猪SERPINC1基因对PCV2复制的效应及其转录调控

Effect of SERPINC1 Gene on PCV2 Replication and Its Transcriptional Regulation

旨在研究猪SERPINC1基因对PCV2复制的影响,并探究SERPINC1基因的转录调控。本研究以15头纯种的莱芜猪(LW)和15头大约克夏-长白杂交的商品猪(YL)为试验动物。每个品种分为2组:接毒组(10头)和对照组(5头)。对接毒组的猪肌肉注射3 mL 6.3×10^-3 TCID50的PCV2-SD毒株,对照组的猪肌肉注射3 mL的磷酸盐缓冲液。在前期转录组测序的基础上,分析SERPINC1基因对PCV2复制的效应,并分析检测SERPINC1基因启动子和转录活性。结果表明,过表达SERPINC1能显著抑制猪肺泡巨噬细胞(PAM)中PCV2的复制。分别克隆了LW和YL猪SERPINC1基因转录起始位点上游3 854 bp的序列,并进行启动子活性的分析,发现PCV2接毒后LW猪SERPINC1基因的启动子活性显著升高(P<0.05),而YL猪的启动子活性无显著变化。在SERPINC1基因的5′调控区找到4个调控该基因转录的关键区段,并对上述关键调控区中的4个多态性位点对SERPINC1基因启动子活性的影响进行了分析。综上表明,这4个多态位点均不影响SERPINC1基因的启动子活性。该研究结果为找到与猪抗PCV2有关的基因及分子标记奠定了基础。

The aim of this study was to investigate the effect of porcine SERPINC1 gene on PCV2 replication and to explore the transcriptional regulation of SERPINC1 gene.In this study,15 purebred Laiwu pigs(LW)and 15 commercial Yorkshire×Landrace hybrid pigs(YL)were used.There were 2 groups in each breed,one for the challenged group(10)and one for the control group(5).Pigs in the challenged group were intramuscularly injected with 3 mL of 6.3×10-3TCID50 PCV2-SD strain,and pigs in control group were intramuscularly injected with 3 mL of phosphate buffer.Based on the previous transcriptome sequencing,the effect of SERPINC1 gene on PCV2 replication was analyzed,and the promoter and transcriptional activity of SERPINC1 gene were detected.The results indicated that over-expression of SERPINC1 could significantly inhibit the replication of PCV2 in PAM cells.The 3 854 bp sequence of the 5′regulatory region of the SERPINC1 gene in LW and YL pigs was cloned respectively,and the promoter activity was analyzed.It was found that the promoter activity of SERPINC1 gene in LW pigs significantly increased after PCV2 infection(P<0.05),while that of YL pig didn’t significantly change.Four critical regulatory regions were found in the 5′regulatory region of the SERPINC1 gene.Four polymorphic sites were identified in the critical regulatory regions in LW and YL pigs.By site-directed mutation and dual luciferase reporter gene assay,we found that the activity of the SERPINC1 promoter was not affected by any one of the polymorphic sites in the above regions.The results laid a foundation for finding the gene and molecular markers related to the resistance of PCV2.