Cloning Of Porcine Siglec-1Gene Promoter And Its Verification Of PAM-Specific Expression

PAMs are the target cells infected by PRRSV in porcine. It is very important to screen the promoter of porcine PAM-specific expression gene, which could iniciate efficient and specific expression of anti-PRRSV exogenous genes in target cells. Without transcriptional factors, the polymerase can’t initiate transcripiton, and eukaryotic genes would not express. Only if the Transcriptional fators recognize and bind to the DNA sequences, the genes begin to transcript. Therefore, the investigation of specific gene promoter expression includes both definition of cis-acting elements regions and regulation of transcription factors in vitro.Accumulating evidence indicates that the sialoadhesin (Sn, also called Siglec-1or CD169) was an essential PRRSV receptor that mediates both attachment and internalization on macrophages, highly expressed on porcine alveolar macrophages (PAM). In this study, The different length promoters of Siglec-1gene were cloned and their function was verified in PAM, Preadipocyte, PK-15and PEF, by dual-luciferase reporter assay system. We obtained the promoter region with specific regulatory function and did some insight into the mechanisms underlying the highly expression in PAM. The approaches are as follows:1. The Siglec-1gene transcription start site is determined by rapid amplification of cDNA ends (RACE) technique.The Siglec-1gene mRNA sequence was downloaded from the NCBI, and gene specific primers were designed for5’-RACE. According to Smart RACE kit, Siglec-1gene5’untranslated region (5’-UTR) was cloned and C was determined as its transcription start site, which located at88bp upstream of initiation codon ATG. Siglec-1gene transcription start site settles a basis for the study of transcriptional regulation of the gene.2. Deletion expression vector construction of Siglec-1gene promoterFirst, the use of bioinformatics methods to find porcine Siglec-1gene5’regulatory sequences and predict the location of promoter as well as transcription factor binding sites; Second, application of different lengths of promoters fragments by PCR, which connected to the firefly luciferase reporter gene vector pGL3-Basic. Nine recombination expression vectors, such us pLUC173, pLUC574, pLUC691, pLUC740, pLUC869, pLUC90, pLUC1444, pLUC2188, pLUC3412were successfully constructed identified by restriction enzymes digestion and DNA sequencing.3. Activity analysis of deletion promoter and cell-specific verificationDeletion expression vectors and Renilla luciferase reporter vector as20:1were co-transfected porcine alveolar macrophages. After24hours, the luciferase activity was detected by dual-luciferase reporter assay system, the value of pRL-TK luciferase activity as an internal control and pGL3-Basic luciferase activity as a basis value, pLUC173as core promoter region was screened by promoters activity analysis, while the pLUC869exhibited the highest activity. The cell-specific promoter was verified by transient transfection in other non-macrophages such as kidney cells, fat precursor cells and porcine fetal fibroblast cells.4. Site-directed mutagenesisThe combination of bioinformatic approaches and experimental process were led to understand the mechanisms of the transcription factor PU.1in regulating the higly expression of Siglec-1gene. Using site-directed mutagenesis, we found that the Siglec-1gene was regulated by transcription factor PU.1. In addition, mutation of the PU.1consensus remarkably decrease Siglec-1expression. This prompted that the transcription factor PU.1may play an important role in the regulation of highly expression in PAM and PAM-specific expression of Siglec-1gene.