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Extraction of duck enteritis virus genom DNA and sequence thereof

A duck enteritis virus, DNA sequence technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve the problem of lack of whole genome sequence and so on

Inactive Publication Date: 2008-05-21
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

DEV is spherical, the diameter of the core is about 60nm, the diameter of the capsid is about 90nm, the diameter of the mature virus is 150-380nm, the floating density in CsCI is 1.272g / ml, and the average molecular weight of the nucleic acid measured by the restriction endonuclease map is 93.93 ×106 D, but there is no report on the whole genome sequence of DEV

Method used

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  • Extraction of duck enteritis virus genom DNA and sequence thereof
  • Extraction of duck enteritis virus genom DNA and sequence thereof

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Embodiment Construction

[0017] (1) Proliferation of Duck Enteritis Virus and Extraction of Genomic DNA

[0018] 1. Proliferation of Duck Enteritis Virus

[0019] Take SPF chicken embryos hatched to 9 days old, disinfect the eggshell with iodine, take out the embryo body aseptically, put it in a petri dish, and remove the head, limbs and viscera. The embryo body was washed 3 times with Hanks solution, transferred to a small beaker with a moderate volume, cut into small pieces of 1-2 mm, washed 3 times, added an appropriate amount of trypsin, and digested at room temperature for 20-30 minutes. Add an appropriate amount of cell growth solution, pipette repeatedly with a wide mouth pipette to disperse the cells and form a suspension, transfer to a centrifuge tube, centrifuge at 1000r / min for 10min, discard the supernatant, and wash the cell pellet twice with Hanks solution. Add cell growth solution, blow with a pipette to make a cell suspension, count, and dilute to 10 6 cells / ml, aliquoted into cell c...

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Abstract

The invention discloses a DNA extraction method of Duck Enteritis Virus genome and the genome sequence. Firstly, the pyrolysis of chicken embryo fibroblast which is infected with DEV is processed by hypertonic buffer; then the nuclease is added for the digestion of cell DNA; and the virus genome DNA is obtained through phenol chloroform extracting method. The obtained sample is broken randomly into 2kb to 2.5kb fragments; the pUC19 vector is linked to transform host E. coli and construct genomic library. The positive clones in the library are randomly sequenced; the total sequence length has 6 times coverage rate for the virus genome. The sequence result is assembled and clustered to obtain the whole DEV genome sequence with the length of156512bp. The analysis shows that the genome structure is the same with that of the members of Varicella virus in Alpha- herpesus subfamily, which provides theoretical basis for the classification of DEV.

Description

technical field [0001] The invention relates to the extraction of genome DNA of duck enteritis virus, and also relates to the genome sequence of duck enteritis virus, which belongs to the field of biotechnology. Background technique [0002] Duck viral enteritis (duck virus enteritis, DVE), also known as duck plague (duck plague, DP), is an acute, contact, septic infectious disease that harms ducks, geese and other birds of Anseriformes. Gastrointestinal hemorrhage and necrosis, lymphoid organ damage, and degenerative changes in parenchymal organs are the main features. The disease has been prevalent in my country for nearly 40 years, but the epidemic characteristics have undergone some changes during this period. According to reports before 1963, the morbidity and mortality of the most susceptible ducks were above 90%, but there was a significant decline in the following 20 years. Since 2000, the epidemic characteristics of DVE have undergone new changes. The virus mainly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33
Inventor 李玉峰宋敏训史玉颖黄兵马秀丽吴静于可响
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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