新城疫病毒疫苗株F蛋白裂解位点改造对诱导HepG2细胞发生自噬的影响  

The effect of cleavage site of Newcastle disease virus F protein on the induction of autophagy in HepG2 cell line

作　　者：吴云舟[1] 安莹[1] 孙田[1] 何金娇[1] 王卉[1] 朱升龙[1] 田贵游[1] 尹杰超[1] 张天援[1] 牛泽杉[1] 倪霁[1] 曲素素[1] 刘畅[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院/生物制药学教研室,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2013年第9期698-701,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省青年基金(QC2012C099);东北农业大学博士启动基金(2012RCB43)

摘　　要：新城疫病毒(NDV)通过启动不依赖p53的内源性凋亡通路特异性诱导肿瘤细胞凋亡。最新研究表明NDV可以引起U251肿瘤细胞发生自噬,并在后期导致细胞凋亡,但其机理尚不清楚。本研究通过反向遗传操作技术对NDV Clone30疫苗株F蛋白的碱性裂解位点进行突变,将F蛋白的裂解位点由弱毒株的GGRQGR↓L突变为强毒株的GRRQRR↓F基序,并成功拯救出突变改造病毒rC30-FmF。分别将Clone30野生型病毒株rC30-wt与rC30-FmF感染HepG2肝癌细胞,通过透射电镜检测细胞超微结构显示,rC30-FmF感染4 h后细胞内出现大量自噬小体及自噬溶酶体。通过western blot检测自噬标志蛋白LC-3 II表明,rC30-FmF感染4 h后LC3II表达量显著上调,与rC30-wt对照组相比差异极显著(p<0.01)。研究表明,rC30-FmF可以在感染早期增加HepG2细胞自噬程度。从而初步证明F蛋白的裂解位点在诱导HepG2细胞自噬过程中具有关键作用。Newcastle disease virus （NDV） specifically elicits tumor apoptosis through the p53 independent innate pathway. The newest research indicated that NDV induced autophagy in U251 cell and resulting in cell apoptosis, but the underline mechanism remains unknown. In the present study, the recombinant NDV （rC30-FmF） was rescued by reverse genetics technology, which the cleavage site of the F protein of Clone 30 vaccine strain was replaced from to the GGRQGR↓L to the GRRQRR↓F of velogenic NDV. Then the HepG2 cell line was infected with the rC30-FmF and wild type Clone 30 strain （rC30-wt）, respectively. Transmission electron microscopy examation shown that a large unmbers of autophagosomes and autolysosomes appeared in rC30-FmF infected HepG2 cells at 4 hours post infection. In addition, the expression of the autophagy marker protein （LC3II） was also significantly up-regulated in rC30-FmF infected cells, comparing with the rC30-wt infected cells （p〈0.01） detected by western blot. These results indicated that rC30-FmF was able to induce the formulation of autophagosomes in the early infection stage and the F protein cleavage play a key role in inducing autophagy in HepG2 cells.

关 键 词：重组新城疫病毒 F蛋白 自噬 

分 类 号：S852.65[农业科学—基础兽医学]