MUTAGENESIS OF THE DENGUE VIRUS PROTEASE

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)中的大多数卵裂 蛋白质区域受病毒编码的蛋白酶的影响，该酶包括 两种蛋白质，NS2B和NS3。NS3含有蛋白水解酶活性部位，但 人们对NS2B的作用知之甚少。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 一直在寻求两种方法来诱变4型登革热病毒 (DEN4)NS2B和NS3，努力更好地理解这两个 组成蛋白酶。通过选择进行诱变。之前，我们克隆了 在tac启动子控制下，将DEN4插入到pKK223-3载体中。 该表达载体可以在没有IPTG的情况下在大肠杆菌JM110中生长，但 在大肠杆菌HB101中，只会形成非常微小的菌落。我们推论说 当表达该酶时，该酶具有活性，对E. Coli.我们选择了HB101上罕见的大菌落，对它们的序列进行了测定 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‘基因被克隆到框内 在酶的下游。我们用该质粒转化JM110，并 在IPTG和XGAL存在的情况下，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 移码或胡说八道，因为它们是蓝色的，因此表达了紫胶 Z‘片段。然而，几个这样的假定突变体的测序 质粒显示的大部分是野生型的蛋白酶序列，有两个例子 无意义的突变。我们不明白为什么选拔不是 按计划工作。NS2B的定点突变。我们已经有了 制造了20个寡核苷酸，每个都可以用来随机突变两个 NS2B的40个氨基酸必需结构域的连续残基。 突变正在使用一种商业试剂盒在M13克隆中进行。 然后，必须将突变体亚克隆到pTM1载体中进行分析 它们的表型。到目前为止，我们已经成功地进行了突变 与我们尝试的前两个寡聚和几个突变体的反应 已经被确认，并正在亚克隆。

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.