猪胎盘PPARγ编码序列的克隆、序列分析和表达  

Cloning,sequence analysis and expression of PPARγ coding sequence of pig placentas

作　　者：张居作[1] 徐君飞[2] 薛立群[1] 谢杨[1] 聂昂 王婕敏 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]怀化学院生命科学系,湖南怀化418000

出　　处：《中国兽医学报》2016年第8期1318-1323,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31171438;31272630);国家重点基础发展规划项目("973"计划)资助项目(2012CB126310);教育部科学技术研究重点资助项目(21221)

摘　　要：过氧化物酶增殖物激活受体r(peroxisome proliferator-activated receptor type gamma,PPARγ)对人和小鼠的胎盘形成起重要作用,但在猪胎盘中其编码序列和作用还不明确。为了掌握母猪胎盘PPARγ剪辑方式和准确的编码序列,分析其结构与功能的关系,应用高保真PCR技术从4个不同妊娠时期的7份胎盘样品(61个胎囊)克隆出了一致性的PPARγ目的片段(KM382175),其编码序列和预测蛋白序列与人、鼠、牛、羊的具有较高的相似性;同时发现胎盘PPARγ蛋白与猪卵巢来源和猪皮下脂肪来源PPARγ蛋白在配体结合区分别存在1个和2个氨基酸残基差异。构建pET28α(+)-PPARγ表达质粒,重组蛋白应用兔抗PPARγ和鼠抗His单克隆抗体2种抗体Western blot双重检测阳性。研究表明,妊娠期猪胎盘PPARγ剪辑方式一致(1 428bp),稳定的PPARγ结构对正常生理功能的维持具有重要意义,但配体结合区氨基酸的改变可能意味着组织差异性的存在;本研究同时实现了PPARγ的原核表达,将为进一步研究PPARγ对胎盘的作用及其与母猪产仔性能的关联性提供基础理论指导。Peroxisome proliferator-activated receptor gamma （PPARγ） plays important roles in pla- cental development of human and mouse, but is still elusive in pig. To acquaint the splicing way of PPARy and precise coding sequence of pig placentas,and analyze the relationship of structure and function,the same fragment was found from seven placental samples of four pregnant stages using high fidelity PCR. Its coding sequence （KM382175） was high similar to that from Homo sapiens, Mus musculus ,Bos taurus and Caprahircus. But there was one two amino acid residue differences compared with that from ovary and subcutaneous fat respectively. The placentas PPARγ was successfully expressed in E. coli BL21 （DE3） using constructed pET28a（＋）-PPARγ. The recombi- nant PPARγ was determined using Western blot hybridized with both rabbit anti-PPARy antibody and mouse anti-His monoclonal antibody,the successfully prokaryotic expression of EPARγ. The present study showed that the clip way of placental PPARγ was kept during pregnancy,in which stable structure was significant for maintaining normal physiological function,the change of amino acid residue might mean tissue variability. These results laid a foundation for further investigation of PPAR）＇ function for placenta and the correlation with farrowing performance.

关 键 词：猪胎盘 PPARΓ 序列分析 

分 类 号：S852.1[农业科学—基础兽医学]