【作者】 李靖；

【导师】 赵蔚明；

【作者基本信息】 山东大学 ， 病原生物学， 2009， 博士

【摘要】 人乳头瘤病毒(Human papillomavirus,HPV)是能引起人类皮肤粘膜多种良、恶性增殖性疾病的一类肿瘤相关病毒。目前,人们已经成功分离出了100多个型别的HPV。根据致病性的不同,又可将其分为低危型和高危型。低危型包括1,2,6,11等型,主要引起良性瘤或疣,如扁平疣和尖锐湿疣;高危型包括16,18,31,58等型,主要引起皮肤粘膜的恶性肿瘤,如宫颈癌等。对HPV生物学特征的研究表明,HPV为无包膜的8kb左右的双链DNA病毒,基因组可分为3个区:长调控区(long control region,LCR),早期基因区(early gcne region)和晚期基因区(late gene region)。HPV的生活周期依赖于表皮宿主细胞的分化,晚期基因转录翻译和病毒颗粒装配只能在上皮终末分化细胞中进行。HPV的这一特性限制了病毒的体外培养,延缓了对HPV生活周期和各基因功能的研究。人乳头瘤病毒58型(Human papillomavirus type 58,HPV58)是一种重要的高危型HPV。流行病学研究表明,虽然HPV58在世界范围内的总体感染率较低但在东亚地区宫颈癌的发生中却扮演着重要角色;在我国HPV58仅次于HPV16,与宫颈癌发生密切相关。然而,迄今为止国内外对HPV58的研究较少,尽管目前已经建立了含有HPV16,18等型别的稳定细胞系用来研究病毒基因组的复制转录和致病机制。但是目前还没有含有HPV58基因组的稳定细胞系,这在很大程度上限制了对HPV58生活周期和致病机制的研究。因此,为进一步研究HPV58型的基因功能,为病毒感染及肿瘤的防治奠定基础,急需构建一种HPV58的细胞模型。酿酒酵母(Saccharomyces cerevisiae)是一种单细胞真核生物,因其遗传背景清晰,易于培养,内在的分子生物学和细胞生物学机制与哺乳动物细胞有诸多相似之处。因此,被人们誉为真核生物中的大肠杆菌,作为模式生物广泛用于细胞生物学和分子生物学方面的研究。在此研究中,我们建立了HPV58的酵母复制系统,将HPV58基因组导入酵母细胞中,发现HPV58基因组在酵母细胞中以游离体形式存在于酵母细胞染色体外,并且可以稳定复制,在酵母有丝分裂时HPV58基因组可以均衡稳定地分配到子代细胞核中。我们同时还发现,HPV58基因组在酵母系统中可以有早期和晚期基因的转录,成功建立了HPV58酵母复制系统。我们还构建了带有E2基因突变的HPV58基因组和HPV58 E2基因酵母表达载体,借助于此酵母复制系统研究了E2蛋白在HPV58基因组复制,稳定性维持和基因转录调控方面的功能。针对酿酒酵母细胞破壁困难,RNA难以提取的技术难题,我们在研究中还建立了一种简便经济的酵母总RNA提取新技术。一.HPV58酵母复制系统的建立本部分研究中,我们将1.1kb的酵母营养缺陷筛选标记Ura3亚克隆到HPV58基因组L2 ORF中,在不破坏L1基因的同时也保证了病毒基因组LCR区的完整性。构建带有酵母选择标记Ura3的HPV58基因组HPV58-Ura,连接环化后转化入酿酒酵母中。涂布营养缺陷选择平板,并在选择培养基上连续传代5次以后提取酵母细胞DNA和总RNA。荧光定量PCR法检测不同代时酵母DNA中HPV58基因组的含量,发现HPV58基因组可以在酵母细胞中稳定复制传代;Southern blot验证病毒基因组以游离体形式存在于酵母细胞中,没有病毒基因的整合或重组;DNA稳定性实验表明HPV58基因组在酵母细胞中稳定很高,每代丢失率为1.9±0.31%,甚至略低于酵母质粒pRS316的丢失率2.1±0.25%;利用RT-PCR以及实时荧光定量PCR技术检测到HPV58基因组在酵母细胞中早期基因(E1,E2,E6,E7)和晚期基因(L1,L2)的转录。以上结果说明,HPV58在酵母细胞中以染色体外游离体的形式存在,并且可以稳定复制传代;HPV58可以在酵母细胞中有其早期和晚期基因的转录。表明HPV58酵母细胞复制系统构建成功。这一新系统的建立有助于我们探索HPV的生活周期中各个基因的功能,对于进一步揭示HPV58致癌机制、寻找有效的防治策略有重要的理论意义和应用价值。二、HPV58酵母复制系统中病毒E2基因功能的研究在成功构建HPV58酵母复制系统的基础上,我们进一步利用此系统研究了HPV58早期基因E2的功能。利用基于PCR的盒式突变(cassette mutagenesis)技术,将HPV58E2基因翻译起始密码子下游79-87bp处的密码子突变为三个终止密码子,使E2基因功能缺失,构建E2突变体HPV58-Ura-E2mt。同时构建HPV58 E2蛋白酵母表达载体pDBLeu-E2。将HPV58-Ura-E2mt转化酵母细胞。发现含有HPV58-Ura-E2mt的酵母细胞在选择培养基上生长缓慢;E2功能缺失导致HPV58基因组在酵母细胞中的复制效率低下,稳定性差,传代困难。此时将pDBLeu-E2导入HPV58-Ura-E2mt转化的酵母细胞中。结果发现在HPV58-Ura-E2mt/pDBLeu-E2共转化的情况下,HPV58-Ura-E2mt在酵母细胞中的复制水平及稳定性均得到不同程度的恢复。至此,利用已经构建的三个不同的HPV58/酵母复制系统:HPV58-Ura/酵母,HPV58-Ura-E2mt/酵母,HPV58-Ura-E2mt/pDBLeu-E2/酵母。我们分别比较了三种情况下HPV58基因组在酵母细胞中的复制效率,在宿主细胞有丝分裂时的稳定性。结果表明,在HPV58/酵母系统中E2蛋白具有正常功能,不仅可协助HPV58基因组的复制,而且维持病毒基因组在宿主细胞有丝分裂时的稳定性,使HPV58基因组在宿主细胞有丝分裂时能够被正确均匀地分配到子代细胞核中而不被丢失。我们的研究发现HPV58基因组在酵母细胞中稳定性的维持机制不同于HPV16基因组,即HPV58基因组稳定性的维持是E2蛋白绝对依赖性的。而据以往的研究报道HPV16的E2蛋白对于病毒在酵母细胞中的稳定性的维持并不是一个绝对必备的因素。此发现为我们干预HPV58的早期感染提供了线索,有重要理论意义和实际应用价值。我们还进一步探讨了HPV58 E2蛋白对病毒早期和晚期基因的转录活性调控作用。利用荧光定量RT-PCR技术比较在有或无E2蛋白的情况下病毒早期基因(E6、E7)和晚期基因(L1、L2)转录水平。研究结果表明在HPV58基因组以游离体存在的形式下,E2蛋白对E6和E7早期基因的转录起到抑制作用。其机制可能是通过E2蛋白和早期基因启动子TATA框附近的E2结合位点结合,这一物理性的空间占位作用阻止细胞转录因子和启动子序列的结合,下调了E6、E7早期基因的转录水平。我们还在HPV-酵母系统中观察到了E2功能缺失对HPV58 L1和L2晚期基因转录的影响。研究结果发现E2功能缺失导致酵母细胞中L1和L2晚期基因转录水平的下降。说明E2蛋白对HPV58 L1和L2晚期基因转录起到促进作用,具体的调控机制还有待进一步的实验研究。三、一种提取酵母细胞总RNA的新方法:水浴法在本部分研究中,我们建立了一种提取酵母细胞总RNA的新方法。传统的酿酒酵母细胞总RNA的提取方法主要有两种,一种是玻璃珠配合强蛋白变性剂Trizol强烈震荡致酵母细胞壁破裂,RNA溢出,再通过核酸沉淀方法提取,另一种是热酸酚法,将Tris饱和酚用AE buffer(50 mM sodium acetate,pH 5.3,10 mM EDTA)平衡,然后用于酵母细胞破壁,但这两种方法都较为繁琐,不适合一次处理大量样品,且接触有害性物质如强蛋白变性剂Trizol和酚的机会较多。我们建立的新型的酵母RNA提取方法,不需要玻璃珠震荡破壁,不需要AE buffer平衡酚,可一次处理大量样品。且因为破壁过程不需要强蛋白变性剂,有毒试剂对操作者和环境的危害得以减少。具体方法为:将酵母细胞离心收集后在酵母RNA提取缓冲yeast RNA isolation buffer(10 mM EDTA,50 mM Tris-HCl,5%SDS,pH 6.0)中65℃水浴5分钟后即可使酵母细胞完全破壁,然后常规酚氯仿抽提,乙醇沉淀,提取完整的酵母细胞RNA。所提取的RNA完整性好,无明显降解,可以用于下游实验如Northern blot和RT-PCR。综上所述,本课题的特色和创新之处在于:(1)以高危型HPV58为研究对象,首次建立了HPV58/酵母复制系统,为HPV58的相关研究奠定了基础。(2)应用HPV58/酵母复制系统对HPV58 E2基因的功能进行了研究。研究了HPV58 E2蛋白在基因组复制,稳定性维持以及转录方面的功能。首次发现HPV58和HPV16维持基因组在宿主细胞中稳定性的不同机制,即HPV58基因组稳定性的维持是E2蛋白绝对依赖性的。说明此HPV-酵母系统为我们提供了一个研究HPV58基因功能的良好平台。(3)建立了一种提取酵母细胞RNA的方法,为重要模式生物酵母菌的相关研究提供了方法学上的支撑。以上研究目前国内外尚未见报道。更多还原

【Abstract】 Human Papillomaviruses(HPVs) are DNA viruses that infect keratinocytes in differentiating epithelia and induce a series of benign and malignant hyperproliferative lesions.The viruses are classified as either high risk or low risk viruses.Low-risk HPV genotypes,such as HPV1, 2,6,11,induce benign warts such as common warts and condyloma acuminata.In contrast, infections by high-risk HPV types including HPV 16,18,31,58 can lead to the development of cervical cancer.HPVs are a family of approximately 8kb,non-enveloped and double-stranded DNA viruses.The genome of HPV can be divided into three regions:the long control region,the early and late regions.The productive life cycle of HPVs is intimately linked to epithelial growth and cell differentiation.The link has posed a substantial barrier to the study of HPV in the laboratory because HPVs cannot be propagated in conventional cell lines.HPV 58 is an important HPV type tightly associated with cervical cancer.Several studies have suggested that HPV58 plays a prominent role in cervical cancer in Asia,and it is also the second most prevalent HPV type found in cervical cancer patients in China except HPV16.So far,cell lines containing the DNA of high-risk HPVs such asHPV16,18 and 31 in an episomal state are available for biological studies,neither cell lines nor model system containing HPV58 have been established for the study of HPV58.S.cerevisiae is a single-cell eukaryotic organism.The cellular mechanism required for DNA replication in human cells is sufficiently similar to those in yeast.In the present study,we inserted a yeast selectable marker Ura into the HPV58 genome to generate a recombinant HPV58-Ura construct and transformed the circular HPV58-Ura construct into yeast.We discovered that HPV58 genome can replicate stably as an episome and transcribe its early and late open-reading frames(ORFs) in transformed yeast cells. However,when the E2 gene of HPV58-ura was interrupted by stop codons in the 5’ of the ORF to construct the recombinant HPV58-Ura-E2mt,the HPV 58 genome can not replicate stably in the yeast system and the transcription pattern of early genes(E6 and E7) and late genes(L1 and L2) was also changed,which means that E2 protein can not only facilitate the replication and stability of HPV DNA but also regulate the gene transcription level.The HPV58/Yeast system described here provides us with a powerful model to further study HPV genome replication and viral gene transcription in future.Furthermore,we have developed a simple and efficient method for the preparation of total RNA from Saccharomyces cerevisiae.Yeast cells were incubated at 65℃for 5 min in yeast RNA isolation buffer(10 mM EDTA,50 mM Tris-HCl,5%SDS,pH 6.0),and the RNA was isolated and purified.The yield and quality of the isolated RNA was consistently high,and the isolated RNA was suitable for downstream applications,such as Northern blot hybridization and reverse transcription PCR(RT-PCR).1.Establishment of HPV58/Yeast systemA 1.1kb yeast selective marker Ura3 was subcloned into the L2 ORF of HPV58 genome to construct the recombinant HPV58-Ura.The HPV58-Ura was recircularized and transformed into yeast.The HPV58-Ura transformed yeast was cultured in selective medium for 5 passages,then yeast DNA and total RNA was isolated.The result of real time quantitative PCR indicated that HPV58 can replicate its genome in yeast cells,and Southern blot analysis revealed that viral genome can replicate episomally in yeast cells without integration or recombinantion.The DNA stability assay indicated that HPV58-Ura was very stable in the yeast cells with a loss rate of 1.9±0.31%per cell generation.RT-PCR analysis reveals that HPV58-ura can transcribe its early(E1,E2,E6,E7) and late genes(L1 and L2) in yeast cells. HPV58/yeast system described here was able to direct the stable replication of HPV58 genome and induce the transcriptions of both early late genes.Therefore,the established system provides us with the opportunity to study the mechanisms that HPVs can complete their multiplication life cycle in such a simple eukaryote.2.Studies of HPV58 E2 gene functions in yeast systemA recombinant HPV58-Ura-E2mt was constructed by introducing three stop codons in the 5’ of E2 ORF through a PCR based cassette mutagenesis.The HPV58 E2 expression plasmid pDBLeu-E2 was constructed at the same time.HPV58-Ura-E2mt transformed yeast cells grow poorly in the selective plates.When the pDBLeu-E2 plasmid was transformed into yeast cells harboring HPV58-Ura-E2mt.Then the HPV58-Ura-E2mt/ pDBLeu-E2 transformed yeast cells can grow stably in selective medium.E2 protein is a auxiliary replication protein and mitotic stability factor.HPV58 E2 protein can not only facilitate the replication of HPV58 genome but also help to distribute the viral genome to daughter cells’ nucleuses during cell division.We also found the different mechisms of viral genome maintenance between HPV58 and HPV16 in the yeast system.In order to explore the regulation function of E2 on viral early promoter which promote the E6 and E7 gene transcription and late promoter which promote the L1 and L2 gene transcription.The E6,E7,L1 and L2 gene transcription level was compared through qRT-PCR.The results of qRT-PCR indicated E2 protein can repress the early gene transcription of episomal HPV58 genome in yeast.However,we found that E2 protein can stimulate the late gene transcription of episomal HPV58 genome in S.cerevisiae,the detailed mechanism needs further research..3.A waterbath method for preparation of RNA from Saccharomyces cerevisiaeYeast RNA has traditionally been extracted by tedious vortex mixing with glass beads or with hot acid/phenol.Although the glass beads method results in efficient breakage of the cells,the procedure itself is complicated,especially for multiple samples.The hot acid/phenol method is simpler than the glass beads method,but it requires the equilibration of phenol with AE buffer(50 mM sodium acetate,pH 5.3,10 mM EDTA).Here,we describe a novel,simple and reliable method for preparation of total RNA from baker’s yeast.The yeast cells were incubated at 65℃for 5 min in yeast RNA isolation buffer(10 mM EDTA,50 mM Tris-HCl, 5%SDS,pH 6.0),and then the SDS and cell debris were precipitated by the addition of KCl. The RNA was isolated by extraction with phenol/chloroform and precipitation in ethanol.The RNA waterbath extraction method gave good yields of high-quality RNA from S.cerevisiae within 1 h without any vortex mixing or expensive Trizol reagent.This method is very suitable for dealing with large quantities of yeast cultures.The RNA is suitable for downstream applications,such as RT-PCR and Northem blot hybridization.To conclude,we are the first to establish a HPV58/yeast system,which is potential for the studies on the HPV58 life cycle;secondly,we studied the HPV58 E2 gene function in the yeast system and found its important roles in DNA replication,maintenance and transcription regulation;thirdly,we have developed a novel method for preparation of intact yeast RNA.更多还原