Expression of RNase H of human hepatitis B virus polymerase in Escherichia coli.
OBJECTIVE
To amplify HBV-RNase H gene fragment and expression of RNase H for further use in the studies of HBV associated liver diseases.


METHODS
The encoding gene of HBV-RNase H was separately amplified for the first half and second half (H1 and H2) by PCR from full length HBV gene and cloned into pT7Blue-T vector. Clones were first screened by digestion with XbaI and Hind III enzyme for the correct size, and analyzed further by DNA sequencing. The RNase H1 and H2 fragments isolated from XbaI and Hind III digestion products of pT7 Blue-RNase H plasmid were ligated to the GSTag expressing vectors separately, and expressed in E.coli BL21. The expressed proteins were checked by PAGE gel and Western blot.


RESULTS
Both H1 and H2 nucleotide seqences consisting of known genes and proteins, in correct size, were further confirmed by Western blot to be the GST and RNase H1 or H2 fusion proteins.


CONCLUSIONS
The successful cloning and expression of HBV-RNase H will contribute to further research and application in HBV-associated diseases.
Cheng
Hong
H
Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China. nelson@fmmu.edu.cn
Zhang
Hui-Zhong
HZ
Shen
Wan-An
WA
Liu
Yan-Fang
YF
Ma
Fu-Cheng
FC
D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006515:Hepatitis B virus / Q000378:metabolism*; D006801:Humans; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*
D004259:DNA-Directed DNA Polymerase; C091139:ribonuclease HII; D016914:Ribonuclease H; C078856:ribonuclease HI