Constructing series of clones with different functional regions deletion of PEPV 5′Untranslated region and sequence analysis

To analyze the transcriptional regulation of PERV untranslated region,A series of functional region deletion of PERV 5′UTR were cloned,including completed 5′UTR,5′UTR lacking of PBS and Leader(5′UTR),5′UTR lacking of U3 region,5′UTR lacking of R region,5′UTR devoid of the U3 repeat box.First,we use PCR to amplify the above five fragments,then ligated them to pMD18-T vector,chose the positive clones,restricted them with enzymes to identify the orient of the inserts.The plasmids LTR-PMD,UTR-PMD,UTR-U3-PMD,UTR-R-PMD,UTR-U-PMD were constructed for further use,the 5′UTR sequenced and the potential binding of transcription factors(TFS) predicted by the Kyoto Encyclopedia of Genes and Genomes(KEGG).It is concluded that there have different potential binding of TFS different functional region of PERV 5′UTR;When passaging the WZSP-PERV in HEK293 cells,not only the number of U3 repeat box increased but also the subtype changed.