东方粘虫中肠V-ATPase H亚基原核表达及纯化  

Prokaryotic Expression and Purification of V-ATPase Subunit H in the Midgut of Mythimna separate

作　　者：李秋利[1] 赵吉建 祁志军[1] 吴文君[1] 

机构地区：[1]西北农林科技大学农药研究所,陕西杨凌712100

出　　处：《西北农业学报》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)

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.

关 键 词：东方粘虫 V-ATPASE H亚基 原核表达 纯化 

分 类 号：Q786[生物学—分子生物学]