Construction method and use of duck enteritis virus bacterial artificial chromosome

A technology of duck enteritis virus and artificial chromosome, which is applied in the direction of virus/bacteriophage, resistance to vector-borne diseases, and the use of vectors to introduce foreign genetic material. cycle effect

Inactive Publication Date: 2014-01-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the homologous region required for homologous recombination mediated by RecA is as long as about 1000bp, which is difficult to operate and relatively time-consuming, which limits its application to a large extent.

Method used

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  • Construction method and use of duck enteritis virus bacterial artificial chromosome
  • Construction method and use of duck enteritis virus bacterial artificial chromosome
  • Construction method and use of duck enteritis virus bacterial artificial chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0030] The construction of embodiment 1BAC homology arm and the formation of multiple copies

[0031] 1. Extraction of duck viral enteritis (DEV) virus genome

[0032] The diseased cells of the frozen duck enteritis virus vaccine strain C-KCE (purchased from the China Veterinary Medicine Supervision Institute, the genome DNA sequence of the vaccine strain C-KCE has been submitted (registration), the accession number is temporarygenebankaccessionNo.KF263690) were frozen and thawed continuously 3 times, centrifuge at 8000rpm at 4°C for 20min to pellet cell debris. The supernatant was collected, 30% (w / w) sucrose solution was placed at the bottom, and the virus particles were precipitated by centrifugation at 16000 rpm at 4° C. for 60 min. Discard the supernatant and add double distilled water to suspend the virus particles. Add proteinase K to a final concentration of 500 μg / rnL and sodium dodecyl sulfate (SDS) to a final concentration of 1%. Place in a water bath at 56°C for...

Embodiment 2

[0037] Screening and purification of embodiment 2 recombinant virus

[0038] 1. Preparation of CEF cells

[0039] Take 9-10-day-old SPF chicken embryos, disinfect them with alcohol cotton balls, wipe the air chamber with tincture of iodine for deiodination, take out the chicken embryos aseptically, place them in sterile glass bottles, and remove the head, limbs and viscera Shred with ophthalmic scissors. Use sterilized phosphate buffer saline (referred to as PBS, formula: Na 2 CO 3 1.59g,NaHCO 3 2.93g, with ddH 2 Dilute the volume to 1000ml, adjust the pH to 7.4) Wash twice in the bacterial bottle, add an appropriate amount of 0.25% trypsin to digest in a 37°C water bath for 30 minutes, discard the trypsin, and use an appropriate amount of serum (10%) and double antibiotics (Penicillin 100u / mL, Streptomycin 100u / mL) in DMEM medium (purchased from GIBCO Company), pipetting to disperse the cells, filtered through six layers of gauze, and mixed with an appropriate density (1...

Embodiment 3

[0046] Example 3 Electric transfer of recombinant virus into Escherichia coli DH10B-IS

[0047] (1) Preparation of competent DH10B-IS: Streak Escherichia coli (DH10B-IS, gifted by Professor Ma Lixin, School of Life Sciences, Hubei University) stored at -80°C on LB plates and culture overnight at 37°C. Insert the single colony of activated E. coli into LB or add the corresponding antibiotics (Cam, 34pg / mL, Str50pg / mL) into the LB liquid medium, culture overnight at 37°C with shaking at 200-250r / min, transfer 0.2-1mL overnight culture To a Erlenmeyer flask filled with 200mL LB. Incubate vigorously at 37°C for 2 to 6 hours, and measure OD regularly 600 value. When OD 600 When the value reaches 0.7-1.0, take out the Erlenmeyer flask from the shaker, place it in an ice-water mixture, and bathe in ice for 15 minutes. Collect the bacteria in a pre-cooled sterile centrifuge tube, centrifuge at 6000r / min at 4°C for 10min, and discard the supernatant. Resuspend the cells in pre-coo...

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Abstract

The invention belongs to the technical field of animal gene engineering and relates to a construction method and a use of a duck enteritis virus bacterial artificial chromosome. Through cloning, a duck enteritis virus bacterial artificial chromosome pBAC-C-KCE plasmid is obtained, is preserved in the China center for type culture collection (CCTCC) and has an accession number of CCTCC NO: M2013377. The invention also discloses the use of the duck enteritis virus bacterial artificial chromosome pBAC-C-KCE in recombinant duck enteritis virus packaging.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to the construction and application of an artificial chromosome of duck enteritis virus bacteria. Background technique [0002] Duck virus enteritis (Duck Virus Enteritis, DEV), also known as duck plague (Duck Plague, DP), is an acute and contagious infectious disease of ducks, geese and other Anseriformes caused by duck enteritis virus (Duck Plague, DP). Birds of all ages can be affected. Duck viral enteritis has been reported in many countries, and it is also widely prevalent in our country. Due to its rapid spread, high morbidity and mortality rate, which can reach 50-100%, and even more than 90% in adult ducks, it has become one of the main diseases that endanger the duck industry. In recent years, there have been successive research reports on the genome of duck enteritis virus; in 2009, the genome sequence of duck enteritis virus has been publi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N7/01
CPCY02A50/30
Inventor 金梅林李淑云邹忠
Owner HUAZHONG AGRI UNIV
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