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Expression of the HadV-41 short fiber C-terminal knob domain in baculovirus system

A L Favier F P Yeda; João Santana da Silva; Dolores Ursula Mehnert; E Gout; M J van Raaji; A Kidd; J Chroboczek; Charlotte Marianna Hársi; Congresso do Instituto de Ciências Biomédicas (3. 2001 São Paulo)

Resumos São Paulo : Comissão de Cultura e Extensão Universitária do ICB/USP, 2001

São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP 2001

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  • Título:
    Expression of the HadV-41 short fiber C-terminal knob domain in baculovirus system
  • Autor: A L Favier
  • F P Yeda; João Santana da Silva; Dolores Ursula Mehnert; E Gout; M J van Raaji; A Kidd; J Chroboczek; Charlotte Marianna Hársi; Congresso do Instituto de Ciências Biomédicas (3. 2001 São Paulo)
  • Assuntos: MICROBIOLOGIA
  • É parte de: Resumos São Paulo : Comissão de Cultura e Extensão Universitária do ICB/USP, 2001
  • Descrição: Human adenovirus of Subgenus F, serotypes 40 and 41, cause infantile gastroenteritis. Structural features uniques of these viruses concern the presence of two different fibers in the viral particle: a long and a short, placed one per penton-base. The long fiber, probably, undergoes the viral adsorption to the host cell by interaction with the receptor CAR. The function of the short fiber is not known. The aim of this project is obtain the Ad41 short fiber knob domain purified, to use it as a ligand on virus-host cell interaction studies. The viral DNA sequence corresponding to the C-terminal domain of the short fiber of the HAdV-41(Tak) was amplified by PCR and cloned into pFastBacDual plasmid (Gibco-BRL). E. coli DH10Bac competent cells, which contain a modified baculovirus genome (Bacmid-Gibco-BRL) were transformed with the donor plasmid. The cells were plated on selective medium and the colonies were screened for their lacZ phenotype (white/blue). High molecular weight mini-prep DNA was prepared from selected E.coli clones. Verification of the insertion of the Ad41-short fiber sequence into the Bacmid DNA was done by PCR with the pUC/M13 (Gibco-BRL) amplification primers. The recombinant Bacmid DNA was then used to transfect Sf21 insect cells. The recombinant baculovirus was recovered from the cultures. Expression trials were done on Sf21, HighFive and Sf+ insect cell lines. The expressed protein was detected by Western-blot assays using an anti-Ad41
    short-fiber rabbit serum. Expression kinetics assays showed best results on Sf21 cells, collected three days post-infection
  • Editor: São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP
  • Data de criação/publicação: 2001
  • Formato: 1 v., pôster 240.
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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