摘要
对东方粘虫Mythimna separate(Walker)中肠V-ATPase H亚基基因(VATPH)进行克隆、原核表达及纯化。首先采用RT-PCR技术克隆基因,再构建原核表达载体(pET15b-VATPH)并转入E.coil BL21(DE3),经异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达,用Ni-NTA柱及S-200分子筛纯化,最后进行SDS-PAGE分析。结果表明,pET15b-VATPH可高效表达V-ATPase H亚基,纯化后可获得单一条带且分子质量约为55ku的重组蛋白。该结果为进一步研究V-ATPase H亚基的晶体结构,药物小分子与H亚基大分子的相互作用,尤其是以H亚基为筛选模型创制新农药奠定基础。
A V-ATPase subunit H gene （VATPH） in the midgut of Mythimna separate was cloned into pET15b vector containing histidine. A pure V-ATPase subunit H was obtained after being trans- ferred into E. coli BL21 （DE3）, induced with IPTG and purified with a histrap chromatogram column and S-200 orderly. SDS-PAGE was used to analysis the recombinant protein expression, and a parti cular 55 ku protein band was visualized successfully. The results will facilitate the investigation of crystal structure of V-ATPase subunit H, interactions of small-molecule drugs with protein and the creation of environmental friendly pesticides acted on the target of V-ATPase subunit H.
出处
《西北农业学报》
CAS
CSCD
北大核心
2014年第7期102-106,共5页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金项目(31371958)
中央高校基本科研业务费专项资金(QN2011008)
关键词
东方粘虫
V-ATPASE
H亚基
原核表达
纯化
Mythimna separate
V-ATPase subunit H
Protein prokaryotic expression
Purification
