Preparation And Identification Of Recombinant Adenoviruses Carrying Short Hairpin Rna Targeting PTHrP Of Goat

PTHrP (Parathyroid hormone related protein) has extensive biological functions and plays an important role in regulating bone turnover during lactation. The aim of this study was to silence the expression of PTHrP by the RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. the main results were as follows:The Block-iT shRNA interference system was used in this experiment. According to the goat PTHrP sequence, we designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322,shRNA-357), then oligonucleotides were cloned into shuttle vector pENTR/CMV-GFP/U6. The western blotting test of PTHrP proteins in HEK-293 cells, cotransfected with pAdTrack-CMV-PTHrP, has shown pENTR/CMV-GFP/ U6-322 has better interference effects. The recombinant vector pAD/PL-DEST/CMV-GFP/ U6-322, which can express the Short hairpin RNA of PTHrP gene, produced by the LR reaction between recombinant vector pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST.Linear products of pAD/PL-DEST/CMV-GFP/U6-322, digested by PacⅠrestriction enzyme, was used for transfecting HEK-293 cells. After 12 days, the first generation adenovirus were collected when most of cells began to appear cytopathic effect, then AD-PTHrP-322 adenovirus were obtained by repeated the processing several times. The titer of adenovirus reached 2.0×109 PFU/mL determined by TCID50 assays. We proved the optimum MOI of AD-PTHrP-322 was 200 by infecting goat mammary gland epithetlial primary cells. The Real-time Quantitative PCR results corroborated its results and shown that AD-PTHrP-322 infect goat mammary epithelial cells after 24 h, 48 h and 72 h, the mRNA of PTHrP gene expression levels were reduced 29.2%, 68.1% and 82.6%. The detection of PTHrP protein expression used Western blotting was consistent with the quantitative. AD-PTHrP-322 has obvious interference effect within 72 h and can be used in the further study of gene function.