RESUMEN
Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P < 0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.
Asunto(s)
Animales , Femenino , Humanos , Adenoviridae , Genética , Metabolismo , Células Epiteliales , Metabolismo , Vectores Genéticos , Genética , Cabras , Células HEK293 , Glándulas Mamarias Animales , Biología Celular , Proteína Relacionada con la Hormona Paratiroidea , Genética , Interferencia de ARN , ARN Mensajero , Genética , ARN Interferente Pequeño , Genética , Proteínas Recombinantes , Genética , TransfecciónRESUMEN
deltaFosB, a naturally occurring truncated isform of fosB gene, existed in many tissues stably and played an important role in formation and differentiation of adipocyte and osteoblast. deltaFosB may be related to the metabolism of calcium in bone and mammary gland and regulate the signal pathway of calcium transfer from bone to mammary gland. We first sub-cloned deltafosB gene of goat into the vector pET32a to construct prokaryotic expression vector pET32a-deltafosB. Then we induced for deltafosB gene expression efficiently by IPTG. Finally we immunized the adult rabbits with purified recombinant deltaFosB to prepare rabbit anti-goat deltaFosB polyclonal antibody. iELISA analysis showed the antibody with the titer of 1:51 200, and Western blotting result showed that the antibody could specifically detect the deltaFosB protein expressed in prokaryotic cell and HEK-293 cell, respectively. Further Western blotting assay showed that deltaFosB expressed in various tissues of goat in vivo.