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NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof

A DHAV-1, DHAV-3 technology, applied in the field of molecular biology, can solve the problems of inability to protect DHAV-3 virus, immune prevention of unwell ducklings, and high production costs, and achieve healthy development, reduce immune stress, and reduce The effect of production costs

Active Publication Date: 2016-07-13
POULTRY INST SHANDONG ACADEMY OF AGRI SCI SHANDONG SPECIFIC PATHOGEN FREEN CHICKS RES CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current prevention of duck hepatitis A virus (DHAV) mainly relies on traditional vaccines. Traditional inactivated vaccines not only have high production costs, but also produce antibodies slowly. Breeding experiments have proved that they are not suitable for immune prevention of ducklings.
The existing DHAV-1 attenuated vaccines cannot protect the increasingly serious DHAV-3 virus at all, and the DHAV-3 attenuated vaccines have not yet obtained the approval number. Therefore, in addition to the urgent demand for DHAV-3 attenuated vaccines, the The demand for multivalent hepatitis A virus vaccine is also very urgent

Method used

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  • NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof
  • NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof
  • NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0062] The connection of the VP1 gene of embodiment 1DHAV-1 type, DHAV-3 type

[0063] Referring to the nucleotide sequences of duck hepatitis virus DHAV-1VP1-2A gene and DHAV-3VP1 gene recorded in GenBank, DHAV-1 and DHAV-3 were used as materials respectively, and DHAV-1VP1-2A was amplified by conventional RT-PCR method gene and DHAV-3VP1 gene. According to the instructions, use Trizol reagent to extract DHAV-1 and DHAV-3 viral RNA, and use the following 1VP1-2A-F / 1VP1-2A-SOE-R, 3VP1-SOE-F / 3VP1-R primers to amplify respectively to obtain DHAV- 1VP1-2A and DHAV-3VP1 genes.

[0064] Described DHAV-1 carries out PCR amplification and is the primer P1 of DHAV-1VP1-2A gene as:

[0065] 1VP1-2A-F: 5'-TGGAATTCGGTGATTCTAACCAGTTG-3' (BamHI),

[0066] 1VP1-2A-SOE-R: 5'-CTGATTGGAATCACCTTGATCTGTAGTAAT-3';

[0067] The fragment size of the amplified product DHAV-1VP1-2A is 1652bp; the nucleotide sequence of the amplified product DHAV-1VP1-2A is shown in SEQ ID NO:1.

[0068] Describe...

Embodiment 2

[0078] Example 2 Construction of recombinant Newcastle disease virus expressing DHAV-1VP1-2A-3VP1

[0079] Using the recombinant plasmid pLS-RFP containing the full-length cDNA of LaSota (the RFP reporter gene is inserted between the P gene and the F gene) as a template, the self-designed primers LS-P-up and LS-F-down were used in the plasmid pLS-RFP The two ends of RFPORF were amplified by reverse PCR to obtain a linearized vector containing the full-length cDNA of LaSota:

[0080] LS-P-up: 5'-GGTGGCTACAACTATCAACTAAACT-3',

[0081] LS-F-down: 5'-GTGTGTAACTACCGTGTACTAAGC-3';

[0082] The fragment size of the amplified product is 18.524kb; the nucleotide sequence of the amplified product is shown in SEQ ID NO:4.

[0083] Using specific primers plant-VP1-F and plant-VP1-R to amplify the DHAV1VP1-2A-3VP1 gene, the end of the gene obtained contains the same 15bp extended sequence as the end of the LaSota linearized vector:

[0084] plant-VP1-F: 5'-atagttgtagccaccATGGGTGATTCTAAC...

Embodiment 3

[0091] Example 3 Rescue of recombinant virus rLS-1VP1-2A-3VP1

[0092] The recombinant vaccinia virus MVA / T7 (MOI=3) expressing T7 polymerase was inoculated in a 24-well plate with 90% monolayer HEp-2 cells, and after incubation for 1 h, 1.0 μg pLS-1VP1 HEp-2 cells were co-transfected with 2A-3VP1, 0.5μgpTM-NP, 0.25μgpTM-P and 0.05μgpTM-L helper plasmids. Six hours after transfection, the transfected cells were washed once with PBS, and DMEM medium containing 2% FBS and antibiotics was added. After 72 hours of transfection, the transfected cells were repeatedly frozen and thawed 3 times, the rescued recombinant virus was harvested, and it was inoculated into 9-day-old SPF chicken embryos, the allantoic fluid was harvested, and the allantoic fluid that was positive for hemagglutination (HA) was used A 0.22um filter was used to filter out the poxvirus, and it was continuously passaged on 9-day-old SPF chicken embryos. The harvested virus was stored at -80°C, and the recombinant...

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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes.The recombinant virus is recorded as rLS-1VP1-2A-3VP1 and is obtained inserting serially connected DHAV-1 and DHAV-3 VP1 genes into an NDV vector and carrying out saving.The NDV (Lasota strain) is used as the vector for the recombinant virus, the serially connected DHAV-1 and DHAV-3 VP1 genes are inserted into the NDV (Lasota strain) to obtain a vector, and the DNV recombinant virus co-expressing DHAV-1 and DHAV-3 VP1 genes is obtained by determining an optimal insertion site to insert the DHAV VIP1 gene; the recombinant virus is useful in preventing duck hepatitis A viruses (type 1 and type 3) and duck Newcastle disease and filling the current blank of DHAV-3 vaccines.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, specifically relates to an NDV recombinant virus co-expressing VP1 genes of DHAV-1 and DHAV-3, and further discloses a preparation method thereof. Background technique [0002] As the largest duck raising country in the world, China has frequent outbreaks of duck hepatitis A. Due to the unstable prevention and control effect of the yolk antibody currently used, the prevention and control status of duck hepatitis A is not optimistic, which not only causes great harm to the duck industry. It also poses certain hidden dangers to my country's food safety. [0003] Duck hepatitis A is mainly caused by duck hepatitis A virus type 1 (DHAV-1), type 2 (DHAV-2) and type 3 (DHAV-3). and DHAV-3 based. As the main protective protein of DHAV, the VP1 gene encodes the main antigenic site and has the main type-specific neutralization site, and has also become the gene of choice for DHAV research. In ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85A61K39/295A61K39/29A61K39/17A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/70C07K14/005C12N7/00C12N2760/18121C12N2760/18134C12N2760/18151C12N2770/32422C12N2770/32434
Inventor 马秀丽宋敏训黄兵李玉峰于可响刘存霞胡峰
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI SHANDONG SPECIFIC PATHOGEN FREEN CHICKS RES CENT
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