Duck adenovirus type 1 virus replication nonessential region fragment screening and its general transporter and recombination obtained therefrom

A virus replication and non-essential region technology, applied in the field of genetic engineering, can solve problems such as difficulties in the application of recombinant viruses

Inactive Publication Date: 2007-03-28
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
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Problems solved by technology

If the constructed recombinant virus genome contains these reporter genes...

Method used

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  • Duck adenovirus type 1 virus replication nonessential region fragment screening and its general transporter and recombination obtained therefrom
  • Duck adenovirus type 1 virus replication nonessential region fragment screening and its general transporter and recombination obtained therefrom
  • Duck adenovirus type 1 virus replication nonessential region fragment screening and its general transporter and recombination obtained therefrom

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Embodiment Construction

[0028] 1 Amplification and cloning of recombinant arm

[0029] Primer design: the positive strand primer is 5′-CCGCGGAAGCATTGAATTCTGGAT-3′, and the negative strand primer is 5′-CGCGTGCTGTAACAGGCATTCGTA-3′. Amplify a fragment with a span of 2.73kb, which is located in the E4 region on the right side of the duck adenovirus type 1 virus genome (refer to the GENBANK accession number as Y09598 There are adenovirus sequences, located between 26462 and 29162), as shown in Figure 1.

[0030] Extraction of total viral genome DNA: inoculate duck embryo allantoic fluid with a strain of duck adenovirus type 1 virus QU strain isolated from healthy quails that has no pathogenicity to chickens, ducks and other poultry, centrifuge at 6000g for 10min, and collect the supernatant. Add an equal volume of lysate (containing 0.2mg / mL proteinase K, 1% SDS, 10mmol / L NaCl, 10mmol / L Tris-HCl pH8.0, 1mmol / LEDTA), and place in a 37°C water bath for 3-4 hours. The lysate was extracted with phenol, phen...

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Abstract

The invention relates to a selecting method for duck adenovirus 1 type virus replicating non-necessary section segment and the recombination fowl adenovirus gained through transporter gene and the universal transporter gene. It uses duck adenovirus 1 type virus as material to expand the sequence at right side of E4 area by PCR method. Inducing loxP sequence to the two sides of GFP gene expression box and inserting into the expanded segment, the universal transporter gene would be constructed. The transporter gene would gain the recombination adenovirus of stable expression GFP, thus, a replicating non-necessary area would be determined. The universal transporter gene loxP-GFP-loxP sequence side contains a unique enzyme Restriction Enzyme cutting site, and cold insert relative target gene to gain recombination virus without GFP gene. The gene engineer medicine developed from the universal transporter gene would not contain selecting marker gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering in the biotechnology pharmaceutical industry, and is specially used for the construction of exogenous gene recombinant duck adenovirus type 1 virus. Background technique [0002] Avian adenovirus can infect the digestive tract and respiratory tract and stimulate local mucosal immunity in the body. The use of avian adenovirus as a carrier can also be used in the construction and research of viral vector recombinant vaccines such as avian adenovirus type 1 (CELO), type 8, and type 10. Group I poultry adenovirus has some difficulties in cell culture and production technology, and most commercial chicken flocks have recessive infection of CELO virus, so the immune efficacy of the virus vector vaccine constructed on this basis will be interfered to some extent. Duck adenovirus type 1 virus and group I poultry adenovirus have a small common antigen, and have a wide range of host tropism, an...

Claims

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Application Information

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IPC IPC(8): C12N15/33C12N15/85C12N15/861
Inventor 黄兵艾武李峰李玉峰逯岩
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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