Chicken infectious laryngotracheitis recombinant virus strain and application thereof
A tracheitis and infectious technology, applied in the field of recombinant virus strains of chicken infectious laryngotracheitis, can solve the problems of unstable virulence and difficulty in controlling the degree of attenuation of weak virus strains, and achieve a good protective effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] Example 1 Isolation and identification of chicken infectious laryngotracheitis virulent strain
[0034] In October 2019, the trachea, throat and other tissues of diseased chickens were obtained from a farm in Weifang, Shandong (not immunized with ILTV vaccine), and PBS solution with pH 7.2 was added (Penicilin 10000IU / mL, Streptomycin 10mg / mL, Geamicin250μg / mL, Kanamycin 250 μg / mL) for grinding. Centrifuge at 8000r / min for 10min, take 0.2mL of the supernatant and inoculate it into 10-day-old SPF chicken embryos, and inoculate the allantoic membrane. The chicken embryos that died within 48 hours after inoculation were discarded, and the chicken embryos that died after 48 hours were temporarily stored in a refrigerator at 4°C. After 120 hours of collection, the undead chicken embryos were also placed in a refrigerator at 4°C for 4 hours, and the allantoic membrane and allantoic fluid of all the dead chicken embryos after 48 hours and 120 hours after 120 hours were collec...
Embodiment 2I
[0046] The animal regression test of embodiment 2ILTV / Q strain
[0047] 30 35-day-old SPF chickens were randomly divided into 3 groups, 10 in each group, of which 2 groups were virus inoculated groups, and 1 group was inoculated with PBS virus, nasal drops, eye drops, 0.1ml per chicken, see the inoculation dose and grouping situation Table 1 shows. Animals in the 3 groups were kept in isolation under the same conditions, and were observed continuously for 14 days after inoculation, and the disease status of the chickens was observed and recorded every day, including symptoms such as death, tearing, coughing, runny nose, and head shaking.
[0048] Table 1: Animal regression test table of ILTV / Q strain
[0049]
[0050] The results showed that the ILTV / Q strain was a highly virulent strain.
Embodiment 3
[0051] Example 3 Constructing the ILTV Recombinant Strain with ORFC Gene Deletion
[0052] Using homologous recombination technology, the transfer vector was constructed, and the transfer vector, transcription activator protein ICP4, UL48 plasmid and ILTV genome were co-transfected into LMH cells, and the ORFC gene-deleted recombinant virus containing fluorescent marker was screened by limiting dilution. Since the Loxp site was introduced during the construction of the transfer vector, the sequence between the Loxp sites can be eliminated by using cre recombinase to achieve the purpose of deleting the fluorescent gene and obtain the ORFC gene-deleted recombinant virus without fluorescent markers.
[0053] The specific steps are described below.
[0054] (1) Construction of transfer vector
[0055] According to the complete genome sequence of ILTV (MF417808.1) published by GenBank, primers were designed to amplify the ORFC gene and its flanking genes. The primer sequences are ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More