MUTAGENESIS OF THE DENGUE VIRUS PROTEASE

登革热病毒蛋白酶的诱变

• 批准号: 3792563
• 负责人: B FALGOUT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792563
• 关键词: dengue virus; endopeptidases; gene mutation; molecular cloning; protein biosynthesis; transfection /expression vector; virus protein

Dengue virus encodes a polyprotein that is processed to mature gene products. The majority of the cleavages in the nonstructural (NS) protein region are effected by a virus-encoded protease, consisting of two proteins, NS2B and NS3. NS3 contains the protease active site, but the role of NS2B is less well understood. Recently, we showed that a forty amino acid domain in NS2B is required for protease activity. We have been pursuing two approaches to mutagenize dengue virus type 4 (DEN4) NS2B and NS3, in an effort to better understand this two component protease. Mutagenesis by selection. Previously, we cloned the DEN4 into the pKK223-3 vector under control of the tac promoter. This plasmid could be grown in E. coli JM110 in the absence of IPTG, but in E. coli HB101 only very tiny colonies would form. We reasoned that when the protease was expressed it was active and detrimental to E. coli. We selected rare large colonies on HB101, sequenced their plasmids, and found that they were all frameshift or nonsense mutations. To try to get around this problem, the lac Z' gene was cloned in-frame downstream of the protease. We transformed JM110 with this plasmid and selected large blue colonies in the presence of IPTG and XGAL. These should have been protease mutants, since they were large, and not frameshift or nonsense, since they were blue and thus expressed the lac Z' fragment. However, sequencing of several such putative mutant plasmids revealed mostly wild-type protease sequences, with two examples of nonsense mutations. We do not understand why the selection is not working as planned. Site-directed mutagenesis of NS2B. We have had made 20 oligos that can each be used to randomly mutagenize two consecutive residues of the forty amino acid essential domain of NS2B. Mutagenesis is being done in an M13 clone using a commercial kit. Mutants will then have to be subcloned into a pTM1 vector for analysis of their phenotypes. So far, we have had successful mutagenesis reactions with the first two oligos we have tried, and several mutants have been identified and are being subcloned.

登革热病毒编码一种多蛋白，可加工成成熟基因 产品。 非结构 (NS) 中的大部分裂解 蛋白质区域受到病毒编码的蛋白酶的影响，包括 两种蛋白质，NS2B 和 NS3。 NS3含有蛋白酶活性位点，但是 NS2B 的作用尚不清楚。 最近，我们展示了一个 NS2B 中的 40 个氨基酸结构域是蛋白酶活性所必需的。我们 一直在寻求两种诱变 4 型登革热病毒的方法 (DEN4) NS2B 和 NS3，为了更好地理解这两个 成分蛋白酶。 通过选择诱变。 之前我们克隆了 将 DEN4 插入到 tac 启动子控制下的 pKK223-3 载体中。 该质粒可以在没有 IPTG 的情况下在大肠杆菌 JM110 中生长，但是 在大肠杆菌 HB101 中，只会形成非常小的菌落。 我们推断 当蛋白酶表达时，它是活跃的并且对大肠杆菌有害。 大肠杆菌。 我们在 HB101 上选择了罕见的大菌落，对它们进行了测序 质粒，发现它们都是移码突变或无义突变。 为了解决这个问题，lac Z' 基因被框内克隆 蛋白酶的下游。 我们用该质粒转化 JM110， 在 IPTG 和 XGAL 存在下选择大的蓝色菌落。 这些 应该是蛋白酶突变体，因为它们很大，而不是 移码或废话，因为它们是蓝色的，因此表达了 lac Z' 片段。 然而，对几个这样的假定突变体进行测序 质粒显示大部分野生型蛋白酶序列，有两个例子 无义突变。 我们不明白为什么选择不 按计划工作。 NS2B 的定点诱变。 我们有过 制作了 20 个寡核苷酸，每个寡核苷酸可用于随机诱变两个 NS2B 四十个氨基酸必需结构域的连续残基。 使用商业试剂盒在 M13 克隆中进行诱变。 然后，必须将突变体亚克隆到 pTM1 载体中进行分析 他们的表型。 到目前为止，我们已经成功诱变 与我们尝试过的前两个寡核苷酸以及几个突变体的反应 已被鉴定并正在被亚克隆。