCR method for the duck virual hepatitis virus type I and the duck plague virus are determined. With the application of the dual-fluorescent quantitative PCR method provided by the invention, two viruses, namely the duck virual hepatitis virus type I and the duck plague virus, can be simultaneously detected and identified; the method has the advantages of being simple to operate, high in sensitivity, strong in specificity, good in repeatability and the like; the method not only can reduce cost but also can save precious time for epidemic surveillance and epidemic control; and in addition, effective means can be provided for researches on early rapid diagnosis and surveillance of of infectious duck diseases caused by the two viruses, epidemiological survey and the like.

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus (DPV) antibodies. The method specifically comprises the steps of preparation of solid phase antigens, primary antibody combination, secondary antibody combination, color development, detection, judgment and the like. The method is a purified recombinant UL55 protein-based indirect enzyme-linked immuno sorbent assay (ELISA) method, and has good specificity; the results of detecting the positive serums of duck virus hepatitis viruses (DHV), riemerella anatipestifer (RA), duck escherichia coli (E.coli), duck-derived salmonella, duck swollen head hemorrhagic disease viruses and duck-derived influenza viruses are negative; and the repeated tests in enzyme-labeled plates or between enzyme-labeled plates display that variation coefficients are less than 10 percent, and the method can detect out the positive serums of DPV weak virus vaccine immune ducks, wherein the DPV weak virus vaccine are diluted according to a ratio of 1:6400.

The present invention relates to the field of animal medicine, particularly to an ELISA kit for detecting duck plague virus antibody. The ELISA kit comprises a solid-phase support, an antibody capturing agent, enzyme-labelled second antibody, a substrate and a blocking solution. The antibody capturing agent comprises recombinant duck plague virus UL16 protein, and the nucleotide sequence of the recombinant duck plague virus UL16 protein is represented by SEQIDNO:3. The method for detecting the duck plague virus antibody through the ELISA kit comprises the following concrete steps: preparation of the solid phase antigen, combination of the first antibody, combination of the secondary antibody, color reaction, detection and determination. According to the present invention, the kit provided by the present invention has high specificity and high sensitivity; the intra-assay or inter-assay repeated test results of the method show that: the variation coefficients are less than 10%; the positive serum of the DPV attenuated vaccine immunized duck can be detected, wherein the positive serum is diluted by 5120 folds.

The invention belongs to the field of animal medicament and biotechnology and particularly relates to a truncated duck plague virus (DPV) recombinant envelope gI protein. The DNA sequence of the truncated and expressed DPV recombinant envelope gI protein is shown as the SEQ ID No.1, and the truncated and expressed DPV recombinant envelope gI protein has a peptide sequence shown as the SEQ ID No.2. The preparation method of the truncated and expressed DPV recombinant envelope gI protein comprises the following steps: acquiring genes of the truncated and expressed DPV recombinant envelope gI protein, preparing a recombinant prokaryotic expression vector of the truncated and expressed DPV recombinant envelope gI protein, and preparing the truncated and expressed DPV recombinant envelope gI protein. The invention also relates to the application of the truncated and expressed DPV recombinant envelope gI protein to preparation of a DPV antigen or vaccine, in particular to the application of the truncated and expressed DPV recombinant envelope gI protein to the preparation of a DPV ELISA (enzyme-linked immuno sorbent assay) antibody test kit. The truncated and expressed DPV recombinant envelope gI protein has high purity, and the preparation method of the truncated and expressed DPV recombinant envelope gI protein is easy to operate, adopts the truncated and expressed DPV recombinant envelope gI protein to carry out ELISA on a DPV antibody in serum, and is simple, efficient and accurate.

The invention discloses a recombinant duck plague virus of expressing duck tembusu virus E protein as well as a construction method and application of the recombinant duck plague virus, wherein the gene of the duck tembusu virus E protein is interpolated inside the genome of the recombinant duck plague virus; and the nucleotide sequence of the duck tembusu virus E protein gene is as shown in SEQ ID NO. 9. The construction method comprises the following steps: (1) substituting the CMV promoter of gfp gene in pDEV-vac with an EF1 promoter so as to obtain pDEV-EF1; (2) interpolating a Pcmv-E-BGH-pA expression cassette into the pDEV-EF1 so as to obtain pDEV-E; and (3) transfecting the pDEV-E with chicken embryo fibroblasts and rescuing so as to obtain the recombinant duck plague virus. The recombinant duck plague virus, compared with a parent strain, has no significant difference in the size of virus plaque, showing that the diffusion of the duck plague virus on the chicken embryo fibroblasts is not affected by the interpolation of the duck tembusu virus E protein gene. After transfecting with the chicken embryo fibroblasts, the recombinant duck plague virus can successfully express E protein, so as to lay a foundation for developing duck plague virus-duck tembusu virus bivalent vaccine.

The invention discloses a duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein (EGFP) genes and a constructing method and application of the duck plague virus recombinant vaccine strain rDEVTK-EGFP. The microorganism preservation number of the vaccine strain rDEVTK-EGFP is CGMCC No. 9456. According to the duck plague virus recombinant vaccine strain rDEVTK-EGFP, the constructing method and the application, a recombinant clone technology is adopted; a gene segment sCMV-EGFP containing enhanced green fluorescent proteins (EGFP) and sCMV promoter sequences is inserted into a duck plague virus TK gene region; recombinant EGFP duck plague viruses with an sCMV-EGFP expression cassette inserted into the corresponding position of a TK gene is constructed; the EGFP genes are stably expressed by the recombinant viruses. The invention further relates to a method for constructing the recombinant duck plague virus vaccine strain for stably expressing other poultry pathogeny exogenous genes and the application of the recombinant duck plague virus vaccine strain to preparation of vaccines for preventing duck plagues and other poultry infectious diseases.

The invention relates to the field of biological technology and animal immunology, in particular to a duck plague virus cyst membrane gI protein polyclonal antibody. The polyclonal antibody is obtained from a duck plague virus cyst membrane gI protein immune animal which has a DNA sequence shown as SEQ ID No.1 and a polypeptide sequence shown as SEQ ID No.2; the polyclonal antibody can be used for preparing a duck plague virus detecting reagent; and the polyclonal antibody has high antibody titer and high specificity, can meet the duck plague virus and duck plague virus cyst membrane gI protein-associated detecting test and fills the research blank of duck plague virus cyst membrane gI protein polyclonal antibody.

The invention discloses a duck plague virus (DPV) UL13 intercepted recombinant protein of which an amino acid sequence is shown as SEQ ID No.1, an encoding gene, a recombinant expression vector and engineering bacteria of the intercepted recombinant protein, as well as a method for preparing the intercepted recombinant protein by utilizing the engineering bacteria. The method is simple and convenient to operate, low in cost and suitable for industrial large-scale production. The product standardization can be realized, and results in different labs are compared. The obtained intercepted recombinant protein has high immunoreactivity, can be specifically bound to a DPV antibody, can serve as a detection antigen for detecting the DPV antibody, and is high in detection accuracy, high in specificity, free of cross reaction and free of toxic danger. A polyclonal antibody is successfully obtained for an antigen immune animal through the DPV UL13 intercepted recombinant protein, can serve as a detection antibody for detecting the DPV, and has the advantages of high detection specificity, no cross reaction and the like.

The invention discloses a primer set used in duck plague virus detection, and a LAMP reaction system composed thereof. The detection primer set comprises a forward outer primer F3:SEQ ID NO:1, a backward outer primer B3:SEQ ID NO:2, a forward inner primer FIP:SEQ ID NO:3, and a backward inner primer BIP:SEQ ID NO:4. The LAMP reaction system comprising the primers also comprises a reaction buffer, dNTPs, Mg<2+>, betaine, BstDNA polymerase, detection sample genome DNA, deionized water, and the like. The reaction system has advantages of simple operation method, high specificity, high sensitivity, and convenient detection result identification. The system has wide prospect to be applied in small institutes, veterinary stations and common raising households.

The invention discloses a full suspension culture method of a duck plague virus, comprising the following steps: step 1: resuscitation and passage culture of passage cell lines derived from duck embryo cells; 2, inoculation of the duck plague virus into a passage cell line derived from a fully suspend culture duck embryo cell by adopting a second-order culture method to realize large-scale cultureof that duck plague virus. 3, after that virus is inoculated, the TCID50 of the virus is sampled and detected every 12 hours, the virus is harvested when the TCID50 of the virus reach the maximum, and the virus is stored to obtain the cultured duck plague virus. The full suspension culture method of the invention improves virus culture scale and efficiency, accords with the trend of vaccine production in the future, and has wide application prospect and good economic benefit.

The invention discloses a double-PCR (Polymerase Chain Reaction) detection kit for newcastle disease virus and duck plague virus. A primer group provided by the invention is composed of a primer 1, a primer 2, a primer 3 and a primer 4. Nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence table sequentially. Experiments show that newcastle disease and duck plague can be detected and identified simultaneously by one-time PCR reaction. Compared with the conventional pathogen separation identification and serological experiments, double PCRs have the characteristics of good specificity, high sensibility, fastness, simplicity, convenience, and the like, and has a high clinical application value.

The invention provides a duck plague live vaccine and a preparation method thereof, and belongs to the technical field of biological product preparation. A chicken embryo fibroblast line DF-1 is takenas the host of duck plague viruses to produce a duck plague virus solution, and then a heatproof protective agent is added into the duck plague virus solution to prepare the duck plague live vaccine.DF-1 is used to culture duck plague viruses to avoid the safety hazard that the duck plague viruses cultured by (chicken embryo fibroblast) CEF cells are contaminated by exogenous viruses; the DF-1 cells are available at any time, time and labor are saved, and the production cost is greatly reduced. The prepared duck plague live vaccine has the advantages of good safety and high immunity effect,and can protect ducks from strong duck plague viruses. Furthermore, the live vaccine also comprises an improved heatproof protecting agent, thus the duck plague live vaccine can be stored for 24 months at a temperature of 2-8 DEG C, the virus content an titer are not reduced, the storage conditions are lowered, the storage and transportation of the live vaccine become convenient, and the production cost is reduced.

The invention discloses a UL55 recombinant prokaryotic expression protein for detecting a duck plague virus (DPV) antibody and a preparation method thereof. The recombinant prokaryotic expression protein for detecting the DPV antibody is defined by an amino acid sequence table shown as SEQ ID NO.2 and is a prokaryotic expression product of a nucleotide sequence shown as SEQ ID NO.1. The preparation method comprises the following steps of: (1) designing a specific primer for amplifying a DPV-UL55 gene; (2) performing polymerase chain reaction (PCR) amplification to obtain a UL55 gene fragment; (3) cloning, sequencing and identifying a UL55 gene; (4) directionally subcloning the UL55 gene to a pET32a prokaryotic expression vector; (5) performing inducible expression on the UL55 gene in Escherichia coli; (6) purifying a UL55 gene expression protein; and (7) renaturing and detecting a recombinant UL55 protein. The product can be used as a detection antigen for detecting the DPV antibody by indirect enzyme linked immunosorbent assay (ELISA) and a western blot method, and also can be used as a gene engineering subunit vaccine for preventing DPV infection.

The invention provides a primer probe combination for simultaneously detecting duck circovirus, duck plague virus and duck adenovirus 3, and belongs to the technical field of livestock and poultry disease diagnosis. The primer probe combination comprises a primer pair and a probe, a nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and a nucleotide sequence of adownstream primer of the primer pair is shown as SEQ ID No.2; the probe comprises a duck circovirus probe, a duck plague virus probe and a duck adenovirus 3 probe, a nucleotide sequence of the duck circovirus probe is shown as SEQ ID No.3, a nucleotide sequence of the duck plague virus probe is shown as SEQ ID No.4, and a nucleotide sequence of the duck adenovirus 3 probe is shown as SEQ ID No.5.By adopting the primer probe combination provided by the invention, the duck circovirus, the duck plague virus and the duck adenovirus 3 can be simultaneously detected.

The invention discloses a duck flavivirus and duck plague virus duplex RT-PCR kit. The kit contains two pairs of specific primers. The kit has the advantages of simple operation, high sensitivity, strong specificity, good repeatability and the like, can establish a duck flavivirus and duck plague virus duplex RT-PCR detection method, can simultaneously detect and identify the two pathogens of the duck flavivirus and the duck plague virus, can realize the time cost saving and pollution reduction when the kit is used in the duck plague virus mixed infection caused by the immunity reduction caused by the duck flavivirus infection.

The invention discloses a duck plague virus recombinant vaccine strain expressing an enhanced green fluorescent protein (EGFP) gene, a constructing method thereof and applications of the recombinant vaccine strain. In particular, by utilization of a recombinant clone technology, a gene fragment CMV-EGFP containing an enhanced green fluorescent protein (EGFP) and a CMV promoter sequence replaces a US10 gene of a duck plague virus, and a recombinant EGFP duck plague virus lacking the US10 gene with a CMV-EGFP expression cassette being inserted into the corresponding position is constructed. The recombinant virus can stably express the EGFP gene. The duck plague virus recombinant vaccine strain is named as rDEVUS10-EGFP. The microbial accession number is CGMCC No.8660. The invention also relates to constructing methods of duck plague virus recombinant vaccine strains capable of stably expressing other poultry pathogeny exogenous genes, and applications of the recombinant vaccine strains in preparation of vaccines preventing duck plague and other poultry infectious diseases.

The invention relates to the technical field of veterinary pathogeny antibody test, in particular to a duck parvovirus indirect ELISA (enzyme-linked immuno sorbent assay) detection reagent kit. The inventor firstly provides a duck parvovirus virus VP3 protein; the amino acid sequence of the protein is shown as SEQ ID No.1; the amino acid sequence expressing the protein is shown as SEQ ID No.2. Study shows that the protein has good immune response original performance, and can be used for DPV (duck plague virus) antibody detection; further, the protein is used for providing the duck parvovirus indirect ELISA detection reagent kit. The reagent kit has the advantages of high speed, high stability, high specificity and high sensitivity, can be used for fast detecting the antibody level of the duck parvovirus virus in the serum, and achieves the obvious promotion effect on the prevention and the control of the disease.

The invention provides a recombinant duck plague virus vaccine strain as well as a construction method and application thereof. The recombinant duck plague virus vaccine strain provided by the invention comprises one or more antigen coding sequences inserted in a spacer region between US8 and US1 genes of a duck enteritis virus (DEV) genome. The invention also relates to the method for constructing the recombinant duck plague virus vaccine strain, and the application of the recombinant duck plague virus vaccine strain for preparing a vaccine for preventing diseases caused by duck virus and / orbacterial infection. The recombinant duck plague virus vaccine strain provided by the invention does not affect the immune effect of the DEV, and provides immune protection against diseases caused byother duck virus and / or bacterial infection.

The invention relates to the field of animal preventive medicine, in particular to a method for preparing injection for preventing animal epidemic diseases. The bee glue injection for preventing duck plague consists of bee glue aqueous solution, tissue homogenate of duck embryos which are dead after being inoculated and infected with duck plague virus, and inactivation liquid. The preparation method comprises the step of adding the inactivation liquid into mixed liquid of the duck embryo tissue homogenate and bee glue to obtain the bee glue injection for preventing the duck plague. The bee glue injection for preventing the duck plague, which is prepared by the method, has the obvious effect on the prevention of the duck plague, the protective rate is improved to 90 to 100 percent, the protective period is prolonged to 6 to 12 months, action producing time is shortened to 5 days, and the expected effect can be achieved only by one-time injection.