Protein Localization And Construction And Identification Of Recombined RNA Interference Lentivirus Of Canine ERβ Isoforms

Beagle dog has constitution advantages which is widely used in national novel drugexperience. But Beagle dogs widely appears a number of reproductive issues，such as nooestrus，oestrus but not pregnant，long dioestrus and less the amount of born. In order to explorethe mechanism of decreasing production rate and quality of Beagle dogs,the study discussedBeagle dogs ERβ isoforms protein location and biological function. Recombinant plasmids ofBeagle dogs ERβ isoforms were built and transfected transiently into HEK293T cells to expressand localize ERβ isoforms proteins in HEK293T cells;The stable expression cell lines of ERβisoforms gene were established and retroviral-mediated short hairpin RNA(shRNA) expressionvectors of ERβ isoforms were constructed and to be packaged as lentivirus, the specificinhibition effects of ERβ isoforms were explored in the stable expression cell lines usingqRT-PCR and Western Blot assays.The results showed that recombinant eukaryotic expressionvectors ERβ isoforms were constructed，and were transfected into HEK293T cells successfully.WB results showed that ERβ1293codes430aa and the protein molecular weight is48kDa.ERβ1257codes419aa，the protein molecular weight is47kDa. Subcellular localization of ERβisoforms in HEK293T cells shows that ERβ1293localizes in cytoplasm and ERβ1257localizesin nucleus;Stable expression cell lines of ERβ isoforms gene were establishedsuccessfully;Retroviral-mediated short hairpin RNA(shRNA)expression vectors of ERβ isoformswere constructed to pGLV3/H1/GPF+puro and to be packaged as lentivirus. qRT-PCR andWestern Blot assays identified ERβ isoforms shRNA sequences from mRNA and proteins.Theresults showed ERβ1293-s hRNA1（p<0.01）and ERβ1293-s hRNA3（p<0.01）differed significantly,ERβ1257-shRNA1（p<0.01） and ERβ1257-s hRNA3（p <0.01）differedsignificantly,ERβ1293-shRNA3and ERβ1257-shRNA3are the best choices.The conclusionssuggested that Beagles dogs ERβ1293and ERβ1257proteins molecular weight are respectively48kDa and47kDa, and locate in cytoplasm and nucleus, Successfully identified the best shRNAsequences of target genes and farther explored the biological function of Beagle dogs ERβisoforms.