致鹅卵黄性腹膜炎大肠杆菌30S核糖体蛋白S6的原核表达及纯化  

Prokaryotic expression and purification of 30 S ribosomal protein S6 of salpingitis-peritonitis Escherichia coliisolated from layer geese

作　　者：金文杰[1] 张勇攀[1] 钱文正[1] 邵红霞[1] 钱琨[1] 秦爱建[1] 

机构地区：[1]扬州大学兽医学院教育部禽类预防医学重点实验室,江苏扬州225009

出　　处：《中国兽医科学》2012年第11期1174-1178,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30800822);江苏省"青蓝工程"项目;教育部"长江学者和创新团队发展计划"创新团队项目(IRT0978);江苏省优势学科项目(PAPD)

摘　　要：根据已发表的30S核糖体蛋白S6(RPS6)基因序列,设计合成了1对针对RPS6的特异性引物,用PCR方法从致鹅卵黄性腹膜炎大肠杆菌中扩增出RPS6基因,并将扩增的目的片段克隆至pGEM-TEasy载体中。测序正确后将RPS6基因片段克隆进表达载体pET-32a(+)中,提取pET-32a(+)-RPS6质粒,转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达。结果显示,PCR产物大小为396bp,与GenBank中同源序列的相似性为99.7%。SDS-PAGE分析结果表明,构建的重组RPS6在大肠杆菌中获得了可溶性表达,分子质量约为34ku,大小与预期相一致。HisTrap FF镍柱纯化大量表达的RPS6融合蛋白(His-RPS6),证实得到了高纯度的重组蛋白,为该蛋白功能研究提供了条件。According to the 30 S ribosomal protein S6（RPS6） gene sequence,a pair of specific primers was designed. The genomic DNA was extracted from salpingitis-peritonitis Escherichia coli strain isolated from layer geese and used as template to amplify the RPS6 gene by PCR. The RPS6 fragment was then cloned into the pGEM-T Easy vector and sequenced. The result showed that the RPS6 fragment was 396 bp. Comparing with the sequences of the RPS6 gene deposited in the GenBank,the homology was 99.7% with other E. coli. The RPS6 fragment was digested and cloned into the expression vector pET-32a （＋）, and then transformed into competent E. coli BL21 （DE3） cells. The positive recombinant pET-32a（＋）- RPS6/DE3 clones were identified by double enzyme digestion and then expressed by IPTG induction. SDS-PAGE analysis indicated that the expressed fusion protein was 34 ku and was soluble. Using the HisTrap FF Ni2＋ column,the protein His-RPS6 was purified. This recombinant protein provided basis for function research of RPS6.

关 键 词：致鹅卵黄性腹膜炎大肠杆菌 30S核糖体蛋白S6(RPS6) 原核表达 纯化 

分 类 号：S852.612[农业科学—基础兽医学]