Avian adenovirus 4, 8 and 11 type trivalent vaccine as well as preparation method and application thereof
An avian adenovirus and vaccine technology, which is applied in biochemical equipment and methods, antiviral agents, viruses/phages, etc., can solve the problems of weak cross protection of avian adenoviruses and different avian adenoviruses, etc., and achieve stable immune effect and immunity. The effect of good originality and high virus titer
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Embodiment 1
[0039] Example 1: Isolation and screening of wild strains of avian adenovirus
[0040] 1. Isolation and identification of wild strains of avian adenovirus
[0041] The present invention collects multiple suspected poultry adenovirus-infected tissue samples in Liaoning, Hebei, Heilongjiang, Shandong, Henan, Jiangsu, Yunnan and Ningxia, etc., and the main apparent necropsy lesions of sick chickens: (1) Accumulation in the pericardial cavity can be seen About 5-10mL of light yellow clear water sample or jelly-like liquid; (2) The liver fades to light brown to yellow, brittle and brittle, swollen and fatty degeneration, the liver color is lighter, and there are local necrotic lesions , Kidney pale, swollen, hemorrhage etc.
[0042]The present invention collects the liver tissue with typical symptoms and pathological changes, adds sterilized PBS, shreds and grinds it according to the weight ratio of 1:3 after weighing, centrifuges at 12000r / min at 4°C for 10min, and passes through...
Embodiment 2
[0057] Example 2: Identification and properties of QYH2019-YN strain, QYH2020-HB strain and QYH2020-SY strain
[0058] 1. Sterility test
[0059] Tested according to the appendix of the current "Chinese Veterinary Pharmacopoeia", the F1 generation seed virus samples of QYH2019-YN strain, QYH2020-HB strain and QYH2020-SY strain were inoculated and grew aseptically.
[0060] 2. Determination of virus content
[0061] Dilute the F1 generation virus solution of QYH2019-YN strain, QYH2020-HB strain and QYH2020-SY strain 10 times serially to 10 -10 , take 10 -5 -10 -10 Four dilutions were inoculated into 96-well culture plates with good growth of LMH cells, and placed at 37°C, 5% CO 2 After 2 hours of incubation, maintenance solution was added and culture continued for 168 hours. After 168 hours, the cell lesions were observed hole by hole, and the typical lesions of the inoculated cells were judged as infection, and the virus content was calculated according to the Reed-Muench...
Embodiment 3
[0080] Example 3: Optimum inoculation amount and harvest time of QYH2019-YN strain, QYH2020-HB strain and QYH2020-SY strain
[0081] 1. Optimal inoculation volume
[0082] The seed viruses of QYH2019-YN strain, QYH2020-HB strain and QYH2020-SY strain were inoculated on well-growing monolayer LMH cells according to 1:100, 1:1000 and 1:10000 respectively, and each sample was repeated 3 times, and placed at 37 °C, 5% CO 2 Cultivate and observe cell lesions. When more than 80-90% of the cells are lesions, harvest the virus solution. After repeated freezing and thawing twice, use the real-time PCR method to measure the virus particles in the culture, and select the one with the highest concentration to determine the optimal inoculation dose. . The results showed that: QYH2019-YN strain was inoculated at 1:1000, QYH2020-HB strain and QYH2020-SY strain were inoculated at 1:100, and the virus titer was the highest.
[0083] 2. The best harvest time
[0084] According to the best i...
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