|1396-1||Production and purification of HAdV-2 hexon protein in HEK-293 cells|
|Autores:||Inarei Jose Paulini Junior (UNIFESP - Universidade Federal De São Paulo / USP - Universidade de São Paulo) ; Joselma Siqueira Silva (USP - Universidade de São Paulo) ; Juliana Cristina Marinheiro (USP - Universidade de São Paulo) ; Charlotte Mariana Harsi (USP - Universidade de São Paulo) ; Celso Francisco Hernandes Granato (UNIFESP - Universidade Federal De São Paulo) |
Human adenovirus (HAdV) present 52 serotypes which are causative agents of infections in the gastrointestinal, uro-genital, and neurological systems, both in children and adults. More than one third of the adenoviruses may cause human diseases such as respiratory infections (group B1 HAdV-3, -7, -21, group B2 HAdV- 14, and group E HAdV-4), gastroenteritis (group F HAdV-40 and -41), ocular infections (group D HAdV-8, -9, and -37), pneumonia (group B1 HAdV-3, -7, group C HAdV-1, 2, and group E HAdV-4), keratoconjunctivitis (group B2 HAdV-11, and group D HAdV-8, -19 and HAdV-37), menigoencephalitis (group A HAdV-12, group B1. Although many of these infections are severe requiring hospitalization, HAdV are rarely diagnosed due to the lack of sensitive, practical and cheap diagnostic tests. The aim of this study is to produce monoclonal antibodies against HAdV-2 hexon protein which will be applied in rapid diagnostic test. First step of this project was to purify HAdV-2 hexon protein. For this, 48 cell culture bottles of 300cm2 were seeded with HEK-293 cells and the cell monolayer infected with 0.1 M.O.I of HAdV-2. When the cytopathic effect was evident (between 2-5 days), the cells were harvested and centrifuged at 220 g for 10 min. The supernatants were collected and the HAdV-2 precipitated by ultracentrifugation. The cell pellets were pooled in 10 mM HEPES buffer at pH 7.4. Viruses were released after three cycles of freezing and thawing. Lysed cells suspensions were clarified with equal volume of Vertrel XF (Clarus Technology), followed by vigorous vortexing. Cells debris were removed by centrifugation at 2.619 g for 25min at 4°C. Viral particles and soluble proteins in the supernatant were purified in CsCl gradients prepared in a 10 mM HEPES buffer at pH 7.4. Purified HAdV-2 complete particles were stored at -20°C. Soluble proteins fraction of the gradient were analyses by SDS- PAGE , Electronic Microscopy and hexon protein purified by ultrafiltration and ion exchange columns.
Financial support : CAPES and Fundação de Amparo à Pesquisa do Estado de
São Paulo – FAPESP (number : 2011/50100-6).
Palavras-chave: HAdV-2, HEK-293, Hexon, Monoclonal antibodies, Rapid tests