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Preparation method of newcastle disease glucoprotein virus antigen and product thereof

A chicken Newcastle disease virus, glycoprotein technology, applied in the directions of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effects of good immunogenicity and low production cost

Active Publication Date: 2014-09-24
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the successful expression of NDV antigen in insects using the silkworm baculovirus expression system

Method used

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  • Preparation method of newcastle disease glucoprotein virus antigen and product thereof
  • Preparation method of newcastle disease glucoprotein virus antigen and product thereof
  • Preparation method of newcastle disease glucoprotein virus antigen and product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Expression of the original sequence of chicken Newcastle disease virus F gene in silkworm bioreactor and detection of expression products

[0049] 1 Construction and identification of recombinant plasmid pT-NDV-F

[0050] 1.1 Primer design and synthesis of target gene

[0051] Genomic RNA of LaSota vaccine strain was extracted by TRIzol method. Primers were designed to amplify the original sequence of chicken Newcastle disease virus F gene (SEQ ID NO.1) by RT-PCR. The designed reverse transcription specific primer was: NDV-F-RT: 5'-TCACATTTTTGTAGTGGCTC-3'. The amplification primers of the original sequence of the F gene are: NDV-F upstream: 5'-CGGATCCATGGGCTCCAAACCTTCT-3' (BamHI); NDV-F downstream: 5'-CTCTAGATCACATTTTTGTAGTGGC-3' (Xba1). After PCR amplification, 5.0 μL of the reaction product was electrophoresed on 1.0% TAE agarose gel to obtain bands of expected size. DNA fragments were purified and recovered using glass milk.

[0052] 1.2 DNA fragment li...

Embodiment 2

[0100]Example 2 Expression of the sequence of the F-O gene in the silkworm bioreactor and detection of the expression product after codon optimization of chicken Newcastle disease

[0101] 1 Obtaining sequence F-O after optimization

[0102] Collect the sequences of NDV F gene in recent years, especially compare the differences between current vaccine strains and epidemic strains, predict its evolution trend, and adjust its original sequence ( figure 1 ). Firstly, according to the changing trend of the amino acid sequence, select its conserved site, and compare and analyze the homology by collecting the Newcastle disease F gene sequence in different regions. The results show that although the amino acid sequence is highly conserved, there are still predictable Generally, the widespread use of vaccine strains leads to point mutations in the amino acid positions of the current epidemic strains, which will lead to a decline in the immune effect of the vaccine strains in the lon...

Embodiment 3

[0154] Example 3 Combined expression of chicken Newcastle disease virus F-IRES-HN-O gene sequence in silkworm bioreactor and detection of expression products

[0155] 1 Obtaining the original sequence of chicken Newcastle disease HN gene

[0156] Primers were designed to amplify the original sequence of chicken Newcastle disease virus HN gene (SEQ ID NO.3) by RT-PCR method, and the amino acid sequence encoded by it was shown in SEQ ID NO.4.

[0157] 2 Obtaining of optimized sequences NDV-F-O and NDV-HN-O

[0158] Collect the sequences of NDV HN genes in recent years, especially compare the differences between current vaccine strains and epidemic strains, predict their evolution trends, and adjust their original sequences ( figure 2 ). Firstly, according to the changing trend of the amino acid sequence, select its conserved site; by collecting the Newcastle disease HN gene sequence and the sequence of the vaccine strain in different regions for homology comparison and analys...

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Abstract

The invention discloses a preparation method of newcastle disease glucoprotein virus antigen and product thereof. According to the preparation method, the F gene and HN gene of the newcastle disease virus are optimized according to virus prevalent trend prediction, a newcastle disease virus glucoprotein antigen gene and an optimized antigen gene or series-connected optimized antigen genes are combined in a bombyx mori bioreactor for expression, and the expressed antigen or prepared recombinant virus can provide effective immune protection for animals. The invention also provides a preparation method of a newcastle disease virus antigen gene carrying vector. The preparation method comprises the following steps of: cloning the optimized newcastle disease virus antigen gene or series-connected optimized antigen genes into the baculovirus carrying vector controlled by a mammal promotor in a combined way; and recombining to obtain the gene carrying vector controlled by the mammal promotor. After the newcastle disease virus antigen gene carrying vector enters an animal body in an injection or oral administration mode, the newcastle disease virus antigen gene carrying vector can be used for efficiently carrying the antigen gene in the animal body and effectively defending the attack of newcastle disease virus.

Description

technical field [0001] The invention relates to a method for preparing chicken Newcastle disease virus antigen, in particular to a method for preparing chicken Newcastle disease virus antigen in insects by using recombinant baculovirus and obtaining recombinant antigen by the preparation method. The invention also relates to a chicken Newcastle disease virus antigen gene silkworm rod-shaped The preparation method of the virus presentation vector and its products, the invention further relates to their use in the prevention, treatment or diagnosis of chicken Newcastle disease vaccine or reagent, which belongs to the field of preparation and application of chicken Newcastle disease virus antigen. Background technique [0002] Newcastle disease was first discovered in Indonesia in 1926, and Newcastle in England in the same year. Due to the serious damage caused by Newcastle disease, the International Office for Epizootics (OIE) designated Newcastle disease as a severe infectious...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N15/56C12N15/866C12N9/24C12N7/01C07K14/125A61K39/17A61P31/14
CPCA61K39/12A61K2039/552C07K14/005C12N9/2402C12N15/86C12N2710/14043C12N2760/18122C12N2760/18134C12Y302/01018
Inventor 张志芳李轶女王智权易咏竹李浩洋李田田胡小元
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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