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Construction of recombinant turkey herpesvirus used for expressing F gene of chicken Newcastle disease virus and application thereof

A technology of chicken Newcastle disease virus and turkey herpes virus, applied in the field of animal virology, can solve the problems of epidemic, short vaccine immune protection period, and increased susceptibility to secondary bacterial infection

Active Publication Date: 2017-07-07
北京邦卓生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the short immune protection period of conventional vaccines, although commercial chickens have been immunized many times throughout the feeding period, they may still be infected with NDV virulence, resulting in the prevalence of atypical Newcastle disease virus
In addition, conventional attenuated and moderately virulent vaccines can also replicate in the respiratory tract of chickens, causing mild lesions and increasing susceptibility to secondary bacterial infections

Method used

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  • Construction of recombinant turkey herpesvirus used for expressing F gene of chicken Newcastle disease virus and application thereof
  • Construction of recombinant turkey herpesvirus used for expressing F gene of chicken Newcastle disease virus and application thereof
  • Construction of recombinant turkey herpesvirus used for expressing F gene of chicken Newcastle disease virus and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] ——Construction of rHVT-F recombinant virus

[0039] 1. Preparation of an expression cassette (see sequence 1 for the sequence) containing the F gene of chicken Newcastle disease virus

[0040] Design and amplify NDV F gene primer NDV-F-F / R (SEQ ID NO:2 / SEQ ID NO:3). Using the extracted NDV RNA as a template, the NDV F gene fragment was amplified by RT-PCR, digested with Nhe I and Xba I, and cloned into the eukaryotic expression vector pcDNA3.1, and the obtained positive plasmid was named pcDNA3.1 / NDV-F. According to Lipofectamine2000 (Thermo Fisher Scientific) transfection method, the recombinant plasmid pcDNA3.1 / NDV-F was transfected into CEF cells, and the expression of recombinant plasmid F protein was identified with the monoclonal antibody of NDV F protein as the primary antibody.

[0041] Table 1 Primers used to construct recombinant plasmids.

[0042]

[0043] 2. Construction of recombinant plasmids containing homology arms and expression cassettes

[004...

Embodiment 2

[0052] ——Study on the stability of rHVT-F recombinant virus in cells

[0053] The purified recombinant virus HVT-F1 was continuously passaged on CEF cells, and the 10th, 20th, and 30th generation recombinant viruses were selected for indirect immunofluorescence identification to detect the expression of NDV F gene. Extract different generations of viral DNA, use PCR to amplify the chimeric region of the foreign gene of the recombinant virus, and perform sequencing verification. Using different generations of DNA as templates, specific fragments of the chimeric region of the NDV F gene expression cassette with a length of about 600 bp can be amplified. The results showed that the NDV F gene could stably exist and express in the recombinant virus HVT-F1. The primers used are:

[0054] qianhe-F:5'-GCACATCTGC TCTCATTAC C-3'21 (SEQ ID NO: 14)

[0055] qianhe-R:5'-GTTTCAAATT TTCACGATTC C-3'21 (SEQ ID NO: 15)

Embodiment 3

[0057] ——Evaluation of immune efficacy of recombinant virus rHVT-F in SPF chickens

[0058] Sixty 1-day-old SPF chickens were randomly divided into 3 groups. The first group was vaccinated with rHVT-F, and the immunization dose was 4000PFU / bird. The second group is a non-immune challenge group, and the third group is a blank control (non-immune non-challenge) group. At the age of 21 days, the virulent Beijing strain of NDV was challenged, and the protection rate of rHVT-F reached 90% after 14 days of observation (see Table 2).

[0059] Table 2 The immune protection effect of rHVT-F on SPF chickens

[0060] group challenge strain Deaths (rate) Protection rate (%) rHVT-F vaccine immunization group NDV (Beijing Branch) 2 / 20(10％) 90 Challenge control group- NDV (Beijing Branch) 20 / 20(0％) 0 Blank control group- - 0 / 20(0％) -

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Abstract

The invention relates to construction of a recombinant turkey herpesvirus used for expressing an F gene of a chicken Newcastle disease virus and an application of the construction. The recombinant turkey herpesvirus used for expressing the F gene of the chicken Newcastle disease virus is named as recombinant turkey herpesvirus rHVT-F strain; the virus strain combines an expression cassette containing the F gene of the chicken Newcastle disease virus with the turkey herpesvirus (HVT), that is, and the F gene controlled by a CMV promoter is inserted into an HVT gene to construct to obtain the brand new promoter rHVT-F strain. The rHVT-F virus strain can induce a chicken to produce lasting protective immunity on MD and ND resistance. The virus strain produces a strain to prepare a bivalent recombinant vaccine for the fowl paralysis and the chicken Newcastle disease.

Description

technical field [0001] The invention relates to the construction and application of a recombinant turkey herpes virus expressing chicken Newcastle disease virus F gene, belonging to the field of animal virology. The invention provides a recombinant turkey herpes virus rHVT-F strain, whose genome contains chicken Newcastle disease virus (NDV) F gene under the control of CMV promoter. Background technique [0002] Marek's disease (MD) and Newcastle disease (ND) have caused great harm to the poultry industry. The prevention and control of MD and ND mainly depends on vaccination. [0003] HVT has no pathogenicity to chickens and other animals, and has been widely used for the prevention and control of MDV since 1970. At present, the vast majority of countries use vaccination as the main measure to control Newcastle disease. However, due to the short immune protection period of conventional vaccines, although commercial chickens have been immunized many times throughout the br...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/17A61K39/245A61P31/14A61P31/20C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/70C07K14/005C12N7/00C12N2710/16334C12N2710/16343C12N2760/18134
Inventor 毕建敏
Owner 北京邦卓生物科技有限公司
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