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Infectious bursal disease virus DNA vaccine and construction method thereof

A DNA vaccine and technology for bursal disease, applied in the field of animal medical bioengineering, can solve the problems of weak protection, limited effect, and low antibody level, so as to avoid gene expression inhibition, promote immune response ability, and improve IBD antibody level low effect

Active Publication Date: 2016-12-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IBDV VP2 gene encodes the main structure of the pathogen and the antigenic protein VP2. Most of the DNA vaccines or related genetic engineering vaccines currently developed use VP2 as the target gene or protein. After immunization, it can play a certain effect, but the effect is limited, stimulating the animal body to produce antibodies Low level, weak protection

Method used

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  • Infectious bursal disease virus DNA vaccine and construction method thereof
  • Infectious bursal disease virus DNA vaccine and construction method thereof
  • Infectious bursal disease virus DNA vaccine and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0042] Example 1 Construction of three gene co-expression vector pTVP2-Akirin2-GM-CSF

[0043] 1), according to the sequence optimization of the three gene coding regions of IBDV VP2 (Genebank accession number AF508177), chicken Akirin2 (Genebank accession number NM_001193595) and chicken GM-CSF (Genebank accession number EU939770.1) in the National Biotechnology Information Center of the United States Tandem; the optimization method is to remove the stop codons of the latter two genes, and optimize the codon preference of chickens on the IBDV VP2 gene. The nucleotide sequence of the finally obtained IBDV VP2 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the chicken Akirin2 gene is shown in SEQ ID NO: 2; the nucleotide sequence of the chicken GM-CSF gene is shown in SEQ ID NO : As shown in 3.

[0044] The above-mentioned finally obtained IBDV VP2 gene, chicken Akirin2 gene and chicken GM-CSF gene are connected in series to form a VP2-Akirin2-GM-CSF sequence; every...

Embodiment 2 3

[0074] Example 2 Verification of the validity of the three-gene co-expression vector pTVP2-Akirin2-GM-CSF

[0075] 1) Mix pTVP2-Akirin2-GM-CSF and an appropriate amount of Lipofectamine LTX (purchased from Invitrogen) thoroughly and add them to a cell culture plate covered with 293T cells, using an empty vector (pTriEx-4) and three single-gene recombinant expression vectors pTVP2, pTAkirin2, and pTGM-CSF (constructed by the applicant and sequenced correctly) were carried out in corresponding control experiments at the same time.

[0076] 2) After 48 hours, the cultured cells were collected, and after the cells were lysed and broken, the effectiveness of expressing the three proteins of VP2, Akirin2 and GM-CSF was tested respectively, further confirming that pTVP2-Akirin2-GM-CSF can express the target protein in 293T cells , the detection result is as Figure 4 and Figure 5 shown. From Figure 4 It can be seen that all three proteins are expressed, and the expression levels ...

Embodiment 3

[0077] Embodiment 3 animal experiments

[0078] 1) Randomly group 14-day-old SPF White Legion chickens (purchased from Guangdong Wenshi Dahuanong Biotechnology Co., Ltd. Egg Branch) into 7 groups, with 20 birds in each group. See Table 1 for specific grouping information:

[0079] Table 1 Animal experiment grouping and DNA injection situation

[0080]

[0081] The pTVP2-Akirin2-GM-CSF plasmid solution and 200 μL of four kinds of control plasmid solutions of pTVP2, pTAkirin2, pTGM-CSF, and pTriEx-4 with a DNA concentration of 1000 ng / μL obtained in Example 1 were tested on 14-day-old SPF white legion chickens. Injection at two points in the leg muscles was recorded as the 0th day, and the same treatment was carried out on the 14th day to boost immunization. The changes (feeding activity, mental state, etc.) of the chickens in each group were observed every day. On the 28th day, except for the blank control group 1, all other groups were orally infected with IBDVBC6 / 85 viru...

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Abstract

The invention relates to the field of animal medicine biological engineering, in particular to an infectious bursal disease virus DNA vaccine and a construction method thereof. The DNA vaccine comprises a three gene co-expression vector of IBDV VP2 gene, chicken Akirin2 gene and chicken GM-CSF gene, and independent and equal co-expression of the IBDV VP2 gene, the chicken Akirin2 gene and the chicken GM-CSF gene can be realized by one carrier. When the DNA vaccine is applied, the situations that the produced IBD antibody level and the provided protection rate are low when the VP2 gene performs immunization alone can be improved, chicken Akirin2 and chicken GM-CSF can form a synergistic effect on the biological function, and the immune response ability induced by the IBDV gene and resisting IBDV infection can be promoted jointly.

Description

technical field [0001] The invention relates to the field of animal medicine bioengineering, in particular to an infectious bursal disease virus DNA vaccine and a construction method thereof. Background technique [0002] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease that mainly harms young chickens and chicks. The weakening of response ability seriously endangers the production and development of poultry breeding industry, and is a Class B disease identified by the World Organization for Animal Health (OIE). The mutated strains and super-virulent strains with different pathogenicity and antigenicity that became popular in the 1980s can break through the protection of traditional vaccines and maternal antibodies, making the prevention and control of the disease even more severe. Under the current epidemic situation, all chicken flocks must be immunized with IBD vaccine, but high lethality or immunization failure caused ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/12A61P31/14C12N15/85C12N15/11
CPCA61K39/12A61K2039/53A61K2039/552C12N15/85C12N2720/10034C12N2800/107
Inventor 陈瑞爱刘传高朱娇娇钟楚红
Owner SOUTH CHINA AGRI UNIV
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