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Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof

A technology of IBVH120S1 and recombinant virus, applied in the direction of microorganism-based methods, medical raw materials derived from viruses/bacteriophages, antiviral agents, etc., can solve the problems of exogenous virus pollution costs, small toxin production, long production cycle, etc.

Active Publication Date: 2013-09-04
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the risk of exogenous virus contamination in the existing vaccine production process and the disadvantages of high cost, small toxin production, and long production cycle, and the main epitope of the current IBV H120 vaccine strain used for immunization Replacement of the corresponding gene segment of the IBV Beaudette strain adapted to Vero cell culture by the fragment of the outer membrane region of the S1 gene, and then construct the recombinant virus BeauR-H120 (S1)

Method used

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  • Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof
  • Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof
  • Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of IBV recombinant virus BeauR-H120 (S1) of the present invention

[0054] The specific construction method is completed by the following steps:

[0055] 1. Use Trizol reagent (Invitrogen, USA) to extract total RNA from IBV H120 vaccine strain and Beaudette P65 generation cytotoxicity respectively. The downstream primers in Table 1 were used for reverse transcription, and the primer Beau-H21711R was used for reverse transcription of the S1 gene extra-membrane region of IBV H120 strain. The primers Beau-5752R, Beau-8693R, Beau-15520R, Beau-20422R, Beau-27608R are used for reverse transcription of the backbone fragment of the parent virus Beaudette strain.

[0056] Table 1 Primer pairs used in the construction of the recombinant virus BeauR-H120 (S1)

[0057]

[0058] 2. Using the primer pair Beau-H20421F and Beau-H21711R in Table 1 to amplify the S1 gene extra-membrane fragment of IBV H120 strain, the resulting fragment was labeled H120(S1e). Use other 5...

Embodiment 2

[0073] Example 2 Cultivation of IBV recombinant virus BeauR-H120 (S1) of the present invention

[0074] 1 method

[0075] 1.1 Stable passage of successfully rescued IBV recombinant virus

[0076] The successfully rescued recombinant virus was serially passaged on Vero cells. When Vero cells have just filled the cell flask, inoculate 1 mL of recombinant virus solution that has been repeatedly frozen and thawed three times, and add 4 mL of serum-free DMEM cell maintenance solution, and place it at 37°C, 5% CO 2 Continue to culture in the cell incubator. Harvest the virus when 90% of the cells have cytopathic changes. Place the entire cell flask in a refrigerator at -70°C and freeze-thaw for 3 times, waiting for the next inoculation. In each generation, 1 mL of cytotoxic RNA was extracted, and RT-PCR was used to detect the presence of IBV genome.

[0077] 1.2 Determination of virus titer

[0078] Take the 5th, 10th, 15th, 18th, and 21st generation cytotoxicities of the successfully resc...

Embodiment 3IB

[0100] Example 3 Evaluation of immune efficacy of IBV recombinant virus BeauR-H120 (S1)

[0101] 1 method

[0102] 1.1 Immune protection test of IBV recombinant virus BeauR-H120 (S1)

[0103] Randomly divide 40 1-day-old SPF chicks into two groups A and B (20 birds / group), and rear them in a negative pressure isolator. At the age of 7 days, group A was inoculated with IBV BeauR-H120(S1) cytotoxicity (21st generation) (10 6.25 EID 50 / 0.1mL), each eye drops 0.1mL. Group B was used as the control group, and 0.1 mL of normal Vero cell lysate was injected into each eye. From the day of vaccination, observe and record the morbidity of the vaccinated flocks every day. On 14 and 21 days after immunization, blood was collected from the two flocks and the serum was separated for specific antibody detection. The specific method refers to the instructions of the IBV antibody detection kit from IDEXX, USA. 21 days after immunization, the two groups of chicks were challenged by intranasal dro...

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Abstract

The invention discloses a recombinant virus of a chimeric IBV H120 S1 gene ectodomain suitable for cell culture and a construction method and application thereof. The recombinant virus is constructed by the following steps of: replacing the S1 gene ectodomain segment of an IBV Beaudette strain with an S1 gene ectodomain segment of an IBV H120 strain to construct the recombinant virus; after successful virus rescue, passing from the Vero cell; collecting toxicity when the cell cytopathic effect area is over 90%; and repeatedly freezing and thawing for three times, and continuing passage to obtain the recombinant virus of a chimeric IBV H120 S1 gene ectodomain suitable for cell culture. The recombinant virus disclosed by the invention is used as a vaccine strain to immunize SPF chicken; after counteracting toxicity, the chicken flocks of an immunity group and a control group are continuously observed to discover that the recombinant virus disclosed by the invention can serve as a vaccine strain to provide good immunity protection to the inoculated chicken; and the recombinant virus is safe to various kinds of chicken and avoids side reaction.

Description

Technical field [0001] The present invention relates to a recombinant virus and its construction method and application, in particular to a recombinant virus suitable for cell culture in the extramembrane region of the chimeric IBV H120S1 gene, its construction method and its application in the prevention and treatment of chicken infectious bronchitis The present invention belongs to the technical field of veterinary drug research. Background technique [0002] Avian Infectious Bronchitis (IBV) is an acute and highly contagious infectious disease caused by Avian Infectious Bronchitis Virus (IBV). IBV is a single-stranded positive-stranded RNA virus and belongs to the Coronaviridae family (Coronaviridae). IBV can be infected with chickens of different ages, but the infection mainly affects chicks under 4 weeks of age. Clinically, the diseased chickens have tracheal rales, coughing, and sneezing; the kidneys are pale and enlarged, with a large amount of urate deposition, and fallo...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K35/76A61K39/12A61P31/14C12R1/93
Inventor 郭慧琛孙世琪魏衍全董虎王海明孙德惠刘定祥才学鹏殷宏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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