[阴道毛滴虫沉默信息调节因子2原核表达载体的构建及其表达]

[Construction of prokaryotic expression vector for Trichomonas vaginalis silent information regulator 2 and its expression].

作者信息

Zhang Ke-hao, Huang Li-xia, Fu Yu-cai, Zhang Jia-xin

机构信息

Taizhou Hospital of Zhejiang Province, Taizhou 317000, China.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Jun;24(3):IV-VI.

PMID:17094634

Abstract

Total RNA was isolated from Trichomonas vaginalis and Tv-Sir2-like cDNA was amplified by RT-PCR and cloned into pGEM-T Easy plasmid. A fragment of Tv-Sir2-like cDNA was subcloned into the expression vector pET-41b and expressed in E.coli BL21 with induction of IPTG. The full-length of Tv-Sir2-like cDNA was cloned and sequenced. The prokaryotic expression system of pET-41b/Tv-Sir2-like was constructed. The fusion protein of Tv-Sir2-like was expressed in E. coli BL21, occupying 30% of the total bacterial protein after being induced by IPTG for 5 h. SDS-PAGE analysis showed that the fusion protein was about Mr 59000. The recombinant protein of Tv-Sir2-like is efficiently expressed in E. coli BL21.

摘要

从阴道毛滴虫中分离出总RNA，通过逆转录聚合酶链反应（RT-PCR）扩增出阴道毛滴虫Sir2样cDNA，并将其克隆到pGEM-T Easy质粒中。将阴道毛滴虫Sir2样cDNA片段亚克隆到表达载体pET-41b中，并在异丙基-β-D-硫代半乳糖苷（IPTG）诱导下在大肠杆菌BL21中表达。对阴道毛滴虫Sir2样cDNA全长进行克隆和测序。构建了pET-41b/阴道毛滴虫Sir2样的原核表达系统。阴道毛滴虫Sir2样融合蛋白在大肠杆菌BL21中表达，IPTG诱导5小时后占细菌总蛋白的30%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，融合蛋白的分子量约为59000。阴道毛滴虫Sir2样重组蛋白在大肠杆菌BL21中高效表达。