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Application of duck BCL2L15 gene in livestock and poultry for resisting avian influenza virus (AIV)

A gene and virus technology, applied in the application field of duck BCL2L15 gene in the anti-AIV virus of livestock and poultry, can solve the problem of breaking the balance between waterfowl and influenza virus, and achieve the effect of inhibiting replication

Inactive Publication Date: 2016-06-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the continuous evolution of influenza viruses, the balance between waterfowl and influenza viruses is broken

Method used

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  • Application of duck BCL2L15 gene in livestock and poultry for resisting avian influenza virus (AIV)
  • Application of duck BCL2L15 gene in livestock and poultry for resisting avian influenza virus (AIV)
  • Application of duck BCL2L15 gene in livestock and poultry for resisting avian influenza virus (AIV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Obtaining the sequence of the full-length coding region of the duck BCL2L15 gene by means of molecular biology experiments

[0051] Referring to the duck genome reference sequence on the Ensemble website, and according to the transcriptome splicing sequence, design the duck BCL2L15 gene full-length CDS region cloning primer BF / BR, the sequence is as follows:

[0052] BF: 5'-CAATGACAACGTTTGAGGAACAGAC-3',

[0053] BR: 5'-GTAGTAAAACACTTCCTCTCAGTCA-3'.

[0054] Using the cDNA of duck spleen tissue as a template, NEB company's Q5 high-fidelity polymerase was used for PCR amplification, and the amplified product was recovered by gel and ligated to T carrier for sequencing. After sequence comparison analysis, it was found that the full-length coding region of the duck BCL2L15 gene is 483bp (SEQ ID NO.1), encoding a total of 160 amino acids (SEQ ID NO.2).

[0055] In this example, the full-length coding region sequence of the duck BCL2L15 gene was successfully clone...

Embodiment 2

[0056] Example 2 Transient overexpression of BCL2L15 gene in cells by cytology experiment method

[0057] 1. Construction of duck BCL2L15 gene overexpression vector

[0058] According to the coding region sequence of duck BCL2L15 gene, the eukaryotic expression vector primer eBF / eBR was designed, and the upstream and downstream primers were respectively introduced into MluI and PmeI restriction sites (underlined); in addition, the upstream primer was before the start codon ATG Introduce the kozak (box marked) sequence, and introduce the flag tag (box marked) at the C-terminus of the target gene. The primer sequences are as follows:

[0059] eBF: 5'-CG ACGCGT ATGACAACGTTTGAGGAACAGACGA-3',

[0060] eBR: 5'-GG GTTTAAAC TCA GTCATCCAAGTTTCTCCCATCCTCCA-3'.

[0061] The CDS sequence of the amplified BCL2L15 gene (as shown in the sequence table CDS) was introduced into the original vector PiggyBac-Xgene (the vector map is shown in figure 2 shown), the PiggyBac-BCL2L15 v...

Embodiment 3

[0066] Example 3 Verification of Duck BCL2L15 Gene Resistance to AIV Infection Using Cytological Experiments

[0067] 1. Duck BCL2L15 gene inhibits influenza virus replication

[0068] Transfected PiggyBac-BCL2L15, PiggyBac-Xgene empty vector (NC) and DF1 cell lines with good growth state were mixed with 2×10 5 Cells / ml were seeded in 12-well cell culture plates, and challenged after the cells were stably adhered to the wall. Two strains of H5N1 subtype avian influenza virus, namely DK / 49 and GS / 65, were selected for the challenge experiment, and the challenge dose was MOI=0.001, and the experiment was set to repeat independently in 3 wells. Cell supernatants were collected at 12h, 24h, 36h, 48h, 60h and 72h after challenge for EID50 detection. The cell challenge experiment was completed in the P3 laboratory of Harbin Veterinary Research Institute.

[0069] The challenge results showed that compared with the negative control group (NC), the duck BCL2L15 gene inhibited the r...

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Abstract

The invention relates to the field of anti-virus genes, and particularly discloses application of a duck BCL2L15 gene in livestock and poultry for resisting an avian influenza virus (AIV). The invention provides the new gene BCL2L15 relevant to resisting of avian influenza, and it is verified that the BCL2L15 has the effect of restraining replication of the AIV by utilizing a method of combining molecular biology with cell biology experiments fir the first time. It is shown through analysis that compared with a cell which is not subjected to overexpression of the BCL2L15 gene, the cell subjected to overexpression of the BCL2L15 gene can significantly restrain replication of the AIV, therefore, in-depth function study can be conducted on the duck BCL2L15 gene, and a key protein structure or amino acid of the duck BCL2L15 gene for resisting the AIV is determined. By means of gene editing methods such as the transgenic technology, transgenic livestock and poultry which inducibly and highly express the BCL2L15 gene of ducks or other animals are obtained, and good varieties of transgenic farm animals capable of resisting various viruses such as the AIV are bred.

Description

technical field [0001] The invention relates to the field of antiviral genes, in particular to the application of duck BCL2L15 gene in livestock and poultry anti-AIV virus. Background technique [0002] Influenza viruses are negative-strand RNA viruses containing eight RNA genome segments. Highly pathogenic avian influenza (Highly Pathogenic Avian Influenza, HPAI) is a severe infectious disease mainly in poultry caused by influenza A virus of the Orthomyxoviridae family. Since avian influenza was reported in Italy in 1878, two subtypes of H5 and H7 have continued to cause influenza outbreaks in chickens and turkeys around the world, seriously threatening the development of the poultry industry. Recent studies have shown that three of the four worldwide influenza pandemics in the last century, namely the Spanish flu in 1918, the Asian influenza A2 (H2N2) subtype in 1957, and the Hong Kong influenza A3 (H3N2) subtype in 1968, Closely related to avian influenza virus. These ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C12N5/10A61K48/00A61P31/14A01K67/027
CPCA01K67/0275A61K48/00C07K14/465
Inventor 李宁黄银花荣恩光
Owner CHINA AGRI UNIV
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