Hepatitis B Virus And Adeno-Associated Assessment Answer

Answer:


A pHBV 1.3-mer WT replicon contains 1.3 copies of the HBV genome. This copy was utilized to generate the HBV fragment. However, to successfully conduct the experimental process the fragment is required to get cloned into vector of AAV. Firstly, the p-AAV need to be digested using restriction digestion enzyme Xba1 in order to generate a linear vector. On the other hand, pHBV 1.3-mer WT replicon can be digested using SacI and HindIII restriction enzyme which is ideal for inserting the fragment. Furthermore, the linearized p-AAV backbone and the pHBV 1.3-mer WT replicon can be blunted using the Klenow I. Moreover, the blunting process can be followed by using a ligation method. This ligation method is generally conducted by using the T4 ligase enzyme (Ko et al., 2014). However, there are other restriction enzymes that can also be used to digest pHBV 1.3-mer WT replicon, the donor plasmid, to get the insert the whole HBV genome. It has been noted that, in pHBV 1.3-mer WT replicon, the NcoI resides in the open reading frame of the HBx gene, moreover this gene is represented twice in the 1.3-mer viral genome. On the other hand, PstI-NcoI restriction site is present in the downstream which can be also used to generate a monomeric circular HBV genome containing an intron-like plasmid backbone. This are the possible methods that can be implemented to digest pHBV 1.3-mer replicon to get insert the whole HBV genome and the following restriction digestion enzymes can be used to properly complete the experimental setup (Weber et al., 2014).

Reference

Ko, C., Lee, S., Windisch, M. P., & Ryu, W. S. (2014). DDX3 DEAD-box RNA helicase is a host factor that restricts hepatitis B virus replication at the transcriptional level. Journal of virology, 88(23), 13689-13698.

Weber, N. D., Stone, D., Sedlak, R. H., Feelixge, H. S. D. S., Roychoudhury, P., Schiffer, J. T., ... & Jerome, K. R. (2014). AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

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