Expression and purification of active HBV RNase H and its application in drug screening
10.3760/cma.j.issn.1003-9279.2015.06.002
- VernacularTitle:乙型肝炎病毒活性RNase H酶的表达纯化及在药物筛选中的应用
- Author:
Gaofeng LU
1
;
Yong YU
;
Chenyi SUN
;
Jing CUI
;
Pengyuan ZHENG
Author Information
1. 450014,郑州大学第二附属医院消化内科
- Keywords:
Hepatitis B Virus;
RNase H;
Activity;
Drug
- From:
Chinese Journal of Experimental and Clinical Virology
2015;29(6):470-474
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify active HBV RNase H in vitro candidate for drug screening.Methods Codon-optimized cDNA sequences for genotype C HBV RNase H was cloned by gene synthesis into pMAL-c5xHis with a N-terminal MBP tag and C-terminal hexahistidine tag.HBV RNase H was expressed in E.coli BL21 cells.RNase H proteins were enriched by nickel-agarose affinity chromatography and identified through SDS-PAGE gel electrophoresis and Western blot.An oligonucleotidedirected RNA cleavage assay was used to measure the RNase H activity,the stability to temperature and the sensitivity to HBV RNase H inhibitor.Results Large amounts of soluble recombinant HBV RNase H was expressed and purified.Purified HBV RNase H could be detected by both Coomassie staining and Western blot analysis.Data demonstrate that the purified HBV RNase H has specific activity and good sensitivity to HBV RNase H inhibitor.Conclusions Recombinant HBV RNase H proteins with specific activity can be expressed in E.coli and enriched by nickel-agarose affinity chromatography,and can be used for biochemical screening of HBV RNase H inhibitors.
