本文選題：新城疫病毒　切入點：基質(zhì)蛋白　出處：《微生物學報》2017年01期 　論文類型：期刊論文

【摘要】：【目的】鑒定與新城疫病毒(Newcastle disease virus,NDV)基質(zhì)蛋白(matrix protein,M)入核相關(guān)的細胞蛋白,以闡明NDV M蛋白細胞核定位的分子機制�！痉椒ā繌碾u胚成纖維細胞中分別克隆核轉(zhuǎn)運受體蛋白KPNA1 KPNA6和KPNB1基因,將其構(gòu)建到真核表達載體,并與表達NDV M蛋白的重組真核表達載體分別共轉(zhuǎn)染HEK-293T細胞,通過免疫共沉淀方法鑒定與NDV M蛋白相互作用的核轉(zhuǎn)運受體蛋白。另外,將M蛋白與Ran蛋白突變體或與M蛋白互作的核轉(zhuǎn)運受體蛋白缺失體分別共表達,通過熒光共定位確定M蛋白入核轉(zhuǎn)運相關(guān)的細胞蛋白�！窘Y(jié)果】構(gòu)建的重組真核表達載體在HEK-293T細胞中能夠正確表達;通過間接免疫熒光觀察發(fā)現(xiàn),重組蛋白中除Myc-KPNA2蛋白定位在細胞質(zhì)外,其它核轉(zhuǎn)運受體蛋白均與M蛋白表現(xiàn)出相同的細胞核定位。免疫共沉淀試驗結(jié)果表明,M蛋白與KPNA1蛋白和KPNB1蛋白均存在相互作用。進一步通過熒光共定位觀察發(fā)現(xiàn),M蛋白與KPNA1蛋白缺失體(DN-KPNA1)共表達不改變M蛋白的細胞核定位,而與KPNB1蛋白缺失體(DN-KPNB1)共表達后導致M蛋白變?yōu)榧毎|(zhì)定位,說明M蛋白入核轉(zhuǎn)運需要KPNB1蛋白的參與。另外,將M蛋白與Ran蛋白突變體Ran-Q69L共表達,熒光觀察發(fā)現(xiàn)M蛋白同樣由細胞核定位變?yōu)榧毎|(zhì)定位,說明M蛋白入核轉(zhuǎn)運還需要Ran蛋白的輔助�！窘Y(jié)論】KPNB1和Ran蛋白共同介導NDV M蛋白的入核轉(zhuǎn)運,其過程是KPNB1蛋白首先和M蛋白發(fā)生相互作用并形成復合物,然后通過Ran蛋白的輔助作用完成入核轉(zhuǎn)運。
[Abstract]:[objective] to identify the cellular proteins associated with the entry of Newcastle disease virus (NDV) matrix protein into the nucleus of Newcastle disease virus (NDV). To elucidate the molecular mechanism of nuclear localization of NDV M protein. [methods] the nuclear transport receptor protein KPNA1 KPNA6 and KPNB1 genes were cloned from chicken embryo fibroblasts and constructed into eukaryotic expression vector. The recombinant eukaryotic expression vector expressing NDV M protein was cotransfected into HEK-293T cells, and the nuclear transport receptor proteins interacting with NDV M protein were identified by immunoprecipitation. The nuclear transport receptor protein deletions of M protein and Ran protein mutant or M protein interacting with M protein were co-expressed, respectively. [results] the constructed recombinant eukaryotic expression vector could be correctly expressed in HEK-293T cells. The recombinant protein was located in the cytoplasm except the Myc-KPNA2 protein. Other nuclear transport receptor proteins all showed the same nuclear localization as M protein. The results of immunoprecipitation test showed that the M protein interacted with KPNA1 protein and KPNB1 protein. The nuclear localization of M protein was not changed by co-expression of M protein and DN-KPNA1. However, co-expression with KPNB1 deletion DN-KPNB1 resulted in the transformation of M protein into cytoplasm, indicating that KPNB1 protein was involved in the nuclear transport of M protein. In addition, M protein was co-expressed with Ran mutant Ran-Q69L. Fluorescence observation showed that M protein also changed from nuclear localization to cytoplasmic localization, indicating that M protein transport into the nucleus also needed the assistance of Ran protein. [conclusion] KPNB1 and Ran protein co-mediate the nuclear transport of NDV M protein. The process is that the KPNB1 protein first interacts with M protein and forms a complex, and then transports into the nucleus through the assistance of Ran protein.
【作者單位】： 貴州大學高原山地動物遺傳育種與繁殖省部共建教育部重點實驗室;揚州大學農(nóng)業(yè)部畜禽傳染病學重點開放實驗室;江蘇省動物重要疫病與人獸共患病防控協(xié)同創(chuàng)新中心;
【基金】：國家自然科學基金(31502074) 教育部“促進與美大地區(qū)科研合作與高層次人才培養(yǎng)項目”(教外司美[2014]2029號) 公益性行業(yè)(農(nóng)業(yè))科研專項(201303033) 貴州大學引進人才科研項目(貴大人基合字(2014)10號)~~
【分類號】：S852.65

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