传染性喉气管炎病毒UL56蛋白在不同感染模式中的表达及其亚细胞定位分析

Expression profiling and subcellular localization analysis of infectious laryngotracheitis virus UL56 protein

传染性喉气管炎病毒(ILTV)编码的UL56蛋白在进化上非常保守,其功能尚不清楚。为明确UL56蛋白在鸡胚肾(CEK)细胞和气管环(TOC)两种不同感染模式中的表达特征及其在HEK293T细胞中的亚细胞定位,本研究分别构建了野生型ILTV UL56的重组质粒pUL56(WT)、其关键结构域突变体的质粒pUL56(AY)将UL56氨基酸序列中两个PY基序(PY motif)的脯氨酸(Pro,P)均突变为丙氨酸(Ala,A)及pUL56(ΔTMD)(删除UL56氨基酸序列中的跨膜域TMD),将这3个质粒分别转染DF-1细胞后均经western blot鉴定。结果显示,分别获得了约81 ku的特异性条带,表明野生型UL56及其突变体均获得了表达。以5000 EID_(50)/mL ILTV疫苗株K317分别感染CEK和TOC后不同时间,利用兔UL56多克隆抗体通过western blot检测UL56蛋白表达的变化。结果显示,UL56蛋白在两种不同感染模式中的表达特征明显不同。CEK中的UL56蛋白从感染后24 h开始表达,48 h达到高峰后逐渐下降;而TOC中UL56蛋白的表达量则持续上升,至感染后96 h达到峰值。将pUL56(WT)、pUL56(AY)和pUL56(ΔTMD)分别转染HEK293T细胞后瞬时表达UL56蛋白及其突变体,经高尔基体标志蛋白58k的抗体孵育后,通过激光共聚焦试验观察UL56蛋白的亚细胞定位。结果显示UL56蛋白主要定位于高尔基体,且亚细胞定位不受其PY motif的影响而受其TMD的影响。本研究首次揭示了ILTV感染细胞和离体组织后UL56蛋白表达的特征,并鉴定了影响其亚细胞定位的关键结构域,为后续探究该蛋白的分子作用机制和生物学功能奠定了实验基础。

The UL56 protein encoded by infectious laryngotracheitis virus(ILTV)is highly conserved during evolution,yet its functions remain unclear.In order to determine the expression characteristics in chicken embryo kidney(CEK)cells and tracheal organ cultures(TOC),and subcellular localization of UL56 protein in HEK293T cells.The wild-type pUL56(WT)plasmid and its key domain mutated plasmid pUL56(AY)were constructed(the proline(Pro,P))of the two PY motifs in the UL56 amino acid sequence were mutated to alanine(Ala,A)and pUL56(ΔTMD)(deletion of the transmembrane domain TMD in the amino acid sequence of UL56).These three plasmids were transfected into DF-1 cells respectively,and the expression was verified by western blot analyses.The results showed that specific bands of about 81ku were obtained,indicating that both wild-type UL56 and its mutants were expressed.CEK cells and TOC were inoculated with at 5000EID_(50)/mL of ILTV K317 strain.Within the time course,the changes of UL56 expression were examined by western blot using rabbit anti-UL56 polyclonal antibody.The results showed that the expression characteristics of UL56 protein were significantly different in two different infection modes.In CEK cells,the expression of UL56 protein began at 24 hours after infection,and peaked at 48 hours followed by diminishing levels afterwards.On the contrary,the levels of UL56 protein in TOC were consistently elevated and reached a peak at 96 hours after infection.Transfection of pUL56(WT),pUL56(AY)and pUL56(ΔTMD)plasmids into HEK293T cells led to the transient expression of UL56 and its mutants,respectively.After incubated with Golgi marker protein 58k antibody,the subcellular localization of UL56 protein in HEK293T cells was observed by laser confocal test.The results showed that UL56 protein was mainly located in Golgi apparatus,and the subcellular localization was not affected by PY motif,but by its transmembrane domain.In this study,the expression characteristics of UL56 protein in ILTV infected cells and tissues in vitro were revealed for the first time,and the key domains affecting its subcellular localization were identified,which laid an experimental foundation for further exploration of the molecular mechanism and biological function of the protein.