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Muscovy duck parvovirus infectious clone construction and saving method

A Muscovy duck parvovirus and infectious cloning technology, which is applied to viruses, virus/bacteriophage, single-stranded DNA viruses, etc., can solve the problems of time-consuming preparation of primary cells, great influence on transfection and rescue results, and achieve potential Application value, avoiding cumbersome and inefficient problems, simple and efficient operation

Inactive Publication Date: 2017-08-04
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For parvoviruses, the cells are most suitable for virus replication when they are in the division phase, so the state of the cells at the time of transfection has a great impact on the results of transfection rescue, and it is time-consuming to prepare primary cells for each transfection

Method used

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  • Muscovy duck parvovirus infectious clone construction and saving method
  • Muscovy duck parvovirus infectious clone construction and saving method
  • Muscovy duck parvovirus infectious clone construction and saving method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0042] 1. Construction of MDPV genome-wide clone pYY

[0043] 1.1. Virus amplification

[0044] Dilute the preserved MDPVYY strain (Archives of Virology, 2016, 161:2589-2594) 1:10 with sterile saline, add penicillin and streptomycin to a final concentration of 2000μg or 2000IU / ml, and place at 37°C After incubating in the incubator for 30 minutes, inoculate 12-day-old non-immune Muscovy duck embryos, 0.2ml each. After sealing with paraffin, put them into an incubator at 37°C, illuminate the eggs once every 8 hours, and remove goose embryos that died before 48 hours. The dead muscovy duck embryos were collected 48 hours later, placed in a refrigerator at 4°C for 4-6 hours, and then the allantoic fluid was aseptically collected and stored in a refrigerator at -40°C for later use.

[0045] 1.2. Virus purification

[0046] Centrifuge 300ml of the collected viral allantoic fluid at 11,000g for 20min, take the supernatant, add 1 / 3 volume of chloroform, shake vigorously, centrifug...

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Abstract

The invention relates to the technical field of biology, in particular to a MDPV (Muscovy duck parvovirus) infectious clone construction and saving method. The method includes: cloning an intact MDPV genome into vector plasmids pBSKN to obtain recombinant plasmids; mixing the recombinant plasmids with a transfection agent, and performing inoculation of nonimmune muscovy duck's embryon through the chorioallantoic membrane to save the recombinant plasmids in the muscovy duck's embryon, wherein a pBSKN vector is obtained by replacement of original EcoRV enzyme digestion sites of commercialized plasmid pBluescript II (SK) multiple clone sites with NcoI sites. Generated infectious viruses are lethal to the muscovy duck's embryon, and saving viruses and parent viruses are similar in lethality rate of young muscovy ducks. The saving method is simple, convenient and efficient, problems of low efficiency and complexity caused by transfection of primary cells are avoided, and potential application values in MDPV reverse genetic research and vaccine creation are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the construction of MDPV infectious plasmid clone and a method for transfecting muscovy duck embryos to rescue the virus. Background technique [0002] Muscovy duck parvovirus (MDPV) mainly infects young muscovy ducks within 3 weeks of age, and the infected ducks show clinical symptoms mainly characterized by diarrhea, soft feet and panting, commonly known as "three-week disease" of muscovy ducks. The morbidity rate can reach 27%~62%, and the mortality rate is between 22%~43%. Most of the recovered ducks become stiff ducks. The disease is one of the important diseases in the Muscovy duck breeding industry. The domestic Muscovy duck parvovirus disease was first reported in 1985, and France reported the existence of the disease in 1990. [0003] Muscovy duck parvovirus belongs to the genus Parvovirus in the family Parvoviridae, and its genome is a linear single-stranded DNA, which exist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N7/00
CPCC12N15/85C12N7/00C12N15/66C12N2750/14352C12N2800/106
Inventor 王建业黄钰王志仙凌珏怡朱国强孟霞朱礼倩
Owner YANGZHOU UNIV
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