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Duck circovirus compound vaccine and preparation method of egg yolk antibody

A duck circovirus and vaccine technology, applied in the direction of virus antigen components, virus/bacteriophage, antiviral immunoglobulin, etc., to achieve good application prospects, increased potency, and excellent effects

Inactive Publication Date: 2020-06-12
WEIFANG HUAYING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The recombinant PVAX1-Cap plasmid is transformed into Escherichia coli DH5α, and then undergoes fermentation, DNA extraction and purification and other steps for mass production.

Method used

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  • Duck circovirus compound vaccine and preparation method of egg yolk antibody
  • Duck circovirus compound vaccine and preparation method of egg yolk antibody
  • Duck circovirus compound vaccine and preparation method of egg yolk antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Construction and Preparation of Recombinant PVAX1-ORF C1 Plasmid

[0018] 1 Vector Construction of Recombinant PVAX1-ORF C1 Plasmid

[0019] 1.1 According to the ORF C1 gene sequence of duck circovirus SDFC12 strain (GenBank accession number: KY328304), the whole gene synthesis was carried out after adding Kpn I and Xho I restriction sites to its two ends respectively.

[0020] 1.2 The synthetic duck circovirus ORF C1 gene was double digested with Kpn I and Xho I and then connected to the corresponding restriction site of the PVAX1 vector, CaCl 2 coli DH5α was transformed by the method, the plasmid was extracted and identified by Kpn I and Xho I double enzyme digestion, and then sent to Shanghai Sangon for sequencing.

[0021] 2 In vitro expression and identification of recombinant PVAX1-ORF C1 plasmid

[0022] 2.1 Select well-growing PK-15 cells and inoculate them into 6-well cell culture plates, culture them in a 5% carbon dioxide incubator at 37°C until the cells r...

Embodiment 2

[0042] Preparation and Test of Duck Circovirus Compound Vaccine

[0043] 1 Preparation of recombinant duck circovirus Cap protein

[0044] 1.1 Production strain The recombinant duck circovirus Cap protein production strain DP strain is the N-terminal 21-36 peptide segment rich in arginine in the Cap protein of duck circovirus SDFC12 strain (GenBank accession number: KY328304) with T The cell epitope peptide (TAKSKKFPSYTATYQF) was replaced, and after optimizing the rare codons of Escherichia coli, it was connected with the pET28a vector and transformed into the Escherichia coli expression strain BL21(DE3).

[0045] 1.2 Preparation of recombinant duck circovirus Cap protein High-density fermentation of the recombinant duck circovirus Cap protein-producing strain DP strain, cell crushing, nickel column purification, formaldehyde inactivation, and finally the protein solution was diluted with sterile normal saline to the final Concentration 2.0mg / ml, sterile filtered, ready to us...

Embodiment 3

[0072] Preparation and Test of Egg Yolk Antibody to Duck Circovirus

[0073] 1 Preparation of duck circovirus egg yolk antibody

[0074] According to the method of 3 parts in the embodiment 2, the duck circovirus compound vaccine immunization laying hen is prepared to be highly immune egg, collects qualified highly immune egg (antibody titer is not less than 1:64), carries out yolk separation after eggshell disinfection, Extraction, inactivation, filter sterilization, dilution and subpackaging, etc., the antibody titer of the final product should not be lower than 1:16.

[0075] 2 Safety experiment of duck circovirus egg yolk antibody

[0076] Twenty 7-day-old healthy susceptible ducks were equally divided into two groups, 10 in each group. The first group of leg muscles was injected with the egg yolk antibody prepared by the present invention, 1.0 ml per mouse, and the second group of leg muscles was injected with the same volume of normal saline. Observe the ducks 21 days...

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Abstract

The invention provides a duck circovirus compound vaccine and a preparation method of an egg yolk antibody. A recombinant PVAX1-ORF C1 plasmid containing a duck circovirus ORF C1 gene and a modified recombinant duck circovirus Cap protein are mixed to prepare the compound vaccine, an average antibody titer of eggs collected in 7-120 days after three immunization reaches above 1: 64, and the highest antibody titer can reach 1: 552. An egg yolk antibody product prepared by extracting and purifying hyperimmune eggs can provide complete protection for ducks infected with duck circovirus. The duckcircovirus egg yolk antibody prepared by the method is good in effect and low in cost, and has remarkable economic and social benefits.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a compound vaccine of duck circovirus and a method for preparing egg yolk antibody. Background technique [0002] Duck circovirus (Duck circovirus, DuCV) is a new member of the Circovirus genus (Circovims) in the family Circoviridaeus. In 2018, ICTV listed DuCV as a tentative species in the Circovirus genus. Duck circle is the smallest duck virus known so far. It can invade the immune system of ducks and cause the immune function of the body to decline. It is often associated with duck influenza virus, duck parvovirus, duck reovirus, duck Escherichia coli and duck plague. Merella and other pathogens form mixed infections, resulting in increased mortality and reduced production performance of ducks, causing huge losses to the aquaculture industry. Antiviral chemical drugs have been completely banned in ducks, and traditional Chinese medicine has no definite clinic...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/64A61K39/12A61P31/20C07K16/08
CPCC12N15/70C12N15/64A61K39/12A61P31/20C07K16/081A61K2039/552C12N2750/10034A61K2039/55566C07K2317/11
Inventor 王宏华曹阳张勇王秀云王辉刘磊李佳礼辛瑞祥焦绪娜
Owner WEIFANG HUAYING BIOTECH CO LTD
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