Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting ΔFosB of goat | 靶向山羊ΔFosB基因的shRNA重组干扰腺病毒的制备及鉴定

Chino. ΔFosB作为FosB的一种截短型，具有广泛的生物学功能，在脂、钙沉积和药物成瘾等方面有重要作用。本研究采用RNA干扰技术，通过制备重组干扰腺病毒对山羊(Caprar hircus) ΔFosB基因进行沉默，进一步研究山羊ΔFosB基因在脂、钙沉积方面的相关功能及作用机制。实验采用BLOCK-iT shRNA腺病毒干扰系统，将预先设计好的寡聚shRNA-492/572经退火复性后克隆入穿梭质粒pENTR/CMV-GFP/U6中。经干扰效率检测后，选择pENTR/CMV-GFP/U6-572与病毒骨架质粒pAD/PL-DEST进行同源重组，获得重组干扰腺病毒载体pAD/PL-DEST/CMV-GFP/U6-572，并转染HEK-293细胞，经包装、扩增后即可产生干扰腺病毒AD-ΔFosB-572，TCID50法测定其滴度为1.58×109 PFU/mL。用AD-ΔFosB-572感染山羊的乳腺上皮细胞(MOI=200)，经Western blot及实时定量PCR检测证明在病毒感染24、48和72 h后，山羊乳腺上皮细胞中ΔFosBmRNA表达量分别下调了45%，73%和81%，其蛋白表达水平也明显下调，干扰效果明显，可用于后续的基因功能研究。

Inglés. ΔFosB, a truncated form of FosB, which impacts on fat and calcium deposition, drug addiction and other aspects, has extensive biological functions. The objective of this study was to silence the expression of ΔFosB using the RNA interference by recombinant adenovirus, and provided a material to investigate the relative functions of ΔFosB. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-492/572) which targeting two different sites of ΔFosB mRNA and then oligonucleotides were cloned into shuttle vector pENTR/CMV-GFP/U6. After detection of interference efficiency, we recombined a pENTR/CMV-GFP/U6-572 and adenovirus backbone vector pAD/PL-DEST, to produce recombinant vector pAD/PL-DEST/CMV- GFP/U6-572. The fifth generation recombinant adenovirus particles (AD-ΔFosB-572) were produced and further amplified by transfecting HEK-293 cells. The titer of adenovirus reached 1.58 × 109 PFU/mL determined by TCID50 assays. Western blot and Real-time Quantitative PCR indicated that AD-ΔFosB-572 had better interference efficiency by infecting goat mammary gland epithelial primary cells after 24, 48 and 72 h, in which mRNA expression levels of ΔFosB gene were reduced 45%, 73% and 81%，respectively. Western blot also showed that the expression of ΔFosB protein was reduced by infecting the cells with AD-ΔFosB-572. So AD-ΔFosB-572 has been proved that it has significant interference effect on expression of ΔFosB and can be used for subsequent studies of gene function.