Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof

A technology of IBRV-JN03 and rhinotracheitis virus, applied in antiviral agents, viruses/bacteriophages, medical preparations containing active ingredients, etc., can solve the problems of poor virus protection and achieve good immunogenicity and broad coverage Market application prospect, highly targeted effect

Active Publication Date: 2015-09-23
SHANDONG NORMAL UNIV
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Problems solved by technology

However, there is no relevant vaccine product in my country, and the Ministry of Agriculture has not yet approved foreign IBRV vaccines to enter the Chinese market. M

Method used

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  • Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof
  • Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof
  • Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof

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Example Embodiment

[0029] Example 1 Isolation and identification of bovine infectious rhinotracheitis virus

[0030] 1.1 Isolation of bovine infectious rhinotracheitis virus

[0031] Observing the incidence of cattle herd, the inventor collected samples of nasal secretions from cattle, feces samples of cattle diarrhea, and samples of diseased cattle tissues (liver, spleen, lymph nodes) that were initially diagnosed as bovine infectious rhinotracheitis. For stool and bovine nose swab samples, dilute 1:5 with PBS (100U / mL penicillin, 100g / mL streptomycin). After centrifugation at 3000 r / min for 10 min, the supernatant obtained was filtered and sterilized by a 0.22 μm filter membrane to obtain the disease treatment liquid. The samples of diseased bovine tissues (liver, spleen, lymph nodes) were crushed with a tissue grinder, then repeatedly frozen and thawed 3 times, and diluted 1:3 with PBS (100U / mL penicillin, 100g / mL streptomycin). After centrifugation at 8000r / min for 10min, the supernatant obtain...

Example Embodiment

[0058] Example 2 Comparative analysis of immunogenicity of IBRV isolates

[0059] 2.1 The reproduction of viruses

[0060] Take the MDBK cells in good growth condition and wash them twice with PBS to make the virus titer greater than 10 7.0 TCID 50 / mL, IBRV isolates IBR-JN03 and IBRV-LY9038 after 6 rounds of plaque purification, press 100TCID 50 Inoculate the virus separately, add 2% (v / v) horse serum, pH 7.0 DMEM cell maintenance solution, 37 ℃ 5% CO 2 Cultivation, when the cytopathic effect reaches 70%-80%, the virus culture solution is harvested, freeze-thawed repeatedly 2 to 3 times, and centrifuged at 4°C at high speed to obtain the supernatant to obtain the IBRV antigen.

[0061] 2.2 Comparative analysis of virus immunogenicity

[0062] Add 0.2‰ formaldehyde to the supernatants of the above amplified viruses IBRV-JN03 and IBRV-LY9038 and inactivate them at 37°C for 24 hours. After the inactivation is completed, perform a sterility test, and then combine the virus solution with a...

Example Embodiment

[0063] Example 3 Development of IBRV inactivated vaccine

[0064] 3.1 The reproduction of viruses

[0065] Take the MDBK monolayer cells in good growth condition and wash them twice with PBS. The cell culture medium of the above-mentioned vaccine candidate strain (IBRV-JN03) with good immunogenicity, press 100TCID 50 Inoculate at the same amount of virus and add DMEM cell maintenance solution containing 2% (v / v) horse serum at 37°C with 5% CO 2 Cultivation, when the cell pathological changes are 70% to 80%, harvest the virus culture solution, freeze and thaw repeatedly for 2 to 3 times, and centrifuge at 4°C to obtain the virus supernatant. Take samples for virus content determination, and the titer is 6.75×10 8.5 TCID 50 / mL, among the reported IBRV isolated strains, this virus strain has a higher titer, which is more in line with the requirement of vaccine production for high antigen content.

[0066] 3.2 Preparation of virus inactivated vaccine

[0067] The cell culture supernatant ...

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Abstract

The invention discloses an infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof. The preservation number of the infectious bovine rhinotracheitis virus is CGMCC No.10396 and the infectious bovine rhinotracheitis virus is preserved in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and has relatively high virus titer and good immunogenicity; after calves are immunized by an aluminium hydroxide adjuvant inactivated vaccine prepared through the infectious bovine rhinotracheitis virus, relatively high antibodies are generated, so that the infectious bovine rhinotracheitis virus IBRV-JN03 isolate is a good infectious bovine rhinotracheitis vaccine candidate virus strain. Therefore, an infectious bovine rhinotracheitis diagnostic reagent, inactivated vaccine, attenuated vaccine and genetic engineering vaccine prepared on the basis of the virus can be used for diagnosis and vaccine prevention and control of the endemic infectious bovine rhinotracheitis and a wide market application prospect is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bovine infectious rhinotracheitis virus IBRV-JN03 isolate and application thereof. Background technique [0002] Infectious bovine rhinotracheitis virus (Infectious bovine rhinotracheitis virus, IBRV) is an acute, febrile, contagious infectious disease of cattle. The disease can cause immunosuppression, secondary bacterial infection and lead to more severe respiratory disease. At present, the disease is widely prevalent in the world, and the disease seriously affects the international cattle production and trade, and has been listed as a B-type communicable disease by the World Organization for Animal Health (OIE). IBRV was detected in cows imported from New Zealand in the late 1970s in my country. Since there were no good immune protection measures, the virus was infected in cows in many provinces of my country, and the incidence rate was 10% to 90%. The fattening, milk productio...

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Application Information

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IPC IPC(8): C12N7/00A61K39/265A61P31/22C12Q1/70C12Q1/68G01N33/569C12R1/93
Inventor 王洪梅侯佩莉何洪彬
Owner SHANDONG NORMAL UNIV
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