通过Primer Premier 5.0设计1对特异性引物,用于扩增家蚕核型多角体病毒囊膜蛋白GP64的部分DNA片段,对PCR扩增得到的DNA片段进行纯化,并对纯化后的双酶切DNA片段与经同样双酶切后的原核表达载体pET28a进行连接;对捕获有重组质粒pET28a-G...通过Primer Premier 5.0设计1对特异性引物,用于扩增家蚕核型多角体病毒囊膜蛋白GP64的部分DNA片段,对PCR扩增得到的DNA片段进行纯化,并对纯化后的双酶切DNA片段与经同样双酶切后的原核表达载体pET28a进行连接;对捕获有重组质粒pET28a-GP64的大肠埃希菌BL21(DE3)细胞进行IPTG诱导,通过SDS-PAGE对诱导产物进行电泳分析,结果表明扩增的目的片段获得了表达;通过His单抗对诱导产物进行Western Blot印迹分析,其结果表明诱导蛋白带为融合有组氨酸的目的蛋白。对原核表达的GP64截短蛋白和免疫佐剂进行充分研磨,将充分研磨后的匀浆液对昆明小鼠进行皮下多点注射,以制备的GP64多抗对家蚕核型多角体病毒粒子感染的BmN细胞总蛋白进行Western Blot印迹分析,结果在杂交膜上出现一条特异杂交带、其分子量大小约为64 ku,表明制备的GP64多抗可用于其功能的进一步研究。展开更多
【目的】了解广西家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)gp64基因的遗传多态性及进化特点,掌握BmNPV在广西蚕区的流行和传播情况,揭示BmNPV种群维持遗传多样性的模式与机制。【方法】对20株广西BmNPV毒株的gp64...【目的】了解广西家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)gp64基因的遗传多态性及进化特点,掌握BmNPV在广西蚕区的流行和传播情况,揭示BmNPV种群维持遗传多样性的模式与机制。【方法】对20株广西BmNPV毒株的gp64基因进行测序分析,根据gp64基因构建遗传进化树及绘制毒株流行分布图,并比对不同毒株的致病力。【结果】广西BmNPV毒株gp64基因开放阅读框(ORF)长度存在3种情况(1590、1593和1599 bp),分别编码含529、530和532个氨基酸残基的GP64蛋白。20株广西BmNPV毒株与标准参考T3株的gp64基因核苷酸序列同源性在97.6%~99.2%,其推导氨基酸序列同源性在96.4%~99.6%。在广西BmNPV毒株gp64基因近N端分别出现GCG缺失和GCGCCG/GTGCCG插入突变,发生核苷酸替换突变的位点数目在13~28个,但大部分为同义替换,对编码蛋白的三聚体空间构象无明显影响。广西BmNPV毒株gp64基因编码蛋白的N-糖基化位点为3~4个;除GXZS株外,所有毒株的O-糖基化位点均为2个,且预测位点一致。基于gp64基因构建的遗传进化树显示,几乎所有的广西BmNPV毒株聚类于Clade Ⅰ分群,其又被分为2个主要亚群(Sub-clade Ⅰ和Sub-clade Ⅱ);而几乎所有的国外参考毒株聚类于Clade Ⅱ分群。广西蚕区的BmNPV流行分布呈集中性与分散性并存;GXUA株对四龄和五龄起蚕的半数致死量(LD_(50))分别为3.3和3.1,而GXZZ株对四龄和五龄起蚕的LD_(50)分别为5.5和5.3,说明GP64蛋白糖基化位点较少的BmNPV毒株表现出较弱的致病力。【结论】广西BmNPV毒株gp64基因在进化过程中其信号肽区出现明显变异,发生同义突变的频率较高,形成较独立的进化分群,毒株间的致病力差异可能与GP64蛋白糖基化位点不同有关,说明广西BmNPV毒株具有不同的基因型和表型,在一定程度上维持了BmNPV野生群体的遗传多样性。展开更多
GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy an...GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.展开更多
基金supported by aBBSRC grant (LAK, RDP)a BBSRC-funded PhDstudentship (FJH)
文摘GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.
