Novel method for cloning of hepatitis B virus DNA using the In-Fusion enzyme

Abstract

Hepatitis B virus (HBV) infection is a great health problem not only in Egypt but also worldwide. High rate of mutations is a characteristic feature of HBV during the replication cycles. Mutations in different genes of HBV may result in vaccine escape, immune evasion, diagnostic failure and hepatocellular carcinoma. Therefore, continuous and updated reporting of mutations of whole genome of viruses is of great concern. This can be achieved through cloning of the whole genome into a suitable vector for further analysis of each gene separately. In this study, we cloned the DNA of Egyptian isolate of HBV in pUC19 vector using the In-Fusion enzyme. The tested isolate was confirmed to be serotype ayw3 and subgenotype D3. Utilizing the In-Fusion enzyme allowed fusion of the whole genome of the HBV isolate in the vector precisely in the correct orientation and without the use of any restriction enzymes. Depending on the number of the fused fragments, two strategies (A and B) were followed to ligate the PCR amplified fragments. Both strategies were successful in cloning, however strategy “A” (fusion of 3 fragments) resulted in a greater number of transformed colonies than strategy “B” (fusion of 4 fragments). To the best of our knowledge, our work is the first to use the In-Fusion technique as a novel cloning method for cloning of a whole viral genome without the use of any restriction enzymes.