Abstract
Hepatitis B virus (HBV) mutants can lead to vaccine failure, diagnostic failure of HBV detection, increase viral replication and resistance to antiviral agents. To study the biological characteristics of these mutations may contribute to our knowledge on viral pathogenesis. Therefore, it is essential to isolate and characterize HBV strains from patients. Here we describe the experimental methods to isolate and clone HBV DNA from patient serum. The method will facilitate isolation and functional analysis of new HBV variants.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Schematic diagram of cloning for HBV DNA. HBV DNA was extracted from patient serum as the PCR template. Primer P1 and P3 are used to amplify the 2.05 kb-HBV DNA fragment; Primer P2 and P4 are used to amplify the 1.15 kb-HBV DNA fragment. The two fragments were ligated into pGEM®-T Vector, respectively and subjected to sequence analysis. The 2.05 kb-HBV DNA fragment and 1.15 kb-HBV DNA fragment were digested from the right recombinant plasmid and were ligated into the pUC19 plasmid to form the recombinant pUC/HBV plasmid. Figure 2. Construction and Identification of pUC/HBV. A. Amplification of 1.15 kb HBV DNA fragment. Lane 1: Marker; Lane 2,3: samples. B. Amplification of 2.05 kb HBV DNA fragment. Lane 1: Marker; Lane 2,3: samples. C. Identification of pUC/HBV by Sac I and Spe I digestion. Lane 1: Marker; Lane 2: negative clone; Lane 3: positive clone.
Recipes
Acknowledgments
This protocol was modified from the previous work by Hui Shi and Liang Cao. This study was supported by the National Nature Science Foundation of China (31200699).
References
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