30 results about "Avian leukemia virus" patented technology

The invention relates to the technical field of antibodies, in particular to an antibody or an antigen binding fragment specifically binding to avian leukosis virus p27 protein and application of the antibody or the antigen binding fragment. The antibody or the antigen binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ.ID NO.1 or SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ.ID NO.3 or SEQ.ID NO.7. The antibody or the antigen binding fragment is positive in reaction with the avian leukosis virus, so that the antibody or the antigen binding fragment has good reaction characteristics with the avian leukosis virus.

The invention provides a reagent for detecting avian leukosis virus, a nano-enzyme immunochromatography test strip and application of the nano-enzyme immunochromatography test strip, and belongs to the technical field of immunology. Aiming at the problems that the existing avian leukosis virus detection method is complicated in operation, time-consuming or strong in equipment dependence, the reagent comprises a capture antibody and a detection antibody; a heavy chain variable region of the monoclonal antibody A comprises an amino acid sequence as shown in SEQ ID NO: 1, and a light chain variable region of the monoclonal antibody A comprises an amino acid sequence as shown in SEQ ID NO: 2; a heavy chain variable region of the monoclonal antibody B comprises an amino acid sequence as shown in SEQ ID NO: 5, and a light chain variable region of the monoclonal antibody B comprises an amino acid sequence as shown in SEQ ID NO: 6; the antigen binding fragment is a Fab fragment, an F (ab) 2 fragment or a single-chain Fv fragment. The reagent is mainly used for detecting avian leukosis virus, and is suitable for detecting samples such as cloaca swab, meconium, egg white or serum of poultry.

The present invention provides primers and methods for amplifying the entire genome of avian leukosis virus. The detection primer set for avian leukosis virus includes 18 pairs of amplification primers, the specific sequences of which are shown in SEQ ID NOs. 1 to 36. The detection primer set provided by the present invention can achieve relatively uniform coverage of avian leukosis virus, and in nanopore sequencing, 100% regional coverage of the genome is achieved. The method for preparing sequencing fragments of the entire genome of avian leukosis virus provided by the present invention is simple and easy to operate, with good amplification effect. Nanopore sequencing has the advantage of real-time analysis while sequencing, which can greatly shorten the detection time and can quickly identify and diagnose avian leukosis virus infection. At the same time, its entire genome sequence can be obtained. The obtained genome sequence can provide a scientific basis for virus tracing, pathogen mutation tracking, new strain identification and early warning, etc.

The invention discloses application of a LOXL2 gene in resisting avian leukosis virus infection, and belongs to the technical field of biology. The nucleotide sequence of the LOXL2 gene is as shown in SEQ ID NO. 1. The invention provides a new gene target LOXL2 related to avian leukosis virus infection resistance, and proves the effect of the LOXL2 gene in regulating and controlling avian leukosis virus replication. Replication of the avian leukemia virus can be promoted by inhibiting expression of the LOXL2 gene in a host cell, and replication of the avian leukemia virus can be inhibited by overexpressing the LOXL2 gene in the host cell. According to the invention, favorable theoretical guidance and technical support are provided for the application of the LOXL2 gene as a target in the aspects of development of drugs for treating avian leukemia, breeding of transgenic animals for resisting avian leukemia, construction of avian leukemia virus infection models and the like, and the LOXL2 gene has wide application prospects and extremely high market value.

The invention relates to the field of avian virus detection, and discloses a method for rapidly identifying avian leukosis virus, which comprises the following steps: S1, sample collection and pretreatment: firstly collecting a poultry sample, S2, loading a micro-fluidic chip, and then injecting the pretreated sample into the micro-fluidic chip; s3, performing micro-fluidic LAMP amplification on the micro-fluidic chip to obtain enough products for subsequent detection; s4, a CRI SPR specific detection amplification product automatically enters a CRI SPR reaction chamber in the chip; and S5, collecting and analyzing a detection signal, and reading a fluorescence signal on the chip by using a portable fluorescence detector. By fusing the rapid amplification capability of the microfluidic LAMP and the high-specificity detection advantage of CRISPR / Cas12a, the avian leukosis virus can be rapidly, sensitively and specifically recognized, the portable and field application is realized, the target sequence in the amplification product is recognized, the cleavage function is activated, and the detection sensitivity is high. In use, whether a sample reacted by a fluorescence signal contains viruses or not can be quickly identified.

The invention relates to a primer probe combination for identifying E, J and K subgroups of avian leukosis viruses, a detection method and application of the primer probe combination. The detection system for identifying the avian leukosis virus subgroup comprises an upstream primer ALV-E-F, a downstream primer ALV-E-R and a probe ALV-E-P of a gp85 gene conserved region of targeted ALV-E, and an upstream primer ALV-J-F, a downstream primer ALV-J-R and a probe ALV-J-P of an env gene conserved region of targeted ALV-J, the upstream primer ALV-K-F, the downstream primer ALV-K-R and the probe ALV-K-P are used for targeting a gp85 gene conserved region of ALV-K. The detection system for identifying the avian leukosis virus subgroups has no cross reaction with other ALV subgroups and common avian pathogens, and has strong specificity.

The invention discloses a method and a kit for simultaneously detecting avian leukosis virus, mycoplasma synoviae and salmonella pullorum antibodies, and belongs to the technical field of animal epidemic disease detection. According to the method, an avian leukosis virus p27 protein, a chicken mycoplasma synoviae p50 protein and a truncated GroEL-delta8-1 protein of a dominant antigen GroEL of salmonella pullorum are taken as coating antigens, synchronous detection of the three antibodies is realized by optimizing ELSA reaction conditions, and a kit capable of simultaneously detecting the three antibodies is developed at the same time. The kit has the characteristics of strong specificity, high sensitivity and simplicity and convenience in operation, is suitable for rapid screening and purification of poultry epidemic diseases, and reduces the detection cost.

The application discloses application of a LOXL2 gene in resisting avian leukemia virus infection and belongs to the technical field of biotechnology.The nucleotide sequence of the LOXL2 gene is shown as SEQ ID NO.1.The application provides a new gene target point LOXL2 related to resisting avian leukemia virus infection, and proves the role of the LOXL2 gene in regulating avian leukemia virus replication.Inhibition of LOXL2 gene expression in host cells can promote avian leukemia virus replication, and overexpression of the LOXL2 gene in host cells can inhibit avian leukemia virus replication.The application provides favorable theoretical guidance and technical support for application of the LOXL2 gene as a target point in developing drugs for treating avian leukemia, breeding transgenic animals resisting avian leukemia, and constructing an avian leukemia virus infection model, and has a wide application prospect and extremely high market value.

The present invention discloses an avian leukosis antigen detection kit and a preparation method thereof. The kit comprises an anti-avian leukosis virus P27 protein monoclonal antibody and a horseradish peroxidase-labeled sheep anti-P27 polyclonal antibody as a sandwich secondary antibody. The kit is based on a double-antibody sandwich method and has good specificity for A / B, J, and K subtype avian leukosis viruses prevalent in breeder chickens. The minimum detection limit is 1 ng / mL, the kit has strong anti-interference ability, a low false positive rate, and is easy to operate and highly stable. It is suitable for scenarios such as breeder chicken flock purification, SPF embryo quality monitoring, and epidemiological surveys. It effectively solves the problem of expensive imported products and limited domestic promotion, and has significant economic and social benefits.

1. The name of the design product: kit (virus antigen detection). 2. The use of the design product: for containing each component of the kit for detecting avian leukosis virus. 3. The design points of the design product: the combination of shape, pattern and color. 4. The picture or photo that best shows the design points: perspective view 1. 5. The design for which protection is sought contains color.

Provided is a kit for rapid detection of avian leukosis virus subgroup J (ALV-J) based on a CRISPR / Cas13a system. The method is based on the combination of the CRISPR / Cas13a system and recombinase aided amplification (RAA) for ALV-J detection. An oligonucleotide probe is designed as a substrate for CRISPR / Cas13a trans-cleavage and produces a detectable signal. The method can substantially improve detection sensitivity by amplifying a detection signal twice by RAA and T7 transcription. The detection method further exhibits excellent specificity, allowing for clear differentiate from other avian viruses. It does not require expensive experimental equipment and special laboratory environment, and it is rapid and efficient. The method is of great significance for biological research and on-site detection of ALV-J.

The application provides an avian leukosis virus multi-subtype antigen epitope fusion protein, a multi-subtype recognition antibody, a detection kit and application thereof. The amino acid sequence of the avian leukosis virus multi-subtype antigen epitope fusion protein is shown as SEQ ID NO:1. Experiments show that the avian leukosis virus multi-subtype antigen epitope fusion protein is reactive to avian leukosis virus multi-subtype serum and is not reactive to other common chicken infectious virus or bacterial serum. The avian leukosis virus multi-subtype antigen epitope fusion protein can be used for preparing multi-subtype recognition antibodies by immunizing animals. The multi-subtype recognition antibody can be specifically recognized with multi-subgroup avian leukosis specific antigen epitopes, a detection kit is developed, and the detection kit is used for rapid screening and purification of chicken population avian leukosis virus, and has a good application prospect.

The invention relates to the technical field of virus isolated culture, in particular to a method for promoting avian leukosis virus proliferation. The invention provides a method for promoting proliferation of avian leukosis viruses. The method comprises the step of culturing the avian leukosis viruses by taking STAT1 gene inactivated chicken DF-1 cells as host cells. The DF-1 cell strain with the STAT1 knocked out is established, when the cell strain is used for isolated culture of ALV, the proliferation rate of the ALV can be effectively increased, compared with an original DF-1 cell, the proliferation process of the virus can be remarkably accelerated, and the proliferation time is shortened from at least 7 days to 4 days. The DF-1 cell strain of which the STAT1 is knocked out can be used as a host cell to be applied to separation, amplification or detection of ALV in the industry, and shows an ideal amplification effect for poultry plasma samples under different health conditions.

The invention discloses an avian leukosis tetravalent epitope mRNA vaccine and application, and belongs to the technical field of immunology and biology. According to epidemic strains of A, B, J and K subgroup avian leukosis viruses in recent 10 years, epitopes with relatively high antigenicity and conservative property in surface proteins (gp85 gene coding) are screened and connected in series, so that immune escape caused by self gene mutation of the avian leukosis viruses is avoided, the protection range is wider, and the kit has a good application prospect. And a molecular adjuvant is also added into an mRNA vaccine sequence to enhance the cellular immunity of the vaccine, so that the problem of low antigenicity of other vaccines is solved. Compared with purification investment, the vaccine has the advantages that the capital cost is saved by more than 50%, the consumption of time, manpower and material resources is greatly reduced, and the vaccine has a good application prospect.

The present invention discloses a molecular marker, screening / identification method and application of chicken genetic resistance to subgroup B avian leukosis virus infection, the method comprising: extracting genomic DNA of a sample to be tested, amplifying a TVB receptor gene fragment, and then sequencing to determine whether there is a deletion of base G at position 3747 of the TVB receptor gene. The method can quickly and accurately determine whether the test sample is an ALV-B resistant chicken or a susceptible chicken, and can screen breeding materials for breeding chicken varieties / lines resistant to ALV-B infection from Chinese chicken breeds (including local chicken breeds and commercial broiler strains), providing technical support for breeding chicken varieties / lines resistant to ALV-B infection.

The invention belongs to the technical field of biological detection, and discloses an avian leukosis virus detection kit based on a magnetic particle chemiluminescence method and application thereof. The kit comprises a liquid A, a liquid B, a liquid C, a liquid D calibration product and a quality control product, the liquid A comprises magnetic particles coated with an avian leukosis virus antibody, and the liquid B comprises an acridinium ester labeled avian leukosis virus antibody. According to the kit disclosed by the invention, the avian leukosis virus antibody is coated on the magnetic particles, and the magnetic particles have a relatively large surface area, so that the antigen capture efficiency is greatly improved, and the detection specificity and sensitivity are enhanced. In addition, acridinium ester is adopted for labeling, the cost is low, enzyme catalysis is not needed, the influence of environment temperature and humidity on the reaction is small, luminescence is stable, the luminescence efficiency is high, background luminescence is low, the signal-to-noise ratio is high, the detection time is short, the detection efficiency and stability are greatly improved, and rapid detection of large-scale samples in production is facilitated.

The application provides a reagent for detecting avian leukemia virus, a nanozyme immunochromatography test strip and application thereof, and belongs to the technical field of immunology. In view of the problems of complicated operation, time consumption and strong equipment dependence of the existing avian leukemia virus detection method, the reagent comprises a capture antibody and a detection antibody; the heavy chain variable region of a monoclonal antibody A comprises an amino acid sequence as shown in (SEQ ID NO: 1), and the light chain variable region comprises an amino acid sequence as shown in (SEQ ID NO: 2); the heavy chain variable region of a monoclonal antibody B comprises an amino acid sequence as shown in (SEQ ID NO: 5), and the light chain variable region comprises an amino acid sequence as shown in (SEQ ID NO: 6); and the antigen binding fragment is a Fab fragment, a F(ab)2 fragment or a single-chain Fv fragment. The reagent is mainly used for detecting avian leukemia virus and is suitable for detecting samples such as avian cloaca swabs, fetal meconium, egg white or serum.

This invention discloses a mutated A subgroup A avian leukosis virus (ALV-A) receptor gene and its application in combating A subgroup A avian leukosis virus infection. This invention identified L55 and W69 as key functional amino acid sites on the receptor protein Tva, which mediates ALV-A infection of chicken cells. Mutation of these two sites resulted in ALV-A's inability to infect host cells. Furthermore, this invention discloses a method for constructing a DF-1 cell line resistant to ALV-A infection. This method utilizes CRISPR / Cas9 and flow cytometry screening to mutate the key amino acid sites L55 and W69 of the Tva gene as the ALV-A receptor. Results show that substitution sequences containing mutations at L55 and W69 can efficiently and site-specifically replace the corresponding sequences in the Tva gene; the Tva gene-edited cell line can resist ALV-A infection without affecting cell proliferation. This invention lays the foundation for further research on the function of the Tva gene and the establishment of new technologies for combating ALV-A infection.

The invention discloses a conservative functional small peptide LL10 on the surface of avian leukosis virus (ALV) envelope protein gp37, a nucleic acid molecule, a recombinant vector and application of the conservative functional small peptide LL10 in diagnosis and detection of different subgroup ALV infection. The recombinant bacteria provided by the invention can specifically recognize different subgroup ALV infectious antibody serum samples. The recombinant thallus is taken as an agglutination antigen and can be subjected to specific reaction with serum infected by different subgroups of ALV to form clear and visible agglutination particles, and meanwhile, the agglutination antigen does not react with serum infected by other pathogens. A rapid and convenient detection method for different subgroup ALV infection is established, qualitative and quantitative detection of specific antibodies after ALV infection can be achieved, and a reliable novel technical method is provided for ALV diagnosis and purification.

The invention discloses an epitope peptide of avian leukosis virus J subgroup envelope protein GP85, a hybridoma cell strain 3G10E12C4, a monoclonal antibody (mAb) generated by the hybridoma cell strain 3G10E12C4 and application of the monoclonal antibody (mAb). An ALV-J subgroup GP85 protein FE10 epitope peptide is expressed on the surface of an engineering bacterium DH5alpha through a MisL protein passenger-carrying structural domain of a salmonella V-type secretion system in an exhibiting manner and serves as an immunogen to immunize a BALB / c mouse, meanwhile, the FE10 epitope peptide is inserted into a peg pilus operon, an inert carrier bacterium S9H is introduced, the FE10 epitope peptide serves as a detection system, mouse splenocytes and SP2 / 0 cells are fused, and a detection result is obtained. And the monoclonal antibody is obtained through double screening of ELISA (enzyme-linked immuno sorbent assay) and detection targets. The mAb prepared from the hybridoma cell can be specifically combined with the ALV-J strain infected with the HD-11 cell, and has potential application value and prospect in detection, treatment and purification of the ALV.

The invention discloses a hybridoma cell strain capable of secreting a monoclonal antibody for recognizing K subgroup avian leukosis virus and application of the hybridoma cell strain, and belongs to the technical field of biology. The preservation number of the hybridoma cell strain provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO.23876. The monoclonal antibody generated by the hybridoma cell strain can recognize the antigen epitope (204-DWSRPQL-210) of the K subgroup avian leukosis virus gp85 envelope protein, the subtype of the antibody is IgG1, the monoclonal antibody can be used for developing a diagnostic reagent or medicine for the K subgroup avian leukosis, and a new substance is provided for clinical differential diagnosis and laboratory research of the K subgroup avian leukosis.

The application belongs to the technical field of biological genetic engineering, and relates to application of PRMT7 as a target point in preparation of a medicine for inhibiting proliferation of subgroup J avian leukemia virus, and application of a PRMT7 gene polymorphism site as a molecular marker for evaluating resistance of poultry to subgroup J avian leukemia virus. Research shows that PRMT7 is involved in immune regulation of a host to infection of subgroup J avian leukemia virus, and inhibition of PRMT7 expression can enhance activity of a type I interferon related path and reduce a virus replication level. Further, a plurality of nucleotide polymorphism sites in the PRMT7 gene are screened to obtain a significant correlation with an ALV-J related phenotype, and the related phenotype includes a pancreas tissue index, a spleen tissue index, a bursa of fabricius tissue index, an IL-6 level and an IFN alpha level. The application provides a new technical scheme for prevention and control of subgroup J avian leukemia virus, development of an antiviral target point and poultry disease resistance molecular breeding.

The application discloses an avian leukosis virus fluorescent quantitative PCR detection kit based on a pol gene and application thereof. The application is based on a fluorescent quantitative PCR technology, and specific primers and a TaqMan probe aiming at a pol gene conservative region of avian leukosis virus are designed to realize specific amplification and detection of virus nucleic acid. The core improvement of the application is that after nucleic acid is extracted from animal tissue, plasma and other samples, a DNA enzyme is added to perform digestion treatment, and residual host genome DNA in the sample is specifically degraded, so that a false positive problem caused by amplification of an endogenous pol gene is completely eliminated, and the specificity and accuracy of detection are obviously improved.

The invention provides a polypeptide for detecting an avian leukosis virus (ALV). The polypeptide at least comprises amino acid fragments from the 6th site to the 39th site or amino acid fragments from the 177th site to the 204th site of an amino acid sequence as shown in SEQ ID NO: 1. When the polypeptide is used for detecting the antibody in avian leukosis virus B subgroup serum, positive serum and negative serum can be obviously separated.

The invention discloses an avian leukosis antigen detection kit and a preparation method thereof. The kit comprises an anti-avian leukosis virus P27 protein monoclonal antibody and a goat anti-P27 polyclonal antibody labeled by horse radish peroxidase, wherein the monoclonal antibody and the goat anti-P27 polyclonal antibody are used as sandwich secondary antibodies. The kit is based on a double-antibody sandwich method, has good specificity on epidemic A / B, J and K subtype avian leukosis viruses of breeding hens, has the lowest detection limit of 1 ng / mL, has strong anti-interference capability, low false positive rate, simple operation and high stability, is suitable for breeding hen flock purification, SPF embryo source quality monitoring, epidemiological investigation and other scenes, effectively solves the problems of high price of imported products and limited domestic popularization, and has a wide application prospect in the fields of breeding hen flock purification, SPF embryo source quality monitoring, epidemiological investigation and the like. And the method has remarkable economic benefits and social benefits.

The invention relates to the field of virus detection, in particular to a primer probe combination, a detection method and a kit for identifying and detecting avian leukosis virus A, B and K subgroups. The invention provides a primer combination for identifying and detecting avian leukosis virus A, B and K subgroups, wherein the nucleotide sequences of the primer combination are as shown in SEQ ID NO: 1-SEQ ID NO: 9. The fluorescent quantitative RT-PCR detection method has the advantages of strong specificity, high sensitivity, good repeatability and the like, can accurately identify whether a sample contains the avian leukosis virus A subgroup, the avian leukosis virus B subgroup and the avian leukosis virus K subgroup or not, and can effectively solve the problem that infection of different strains is difficult to distinguish in the process of identifying, preventing and controlling the avian leukosis virus A / B / K subgroups.

The application relates to the technical field of antibodies, in particular to an antibody or antigen binding fragment specifically binding to p27 protein of avian leukosis virus and application thereof. The antibody or antigen binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ.ID NO.1 or SEQ.ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ.ID NO.3 or SEQ.ID NO.7. The antibody or antigen binding fragment is positive to avian leukosis virus, which indicates that the antibody or antigen binding fragment has good reaction characteristics with the avian leukosis virus.

The application discloses a mutated K subgroup avian leukosis virus (ALV-K) receptor gene (Tva) and application thereof in resisting K subgroup avian leukosis virus infection. The application identifies key functional amino acid sites E53, L55, H59 and G70 of a receptor protein Tva mediating ALV-K infection of chicken cells, and finds that after the four sites are mutated, ALV-K cannot infect host cells. Further, the application also discloses a method for constructing a DF-1 cell line resisting ALV-K infection, which utilizes a CRISPR / Cas9 method and flow cytometry screening, and mutates the key amino acid sites E53, L55, H59 and G70 of the Tva gene as the ALV-K receptor. Results show that a replacement sequence containing the above site mutation can efficiently replace the corresponding sequence of the Tva gene; the Tva gene edited cell line can resist ALV-K infection without affecting the cell proliferation level. The application lays a foundation for further studying the function of the Tva gene and establishing a new technology for resisting ALV-K infection.

The invention belongs to the technical field of biology, and discloses a primer group and application thereof, and a kit for detecting RPA-CRISPR / Cas12a of ALV-F. According to the technical scheme, the primer group for specifically amplifying an ALV-F sequence is characterized by comprising an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 2; based on a conserved region of an avian leukosis virus (ALV) F subgroup genome, a set of RPA primer group and crRNA with high specificity and high amplification efficiency are autonomously designed and verified. According to the design, the genetic diversity of target viruses is effectively covered, high specificity of detection on ALV-F subgroups is ensured, and the risk of cross reaction with other poultry pathogens is reduced to the maximum extent.

The invention provides a primer probe group for detecting J and K subgroup avian leukosis viruses, a kit and application, and relates to the field of avian leukosis virus detection. A J type primer group and a K type primer group designed by the invention comprise primers with sequences as shown in SEQ ID NO.1-2, a probe with a sequence as shown in SEQ ID NO.3, primers with sequences as shown in SEQ ID NO.4-5 and probes with sequences as shown in SEQ ID NO.6-7, and a preserving fluid formula suitable for sample direct expansion and an amplification reagent with strong stress resistance are screened out through a large number of optimization experiments. The kit has the advantages of simplicity and convenience in operation, high detection speed, high specificity, high sensitivity and the like, can save time, manpower and material costs, and is suitable for detecting batch samples.