8 results about "Avian leukemia virus" patented technology

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.

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 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.

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 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 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 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.