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Method for blocking vertical transmission of avian leukosis virus and application of method

A technology of vertical transmission of avian leukosis virus, applied in the field of veterinary medicine, can solve problems such as time-consuming, high cost, and technical complexity, and achieve the effects of improving production efficiency, improving utilization rate, and reducing production links

Active Publication Date: 2019-07-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to time-consuming, high cost and complex technology, it is still difficult for general breeder farms to implement

Method used

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  • Method for blocking vertical transmission of avian leukosis virus and application of method
  • Method for blocking vertical transmission of avian leukosis virus and application of method
  • Method for blocking vertical transmission of avian leukosis virus and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Confirmation of inclusions (ALV-J virus components) in semen exosomes

[0048] 1) Extraction and purification of exosomes in semen samples: cocks were infected with ALV-J, and blood was collected to detect viremia and p27 antibodies to confirm that the roosters were continuously infected. The semen of 3 ALV-J infected roosters and 3 healthy roosters were collected and stored at -80°C for future use. In the following examples, SEP is healthy exosomes, and SEN is positive exosomes.

[0049] Semen samples were subjected to gradient centrifugation at 4°C, respectively at 1000g, 10min; 2400g, 10min; 10000g, 30min to remove cells and cell debris, and filter macromolecular impurities with a 0.22μm filter membrane, and use the Exo-quick kit (exosome extraction Kit) mix the supernatant of the semen sample at a ratio of 4:1, incubate overnight (>12h), collect the precipitate after centrifugation at 1500g, 30min, 4°C, and resuspend with 1 / 10 the volume of the original solution in...

Embodiment 2

[0087] Infectivity of ALV-J positive chicken semen exosomes

[0088] 1) Positive exosomes infected DF-1 cells in vitro:

[0089] (1) According to 1×10 6 Inoculate DF-1 cells into a six-well plate at a ratio of / well, and the cell density reaches about 60-70%, add 50 μg and 100 μg of the purified positive semen exosomes obtained in Example 1, and incubate for 2 hours;

[0090] (2) Change 2% maintenance solution for 5 days;

[0091] (3) Cell RNA was extracted with TRIZOL Reagent for PCR detection.

[0092] Table 5: Primer sequences and parameters for ALV-J specific detection

[0093]

[0094] Table 6: Amplification reaction system

[0095]

[0096] Reaction conditions: 50°C, 30min; 94°C, 5min; 94°C, 30s; 56°C, 30s; 72°C, 30s, 34 cycles; 72°C, 5min; stop the reaction at 4°C; use 1% agarose gel electrophoresis Identification.

[0097] The results show that see Image 6 , After DF-1 cells were co-incubated with positive exosomes, ALV-J virus RNA was detected in the cel...

Embodiment 3

[0101] A blocking agent for vertical transmission of ALV-J virus

[0102] (1) According to 1×10 6 Inoculate DF-1 cells to a six-well plate at a ratio of 50 μg per well, and the cell density reaches about 60-70%, and divide them into 9 groups, which are respectively the experimental group (4 Group) and the control group (group 4) that only added positive semen exosomes without adding the corresponding marker protein blocker, incubated the cells, and collected ALV-J virus RNA and protein in the cells at 12h, 24h, 36h and 72h for detection. Among them, in this example, the common marker proteins CD9, CD63, CD81 and HSP70 of exosomes were selected for blocking. The blank group was cultured pure DF-1 cells without adding positive exosomes or blockers.

[0103] see closing effect Figure 8 , compared with the control group, the addition of CD9, CD63, CD81 and HSP70 protein blockers in the experimental group can effectively reduce the proliferation of ALV-J virus in cells, and red...

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Abstract

The invention discloses a method for blocking vertical transmission of avian leukosis virus and an application of the method. The inventor discovers for the first time that vertical transmission of avian leukosis virus is carried, mediated and infected by exosomes derived from a reproductive system of avian leukosis positive chickens to infect receptor chickens and progeny chicks, and provides a method and a blocking agent for blocking vertical transmission of ALV by interfering with seminal exosomes of the avian leukosis positive chickens. The method has the advantages that the exosomes related to the vertical transmission of the avian leukosis virus provide a new target point for treating or preventing the avian leukosis; and the provided blocking method is not affected by virus strain subpopulation and variation, is uniformly cut off from the transmission route, has a wide range of targets, is simple and rapid, and has a good effect.

Description

technical field [0001] The invention belongs to the technical field of veterinary medicine, and more specifically relates to a method for blocking the vertical transmission of avian leukemia virus and its application. Background technique [0002] Avian leukosis is a collective designation for various neoplastic diseases of poultry caused by retroviridae, αretrovirus genus Avian Leukosis Virus (ALV) and sarcoma virus group. Tumor diseases dominated by excessive proliferation of cell components, including myeloid leukemia, myeloblastic leukemia, erythroblastic leukemia and lymphocytic leukemia, are one of the major diseases threatening the poultry industry in the world to a huge loss. The avian leukosis / sarcoma viruses are divided into 11 subgroups, A-K, and the avian leukosis viruses infecting chickens include 7 subgroups, ALVA-E, J and K. [0003] Subgroup J has the characteristics of high infection rate, fast transmission speed, wide host range, and high lethality rate. ...

Claims

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Application Information

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IPC IPC(8): A01K67/02
CPCA01K67/02
Inventor 谢青梅廖立钦陈伟国蔺文成张新珩
Owner SOUTH CHINA AGRI UNIV
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