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Construction and application of recombinant virus vector for expressing infectious bursal disease virus VP2 protein

A recombinant vector, bursal disease technology, applied in the direction of virus/phage, application, virus, etc., can solve the problems of unsatisfactory immune protection effect, damage to bursal, and strong virulence.

Inactive Publication Date: 2020-11-10
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The attenuated vaccine is safe for chickens, but the immune protection effect on chickens with maternal antibodies is not ideal
Although the immune protection of moderately virulent and highly virulent vaccines is better, but due to certain pathogenicity, it will damage the bursa of Fabricius, and there is also a risk of virulence returning to strong
In addition, due to the interference of maternal antibodies and the characteristics of IBDV, traditional vaccines can form an inevitable immune blank period in practical applications.

Method used

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  • Construction and application of recombinant virus vector for expressing infectious bursal disease virus VP2 protein
  • Construction and application of recombinant virus vector for expressing infectious bursal disease virus VP2 protein
  • Construction and application of recombinant virus vector for expressing infectious bursal disease virus VP2 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of recombinant turkey herpes virus (rHVT-U3VP2 strain)

[0048] The construction method of recombinant turkey herpes virus (rHVT-U3VP2 strain) of the present invention is as follows:

[0049] (1) Construction of transfer vector containing RFP expression cassette

[0050] The pUC19 vector, used for cloning, sequencing and construction of transfer vectors, was purchased from Bao Biological Engineering (Dalian) Co., Ltd. RFP reporter gene vector p19-UL3 / 4-RFP containing pHCMV promoter, BGH polyA (see figure 1 ), constructed and preserved by the inventor's laboratory.

[0051] (2) Primer design

[0052] The primer design sequence of the present invention is shown in Table 1

[0053] Table 1 The relevant primer information involved in the construction of the present invention

[0054]

[0055] (3) Construction of turkey herpesvirus rHVT-U3R containing RFP marker

[0056] 1) Construction of transfer vector p19-UL3 / 4-RFP

[0057] According ...

Embodiment 2

[0069] Embodiment 2: Identification of recombinant turkey herpes virus (rHVT-U3VP2 strain)

[0070] 1. PCR identification of recombinant virus rHVT-U3VP2

[0071] UL3F and UL4R primers were used for PCR amplification identification, and PCR product sequencing confirmed that the VP2 gene was successfully inserted between the UL3 and UL4 genes (see image 3 ).

[0072] 2. Indirect immunofluorescence identification of recombinant virus rHVT-U3VP2

[0073] The recombinant virus rHVT-U3VP2 and its parent virus were inoculated into CEF respectively, and when the cytopathic effect reached 80%, the expression of the VP2 gene was detected by indirect immunofluorescence (see Figure 4 ).

[0074] Indirect immunofluorescence step: discard the cell culture medium, lightly wash the cell surface once with PBS (pH7.2), then add 500 μL of 80% pre-cooled acetone solution to each well, place at room temperature for 20 minutes, discard the liquid, and dry naturally; Wash the cell surface onc...

Embodiment 3

[0087] Embodiment 3: the preparation of vaccine

[0088] (1) Vaccine preparation

[0089] 1) Cell preparation: select well-developed 9-11-day-old SPF chicken embryos, prepare CEF according to the conventional trypsinization method, and culture them for about 24 hours to grow into a single layer for later use.

[0090] 2) Preparation of virus liquid: inoculate the CEF cell monolayer with the rHVT-U3VP2 strain of the production seed virus at a volume ratio of 0.01% to 0.5%. 2 Cultivate in a 37°C incubator for 36-72 hours, harvest the cells when the lesion rate reaches over 85%, and add cell freezing solution to make a cell suspension.

[0091] 4) Mixing and subpackaging: add 10% DMSO content (V / V) and 20% bovine serum content (V / V) to the collected cell suspension, mix well, subpackage according to 2.0ml per ampoule and seal.

[0092] 5) Preservation Immediately place the subpackaged and labeled vaccine ampoules in a programmed cooling device for freezing. Take it out and pu...

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Abstract

The invention constructs a recombinant turkey herpesvirus expressing chicken infectious bursal disease virus VP2 gene; the recombinant virus strain is prepared into a live vaccine, so that Marek's diseases and infectious bursal diseases can be prevented; due to a homologous recombination method, the vector is used to express exogenous gene; and a foundation is laid for constructing a novel recombinant vaccine for resisting Marek's disease and other pathogens by taking the turkey herpesvirus as the vector.

Description

technical field [0001] The invention relates to a vaccine and a method for VP2 protein of poultry infectious bursal disease virus. Background technique [0002] Without limiting the scope of the invention, its background is described in connection with the morbidity and mortality of chicken Infectious bursal disease (IBD) virus. Chicken infectious bursal disease is an acute, highly contagious, contagious disease caused by infectious bursal disease virus (IBDV). The disease can cause immunosuppression by damaging the bursa of Fabricius and cause high mortality in chicks. Known IBDV has two serotypes, namely serotype I and type II, wherein serotype II is not pathogenic or has very weak pathogenicity, and serotype I can cause disease in chickens. In the decades since the discovery of the disease, serum type I IBDV has mutated, and classic strains, variant strains and super-strong strains have emerged one after another. Since the 1990s, a very virulent IBDV strain with high l...

Claims

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Application Information

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IPC IPC(8): C12N15/869C12N15/40A61K39/12A61K39/245A61P31/14A61P31/22
CPCA61K39/12A61K2039/5256A61K2039/53A61K2039/552A61K2039/70A61P31/14A61P31/22C07K14/005C12N15/86C12N2710/16334C12N2710/16343C12N2720/10022C12N2720/10034
Inventor 蒋桃珍张勇宋亚芬杨宵玥陈玲陈光华杨承槐
Owner CHINA INST OF VETERINARY DRUG CONTROL
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