【作者】 王飞；

【导师】 卢春；

【作者基本信息】 南京医科大学 ， 微生物学， 2018， 硕士

【摘要】 背景:卡波氏肉瘤病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)是一种γ2型疱疹病毒,基因组全长约140 kb,编码约90个开放阅读框(open reading frame,ORF)。其中,KSHV K13基因编码的病毒Fas相关死亡结构域白介素-1β转换酶抑制蛋白(viral Fas-associated death domain-like interleukin 1βconverting enzyme inhibitory protein,v FLIP)和K9基因编码的病毒干扰素调节因子1(viral interferon regulatory factor 1,v IRF1)已被证明能够调控多种信号通路促进KSHV相关肿瘤的发生与发展。目前,关于这两个致瘤蛋白的功能和机制研究大部分都借助于过表达系统,在KSHV全基因组中删除K13或K9是否影响KSHV诱导肿瘤形成的能力,以及其中涉及的分子机制等,至今仍不清楚。目的:构建缺失K13或K9基因的KSHV突变株,为后续进一步研究v FLIP或v IRF1功能提供有力工具。方法:利用E coli GS1783工程菌通过两步热重组法删除KSHV红-绿-蓝细菌人工染色体(red-green-blue-bacterial artificial chromosome,RGB-BAC16)中的K13或K9的编码区(coding sequence,CDS)序列进行突变质粒构建,并结合聚合酶链式反应(polymerase chain reaction,PCR)、测序、酶切等方法对突变的质粒进行验证。将成功构建的突变质粒转染i SLK-puro细胞,运用潮霉素B进行细胞筛选,使细胞的荧光率达90%以上,获得稳定转染RGB-BAC16质粒的细胞。诱导i SLK细胞获得野生型或突变型的KSHV病毒,分别感染人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs),利用荧光实时定量PCR(real-time quantitative polymerase chain reaction,RT-q PCR)、免疫印记(Western blot)或双荧光素酶报告等实验验证K13或K9基因缺失是否成功。结果:成功构建了缺失K13或K9 CDS区的KSHV突变质粒,以及稳定转染KSHV野生型或突变型质粒的i SLK细胞系。诱导i SLK细胞获得的KSHV野生型或突变型病毒能够成功感染HUVECs细胞。结论:本研究成功构建了KSHV K13或K9基因缺失突变株,有助于进一步研究致瘤蛋白v FLIP和v IRF1的功能及其中涉及的分子机制。更多还原

【Abstract】 Background: Kaposi’s sarcoma-associated herpesvirus(KSHV)is a γ2 typeherpesvirus and encodes about 90 open reading frames(ORFs).KSHV K13 coded viral Fas-associated death domain-like interleukin 1β converting enzyme inhibitory protein(v FLIP)and K9 coded viral interferon regulatory factor 1(v IRF1)have been demonstrated to participate in KSHV induced tumorigenesis through virous pathways.Previously,most studies are based on overexpression systems,while the influence when K13 or K9 deleted from KSHV genome on KSHV tumorigenesis is not clear.Objective: To construct K13 or K9 deleted KSHV mutants and apply them for the functional research of K13 or K9 based on the KSHV whole genome.Methods: In this study,we generated recombinant viruses lacking the CDS of K13 or K9 gene from KSHV RGB-BAC16 genome through two-step red-mediated recombination which is manipulated in E.coli GS1783.PCR,sequencing and digestion by restriction endonuclease were used to verify whether the mutant was constructed successfully.The RGB-BAC16 were transfected into i SLK-puro cells and screened by antibiotics until the rate of fluorescence was up to 90%.The wide type and mutant KSHV viruses was induced in i SLK cell lines and the viruses were used in de novo infection of HUVECs.RT-q PCR,western blot and dual luciferase reporter assay system have been used to verify the function of KSHV infected HUVECs.Results: In this project,we generated recombinant viruses lacking the CDS of K13 or K9 gene from KSHV RGB-BAC16 genome.The mutant viruses producer,i SLK cells were stably transfected with K13 or K9-deleted mutant bacmids.Importantly,the v FLIP or v IRF1 knockout viruses possessed the ability of de novo infection of HUVECs.Conclusions: This project generated recombinant KSHV viruses lacking the CDS of K13 or K9,and it can help study the function and molecular mechanism of both oncoprotein v FLIP and v IRF1.更多还原