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A kind of recombinant Marek virus strain sca13 strain and application thereof

A technology of Marek's virus and chicken Marek's disease, applied in the field of animal virology, can solve the problem of poor immune effect and achieve a good level of transmission ability

Active Publication Date: 2021-07-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Witter et al. compared 10 new vaccine candidate strains but the immune effect was not as good as CVI988 / Rispens

Method used

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  • A kind of recombinant Marek virus strain sca13 strain and application thereof
  • A kind of recombinant Marek virus strain sca13 strain and application thereof
  • A kind of recombinant Marek virus strain sca13 strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Isolation and identification of recombinant MDV

[0045] Select well-developed SPF chicken embryos at the age of 9-10 days, and disinfect the eggshells at the air chamber with iodine cotton in the ultra-clean bench, and prepare chicken embryo fibroblasts (CEF) by conventional methods, according to 5 million / cell The concentration of the bottle was inoculated in the square bottle of cell culture (35cm 2 ), placed at 37°C, 5% carbon dioxide (CO 2 ) in an incubator, and after the cells grow into a single layer, they are replaced with a maintenance solution (1% newborn bovine serum, 500 IU / mL penicillin and streptomycin, 2 IU / mL amphotericin) for later use.

[0046] 1ml of anticoagulant blood was aseptically collected from chickens with obvious clinical symptoms of MD in flocks. Thoroughly mix the blood with an equal volume of PBS buffer, slowly add 2ml of lymphocyte separation solution along the tube wall, and centrifuge at 400g for 15min. Collect lymphocyte ...

Embodiment 2

[0047] Embodiment 2: Pathogenicity test of recombinant MDV SCA13 strain

[0048] Sixty-five 1-day-old SPF chickens were randomly divided into four groups and housed in positive pressure isolation hoods. In the first group of 20 SPF chickens, 15 SPF chickens were intraperitoneally injected with SCA13 strain MDV at a dose of 2000 PFU / bird at the age of 1 day, and immunized with NDV and AIV-H9 inactivated vaccine at the age of 7 days. The other 5 chickens were left without any treatment and marked for detection of the lateral transmission ability of the SCA13 strain and the stability of the ALV LTR fragment in the genome of the SCA13 strain during the contact infection process. In the second group, 15 SPF chickens were vaccinated with NDV and AIV-H9 inactivated vaccine at the age of 7 days, and served as the blank control group. After 4w and 5w after immunization, the blood separation serum of the two groups of chickens was collected, and the antibody titers of AIV-H9 and NDV of...

Embodiment 3

[0054] Embodiment 3: Construction and identification of recombinant MDV SDL161 strain

[0055] 1. Construction of BAC clone of MDV SCA13 strain

[0056] Take 1 μg of CEF DNA infected by SCA13 strain and 2.5 μg of pDS-pHAI-US2 plasmid DNA to co-transfect primary CEF. After 6 days, the transfected diseased cells were digested with 0.05% trypsin, and inoculated into culture medium containing 8× 10 6 In a T75 cell flask of CEF, 37°C﹑5%CO 2 After culturing for 5 days, the infected CEFs were digested and inoculated again in fresh CEFs. This was repeated four times. When 60% of the cells were damaged, the cells were digested and the genomic DNA of the cells was extracted. Put 1 μg of virus-infected CEF genomic DNA and 50 μL of DH10B competent cells in a pre-cooled 0.1 cm electroporation cuvette, and perform electroporation under the conditions of 2000V, 100Ω, 25uF. After electric shock, quickly add 1 mL of SOC liquid medium (containing 0.2 mmol / L glucose, 0.1 mmol / L Mg 2 SO 4 ,...

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Abstract

The invention discloses a recombinant Marek virus strain SCA13 strain, whose biological deposit number is CGMCC NO.15285. ALV LTR is integrated in the genome of the SCA13 strain and can exist stably. The present invention also uses Red E / T recombination technology to delete meq and vTR genes based on its BAC clone, and constructs MDV SCA13 strain meq and vTR gene deletion strains. Its biological deposit number is: CGMCC NO.15286. The double gene deletion strain has completely lost its lethality and tumorigenicity to chickens, and its immunosuppressive properties have also been lost. It has better biosafety and better horizontal transmission ability, which can solve the problem of chickens in the process of MD vaccine immunization. Lack of immunity leads to the problem of morbidity, and it has better immune protection for chickens in clinical practice.

Description

technical field [0001] The invention relates to the field of animal virology, in particular to a recombinant Marek virus strain SCA13 and an application thereof. Background technique [0002] Marek's disease (MD) is a neoplastic disease of chickens caused by chicken Marek's disease virus (MDV), characterized by lymphocyte proliferation and tumor formation, peripheral nerve Mononuclear cell infiltration in gonads, viscera, and immune organs, as well as semi-paralysis or complete paralysis caused by the accumulation of peripheral nerve tumor cells are typical features (Marek, 1907). MDV belongs to the Marek virus genus in the α-herpesvirus subfamily, which is divided into three closely related but different serotypes. Serum type 1 virus is pathogenic and can cause T-cell lymphoma and other lesions; Serum type Ⅱ and Ⅲ viruses were isolated from chickens and turkeys respectively, and they were not pathogenic. Isfort et al. found for the first time that chicken embryo fibroblas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12Q1/70C12Q1/686A61K39/255A61P31/22C12R1/93
CPCA61K39/12A61K2039/525A61P31/22C07K14/005C12N7/00C12N2710/16321C12N2710/16322C12N2710/16334C12N2740/11022C12Q1/686C12Q1/701C12Q2531/113
Inventor 苏帅
Owner SHANDONG AGRICULTURAL UNIVERSITY
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