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Cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of cell adaptation strain MJ

A technology for adapting strains and cells, applied in the field of chicken infectious bronchitis virus, can solve the problems of lack of IBV, lack of Vero cell adaptation strains, hidden dangers in biological safety, etc.

Inactive Publication Date: 2019-03-22
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cell-adapted strain obtained in this way is not a natural strain, and there are hidden dangers in biosafety
[0005] Therefore, there is a lack of IBV that can be cultured on Vero cells at home and abroad, especially the lack of Vero cell-adapted strains for the main popular QX strains in China.

Method used

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  • Cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of cell adaptation strain MJ
  • Cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of cell adaptation strain MJ
  • Cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of cell adaptation strain MJ

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The isolation and identification of embodiment 1 chicken infectious bronchitis virus

[0038] 1 Virus isolation

[0039] The disease material came from a large-scale farm in Jiangsu Province. Kidney tissues of dead chickens were aseptically collected, chopped and ground, and sterilized saline with double antibody content of 1000IU / mL was added at a ratio of 1:5 (v / v), 5000r / min Centrifuge for 5 minutes, take the supernatant and inoculate four 11-day-old SPF chicken embryos through the allantoic cavity, 0.2 mL / piece, and incubate at 37°C for 48 hours; discard the dead chicken embryos within 24 hours, and collect the allantoic fluid of 24-48 hours chicken embryos as The subcultured virus was named as IBYZ isolate and stored at -20°C.

[0040] 2 Identification of the virus

[0041] 1) Dwarfing test of chick embryos

[0042] After diluting the isolated IBYZ isolate strain 1:100 times with PBS, inoculate into the allantoic cavity of 5 11-day-old SPF chicken embryos, 0.2mL...

Embodiment 2

[0047] Embodiment 2 The cultivation of QX type chicken infectious bronchitis virus Vero cell adaptation strain MJ strain

[0048] 1 Continuous subculture of chick embryos isolated from IBYZ

[0049] Inoculate the allantoin of IBYZ isolates into the allantoic cavity of 4 SPF chicken embryos aged 9-11 days for subculture, 0.2mL / piece, and incubate at 37°C for 48 hours; discard the dead chicken embryos within 24 hours, and collect them for 24-48 hours Chicken embryo allantoic fluid was used as the subcultured virus. The harvested allantoic fluid was inoculated into chicken embryos again, and the above-mentioned process was repeated for 60 to 100 generations of continuous passage.

[0050] 2 Passage adaptation of Vero cells

[0051] The harvested allantoic venom of the 60th generation of IBYZ isolates was inoculated with a single layer of Vero cells, and placed in 37°C CO 2 After culturing in the incubator for 72 hours, freeze and thaw three times in the refrigerator, harvest t...

Embodiment 3

[0064] Example 3 Safety evaluation test of MJ strain

[0065] 1. Pathogenicity test on 3-day-old SPF chicks

[0066] The dosage is 10 6.5 The MJ strain cytotoxicity of TCID50 was used to inoculate 10 3-day-old SPF chicks through nasal drops and eye drops, and a PBS group was set up at the same time with a dose of 10 6.83 EID 50 The IBYZ virulent group was used as negative and positive controls. The results show that the chicks inoculated with IBYZ group began to die on the 4th day, and the mortality rate reached 60% during the test, while the PBS group and the MJ group did not observe obvious clinical symptoms during the whole test period, indicating that the MJ strain is safe and reliable for SPF chicks (Table 2).

[0067] Table 2 Pathogenicity test on SPF chicks

[0068]

[0069] 2. Virulence recovery test

[0070] The dosage is 10 6.5 TCID 50 Inoculate the first batch of 5 7-day-old SPF chicks through nasal drops and eye drops, and 7 days later, put the second ba...

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Abstract

The invention provides a cell adaptation strain MJ of QX type IBV (Infectious Bronchitis Virus) and application of the cell adaptation strain MJ and belongs to the technical field of avian IBV. The cell adaptation strain MJ has the collection number of CGMCC No. 14681. Proven by tests, TCID50 of cell toxicant fluid, gathered after a Vero cell adaptation strain MJ is inoculated with single-layer Vero cells, reaches 106.5 / mL or more; the strain MJ does not have pathogenicity to SPF (specific pathogen free) chicks, and the toxicity of a virus does not become high after the virus is subjected to continuous passage for 5 generations or more in chick bodies; after the SPF chicks are immunized by adopting cell toxicants gathered after the strain MJ is inoculated with Vero cells, the specific antibody level of organisms rises, and good protection is provided for attack of homotype powerful toxicants. The QX type avian IBV can be extensively prepared by using the Vero cell adaptation strain MJprovided by the invention, and the cell adaptation strain MJ is applicable to large-scale industrial production; the cell adaptation strain MJ can also be applied to preparation of QX type avian infectious bronchitis vaccines.

Description

technical field [0001] The invention relates to the technical field of chicken infectious bronchitis virus, in particular to a cell-adapted strain MJ of QX type IBV and its application. Background technique [0002] Chicken infectious bronchitis (Infectious bronchitis, IB) is one of the second-class infectious diseases of poultry stipulated by the International Veterinary Bureau. It is an acute and highly contagious infection caused by infectious bronchitis virus (Infectious bronchitis virus, IBV). disease, causing serious economic losses to the poultry industry, seriously restricting the development of China's poultry industry and even the national economy. According to the main immune protection gene (S1 gene) sequence of the IBV isolate, the genotype of the strain was determined by genetic evolution analysis. Studies have shown that the current IBV prevalent in chickens in China is dominated by strains represented by the QX genotype, while commercial vaccines such as the...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00A61K39/215A61P31/14C12R1/93
CPCA61K39/12A61K2039/541A61K2039/543A61K2039/552A61P31/14C12N5/0686C12N7/00C12N2770/20011C12N2770/20034
Inventor 周生唐梦君俞燕姜逸程旭高明燕赵秀美戴有理
Owner JIANGSU INST OF POULTRY SCI
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