Avian bursal disease virus genetic engineering vaccine as well as preparation method and application thereof

A technology of genetic engineering vaccine and poultry method, which is applied in the field of poultry bursal virus genetic engineering vaccine and its preparation, can solve the problems of high gene mutation and recombination, high endotoxin, side effects, etc., and achieve strong immunity and safety High, high-purity effect

Inactive Publication Date: 2020-08-18
乾元浩生物股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some defects. Among them, the VP2 protein expressed by E. coli mostly exists in the form of inclusion bodies and must be denatured and refolded. However, the antigenic epitope of IBDV is conformation-dependent, and the denatured and refolded VP2 protein loses its ability to induce chicken neutralization. In addition, the VP2 antigen expressed by Escherichia coli often has the problem of high endotoxin, which leads to large side reactions in chickens
Recombinant live vector vaccines not only have safety problems in the laboratory and production process, but also have a higher probability of gene mutation and recombination like ordinary live vaccines

Method used

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  • Avian bursal disease virus genetic engineering vaccine as well as preparation method and application thereof
  • Avian bursal disease virus genetic engineering vaccine as well as preparation method and application thereof
  • Avian bursal disease virus genetic engineering vaccine as well as preparation method and application thereof

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Experimental program
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preparation example Construction

[0038] The preparation method of the vaccine can adopt the conventional preparation method of genetic engineering subunit vaccine in the field.

[0039] Preferably, the preparation method includes:

[0040] The pMD19-T vector containing the target gene was double digested with Bam HI and Hind III to obtain the target fragment, and the target fragment was recovered and purified from the gel. At the same time, the pFastBac 1 vector was digested with double enzymes and recovered by gel. The target gene fragment and pFastBac 1 vector were ligated overnight with T4 DNA ligase. Then it was transformed into DH5α competent cells, picked and cultured and then sent for sequencing. After the sequencing is correct, extract the pFastBac 1 vector containing the target gene.

[0041] The pFastBac 1 vector containing the target gene was transformed into DH10Bac, and the transformed product was cultured in SOC medium at 37°C for 5 hours, then diluted and inoculated on a blue-white spot screen...

Embodiment 1

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. The preparation of embodiment 1 avian bursal virus genetic engineering vaccine

[0046] 1. Screening and cloning of target genes

[0047] Find the gene sequence and protein sequence of avian bursal virus VP2 protein, and compare the RNA extracted from the existing virus.

[0048] Check the restriction endonuclease cutting sites in the optimized sequence to ensure that it does not contain Bam HI and Hind III cutting sites.

[0049] A pair of primers for amplifying the VP2 gene fragment of avian bursal virus was designed ...

Embodiment 2

[0061] The expression characteristic and immunogenicity of embodiment 2 avian bursal virus genetic engineering vaccine

[0062] 1. Expression characteristics

[0063] Sf9 cells were cultured to 5 × 10 in Sf9 serum-free suspension medium 6 cells / ml, dilute the cell density to 2.5×10 with fresh serum-free medium 6 cells / ml, the recombinant baculovirus was inoculated at a ratio of 1‰, and the supernatant was harvested after culturing for 96 hours. Compared with healthy cells, the cells at 96h post-infection were significantly enlarged ( figure 1 ). Determine virus titer by plaque method, recombinant baculovirus P1 generation virus titer 10 8.5 TCID 50 / ml.

[0064] Sf9 cells were cultured with Sf9 serum-free suspension medium to 5 × 10 6 cells / ml, dilute the cell density to 2.5×10 with fresh serum-free medium 6 cells / ml, inoculate the recombinant baculovirus at a ratio of 1‰, culture until 168h and harvest the culture supernatant. Carry out SDS-PAGE electrophoresis ident...

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Abstract

The invention provides an avian bursal disease virus genetic engineering vaccine as well as a preparation method and application thereof. The avian bursal disease virus genetic engineering vaccine isprepared by expressing avian bursal disease protective antigen VP2 protein through an insect cell-baculovirus expression system to form avian bursal disease virus-like particles on spatial conformation, adding an adjuvant, and emulsifying. The vaccine provided by the invention is simple in preparation method, capable of preparing a large amount of avian bursal disease virus antigen protein, shortin time consumption, high in expression quantity and beneficial to large-scale production, and the obtained genetic engineering vaccine is good in immune effect and capable of effectively preventing avian bursal disease virus infection.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to an avian bursal virus genetic engineering vaccine and its preparation method and application. Background technique [0002] Chicken infectious bursal disease (Infectious bursal disease, IBD) is an acute, highly contagious disease caused by chicken infectious bursal disease virus (Infectious bursal disease virus, IBDV). The lymphoid tissue of young chickens, especially the central immune organs such as the bursa, can cause serious pathological damage to the bursa tissue, which in turn leads to severe immunosuppression in the body, which can easily lead to secondary infection or the failure of various vaccines. Causing huge losses to the poultry industry. [0003] IBDV belongs to the genus of double-stranded RNA viruses in the family Double-stranded RNA Virus. The genome consists of two double-stranded RNA fragments, namely A fragment and B fragment. The A...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/40C12N15/866A61K39/12A61K39/39A61P31/14
CPCA61K39/12A61K39/39A61K2039/5252A61K2039/552A61K2039/55511A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2720/10022C12N2720/10034C12N2720/10051C12N2800/105
Inventor 岳建新袁野王飞孙灵睿周蕾蕾陈秋阁向王震张秀美李浩鹏李浩哲朱昊旻魏杰安铁军
Owner 乾元浩生物股份有限公司
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