Generation of recombinant ant adenoviruses carrying the gene coding for interleukin 6 of chicken

  • Vũ Thu Huyền
  • Lê Thị Hồng Minh
  • Nguyễn Thị Kim Cúc
  • Vũ Thị Quyên
  • Nguyễn Mai Anh
  • Lê Thanh Hòa
  • Phạm Việt Cường

Abstract

In our previous study in 2013, we had cloned Vietnamese chicken interleukin 6 gene. In this study, we describe the creation of an recombinant adenovirus containing chIL-6 as an immune stimulation product for poutry.

ChIL-6 gene was amplified total RNA extracted from the spleen of Viemamese chicken cell by RT-PCR. Then ChlL-6 gene was cloned into the shuttle vector pENTR3C that carried attLl and attL2 sites for site-specific recombination of the entry clone with gateway destination vector to generate a recombinant plasmid pAd/CMV-V5-chIL6. pAd/CMV-V5-chIL6 was exposed by Pad enzyme, the purpose of Pad digestion is to liberate the two ITRs (left and right) of adenoviral genome.

Finally, the recombinanted adenoviruspAd/CMV-V5-chIL6 was transfected into HEK293A cells for packaging and amplification. ChIL-6 expression in transfected HEK293A cells could be observed at 48 - 72 hours post. Adenovirus and ChlL-6 gene was detected in infected REK293A cells by using PCR. ChEL-6 protein was detemined by ELISA with chicken Interleukin 6 antibody (Cusabio, USA), The titer of recombinant adenovirus is 5x10^ PFU/ml after purification by using ibiPureTM adeno kit (Germany).

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Published
2017-09-28
Section
Articles