Eosinophilic kidney type avian infectious bronchitis virus (IBV) virulent vaccine strain and application thereof
A chicken infectious and bronchitis technology, applied in the direction of antiviral agents, viruses/bacteriophages, medical preparations containing active ingredients, etc., to achieve good immunogenicity
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Embodiment 1
[0022] The acquisition and characteristic determination of embodiment 1 virus
[0023] In this example, the suspected nephrophilic chicken infectious bronchitis disease material isolated from the diseased chicken flock in Jiangsu, my country, was inoculated with specific pathogen free (Specific Pathogen Free, SPF) chicken embryos to isolate the virus, and the virus strain was tested in SPF. The nephrophilic chicken infectious bronchitis virulent vaccine strain was obtained by cloning and purifying the virus by limiting dilution three times on chicken embryos, named Ck / Jiangsu / DS10 / 2008, DS10 for short. The virus DS10 was returned to inoculate SPF chickens, and the pathogenicity and immunogenicity of the virus were evaluated.
[0024] specific method:
[0025] (1), virus DS10 amplification: virus DS10 is diluted 10 with sterile PBS buffer solution 3 After doubling, 11-day-old SPF chicken embryos were inoculated through the allantoic cavity at an inoculation volume of 0.2 mL / em...
Embodiment 2
[0030] Embodiment 2 chicken embryo neutralization protection test
[0031] In order to verify the cross-protection of virus DS10 to respiratory infectious bronchitis virus, hyperimmune serum was prepared respectively for virus DS10 and virus M41 (respiratory infectious bronchitis virus), and the hyperimmune serum was diluted with virus DS10 and virus DS10 respectively. Virus M41 was tested for cross-protection.
[0032] (1), preparation of virus and serum: virus DS10 and virus M41 (purchased from the China Veterinary Drug Control Institute, referred to as "the Institute") were diluted with sterile PBS buffer for 10 3 After doubling, the 9-11-day-old SPF chicken embryos were inoculated through the allantoic cavity. Within 36-48 hours after inoculation, healthy and alive chicken embryos were frozen in a refrigerator at 2-8°C for 6-8 hours, and the virus allantoic fluid was aseptically collected, subpackaged, and placed in a refrigerator at -70°C. Among them, virus EID 50 Vale...
Embodiment 3
[0040] Embodiment 3 immunogenicity test
[0041] Use the virus DS10 or M41 as the virus strain to prepare the oil emulsion inactivated vaccine respectively, and carry out the cross-challenge protection test.
[0042] (1), virus and vaccine preparation: the preparation of virus allantoic fluid sees embodiment 1, and allantoic fluid detects EID 50 Valence greater than or equal to 10 7.0 EID 50 / mL, inactivated by formaldehyde, and then mixed with white oil adjuvant, the oil emulsion inactivated vaccines of virus DS10 and M41 were prepared respectively according to conventional methods.
[0043] (2) SPF chicken: SPF chicken embryos were purchased from Jinan Saisi Poultry Technology Co., Ltd. of Shandong Poultry Institute, and were hatched by themselves until hatched, and then raised in isolators.
[0044] (3), grouping and immunization of experimental animals
[0045] The 14-day-old SPF chickens were divided into groups, and subcutaneously immunized with 0.2 mL of the corresp...
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