汉坦病毒糖蛋白G1酵母双杂交诱饵载体的构建及初步鉴定

拟利用Ras募集系统(RRS)构建并鉴定含汉坦病毒囊膜糖蛋白GI的诱饵载体。将汉坦病毒76—118株囊膜糖蛋白GI基因与Ras基因连接，构建嵌合基因Ras—G1及Ras—G1’(G1’无前导肽序列)。将两个嵌合基因克隆入酵母表达载体pMet25，并转染至酵母温度敏感株cdc25-2，检测其对RRS系统的激活作用。限制性内切酶酶切鉴定表明，Met—G1和Met—G1’载体构建正确。激活试验为阴性。说明Met—G1和Met—G1’载体可用于从cDNA库中筛选汉坦病毒受体。

The construction and identification of Ras-G1: the bait of Ras recruitment system

To construct and identify bait recombinant vector of hantaan virus(HTNV)glycoprotein G1by using Ras recruitment system(RRS).The glycoprotein of HTNV G1and G1'(mutant of G1without the signal sequence)were ligated with Ras gene at the C terminal of Ras,respectively.Then the two chimeric genes were cloned into pMet25the expressing vector of RRS.The recons were transfected into cdc25-2the temperature sensitive mutant and were tested to detect activation of the recons to RRS.The bait recombinant vectors Met-G1and Met-G1' were proved to be successfully constructed by restriction enzyme analysis.The result of activation test was negative.Met-G1and Met-G1' could be used to screen receptor of HTNV from cDNA library.