Screening of RNA interference sequence and construction and identification of BTN recombinant adenovirus of Xinong Saanen Goat.

[Objective] The aim of this project was to construct RNAi recombinant adenoviral vector specific to BTN gene and to establish foundation on function and mechanism of BTN gene.[Method] The first step was to design and synthesize three pairs of complementary single-strand DNA oligos targeting three various sites of BTN mRNA,and then oligos were cloned into pENTR/CMV-GFP/U6-shRNA,the entry vector,to generate the entry clone to co-transfect HEK 293 cells with pDsRed1-C1-BTN expressing BTN gene to select effective RNAi sequence.Recombination reaction in vitro with the pENTR and pAd/BLOCK-iTTM-DEST,the adenovirus backbone vector,were used to creat the adenovirus plasmid which expressed the interference of BTN gene.Then,we transfected the adenovirus plasmid digested with Pac Ⅰ into HEK 293 cells to produce adenovirus,and infected the 293 cells with the crude adenovirus to amply the adenoviral stock.TCID50 assay was used to titer the adenoviral stock and got a high titer.[Result] The RNAi adenovirus exppression vector targeting to BTN gene of Xinong Saanen Goat was constructed successfully,after being packaged and amplified,the titer of the adenovirus can reach 5×109 PFU/mL.[Conclusion] We got functional adenoviral recombination of pAd/PL-DEST/CMV-GFP/U6-shRNA.