【作者】 董流昕；

【导师】 洪涛； 鲁茁壮；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2010， 博士

【摘要】 人腺病毒F组含有2个成员,Ad40和Ad41,二者均能够引起婴幼儿腹泻。由于它们是天然的肠道病原,因此有改造为肠道靶向基因转移载体的潜力。但是Ad40和Ad41在体外难以培养,被称为难养腺病毒(fastidious adenoviruses),这给载体改构造成困难。Ad40和Ad41难以培养的根本原因还没有阐明。体外培养条件下,病毒早期基因表达不充分,病毒基因组复制效率低,晚期基因表达水平低下,病毒装配和释放困难,难以培养可能是这些因素综合作用的结果。293细胞是Ad5 E1区转化的人胚肾细胞,组成性表达Ad5 E1区基因(包括E1A、E1B19K和E1B55K)。部分Ad41的分离株能够用293细胞进行传代(虽然子代病毒产量很低),这可能是由于Ad41的E1基因表达水平低,而293细胞表达的Ad5 E1蛋白部分补偿了Ad41相应分子的功能。之所以说是部分补偿,而不是完全补偿,是因为E1B55K蛋白可能具有型别特异性,例如Ad5 E1B55K不能完全补偿Ad11p或Ad35 E1B55K的功能。我们前期的工作将Ad41 E1B55K基因转染293细胞,’筛选得到稳定表达Ad41 E1B55K的293E12细胞,结果显示293E12包装Ad41的能力较293细胞提高了约10倍,我们同时建立了E1区缺失的复制缺陷型Ad41载体系统,携带GFP报告基因的重组Ad41-GFP病毒能够在293E12细胞得到拯救和扩增。由于E1区是腺病毒感染细胞后首先表达的基因,E1蛋白为腺病毒复制营造适宜的细胞内环境,并调控腺病毒其他基因的表达,推测E1区表达水平低是Ad41体外难养的主要原因。本研究以293为模型细胞,重点考察了提高Ad41 E1B55K表达对Ad41复制的促进作用,并分析了可能的原因。1.提高包装细胞Ad41 E1B55K的表达,促进了重组病毒的复制293E12中Ad41 E1B55K基因由CMV启动子控制表达,为了进一步提高293细胞中Ad41 E1B55K表达水平,我们克隆了Ad41的三联体前导序列(tripartite leader sequence, TPL)。腺病毒晚期基因mRNA5’非翻译区都含有一段共有序列,这段序列起源于病毒基因组晚期基因的3个外显子,被称为三联体前导序列(tripartite leader sequence, TPL), TPL有促进病毒晚期mRNA转运(由细胞核转移到胞浆)和翻译的功能。为了进一步提高包装细胞中Ad41 E1B55K的表达水平,我们在其mRNA5’非翻译区引入TPL。首先,进行Ad41 TPL克隆。Ad41-GFP感染293E12细胞,24h后提取细胞总RNA,逆转录为cDNA;利用巢式PCR方法,扩增晚期基因L1 52K mRNA5’端的部分序列,将其克隆到T载体,得到pMD-TPL质粒；测序,通过序列分析,获得Ad41 TPL序列信息。然后,设计引物,并在引物5’末端添加Hindlll的酶切位点,以pMD-TPL为模板,PCR扩增得到TPL序列,Hindlll酶切后插入到pcDNA3-E1B55K的巨细胞病毒启动子(CMVp)的下游,得到pcDNA3-TPL-E1B55K真核表达质粒。该质粒转染293细胞,G418筛选,获得20株G418抗性的单克隆细胞(293TE)；以等量重组病毒Ad41-GFP感染293TE细胞,以细胞中GFP数量的增多、荧光强度的提高为指标,初步评价293TE的病毒复制能力,获得3株病毒产量提高的单克隆细胞(293TE7,293TE12和293TE20)。最后,重点考察了293TE7单克隆细胞与293E12细胞病毒包装能力的差异。将Ad41 E1B55K基因克隆到原核表达载体pET30a(+),以E.coli BL21(DE3)菌株表达的E1B55K蛋白免疫小鼠,制备抗血清,以该抗血清作为一抗,免疫荧光检测Ad41E1B55K的表达,结果显示293TE7细胞Ad41 E1B55K的表达明显高于293E12细胞。以线性化的腺病毒质粒pAd41-GFP转染293E12或293TE7细胞,滴度测定实验的结果表明,293TE7的病毒拯救能力约为293E12细胞的20倍；以等量Ad41-GFP重组病毒分别感染293E12和293TE7细胞,收集子代病毒并进行滴度测定,结果显示293TE7细胞的病毒产量较293E12细胞提高3-10倍。将Ad41-GFP感染293TE7细胞,收获的子代病毒再次感染293TE7细胞,如此进行重组病毒的连续传代,在传代6次后,利用Hirt法提取病毒基因组,选用多种限制性内切酶进行酶切鉴定,结果显示Ad41-GFP在293TE7细胞传代稳定。总之,我们克隆了Ad41 TPL序列,成功构建了Ad41 E1B55K真核表达载体,建立了Ad41 E1B55K稳定表达的293TE7细胞；TPL的引入提高了Ad41 E1B55K的表达水平,结果使293TE7的病毒拯救和包装能力较原包装细胞明显提高。2.Ad41 E1B55K表达促进病毒包装的可能机理病毒包装的物质基础和前提是病毒基因组的复制和结构蛋白的表达,我们观察了在293细胞表达Ad41 E1B55K对Ad41病毒基因组复制和结构蛋白表达的影响。等量Ad41-GFP感染293、293E12和293TE7细胞,Hirt法提取病毒基因组,琼脂糖凝胶电泳后,溴化乙啶(EB)染色观察,293E12和293TE7细胞病毒基因组的合成量相近,约为293细胞合成量的4倍。E1B55K蛋白的主要功能之一是促进病毒晚期mRNA的运输。病毒感染细胞后,我们利用荧光定量PCR分别检测细胞总RNA中或细胞浆RNA中病毒晚期mRNA表达水平,结果显示,12h、16h和24 h这些时间点中,细胞中总L2和L3区的mRNA的量,293E12细胞与293细胞大致相同,而细胞浆中,293E细胞L2和L3区mRNA的量均高于293细胞。我们又进一步观察了较高水平mRNA的转录和转运是否最终表现为病毒结构蛋白的较高表达。等量的Ad41-GFP分别感染293E和293细胞,Western blot检测结果表明,在内参基因beta-Actin表达量相近的情况下,293E细胞中V蛋白的表达量明显高于293细胞。这些结果表明,在293细胞表达Ad41 E1B55K,促进了Ad41病毒基因组的复制和病毒结构蛋白的表达,并最终提高了子代病毒的产量。3.Ad41载体系统的新探索293细胞中原有的Ad5 E1B55K不能完全替代Ad41 E1B55K的功能,在293细胞中表达Ad41 E1B55K有利于Ad41重组病毒的包装,这个研究结果促使我们尝试建立新的复制缺陷型Ad41载体系统,在新系统中,E1区的E1A和E1B19K基因被删除,而E1B55K基因保留,产生的重组病毒有可能在293细胞高效复制。在已经构建的Ad41载体系统的穿梭质粒pSh41-GFP中插入Ad41 E1B55K基因,得到新的穿梭质粒pSh41ElB-GFP,再将该穿梭质粒与骨架质粒在E. Coli BJ5183菌株中进行同源重组,得到的重组腺病毒质粒pAd41ElB-GFP, PmeⅠ线性化后经脂质体转染293细胞,荧光显微镜观察GFP和细胞数量的变化,以了解病毒复制情况。Ad41ElB-GFP病毒在293细胞中能够进行复制并传代,这表明Ad41 E1B55K基因能够影响病毒的复制,但得到病毒量很低,没有达到预想的实验结果,分析原因可能是由于控制Ad41 E1B55K的启动子启动效率低,E1B55K表达不充分。该研究方案正在改进中。总之,本研究主要观察了在293细胞表达Ad41 E1B55K与Ad41病毒复制的关系,发现表达Ad41 E1B55K能够提高病毒基因组的复制和结构蛋白的表达,从而提高病毒产量；如果提高Ad41 E1B55K表达水平,则能够进一步提高包装细胞的病毒包装效率；我们尝试了构建新的重组Ad4l载体系统,建立的293TE7细胞可以用于野生型或重组Ad41的包装,同时,本研究为Ad41载体系统的进一步改进奠定了基础。更多还原

【Abstract】 Adenoviruses type 40 (Ad40) and 41 (Ad41) are natural pathogens infecting human digestive tract and can cause diarrhea. There have been great interests in developing Ad40 or Ad41 as a gene delivery vector targeting gastrointestinal tract, which is hampered by that their viruses are hard to cultivate in vitro. Many possible reasons contribute to the fastidiousness of Ad40 or Ad41, such as weak expression of the early genes, decreased genome replication, insufficient expression of viral structure proteins, and defects in virus assembling and release. However, the fundamental reason remains unknown.HEK293 cell line, also called as 293, is human embryo kidney cell transformed by the El region of Ad5, which constitutively express E1A, E1B19K and E1B55K protein of Ad5. Some of Ad41 isolates can be passaged on 293 cells. Generally, it is explained as El proteins of Ad5 in 293 can partly remedy the insufficient expression of Ad41 El. However, the growth of Ad41 in 293 cells is far from perfect. The yield of progeny viruses was so low that some isolates were just lost after being consecutively passaged on 293 cells. We assumed that adenovirus E1B55K proteins of different serotypes could not totally complement each other. There are some evidences. During the construction of Ad35 replication-defective vector, for example, Ad35 E1B55K-transduced 293 rather than 293 was used as the packaging cell because El-deleted Ad35 could not be rescued and proliferated in 293 cells. In our previous work, we established Ad41 E1B55K-transduced HEp2 (HEp2-ElB) and 293 (293E12) cell lines. HEp2-E1B and 293E12 can be used to amplify wild-type Ad41 isolated from children’s stools. Furthermore,293E12 can be utilized as the packaging cell for E1-deleted Ad41 vectors.The E1 region is the first transcription unit to be activated following Ad infection. They modify the host cell environment to facilitate Ad replication. We assume that poor expression of Ad41 E1 takes the major responsibility for the virus fastidiousness. In this study, we focus on the correlation between the expression of Ad41 E1B55K and the efficiency of Ad41 replication in 293 cells.1. Increased expression of Ad41 E1B55K improved Ad41-packaging ability of 293 cells.We have established Ad41 E1B55K-transduced 293 cell line (293E12), in which the expression of Ad41 E1B55K was controlled by CMV promoter. In this study, we attempt to further enhanced Ad41 E1B55K expression by employing Ad41 tripartite leader sequence (TPL). Adenovirus late mRNAs all share a common 5’non-translated region about 200 nucleotides in length. This sequence is termed as TPL because it is coded by three extrons just following the major late promoter in the viral genome. TPL was well known due to its function of modulating mRNA export from the nucleus as well as facilitating mRNA translation during the late phase of adenovirus infection. Therefore, TPL could enhance the efficiency of gene expression when being inserted downstream of a promoter. Firstly, we cloned TPL fragment. RNA was extracted from Ad41-GFP-infected 293E12 cells at 24 h post infection. Reverse transcription was performed to prepare single-stranded cDNA using a specific primer complemented to the L1 52K gene of Ad41. And then nested PCR was employed to amplify the 5’region of L1 52K mRNA sequence with the cDNA as the template. PCR product was inserted into the cloning site of pMD-18T vector, followed by sequencing. TPL sequence information was obtained after bioinformatics analysis. New primers carrying restriction sites on the 5’ends were designed, synthesized and used to amplify TPL of Ad41. PCR product was digested and then inserted into the downstream of CMV promoter of pcDNA3-E1B plasmid, which was a eukaryotic expression vector carrying Ad41 E1B55K. The generated plasmid, named as pcDNA3-TPL-E1B, was purified and mixed with lipofectamine 2000 to transduce 293 cells. The G418-resistant cell colonies (293TE) were isolated and proliferated. Packaging ability of 293TE cell lines were preliminarily evaluated by the increase of GFP-positive cells after being infected with equivalent Ad41-GFP. Three cell lines,293TE7,293TE12 and 293TE20, were relatively more productive and kept for further study. Ad41 E1B55K was also cloned into pET30A(+) vector and expressed in E. Coli strain BL21(DE3). Antiserums to Ad41 E1B55K were collected from Ad41 E1B55K-immunized mice.293TE7 was assayed for the expression of Ad41 E1B55K with the method of immunofluorescence using antiserum against Ad41 E1B55K. The results showed that more Ad41 E1B55K was expressed in 293TE7 cells than in 293E12 cells, suggesting that TPL could substantially enhance E1B55K expression. To compare the ability of virus rescue, adenovirus plasmid pAd41-GFP was linearized by PmeⅠand used to transfect 293TE7 or 293E12 cells. The product of rescued viruses in 293TE7 was about twenty times higher than that in 293E12 cells by virus titration assay. The virus-packaging ability was also studied quantitively. The amount of progeny viruses produced in 293TE7 was three to ten times higher than that in 293E12 cells when equivalent seed Ad41-GFP viruses were incubated. Furthermore, Ad41-GFP still possessed a genetically-stable genome after being passaged six times on 293TE cell line based on the restriction digestion analysis.In summary, TPL of Ad41 was cloned and used to construct eukaryotic expression plasmid pcDNA3-TPL-E1B.293TE cell lines with increased Ad41 E1B55K expression were successfully established, which possessed an enhanced Ad41-GFP-Packaging ability.2. Enhanced virus mRNA export partially accounts for the promoted Ad41-GFP-packaging ability of 293E12 or 293TE7.Virus genome synthesis and structural protein expression are two key events before Ad assembly, which were investigated in this study.293,293E and 293TE cells were infected with Ad41-GFP at an MOI of 100 (MOI was calculated with the viral particle titer). At 24 hours post infection, virus genomes were extracted with Hirt’s method and analyzed on agarose gels before stained with EB. The amount of virus genomes synthesized in 293E12 was equal to that in 293 TE7, and was 4 times more than that in 293 cells. To determine if the increased E1B55K expression resulted in an increased virus late mRNA concentration in the cytoplasm, viral late cytoplasmic and nucleic RNAs were analyzed by quantitative RT-PCR at 12,16 and 24 h after infection. At all times tested, the L2 and L3 mRNAs were expressed at higher levels in cytoplasm of 293E12 cells than that of 293 cells. We further evaluated the V proteins with Western blot.293TE7 produced more these structure proteins than 293E12, which also produced more than 293 cells after being infected by Ad41-GFP. These data were consistent with the results of Real-time RT-PCR. We therefore concluded that Ad41 E1B55K, which can modulate viral mRNAs export from the nucleus, contributed to improved structure proteins expression during the late phase of adenovirus infection.3. Development of new Ad41 vector systemThe Ad41 E1B55K could not be totally complemented by the corresponding gene of Ad5, and expressing Ad41 E1B55K in packaging cells can improve the replication of Ad41. These findings prompted us to establish a new replication-deficient Ad41 vector system, in which only E1A and E1B19K genes were deleted and replaced with transgene while the E1B55K was preserved. The E1B55K gene of Ad41 as well as the corresponding promoter of Ad5 was inserted downstream of the transgene (GFP) expression cassette of Ad41 shuttle plasmid pSh41-CMV. The generated plamid pSh41ElB-GFP was used to co-transform E. coli BJ5183 together with the backbone plasmid pAdbone41. The recombinant, adenovirus plasmid pAd41E1B-GFP, was linearized by Pmel and then used to transfect 293 cells. We had thought that Ad41 E1B55K saved in the virus genome would make the recombinant vector propagate effectively in 293 cells. Ad41E1B-GFP did be rescued from and could be passaged on 293 cells. However, the yield of progeny viruses was very low, which we attribute to the low expression of Ad41 E1B55K. Further efforts will be made to exchange the promoter of Ad41 E1B55K in the vector system.Conclusively, in this study we investigated the correlation of expression of Ad41 E1B55K and virus replication in 293 cells. The synthesis of virus genomes and structure proteins were remarkably enhanced in virus-infected 293 cells when exogenous Ad41 E1B55K was expressed, which finally resulted in promoted yield of progeny viruses. A more effective packaging cell line (293TE7) was established and could be used to amplify wild-type or recombinant Ad41 viruses. This study also laid a foundation for further improvement of Ad41 vector system.更多还原