Fifty ml of plasma sample (HBsAG+ and HBV DNA+) was used in the preparation of hepatitis B virus particle by sucrose density gradient centrifugation. The partially double stranded viral DNA was initially repaired by incubation with DNA polymerase I, then dissociated from its protein coat by treatment with proteinase K and sodium dodecyl sulfate and finally extracted with phenol chloroform. The purified HBV DNA was digested with BamH1 restriction enzyme, then ligated into plasmid vector pBr332 that has been previously treated with BamH1 and dephosphorylated by bacterial alkaline phosphatase. Transformed cells (JP101) bearing hepatitis B virus DNA insert were selected by their resistance/sensitivity to two antibiotics: ampicillin and tetracycline. Nineteen colonies presumably bearing the hepatitis B virus DNA insert were initially identified, but will have to be subjected for further analysis.
Production of a cloned radioactive HBV DNA that can be utilized as a specific probe in molecular hybridization studies for the detection of HBV DNA in serum and liver samples of patients with liver diseases.
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