草地贪夜蛾LIP5基因的克隆及过表达LIP5对感染性AcMNPV产生的影响

发布时间：2018-04-19 01:17

本文选题：杆状病毒 + LIP5　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：在真核细胞中,内吞体分选转运复合体(Endosomal-sorting complex-required for transport,ESCRT)由ESCRT-0,-I,-II,-III,和VPS4五个复合体及一些辅助蛋白如Alix组成。ESCRT系统参与多泡体的形成、胞质分裂、自噬、以及质膜修复等多种生理过程。此外,ESCRT也参与一些囊膜病毒的入侵和/或出芽释放。VPS4属AAA(ATPase-associated with various cellular activities)ATP酶,具有水解和循环利用ESCRT-III组分的功能。近期的研究发现,在酵母和人类细胞中,Vta1(人类细胞中命名为LIP5)与VPS4形成复合体,具有介导VPS4多聚化、激活VPS4 ATP酶活性、以及促进ESCRT-III水解的功能。本文采用RT-PCR方法从草地贪夜蛾(Spodoptera frugiperda)细胞Sf9克隆了LIP5同源基因。序列分析表明,草地贪夜蛾LIP5编码区的长度为969bp,编码由322个氨基酸组成的、预测分子质量为35.35kDa的蛋白,其等电点为5.24。同源性比较发现,草地贪夜蛾LIP5与酵母和人类LIP5的氨基酸相似性分别为20%和48.8%,而与其它昆虫LIP5的相似性为45.9-78.6%。在酵母和人类LIP5中具有重要功能的MIT(Microtubule-interacting and transport)和VSL(Vta1/SBP-1/LIP5)结构域也保守地存在于昆虫LIP5序列中。此外,在昆虫LIP5的MIT与VSL结构域之间的连接区内存在两个富含脯氨酸的模序。通过重叠PCR方法构建了缺失草地贪夜蛾LIP5 N-端MIT1或MIT2,C-端VSL结构域的截断突变体,以及MIT1内的M63A、MIT2内的W146D和VSL结构域内的K287A、K290A、K287A/K290A、L315E等6个点突变,并将这些突变体N-端与GFP融合后连接到昆虫细胞瞬时表达载体。荧光显微镜观察和Western blot分析表明,GFP标签的LIP5及其突变体在Sf9细胞中能正确表达,其中,丙氨酸替换VSL结构域内的K287和K290位点致使Sf9细胞内产生典型的E类囊泡,而缺失LIP5 N-端的MIT2结构域则导致过表达该突变的部分Sf9细胞由典型的圆形变为梭形。进一步将LIP5及其突变体的N-端与双分子荧光互补系统中的mCherry C-末端融合,并将这些表达融合片段的质粒与携带有mCherry N-端的VPS4、VPS46或VPS60瞬时表达质粒共转染Sf9细胞。荧光显微镜观察发现,缺失LIP5 C-端的VSL结构域或用丙氨酸替换K287和K290位点,将导致LIP5与VPS4不能相互作用,而缺失N-端的MIT1或MIT2结构域显著降低LIP5与VPS4的作用。同时,缺失LIP5的两个MIT结构域中的任何一个,都将导致LIP5不能与VPS46或VPS60互作。此外,MIT2内的W146位点在LIP5与VPS60互作中发挥重要作用。最后,通过缺失补偿实验分析表明,在Sf9细胞中瞬时过量表达缺失MIT1、MIT2或VSL结构域,或M63A、K287A/K290A等LIP5突变体将显著降低感染性苜蓿丫纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)的产生,推测LIP5参与杆状病毒AcMNPV的侵染过程,但其详细的分子机理尚需深入研究。
[Abstract]:In eukaryotic cells, endosome sorting complex-required for transport complex (ESCRTT) is composed of five VPS4 complexes, including ESCRT-0- IHI-III-III, and some auxiliary proteins such as Alix, which are involved in many physiological processes, such as vesicular formation, cytoplasmic division, autophagy, and plasma membrane repair.In addition, ESCRT is also involved in the invasion and / or budding release of some envelope viruses. VPS4 belongs to AAA(ATPase-associated with various cellular activities)ATP, which can hydrolyze and recycle ESCRT-III components.Recent studies have found that Vta1 (named LIP5 in human cells) forms a complex with VPS4 in yeast and human cells, which mediates VPS4 polymerization, activates the activity of VPS4 ATP and promotes the hydrolysis of ESCRT-III.LIP5 homologous gene was cloned from Sf9 of Spodoptera frugiperda cells by RT-PCR method.Sequence analysis showed that the length of LIP5 coding region was 969 BP, which encoded 322 amino acids and predicted molecular weight of 35.35kDa protein with isoelectric point of 5.24.Homology analysis showed that the amino acid similarity of LIP5 with yeast and human LIP5 was 20% and 48.8%, respectively, while the similarity with other insect LIP5 was 45.9-78.6.The MIT(Microtubule-interacting and transport and VSLV Vta1 / SBP-1 / LIP5) domains, which have important functions in yeast and human LIP5, are also conserved in insect LIP5 sequences.In addition, there are two proline rich motifs in the junction region between MIT and VSL domain of insect LIP5.The truncated mutants of LIP5 N-terminal MIT1 or MIT2C- terminal VSL domain were constructed by overlapping PCR method, and six point mutations, such as W146D in M63AN MIT2 and K287AN K290Am K287Ar / K290AL315E in MIT1, were constructed.These mutants were fused with GFP and ligated to insect cell transient expression vector.Fluorescence microscopy and Western blot analysis showed that LIP5 and its mutants could be correctly expressed in Sf9 cells. Among them, the substitution of alanine for K287 and K290 sites in VSL domain resulted in typical E vesicles in Sf9 cells.The deletion of the MIT2 domain at the N-terminal of LIP5 resulted in the partial Sf9 cells overexpression of the mutation changing from a typical round to a fusiform.Furthermore, the N-terminal of LIP5 and its mutants were fused with the C-terminal of mCherry in the bimolecular fluorescence complementary system, and the plasmids expressing the fusion fragments were cotransfected into Sf9 cells by transient expression plasmid VPS4VPS46 or VPS60 carrying mCherry N-terminal.Fluorescence microscopy showed that the deletion of VSL domain at the C-terminal of LIP5 or the replacement of K287 and K290 sites with alanine would result in no interaction between LIP5 and VPS4, while the deletion of MIT1 or MIT2 domain at N-terminal could significantly reduce the interaction between LIP5 and VPS4.At the same time, the absence of either of the two MIT domains of LIP5 will result in LIP5 not interacting with VPS46 or VPS60.In addition, W146 site in MIT2 plays an important role in the interaction between LIP5 and VPS60.Finally, the results of deletion compensation assay showed that the transient overexpression of MIT1T 2 or VSL domain in Sf9 cells, or LIP5 mutants such as M63Agna K287A / K290A, could significantly reduce the production of infected californica multiple nucleopolyhedrovirusAcMNPV.It is speculated that LIP5 is involved in the infection of baculovirus AcMNPV, but its molecular mechanism needs further study.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.13

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