XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:92-5

Poster (Painel)

92-5

Use of antisense RNA technology for silencing of PbP27 gene in Paracoccidioides brasiliensis

Authors:

Isaura Torres Gomez (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Orville Hernandez Ruiz (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Jose F Munoz (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Diana Tamayo (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Biology Institute, U de Antioquia, Medellin, Colombia) ; Ana Maria Garcia (BCM, CIB - Molecular and Cellular Biology Unit) ; Angela Restrepo (BCM, CIB - Molecular and Cellular Biology Unit) ; Juan G Mcewen (BCM, CIB - Molecular and Cellular Biology UnitU DE A, MEDELLIN, CO - Facultad de Medicina, U de Antioquia, Medellin, Colombia)

Abstract

P27 is an antigenic protein produced by P. brasiliensistext, the etiologic agent of paracoccidioidomycosis (PCM). This protein has been considered as a putative virulence factor, and proposed as a suitable target for the designing diagnostic tools and vaccines. To evaluate the role of this antigen, we designed four antisense oligonucleotides vectors targeting PbP27 and using the transformation system mediated by Agrobacterium tumefacienstext (ATMT), we transformed yeast cells from the strain Pb339. The Hygromycin resistance was used to select the transformed isolates with the antisense molecules as well as the yeast with empty vector (PbWt+EV). The isolates were evaluated at the transcriptional and translational level: (i) PbP27 gene expression was determined by quantitative reverse transcription PCR (qRT-PCR) performed on a CFX 96 System (Bio-Rad) and (ii) protein synthesis was assayed by western immunoblotting using a monoclonal antibody BJ4, previously reported. ਀䤀渀琀栀椀猀猀琀甀搀礀眀攀最攀渀攀爀愀琀攀搀琀栀爀攀攀搀椀昀昀攀爀攀渀琀倀⸀ 戀爀愀猀椀氀椀攀渀猀椀猀㰀椀㸀琀攀砀琀㰀⼀椀㸀椀猀漀氀愀琀攀猀琀栀愀琀眀攀爀攀洀椀琀漀琀椀挀愀氀氀礀猀琀愀戀氀攀 ⠀倀戀倀㈀㜀愀刀一䄀⤀Ⰰ 眀椀琀栀搀漀眀渀爀攀最甀氀愀琀椀漀渀椀渀倀戀倀㈀㜀最攀渀攀攀砀瀀爀攀猀猀椀漀渀爀愀渀最椀渀最昀爀漀洀㜀　ⴀ㜀㔀─ 挀漀洀瀀愀爀攀搀琀漀倀戀圀琀愀渀搀倀戀圀琀⬀䔀嘀⸀ 吀栀椀猀瀀栀攀渀漀琀礀瀀攀爀攀洀愀椀渀攀搀猀琀愀戀氀攀愀昀琀攀爀㄀㠀洀漀渀琀栀猀漀昀猀甀戀挀甀氀琀甀爀攀椀渀愀瀀瀀爀漀瀀爀椀愀琀攀挀甀氀琀甀爀攀洀攀搀椀愀Ⰰ 眀栀椀挀栀椀渀搀椀挀愀琀攀猀琀栀攀椀渀琀攀最爀愀琀椀漀渀漀昀琀栀攀吀ⴀ䐀一䄀椀渀琀漀琀栀攀倀⸀ 戀爀愀猀椀氀椀攀渀猀椀猀㰀椀㸀琀攀砀琀㰀⼀椀㸀最攀渀漀洀攀Ⰰ 愀氀猀漀猀甀瀀瀀漀爀琀攀搀戀礀琀栀攀愀洀瀀氀椀昀椀挀愀琀椀漀渀漀昀琀栀攀栀瀀栀最攀渀攀⸀ 䤀渀愀搀搀椀琀椀漀渀Ⰰ 琀栀攀爀攀搀甀挀琀椀漀渀漀昀瀀㈀㜀瀀爀漀琀攀椀渀眀愀猀搀攀洀漀渀猀琀爀愀琀攀搀椀渀眀攀猀琀攀爀渀椀渀洀甀渀漀戀氀漀琀琀椀渀最愀猀猀愀礀猀眀椀琀栀䈀䨀㐀洀漀渀漀挀氀漀渀愀氀愀渀琀椀戀漀搀礀椀渀搀椀昀昀攀爀攀渀琀攀砀琀爀愀挀琀猀漀戀琀愀椀渀攀搀昀爀漀洀挀漀渀琀爀漀氀猀愀渀搀倀戀倀㈀㜀愀刀一䄀椀猀漀氀愀琀攀猀⸀ 䤀渀猀甀洀洀愀爀礀Ⰰ 愀刀一䄀椀猀愀猀甀椀琀愀戀氀攀猀椀氀攀渀挀椀渀最洀攀挀栀愀渀椀猀洀昀漀爀倀⸀ 戀爀愀猀椀氀椀攀渀猀椀猀⸀ 吀栀攀椀洀瀀氀攀洀攀渀琀愀琀椀漀渀漀昀琀栀椀猀猀椀氀攀渀挀椀渀最琀攀挀栀渀漀氀漀最礀琀漀猀琀甀搀礀愀渀琀椀最攀渀椀挀瀀爀漀琀攀椀渀猀猀甀挀栀愀猀瀀㈀㜀Ⰰ 挀漀渀猀琀椀琀甀琀攀猀愀渀椀渀琀攀爀攀猀琀椀渀最猀琀攀瀀椀渀琀栀攀攀瘀愀氀甀愀琀椀漀渀漀昀琀栀攀椀爀昀甀渀挀琀椀漀渀猀愀渀搀瀀漀猀猀椀戀氀攀爀漀氀攀猀愀猀瘀椀爀甀氀攀渀挀攀昀愀挀琀漀爀猀椀渀瘀漀氀瘀攀搀椀渀倀䌀䴀瀀愀琀栀漀最攀渀攀猀椀猀⸀ 䄀挀欀渀漀眀氀攀搀最攀洀攀渀琀㨀䌀漀氀挀椀攀渀挀椀愀猀 ⠀瀀爀漀樀攀挀琀一漀㈀㈀㄀㌀㐀　㠀㈀　㐀㐀㜀⤀Ⰰ 䌀漀爀瀀漀爀愀挀椀渀瀀愀爀愀䤀渀瘀攀猀琀椀最愀挀椀漀渀攀猀䈀椀漀氀最椀挀愀猀愀渀搀唀渀椀瘀攀爀猀椀搀愀搀搀攀䄀渀琀椀漀焀甀椀愀猀甀瀀瀀漀爀琀攀搀琀栀椀猀眀漀爀欀⸀ 吀栀攀一愀琀椀漀渀愀氀䐀漀挀琀漀爀愀氀倀爀漀最爀愀洀漀昀䌀伀䰀䌀䤀䔀一䌀䤀䄀匀猀甀瀀瀀漀爀琀攀搀䤀猀愀甀爀愀吀漀爀爀攀猀愀渀搀伀爀瘀椀氀氀攀䠀攀爀渀渀搀攀稀⸀ 圀攀猀椀渀挀攀爀攀氀礀琀栀愀渀欀䐀爀愀䈀攀愀琀爀椀稀䜀洀攀稀戀礀栀攀爀欀椀渀搀愀猀猀椀猀琀愀渀挀攀椀渀瀀爀漀瘀椀搀椀渀最琀栀攀䈀䨀㐀洀漀渀漀挀氀漀渀愀氀愀渀琀椀戀漀搀礀⸀

Keyword: Paracoccidioides brasiliensis, PbP27, Gene Silencing, Real time PCR, Agrobacterium tumefaciens

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