Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers.
Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers.
复制标题
用于将单体乙型肝炎病毒 DNA 构建体有效转化为串联二聚体的双向分子连接
DOI：
10.1016/j.jviromet.2016.03.012
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发表时间：
2016-07
影响因子：
3.1
通讯作者：
Tong S
中科院分区：
文献类型：
--
作者：
Zong L;Qin Y;Jia H;Zhou H;Chen C;Qiao K;Zhang J;Wang Y;Li J;Tong S
3.2-kb的B型肝炎病毒（HBV）基因组的复制是由核中的共价闭合环状（ccc）DNA驱动的，从所述ccc DNA转录四类共末端RNA。基因组复制只需要3.5 kb的前基因组RNA，这是末端冗余的。将全长HBV基因组克隆到载体中破坏了其连续性，从而阻止了前基因组RNA转录步骤中的基因组复制。这可以通过将单体构建体转化为串联二聚体来克服，但是需要将HBV基因组的两个分子与载体DNA连接使得其效率低下甚至不成功。为了克服这个问题，我们用用于克隆的独特限制性内切酶部分消化了单体构建体，并在与HBV基因组的另一个拷贝连接之前将线性化的单体去磷酸化。或者，将单体在HBV基因组内的另一个独特限制性位点线性化，然后将其去磷酸化并与在相同位点线性化的HBV基因组的另一个拷贝连接。这些双向分子连接的方法大大提高了二聚体形成的效率，约50%的细菌菌落筛选窝藏串联二聚体。
Replication of the 3.2-kb hepatitis B virus (HBV) genome is driven by the covalently closed circular (ccc) DNA in the nucleus, from which four classes of co-terminal RNAs are transcribed. Genome replication requires just the 3.5-kb pregenomic RNA, which is terminally redundant. Cloning the full-length HBV genome into a vector disrupts its continuity, thus preventing genome replication at the step of pregenomic RNA transcription. This can be overcome by converting the monomeric construct into a tandem dimer, yet the need to ligate two molecules of the HBV genome with vector DNA makes it inefficient and even unsuccessful. To overcome this problem we partially digested the monomeric construct with the unique restriction enzyme used for cloning, and dephosphorylated the linearized monomer before its ligation with another copy of the HBV genome. Alternatively, the monomer was linearized at another unique restriction site inside the HBV genome, followed by its dephosphorylation and ligation with another copy of the HBV genome linearized at the same site. These approaches of two-way molecular ligation greatly improved the efficiency of dimer formation with about 50% of the bacterial colonies screened harboring tandem dimers.