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Novel duck reovirus compound vaccine and preparation method of egg yolk antibody

A new duck reovirus technology is applied in the field of compound vaccine of new duck reovirus and egg yolk antibody preparation, which can solve the problems of high production cost, easy introduction of other embryo-derived pathogens, high immunity and low titer of eggs, etc. Reduced preparation cost, good clinical protection effect, reduced immune stress effect

Active Publication Date: 2020-07-24
WEIFANG HUAYING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Tang Yi et al. (patent application number: CN201810494056.5, CN201810911067.9), Zhang Liwu et al. (patent application number: CN201810720764.6) conducted duck reovirus The research on egg yolk antibody products, these methods adopt the method of chicken embryo or duck culture to prepare the antigen for immunizing laying hens, the production titer of virus is low, and it is easy to introduce other embryo-derived pathogens. The price is relatively low, and the production cost is relatively high

Method used

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  • Novel duck reovirus compound vaccine and preparation method of egg yolk antibody
  • Novel duck reovirus compound vaccine and preparation method of egg yolk antibody
  • Novel duck reovirus compound vaccine and preparation method of egg yolk antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Construction and Preparation of Recombinant PVAX1-σB Plasmid

[0021] 1 Vector Construction of Recombinant PVAX1-σB Plasmid

[0022] 1.1 The amino acid sequence of the new duck reovirus σB protein in GenBank was homologously compared, and imported into the online MOSAIC vaccine design software to screen for more potential 9-mer epitope covered σB proteins, and finally a new duck reovirus was obtained Orphan virus σB protein, the amino acid sequence is SEQ ID NO:1.

[0023] 1.2 According to the codon usage preference, a novel duck reovirus σB protein gene was designed, one of which has a nucleotide sequence of SEQ ID NO:2.

[0024] 1.3 After adding Kpn I and Xho I restriction sites to the two ends of the obtained novel duck reovirus σB gene, the whole gene was synthesized.

[0025] 1.4 The synthetic new duck reovirus σB protein gene was double digested with Kpn I and Xho I and then ligated into the corresponding restriction site of PVAX1 vector, CaCl 2 Coli DH5α was t...

Embodiment 2

[0047] Preparation of recombinant new duck reovirus σC protein

[0048] 1 production strain

[0049] 1.1 The amino acid sequence of the new duck reovirus σC protein in GenBank was homologously compared, and imported into the online MOSAIC vaccine design software to screen for more potential 9-mer epitope covered σC proteins, and finally a new duck reovirus was obtained Enterovirus σC protein, the amino acid sequence is SEQ ID NO:3.

[0050] 1.2 Using the online biological software DNAWorks, the novel duck reovirus σC protein was optimized for rare codons in Escherichia coli, and a nucleotide sequence SEQ ID NO:4 was obtained.

[0051] 1.3 After adding Nde I and HindIII enzyme cutting sites to the two ends of the obtained novel duck reovirus σC protein nucleotide sequence, the whole gene is synthesized.

[0052] 1.4 The novel duck reovirus σC protein gene synthesized by the whole gene was digested with Nde I and Hind III, and then ligated into the corresponding restriction si...

Embodiment 3

[0072] Preparation and Immunization of New Type Duck Reovirus Compound Vaccine

[0073] 1 Preparation of a novel duck reovirus compound vaccine

[0074] 1.1 Preparation of semi-finished products Properly dilute the recombinant PVAX1-σB plasmid DNA prepared in 3.3 of Example 1 with the recombinant new duck reovirus σC protein prepared in 2.3.5 of Example 2, so that the final content of the plasmid DNA is 45 μg / ml , the final content of σC protein is 0.5mg / ml, fully mixed, it is a semi-finished product.

[0075] 1.2 Vaccine preparation

[0076] 1.2.1 Preparation of oil phase Take 94 parts of high-quality white oil for injection and 2 parts of aluminum stearate. Mix evenly in the oil phase tank, heat and melt until translucent, add 6 parts of Siben-80, keep for 30 minutes when the temperature reaches 125-130°C, cool to room temperature for later use.

[0077] 1.2.2 Preparation of water phase Take 4 parts of sterilized Tween-80, add 96 parts of qualified semi-finished products,...

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Abstract

The invention aims to provide a novel duck reovirus compound vaccine and a preparation method of an egg yolk antibody. The preparation method comprises the following steps: mixing recombinant PVAX1-sigma B plasmid containing a novel duck reovirus sigma B protein gene and a novel duck reovirus sigma C protein according to a certain ratio, and performing emulsifying with a white oil adjuvant to prepare the compound vaccine. The average antibody titer of eggs collected 7-150 days after the three times of immunization reaches 1:1024 or above, and the highest antibody titer can reach 1:4096. The egg yolk antibody product prepared by extracting and purifying hyper-immune eggs can provide complete protection for ducks infected with the novel duck reovirus. The novel duck reovirus egg yolk antibody prepared by the method disclosed by the invention is definite in effect and low in cost, and has remarkable economic and social benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel compound vaccine of duck reovirus and a method for preparing egg yolk antibody. Background technique [0002] Novel duck reovirus (NDRV), a member of the Orthoreovirus genus in the family Reoviridae, mainly causes liver and spleen hemorrhage and necrosis in ducklings. Duck white spot disease", "Duck hemorrhagic necrotizing hepatitis", "Duck spleen necrosis" and so on. The new duck reovirus is completely different from the previous Muscovy duck reovirus serologically, and there is no obvious cross-protection. Around 2005, the disease first appeared in some provinces in southern my country, such as Fujian, Guangdong, Zhejiang and other duck populations, and then spread to the main duck breeding areas in the country, causing huge economic losses to the duck industry in my country. [0003] The new duck reovirus contains a double-layer capsid, in which the σC protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/64A61K39/15A61P31/14C07K16/10C07K16/02
CPCC12N15/70C12N15/64A61K39/12A61P31/14C07K16/10C07K16/02A61K2039/552C12N2720/12234
Inventor 王辉王宏华张勇王秀云李佳礼辛瑞祥刘磊焦绪娜曹阳
Owner WEIFANG HUAYING BIOTECH CO LTD
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