畜牧兽医学报 ›› 2007, Vol. 38 ›› Issue (7): 625-629.doi:

• 遗传繁育 •    下一篇

猪Sar1b基因的分子克隆、序列分析及原核表达

王学敏;刘榜;赵书红;樊斌;朱猛进;余梅;熊统安;李奎   

  1. 1.华中农业大学动物遗传育种与繁殖教育部重点实验室,武汉 430070;2.江苏省农业科学院畜牧研究所,南京 210014;3.中国农业科学院北京畜牧兽医研究所,北京 100094
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-07-25 发布日期:2007-07-25

Molecular Cloning, Sequence Analysis and Prokaryotic Expression of the Porcine Sar1b Gene

WANG Xue-min;LIU Bang;ZHAO Shu-hong;FAN Bin;ZHU Meng-jin;YU Mei;XIONG Tong-an;LI Kui   

  1. 1.Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education,Huazhong Agricultural University, Wuhan 430070, China;2.Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;3.Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2007-07-25 Published:2007-07-25

摘要: 根据GenBank已发表的猪Sar1b基因序列(GenBank登录号:AY819557)设计1对引物,以猪肝脏组织总RNA的反转录产物为扩增模板,用RT-PCR方法扩增出猪Sar1b基因cDNA全长编码区,经过EcoRI-SalI双酶切后定向克隆于pET28a原核表达载体,获得pET28a-Sar1b重组原核表达载体。将携带有重组原核表达载体的大肠杆菌BL21(DE3)通过1 mmol/L ITPG进行诱导表达,经过SDS-PAGE电泳检测,显示诱导表达蛋白大小大约为26 ku,与预期表达蛋白大小一致。Western blot检测显示该蛋白为His融合蛋白,表明重组原核表达载体在大肠杆菌中成功表达出了目的融合蛋白。猪Sar1b基因的克隆和表达研究,为进一步研究该基因的生物学功能奠定了基础。

Abstract: A pair of primers was designed based on the published nudeotide sequence of the porcine Sar1b gene (GenBank accession number: AY819557). Total RNA was isolated from the pig liver tissue and the first-strand cDNA was generated using reverse transcription reactions. The full-length coding sequence of the porcine Sar1b cDNA was amplified by RT-PCR. The product was double-digested by EcoRI and SalI, and then directionally cloned into the pET28a vector. The recombinant expression plasmid in the E. coli BL21 (DE3) was induced with 1 mmol/L ITPG. SDS-PAGE analysis showed that the induced expressed protein was about 26 ku. Western blot revealed that the expressed protein was the His tag fusion protein, which indicated that the recombinant prokaryotic expression vector expressed the fusion protein successfully. The cloning and expression of the porcine Sar1b gene established the foundation for further study on its biological function.