论文部分内容阅读
AIM To amplify HBV-RNase H gene fragment and expressionof RNase H for further use in the studies of HBV associatedliver diseases.METHODS:The encoding gene of HBV-RNase H wasseparately amplified for the first half and second half(H1and H2)by PCR from full length HBV gene and cloned intopT7Blue-T vector.Clones were first screened by digestionwith XbaⅠ and Hind Ⅲ enzyme for the correct size,andanalyzed further by DNA sequencing.The RNase H1 and H2fragments isolated from XbaⅠ and HindⅢ digestion productsof pT7 Blue-RNase H plasmid were ligated to the GSTagexpressing vectors separately,and expressed in E.coliBL21.The expressed proteins were checked by PAGE gel andWestern blot.RESULTS:Both H1 and H2 nucleotide seqences consistingof known genes and proteins,in correct size,were furtherconfirmed by Western blot to be the GST and RNase H1 orH2 fusion proteins.CONCLUSION:The successful cloning and expression ofHBV-RNase H will contribute to further research andapplication in HBV-associated diseases,
AIM To amplify HBV-RNase H gene fragment and expression of RNase H for further use in the studies of HBV associatedliver diseases.METHODS: The encoding gene of HBV-RNase H wasseparately amplified for the first half and second half (H1and H2) by PCR from full length HBV gene and cloned intopT7Blue-T vector.Clones were first screened by digestionwith XbaI and HindIII enzyme for the correct size, andanalyzed further by DNA sequencing. RNase H1 and H2fragments isolated from XbaI and HindIII digestion products of pT7 Blue-RNase H plasmid were ligated to the GSTagexpressing 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 furtherconfirmed byWestern blot to be the GST and RNase H1 orH2 fusion proteins. CONCLUSION: The successful cloning and expression of HBV-RNase H will contribute to further research and application in HBV-associat ed diseases,