Recombinant duck viral enteritis virus, preparation method and applications

A duck virus enteritis, recombinant virus technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of application limitation, poor immunogenicity, slow immunity generation, etc., and achieve a simple construction method. , High protective efficacy, good cross-protection effect

Active Publication Date: 2015-02-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country currently adopts whole virus inactivated vaccines for compulsory immunization of poultry. Although this vaccine has advantages such as good safety, it also shows the following deficiencies in practical application. To carry out large-scale virus culture, there is a biological safety hazard of spreading the virus
Second, its immunity is slow to develop, and a lump is formed at the injection site, which limits the application of fast-growing meat poultry with a short production cycle
3. Although the inactivated vaccine can produce highly effective humoral immunity, the cellular immunity is insufficient to completely remove the virus from the body, which may form a recessive infection
In particular, the inactivated vaccine has poor immunogenicity to ducks, and the immune effect is not good.

Method used

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  • Recombinant duck viral enteritis virus, preparation method and applications
  • Recombinant duck viral enteritis virus, preparation method and applications
  • Recombinant duck viral enteritis virus, preparation method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the construction of GFP transfer vector plasmid

[0038] 1. Obtain the GFP expression cassette

[0039] The GFP expression cassette is an expression cassette of green fluorescent protein (green fluorescent protein), and its sequence is shown in SEQ ID NO:2. The GFP expression cassette contains pHCMV promoter, green fluorescent protein gene GFP and termination sequence. Using the DNA of the recombinant strain pHA2 corr as a template and Primer GFP-UL55 F and Primer GFP-UL55 R as primers, the GFP expression cassette was amplified by gradient PCR.

[0040] Primer GFP-UL55 F (SEQ ID NO: 4): 5'-TATCCCGGGTTAACCGGGCTGCATCCGAT-3';

[0041] Primer GFP-UL55 R (SEQ ID NO:5): 5'-ATACCCGGGCGAAGTTATGCGGCCATTTA-3'.

[0042] The reaction system of the gradient PCR method is: 10 × PCR Buffer 5 μL, dNTPs (2.5 mM each) 4 μL, Mg 2+ (25 mM) 3 μL, each 2 μL of upstream and downstream primers (10 pmol / μL ), 0.5 μL of ExTaq enzyme, 2 μL of DNA template, dd H 2O 31.5 μL (PCR ...

Embodiment 2

[0056] Embodiment 2, the acquisition and identification of GFP recombinant duck viral enteritis virus

[0057] The 9-10-day-old SPF chicken embryos were taken, and primary and secondary chicken embryo fibroblasts (Primary chicken embryo fibroblasts, CEF) were prepared and cultured according to conventional methods. Inoculate a single layer of chicken embryo fibroblasts with duck plague chicken embryonized attenuated strain C-KCE at a multiplicity of infection (MOI) of 0.05, and culture for about 72 hours. Collect the cells when 95% of the cells have lesions, and follow the routine Methods Viral DNA was extracted from diseased cells, DNA was quantified by a gel scanner, and the DNA concentration was adjusted to 0.1-0.2 μg / μL. The DNA of the transfer vector pDEV-GFP (UL55) was extracted from the recombinant bacteria by alkaline lysis, quantified by Eppendorf Biophotometer (or quantified by agarose electrophoresis measurement), and the concentration was adjusted to 700 ng / μL. Tr...

Embodiment 3

[0058] Example 3. Construction of avian influenza virus hemagglutinin (HA) gene expression cassette and its transfer vector

[0059] 1. Obtaining the hemagglutinin (HA) gene of avian influenza virus

[0060] The hemagglutinin (HA) gene sequences of H5N1 avian influenza published on GenBank since 2010 were used for multiple sequence alignment using MEGA software to obtain consensus sequences. The base at each position of the consensus sequence is the one with the highest frequency among the four bases (A, C, G, T) at the same position in the HA sequence. Remove the basic amino acid cleavage site between the HA1 and HA2 fragments in the consensus sequence, and then refer to the H5N1 avian influenza virus hemagglutinin (HA) gene sequence of the newly popular 2.3.2.1 lineage (GenBank: AB700635.1; JN986881 .1; JN986882.1; JN646713.1; JN646716.1; HQ020376.1; CY098758.1; JF975561.1; ), the optimized design obtained the avian influenza virus hemagglutinin (HA) gene of the present inv...

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Abstract

The invention provides a recombinant duck viral enteritis virus, a preparation method and applications, belonging to the field of animal medicine vaccines. The avian influenza virus haemagglutinin gene is 1437th to 3131th nucleotide with the nucleotide sequence of SEQ ID NO:1. According to the recombinant duck viral enteritis virus, an expression cassette containing the avian influenza virus haemagglutinin gene is inserted in a spacer region between LORF11 and UL55 genes of duck viral enteritis virus genome. As a vaccine active ingredient, the avian influenza virus haemagglutinin gene can achieve better cross protection effect. The recombinant duck viral enteritis virus can stably and efficiently express the avian influenza virus haemagglutinin gene, virus is stable and grows well, the prepared live vaccine has higher protective efficacy on duck plague and avian influenza, higher avian influenza antibody is generated, and the duration period of the antibody is long.

Description

[0001] technical field [0002] The invention belongs to the vaccine field of veterinary medicine, and in particular relates to a recombinant duck viral enteritis virus, a preparation method and an application. Background technique [0003] Duck plague was first reported at the beginning of the 20th century. At present, most duck-raising countries in the world have reported this disease. It is caused by duck plague virus (also known as duck viral enteritis virus), and its morbidity and mortality can reach 100%. , Duck plague virus will form a persistent infection in the trigeminal ganglion of ducks, and the attenuated live vaccine of duck plague is widely used in the immune prevention of duck plague, which can effectively prevent the disease. [0004] Avian influenza (H5N1) is a major infectious disease of ducks and chickens. It is not only one of the most important infectious diseases currently threatening the safety of poultry farming in the world, but also has a high fata...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/44C12N7/01A61K39/295A61K39/145A61K39/245A61P31/16A61P31/22
CPCY02A50/30
Inventor 王继春许梦微王志胜乔永峰侯继波
Owner JIANGSU ACAD OF AGRI SCI
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