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Bursal subviral particle vaccine and preparation method thereof

A chicken bursal virus and subvirus technology, applied in the field of biomedicine, can solve the problems of increased susceptibility of chicks, no specific prevention, failure of vaccine immunity, etc.

Pending Publication Date: 2022-02-18
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since 2017, a new variant of infectious chicken bursal virus (nvIBDV) has been circulating in many provinces in China. The bursa is the central immune organ of poultry, and its atrophy can make other vaccines immune failure, resulting in increased susceptibility of chicks to other infectious diseases, causing huge economic losses to my country's chicken industry
[0006] However, the expression systems commonly used in genetic engineering vaccines, such as Pichia pastoris expression system, insect cell-baculovirus expression system and Escherichia coli expression system, etc., have a low level of recombinant expression of IBDV subviral particles, and the yield is lower than 100mg / L , so the cost of large-scale production is high
In addition, there is no specific vaccine product on the market to prevent chicken infectious bursal nvIBDV virus variants

Method used

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  • Bursal subviral particle vaccine and preparation method thereof
  • Bursal subviral particle vaccine and preparation method thereof
  • Bursal subviral particle vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, molecular epidemiological analysis of bursal virus nvIBDV FJ-18 strain

[0043] Bursal virus IBDV is a virus that specifically infects poultry bursa, destroys the central immune organs of poultry, and causes severe immunosuppression in infected animals. In 2017, a new mutant strain of nvIBDV began to break out in chicken farms in many provinces in China. The existing IBDV vaccines are not enough to fully protect against the new mutant strain, and there is currently no commercial vaccine against the new mutant strain on the market. In 2018, a pathogenic virus strain was isolated from a chicken farm in Fujian Province, China, and named FJ-18 strain IBDV. The amino acid sequence of the hypervariable region (aa 206-350) of the VP2 protein of the FJ-18 strain was compared with the representative strains of vIBDV, vvIBDV, cIBDV and nvIBDV collected by NCBI. The results showed that the amino acid sequence homology between the FJ-18 strain and the previously isol...

Embodiment 2

[0044] Embodiment 2, the expression of capsid protein of bursal nvIBDV virus FJ-18 strain in Kluyveromyces marx

[0045] Using the VP2 gene (SEQ ID No.1) of the FJ-18 IBDV strain as the expression sequence and the yeast intracellular expression plasmid pUKDN115 as the expression vector, a recombinant strain expressing the main antigen protein of IBDV in the cell was constructed. In the virus structure of IBDV, VP2 protein is the main antigen protein of IBDV, and VP3 is located inside the capsid as a scaffold protein to form a double-layer structure. The mature VP2 protein (441aa) is produced by the gradual hydrolysis of its precursor proVP2 (512aa), and the recombinant expression of proVP2 protein or some intermediates will form a tubular structure, which will affect the assembly of VP2 protein into virus-like particles. Therefore, the selection of the length of VP2 protein is very important for using VP2 protein to obtain virus-like particle vaccines. In addition, the amphip...

Embodiment 3

[0048] Example 3, Screening and Identification of Bursal Fabricius nvIBDV Virus FJ-18 Strain Highly Expressed Capsid Protein Engineering Bacteria

[0049] In order to increase the expression of nvIBDV strain capsid protein in Kluyveromyces marx, the gene encoding enhanced green fluorescent protein (EGFP) was fused to the 3'-end of nvHVP2 gene with fusion fragment, and then linearized with pUKDN115 The vector is connected, and the recombinant plasmid is transformed into the host cell to construct the recombinant yeast KM-nvHVP2-EGFP (such as Figure 4 ). The recombinant yeast was inoculated into 50mL YD culture based on a shaker at 30°C for 16h, with 20mM H 2 o 2 Mutagens were used to treat KM-nvHVP2-EGFP yeast cells, and the high-expression mutant KM-YDQ1-nvHVP2-EGFP was screened by fluorescence intensity (such as Figure 5 ). The KM-YDQ1-nvHVP2-EGFP mutant strain lost the original plasmid through 5-FOA plate reverse selection and other steps to obtain the expression host ...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a bursal subviral particle vaccine and a preparation method thereof. Directed at chicken infectious bursal disease virus variants, Kluyveromyces marxianus is adopted for recombinant expression of engineering strains of bursal subvirus-like particles, and the effective bursal subvirus-like particle vaccine is prepared. The method comprises the following steps that: firstly, a Kluyveromyces marxianus engineering strain for recombinant expression of virus subvirus-like particles is provided; the recombinant strain is obtained by transforming Kluyveromyces marxianus KM-YDQ1 by a recombinant expression bursal virus capsid protein carrier; according to the bursal virus subvirus-like particle vaccine, bursal subvirus-like particles obtained by culturing, fermenting, separating and purifying Kluyveromyces marxianus recombinant engineering strains are used as active ingredients. The protective effects of the chick immune bursal subviral particle vaccine on a bursal virus variant strain FJ-18 and a classic virulent strain BC6 / 85 both reach 100%.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a bursal subvirus particle vaccine based on Kluyveromyces marxe engineering bacteria and a preparation method thereof. Background technique [0002] Kluyveromyces marxianus is an unconventional yeast that belongs to the Saccharomycetaceae family with Saccharomyces cerevisiae and is GRAS certified (Generally recognized as safe, safety evaluation of food additives) and the European Food Safety Authority (EFSA) QPS certification (Qualified Presumption of Safety, safety qualification certification) of safe yeast, has also been approved as a new food raw material by the National Health and Family Planning Commission of China. Kluyveromyces marx is the fastest growing eukaryotic organism, the specific growth rate can reach 0.99h -1 , while the growth rate of Saccharomyces cerevisiae is 0.37h -1 , the growth rate of Pichia pastoris is 0.18h -1 . In addition, Kluyvero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/40C12N15/81C12N7/04A61K39/12A61K39/39A61K48/00A61P31/14C12R1/645
CPCC07K14/005C12N15/815C12N7/00A61K39/12A61K39/39A61K48/0008A61K48/005A61P31/14C12N2720/10022C12N2720/10023C12N2720/10034A61K2039/54A61K2039/5258A61K2300/00
Inventor 吕红周峻岗余垚杨德强
Owner FUDAN UNIV
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