本文選題：桿狀病毒 + LIP5　； 參考：《西北農(nóng)林科技大學》2015年碩士論文

【摘要】：在真核細胞中,內(nèi)吞體分選轉(zhuǎn)運復合體(Endosomal-sorting complex-required for transport,ESCRT)由ESCRT-0,-I,-II,-III,和VPS4五個復合體及一些輔助蛋白如Alix組成。ESCRT系統(tǒng)參與多泡體的形成、胞質(zhì)分裂、自噬、以及質(zhì)膜修復等多種生理過程。此外,ESCRT也參與一些囊膜病毒的入侵和/或出芽釋放。VPS4屬AAA(ATPase-associated with various cellular activities)ATP酶,具有水解和循環(huán)利用ESCRT-III組分的功能。近期的研究發(fā)現(xiàn),在酵母和人類細胞中,Vta1(人類細胞中命名為LIP5)與VPS4形成復合體,具有介導VPS4多聚化、激活VPS4 ATP酶活性、以及促進ESCRT-III水解的功能。本文采用RT-PCR方法從草地貪夜蛾(Spodoptera frugiperda)細胞Sf9克隆了LIP5同源基因。序列分析表明,草地貪夜蛾LIP5編碼區(qū)的長度為969bp,編碼由322個氨基酸組成的、預(yù)測分子質(zhì)量為35.35kDa的蛋白,其等電點為5.24。同源性比較發(fā)現(xiàn),草地貪夜蛾LIP5與酵母和人類LIP5的氨基酸相似性分別為20%和48.8%,而與其它昆蟲LIP5的相似性為45.9-78.6%。在酵母和人類LIP5中具有重要功能的MIT(Microtubule-interacting and transport)和VSL(Vta1/SBP-1/LIP5)結(jié)構(gòu)域也保守地存在于昆蟲LIP5序列中。此外,在昆蟲LIP5的MIT與VSL結(jié)構(gòu)域之間的連接區(qū)內(nèi)存在兩個富含脯氨酸的模序。通過重疊PCR方法構(gòu)建了缺失草地貪夜蛾LIP5 N-端MIT1或MIT2,C-端VSL結(jié)構(gòu)域的截斷突變體,以及MIT1內(nèi)的M63A、MIT2內(nèi)的W146D和VSL結(jié)構(gòu)域內(nèi)的K287A、K290A、K287A/K290A、L315E等6個點突變,并將這些突變體N-端與GFP融合后連接到昆蟲細胞瞬時表達載體。熒光顯微鏡觀察和Western blot分析表明,GFP標簽的LIP5及其突變體在Sf9細胞中能正確表達,其中,丙氨酸替換VSL結(jié)構(gòu)域內(nèi)的K287和K290位點致使Sf9細胞內(nèi)產(chǎn)生典型的E類囊泡,而缺失LIP5 N-端的MIT2結(jié)構(gòu)域則導致過表達該突變的部分Sf9細胞由典型的圓形變?yōu)樗笮�。進一步將LIP5及其突變體的N-端與雙分子熒光互補系統(tǒng)中的mCherry C-末端融合,并將這些表達融合片段的質(zhì)粒與攜帶有mCherry N-端的VPS4、VPS46或VPS60瞬時表達質(zhì)粒共轉(zhuǎn)染Sf9細胞。熒光顯微鏡觀察發(fā)現(xiàn),缺失LIP5 C-端的VSL結(jié)構(gòu)域或用丙氨酸替換K287和K290位點,將導致LIP5與VPS4不能相互作用,而缺失N-端的MIT1或MIT2結(jié)構(gòu)域顯著降低LIP5與VPS4的作用。同時,缺失LIP5的兩個MIT結(jié)構(gòu)域中的任何一個,都將導致LIP5不能與VPS46或VPS60互作。此外,MIT2內(nèi)的W146位點在LIP5與VPS60互作中發(fā)揮重要作用。最后,通過缺失補償實驗分析表明,在Sf9細胞中瞬時過量表達缺失MIT1、MIT2或VSL結(jié)構(gòu)域,或M63A、K287A/K290A等LIP5突變體將顯著降低感染性苜蓿丫紋夜蛾核多角體病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)的產(chǎn)生,推測LIP5參與桿狀病毒AcMNPV的侵染過程,但其詳細的分子機理尚需深入研究。
[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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S476.13

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