51 results about "Leucosis" patented technology

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 a method for rapidly obtaining chicken flocks capable of simultaneously resisting five kinds of subgroup avian leukosis and application, and belongs to the technical field of poultry disease-resistant breeding. The method comprises the following steps: firstly, obtaining primordial germ cells of a donor chicken containing an anti-avian leukosis subgroup genotype I and an anti-avian leukosis subgroup genotype II through in-vitro separation and cultivation, then injecting the primordial germ cells of the chicken into chick embryo blood vessels of a receptor chicken with wild type tvas / s and tvbs / s genotypes, performing incubation and cultivation, performing genetic typing, and finally obtaining the chicken with the anti-avian leukosis subgroup genotype I and the anti-avian leukosis subgroup genotype II. And finally obtaining chicken flocks capable of simultaneously resisting five kinds of subgroup avian leukosis. The method optimizes a chicken primordial reproduction in-vitro efficient culture system, can quickly obtain different chicken flocks capable of simultaneously resisting five kinds of avian leukosis subgroups, can be applied to utilization and preservation of resistance germplasm resources of excellent chicken breeds in China, can be applied to breeding of disease-resistant chickens of different breeds, greatly shortens the period of traditional poultry anti-avian leukosis breeding, and has a wide application prospect. The novel technology has good application value and popularization prospect.

The present invention relates to Split-Intron Final Self-Inactivating (SIN) retroviral vectors which comprises a viral major splice donor (mSD) comprising mutations, a mouse mammary tumor vector long terminal repeat (MMTV-LTR) or a Type B leukemogenic virus long terminal repeat (TBLV-LTR), a eukaryotic splice acceptor (eSA), and a eukaryotic splice donor (eSD). Furthermore, the invention relation to the use and methods of uses of such vectors in gene therapy.

The application belongs to the technical field of biology and particularly relates to an ELISA kit for detecting chicken astrovirus type II antibodies and a preparation method. The Cell-ELISA experiment is carried out by using LMH cells infected with chicken astrovirus type II to detect the chicken astrovirus type II antibodies. The method has no cross reactivity with positive serum of Newcastle disease virus, avian influenza virus, chicken infectious anemia virus, avian leukosis virus, Marek's virus and chicken infectious bursal virus, which indicates that the method has good specificity. The Cell-ELISA method is used to detect the chicken serum sample to be detected by using whole virus infection, and is assembled into a kit for measuring chicken astrovirus antibodies in serum. The kit can be used for serological diagnosis of chicken astrovirus type II infection, monitoring of the level of antibodies, epidemiological investigation and the like, and provides a novel and effective detection means for prevention and treatment of chicken astrovirus type II.

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 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 provides a composition, a method and an application for improving the titer of mouse leukemia virus, and belongs to the technical field of biotechnology.The method comprises the following steps: before a host cell is infected with xenotropic mouse leukemia virus, the host cell is pretreated by using SC79; during virus infection of the pretreated host cell and during cell passage, mifepristone is added to a culture medium; and during continuous virus harvesting, LR3 IGF-1 is added to the culture medium.Through adding additives with different functional mechanisms at different stages of virus production, the application realizes dynamic optimization of the whole process of virus production.The experimental results show that the combination of SC79, mifepristone and LR3 IGF-1 can increase the titer of xenotropic mouse leukemia virus by 14.32 times, which is significantly better than the effect of single additive or two additives in combination.The application solves the problems of low titer of virus stock solution and low production efficiency in the prior art.

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.

This invention provides a high-affinity rabbit single-chain recombinant antibody, its application, and the resulting reagent kit, belonging to the field of immunology. The high-affinity rabbit single-chain recombinant antibody provided by this invention has the amino acid sequence shown in SEQ ID NO.1, named 111-scFv-Fc; and has the amino acid sequence shown in SEQ ID NO.5, named 86-scFv-Fc. The antibodies provided by this invention can recognize different antigenic determinants on the surface of avian leukosis virus (ALV). When applied to the detection of trace amounts of ALV, they exhibit advantages such as high specificity, high sensitivity, and good stability. The combination of the two single-chain recombinant antibodies shows high efficiency in ALV antigen detection, meeting the requirements for ALV detection in clinical samples, and has significant implications for clinical diagnosis and research applications.

The invention provides a molecular diagnosis method for avian leukosis virus and Marek's disease virus mixed infection. The molecular diagnosis method comprises the following steps: (S01) collecting diseased material tissues; (S02) carrying out nucleic acid extraction on the sick material tissue of the sick chicken; (S03) detecting the nucleic acid by using specific primers of avian leukosis and Marek's disease; (S04) preparing a PCR (Polymerase Chain Reaction) detection system, and respectively adding ALV-env gene amplification primers and MDV-meq gene amplification primers for gene amplification; (S05) carrying out 1% agarose gel electrophoresis detection on the amplification product in the step (4), and carrying out sequence determination after determining a target band; (S06) carrying out BLAST comparison on the sequences obtained by sequencing, carrying out gene sequence analysis and drawing an evolutionary tree; and (S07) determining the virus type according to the homology and the evolutionary branches, so as to realize rapid diagnosis of ALV and MDV mixed infection, strain characteristic analysis and exclusion of other similar epidemic diseases, and provide technical support for accurate prevention and control of AL and MD in the poultry industry, provenance purification and epidemiological investigation.

The invention relates to the technical field of avian virus detection, in particular to a TaqMan fluorescent quantitative primer combination for detecting AJ / BK subtype avian leukosis virus as well as application and a kit of the TaqMan fluorescent quantitative primer combination. The TaqMan fluorescent quantitative primer combination for detecting the AJ / BK subtype avian leukosis virus comprises an AJ primer and a probe as well as a BK primer and a probe, and the sequences are shown as SEQ ID No.1-6. The RNA is directly detected by a one-step method, so that the detection speed is increased. According to the invention, only two fluorescence channels are used to increase sensitivity and convenience, the kit can distinguish endogenous and exogenous viruses, avoids endogenous viruses, and only can detect AJ / BK subtype avian leukosis viruses.

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 siRNA (small interfering Ribonucleic Acid) for inhibiting the expression of a chicken RASGRP3 (Reduced Amplification of Sequence Growth Protein 3) gene and application of the siRNA, the siRNA comprises a positive-sense strand and an antisense strand, and is any one of RASGRP3-Anas-214, RASGRP3-Anas-846 and RASGRP3-Anas-1187. According to the specific siRNA aiming at the chicken RASGRP3 mRNA provided by the invention, in a chicken liver cancer cell line (LMH), after liposome-mediated transfection culture is performed for 48 hours, RT-qPCR is adopted to evaluate the transcript level, and a result shows that the relative expression quantity of the chicken RASGRP3 mRNA is remarkably reduced compared with that of negative control, and the repeatability is good. The polypeptide can be further used for inhibiting ALV-J replication, provides a new molecular intervention target for prevention and control of avian leukosis, and has important scientific research and potential economic values.

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 application discloses application of MARCH2 gene as a target point in inhibition of replication of avian leukosis virus (ALV), and further discloses a MARCH2 gene overexpression cell line, a construction method thereof and application of the MARCH2 gene overexpression cell line in inhibition of infection of different subgroups of ALV. The MARCH2 gene overexpression cell line is successfully constructed by a lentivirus packaging infection technology, and experimental results show that the cell line can significantly inhibit replication of different subgroups of ALV. The application proposes that overexpression of the MARCH2 gene or expression of a coded protein of the MARCH2 gene can effectively inhibit replication of different subgroups of ALV. The finding provides a technical means for future anti-disease breeding work against ALV infection by taking the MARCH2 gene as a target point, and has a wide application prospect.

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 a large-scale chicken flock breeding leukemia purification detection starting opportunity analysis method, and relates to the technical field of poultry breeding, and the key points of the technical scheme are as follows: determining a basic time interval of leukemia detection on a subsystem chicken flock next time according to a first detection result of leukemia detection on the subsystem chicken flock last time; determining a first correction coefficient according to a second detection result obtained by performing leukemia detection on the maternal chicken flocks last time; determining a second correction coefficient according to a detection distribution result obtained by performing leukemia detection on the breeding area to which the subsystem chicken flock belongs last time; and combining the basic time interval, the first correction coefficient and the second correction coefficient to determine a final time interval for performing leukemia detection on the subline chicken flocks next time. According to the method, a linkage correction mechanism of maternal line and child line detection results is constructed, and dynamic coupling calculation of basic time intervals and double correction coefficients is combined, so that the purification effect is guaranteed, and meanwhile, the detection resource consumption is remarkably reduced.

The invention aims to provide a dual fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for feline leukemia virus FeLV and feline human immunodeficiency virus FIV, namely a dual fluorescent quantitative PCR detection method for identifying exFeLV and FIV through one-time PCR is established on the basis of screening to obtain a dual PCR amplification primer pair and a probe. According to the primer pair and the probe for detecting the feline leukemia virus, the sequence of an upstream primer is SEQ ID NO: 3, the sequence of a downstream primer is SEQ ID NO: 4, and the sequence of the probe is SEQ ID NO: 5. According to the primer pair and the probe for detecting the cat human immunodeficiency virus, the sequence of an upstream primer is SEQ ID NO: 6, the sequence of a downstream primer is SEQ ID NO: 7, and the sequence of the probe is SEQ ID NO: 8. According to the invention, primer probe dose screening is carried out through optimization design and orthogonal experiments, and an optimal dose combination is determined, so that the amplification efficiency of each primer pair is close, the detection sensitivity is highest, and the purpose of lowest raw material consumption is achieved.

The utility model discloses an avian leukosis P27 antigen emulsifying device which comprises a working table and an L-shaped plate connected to the top of the working table, the L-shaped plate is connected with a stirring mechanism used for stirring a solvent, the stirring mechanism comprises a motor and a stirring head, the motor is used for driving the stirring head to rotate, the motor is connected with a sealing mechanism used for sealing a container, and the stirring head is connected with the sealing mechanism. The cover sealing mechanism comprises a sealing disc, a plurality of fixing cylinders, a plurality of moving rods and a plurality of springs, one ends of the moving rods are inserted into the fixing cylinders, the other ends of the moving rods are connected with the sealing disc, the springs are used for driving the sealing disc to move in the direction away from the fixing cylinders, and the motor is connected with an electric telescopic rod used for driving the motor to ascend and descend. And the operating platform is connected with a limiting seat for supporting the container. According to the utility model, the stability of the container can be improved, the leakage of a solvent is effectively avoided, the pollution to the working platform is reduced, and meanwhile, the lifting of the stirring head can improve the uniformity of antigen emulsification.

The present invention discloses an analysis method for the starting time of leukemia purification detection in large-scale chicken flock breeding, which relates to the technical field of poultry breeding. The key points of its technical solution are as follows: determining the basic time interval for the next leukemia detection of the offspring chicken flock according to the first detection result of the previous leukemia detection of the offspring chicken flock; determining the first correction coefficient according to the second detection result of the previous leukemia detection of the maternal chicken flock; determining the second correction coefficient according to the detection distribution result of the previous leukemia detection of the breeding area to which the offspring chicken flock belongs; and combining the basic time interval, the first correction coefficient, and the first correction coefficient to determine the final time interval for the next leukemia detection of the offspring chicken flock. By constructing a linkage correction mechanism for the detection results of the maternal and offspring lines and combining the dynamic coupling calculation of the basic time interval and the double correction coefficients, the present invention significantly reduces the consumption of detection resources while ensuring the purification effect.

The invention belongs to the technical field of biology, and discloses a recombinant turkey herpesvirus, and a tandem epitope expression cassette is inserted between UL45 and UL46 of the recombinant turkey herpesvirus; the tandem epitope expression cassette is used for expressing a plurality of B cell epitopes and a plurality of T cell epitopes. According to the recombinant turkey herpesvirus, tandem epitopes are inserted into an HVT (turkey herpesvirus) carrier, and through experimental verification, good cellular immune response can be induced and generated in 1-day-old chicks and 18-day-old chick embryos by immunizing the multi-epitope recombinant HVT vaccine for one time, and compared with inactivated vaccines needing two-time immunization, the recombinant turkey herpesvirus can provide earlier and better challenge protection effect. Meanwhile, the recombinant turkey herpesvirus is verified to be capable of performing intraembryonic vaccination and generating an obvious immune protection effect. Meanwhile, the invention further provides a preparation method and application of the recombinant turkey herpesvirus.

The present disclosure belongs to the field of biotechnology, and specifically relates to a method for efficiently infecting human natural killer (NK) cells and other immune cells with a pseudovirus. Specifically, a viral transfection system provided in the present disclosure has an envelope plasmid with a protein having an X-Y-Z structure. The X is an extracellular (ex) structure of a gibbon ape leukemia virus (GALV) envelope glycoprotein, the Y is a transmembrane (TM) structure of the GALV envelope glycoprotein, and the Z is an intracellular segment portion of a murine virus gene.