关键词: 山羊, PRNP基因, 启动子, 转录活性

Abstract: 【Objective】In order to screen the critical region or transcription factors regulating the expression level of prion in goat, the active region in the promoter of PRNP gene was analyzed. These results will provide a theoretical reference for elucidating the regulation of expression and thought for reducing the infectivity of prion disease with genetic strategy.【Method】The specific primer was designed based on the reference genomic sequence (GenBank Accession: EU870890). The fragment in a 5′ flanking region was amplified and cloned into the vector pEASY-T3. The positive colonies were identified and sequenced. The putative promoter and transcription factor binding sites were predicted with bioinformatic methods and online program. Eleven fragments with different lengths of promoter regions were amplified and cloned into the vector pEASY-T3. The positive colonies and vector pGL3-Basic were digested with two restriction enzymes Mlu I and Bgl II. The digested mixture were purified and ligated with T4 ligase to get the recombinant containing the luciferase reporter gene. The endo-free plamids were isolated after the positive colonies were obtained and sequenced. The transfection to SH-SY5Y was done with a Liposome reagent. The luciferase activity was measured with the dual-luciferase detection kit after 48 hours transfection.【Result】The length of the fragment in 5′ flanking region of PRNP gene in goat was 2 332bp. The predicted active region in the promoter, conserved motifs and multiple transcription factor binding sites were involved in the cloned fragment in the 5′ flanking region. Eleven different lengths of fragments were obtained and ligated with luciferase reported vector. The ratio of transfection reagent and DNA and Firefly luciferase vector and Renilla luciferase were 1﹕0.5 and 50:1 respectively. The core promoter was involved in 5′ flanking region of PRNP gene in goat and this region was from -519bp to +82bp. There were some positive regulatory elements in the region from -220 to +59. The exon 1 played a critical role in regulating the activity of the promoter. The positive regulating elements binding sites were predicted for four conserved motifs. There were many transcription factors, such as Sp1, AP-2 alpha and AP-1, binding sites in the strong active promoter. The transcription factors Foxp3 and COE1 binding sites were also predicted for motif 3 and 4 respectively. 【Conclusion】The core promoter region from -519bp to +82bp was identified in goat PRNP gene. The function of exon 1 in regulating promoter activity was critical.

Key words: goat, PRNP gene, promoter, transcriptional regulation