【作者】 程通；

【导师】 陈睦传； 夏宁邵；

【作者基本信息】 厦门大学 ， 动物学， 2006， 博士

【摘要】 HPV感染是常见的性传播疾病,与宫颈癌及泌尿生殖道相关癌症的发生关系密切,严重危害人类健康。开发安全有效的HPV预防疫苗有望消灭或显著降低HPV感染造成的危害,具有十分重大的社会意义。我实验室正在研究开发HPV基因工程疫苗,迫切需要可简便有效对候选疫苗的保护性进行评估的方法。目前的HPV体外感染模型在实验周期、稳定性、滴度等方面存在各自的缺点,而新型实验技术平台的发展为构建更有效的感染模型提供了有利条件。杆状病毒近几年来被发现可作为一种对哺乳动物细胞的新型基因转移载体,相比其它基因转移方法具有许多独特的优势。本论文即探讨了应用重组杆状病毒在哺乳动物细胞中构建HPV假病毒的可行性。为了更有效的获得HPV假病毒本论文对重组杆状病毒对哺乳动物细胞的基因转移方法进行了研究,建立了更为简便、快捷且高效的基因转移方法。该方法被成功用于建立了有效的HPV16假病毒体外感染模型,以及鉴定杆状病毒滴度的新方法。本研究利用Bac-to-Bac系统、增强型绿色荧光蛋白EGFP和CMV-T7联合启动子构建对哺乳动物细胞的杆状病毒基因转移载体,首先探讨了直接使用重组杆状病毒感染后的昆虫细胞培养上清对哺乳动物细胞进行基因转移的可行性。将重组杆状病毒感染4d后的Sf-9细胞培养上清（≥1.0×10⁷PFU/mL）直接与HepG2细胞37℃作用12h,可获得高于95%的基因转移效率,证明了重组杆状病毒感染后的昆虫细胞培养上清可直接用于高效转导哺乳动物细胞。通过对转导时间和稀释比例的优化,显示用哺乳动物细胞培养基等体积稀释后的带毒上清可兼具高效和低损伤的特点。应用上述结果本研究首次提出并初步建立了重组杆状病毒上清转导法。该方法与脂质体、重组逆转录病毒、重组痘苗病毒进行的对比显示其具有高效、低毒的优点。应用该方法对27种不同类型及组织来源的哺乳动物细胞进行基因转移实验,包括16种人类组织细胞、9种啮齿类组织细胞和2种猴组织细胞,结果显示该方法具有较好的适用性,可有效转导大多数哺乳动物细胞。并发现重组杆状病毒对灵长类来源细胞的基因转移效率显著高于对啮齿类来源细胞,对贴壁细胞的基因转移效率显著高于对悬浮细胞。结合最新的研究进展,本研究进一步对病毒上清转导法的实验条件进行摸索优化,并尝试采用新方法提高转导效率。对使用不同的稀释缓冲液和转导温度的研究结果显示,以PBS作为稀释缓冲液在27℃下进行转导可显著提高转导效率。在PBS环境中对比不同转导时间和稀释比例以及不同血清含量对转导效果的影响结果显示,转导效率与转导时间和病毒用量均呈正相关,并首次发现PBS中存在血清成分不利于提高转导效率。应用不同浓度丁酸钠增强表达的实验结果显示,0.5～1mM的丁酸钠较为合适于在CV1细胞中提高杆状病毒载体的表达效率,更高浓度的丁酸钠对细胞有明显毒性。本研究首次在杆状病毒转导哺乳动物细胞中使用离心方法,结果显示通过600g水平离心1h即可获得高水平的转导和表达效率,优于在PBS环境中27℃孵育8h的效果,并进一步对离心时间、离心后孵育时间、缓冲液进行了摸索优化。应用上述结果本研究首次提出并建立了重组杆状病毒上清离心转导法。病毒上清以PBS为缓冲环境600g水平离心1h进行转导的方法可在获得高转导效率的同时显著缩短实验时间,提高了实验效率。该方法具有简便、快捷和高效的特点,并可作为一种常规操作方法用于日常实验。本研究同时探讨了辅助感染可表达T7 RNA聚合酶的重组痘苗病毒VV-T7RP对表达的影响,显示辅助感染VV-T7RP可显著增强带有CMV-T7联合启动子的重组杆状病毒的表达效果,表明将重组痘苗病毒VV-T7RP与重组杆状病毒结合可建立一种高效瞬时表达方法。本研究首次设计构建了可同时在哺乳动物细胞中表达HPV16L1和L2蛋白的重组杆状病毒,并采用本研究建立的重组杆状病毒上清离心转导法,探讨了通过重组杆状病毒转导方法在哺乳动物细胞中表达组装HPV16假病毒的可行性。细胞感染实验和透射电镜观察结果证实,通过该方法成功获得了具有感染能力的HPV16假病毒。应用已被证实的4株HPV16单抗,证明该假病毒感染模型可应用于进行中和实验。应用该模型,从本实验室构建的18株HPV16单抗中筛选获得2株中和单抗3D10和PD1,同时证明本实验室构建的候选基因工程疫苗HPV16 VLP可激发Balb/c小鼠产生具有中和能力的保护性体液免疫。该模型的成功建立为HPV16中和抗体的筛选和HPV16预防疫苗的研究提供了必要的条件和有力的支持,也为进一步构建其它型别的HPV假病毒体外感染模型提供了基础。本研究还首次尝试了使用HPV16假病毒对昆虫细胞的感染实验,显示HPV16假病毒可能具有将核酸导入昆虫细胞的能力。大量的关于重组杆状病毒表达系统的应用研究实践显示,准确测定病毒滴度对于保持蛋白生产和研究实验的稳定性是十分重要的。本研究综合了应用重组杆状病毒上清离心转导法可高效转导哺乳动物细胞的特点以及辅助感染VV-T7RP可显著增强CMV-T7联合启动子表达水平的特点,提出了新型的可在哺乳动物细胞中测定重组杆状病毒滴度的方法。本研究设计构建了同时具有在昆虫细胞中和哺乳动物细胞中高效表达报告基因的重组杆状病毒。应用该病毒首先在昆虫细胞中准确测定滴度,再通过梯度稀释后离心转导CV1细胞并辅助感染VV-T7RP,在12～24h后直接观察细胞发光情况,以TCID₅₀方法计算病毒滴度。结果显示,以CMV-T7联合启动子引导的EGFP报告基因表达元件在VV-T7RP辅助下具有最高的检测灵敏度,其测得的滴度与昆虫细胞中测得的滴度相当,优于单独使用CMV启动子或T7启动子。本研究首次提出了可将CMV-T7-EGFP表达元件作为一种优良的病毒滴度鉴定元件克隆于杆状病毒载体上,其在昆虫细胞中不会进行有效表达和干扰重组蛋白和病毒的生产,同时又可在哺乳动物细胞中进行高灵敏度和快速、直观的检测,不需要使用流式细胞仪进行分析。该方法为重组杆状病毒滴度的测定提供了一种选择。更多还原

【Abstract】 Human papillomavirus can cause cervical cancer and other related cancers. It is very important to research and develop HPV vaccines. The recombinant HPV vaccine is underdeveloping in our lab. And the simple and efficient method for estimation of the protective ability of the candidate vaccine was needed. However currently available in vitro infection models are technically demanding or relatively low-yield. Recently reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages compared with other gene transfer methods. In this study, the feasibility of constructing HPV pseudovirion in mammalian cells by recombinant baculoviruses was investigated. The transduction protocol of delivering exogenous genes into mammalian cells by recombinant baculoviruses was investigated and optimized. And a new convenient way for rapid and efficient expression of target genes in mammalian cells was founded. With help of the new transduction protocol, the HPV16 pseudovirion and a rapid method for estimation of baculovirus titer were obtained in this research.Based on the Bac-to-Bac system recombinant baculoviruses were constructed, which contain the enhanced green fluorescent protein （EGFP） gene driven by CMV-T7 promoter, to investigate the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of insect cells infected by recombinant baculoviruses. Directly incubating HepG2 cells with the culture supernatant of infected Sf-9 cells （≥1.0×10⁷pfu/ml） for 12 hours in 37℃could achieve hyper 95% efficiency of gene transfer. The optimization of the incubation times and dilution ratios showed that the culture supernatant of infected insect cells l:l（vol:vol） diluted by the mammalian cell complete culture medium could achieve the high efficiency of gene transfer and expression together with the least impairment to cell viability.We first developed a transduction protocol based on the recombinant baculovirus culture supernatant transduction. Comparing with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems, the baculovirus vector could achieve higher transduction rate with no obvious cytopathic effect. Twenty-seven different mammalian cell lines were used to investigate the feasibility of delivering reporter gene into various mammalian cells with the culture supernatant transduction protocol, including sixteen human cell lines, nine rodent cell lines and two monkey cell lines. Results showed that most mammalian cell lines could be transduced with recombinant baculoviruses by this way. And we found that the gene transfer efficiencies in primate cell lines by baculovirus vector were markedly higher than those from rodent, and the efficiencies in adherent culture cells were markedly higher than suspended culture cells.Refer to the recent research reports, the protocol of culture supernatant transduction was further modified and optimized, and new ways were attempted to enhance gene delivery and expression. Different surrounding solutions and incubation temperatures were investigated, and the result showed that transduction occurred at 27℃using PBS as the surrounding solution could achieve the highest efficiency. Different incubation times and dilution ratios and different concentrations of FBS in PBS solutions were investigated, and results showed that transduction efficiencies were consistent to the virus dosages and incubation times. And we first found that the presence of FBS in PBS surrounding solution could influence transduction efficiency. Different concentrations of sodium butyrate were used in the culture medium to enhance gene expression. Results showed that 0.5～1 mM sodium butyrate was comparatively fit for the CV1 cells. In this study, centrifugal method was first used in the transduction of the mammalian cells by baculoviruses. And we found that centrifugal transduction at 600g for 1h could achieve more higher gene delivery and expression efficiencies than transduction in PBS at 27℃for 8h. And different centrifugal times, incubation times post centrifugal transduction and surrounding solutions were further investigated and optimized.In this study, we first developed a new and efficient transduction protocol based on the recombinant baculovirus culture supernatant transduction and centrifugal transduction. Mammalian cells transduced with the virus supernatant at 600g for 1h in PBS surrounding solution could achieve higher transduction efficiency with more fewer time. The centrifugal transduction protocol have notable advantages: simple, rapid and efficient, and could be easily used in daily common experiments. The effect of co-infection of recombinant vaccinia virus VV-T7RP, which could efficiently express the T7 RNA polymerase, on the gene expression in cells transduced with recombinant baculovinis containing the CMV-T7 promoter was investigated, results showed that VV-T7RP coinfection could markedly enhance the gene expression level in the transduced cells. It suggested that the VV-T7RP and recombinant baculovinis vectors could combine to an high efficient transient expression protocol.In this study, the recombinant baculovinis BacCS16L1L2 was constructed and capable of expressing HPV16 L1 and L2 protein in mammalian cells simultaneously. Utilizing the recombinant baculovinis culture supernatant centrifugal transduction protocol developed in this study, the HPV16 pseudovirion was successfully obtained in the 293FT cells. The infection assays and electron microscopy showed that the HPV16 pseudovirion have the infectivity. Four anti-HPV16 Mab, which have been fully characterized, were used to identify the HPV16 pseudovirion. Results showed that the HPV16 pseudovirion obtained in this study could be used for neutralization assay. And this model was used for testing neutralizing antibodies. Two neutralizing antibodies, 3D10 and PD1, was selected from eighteen anti-HPV16 Mab lines obtained in our lab. Utilizing the HPV16 pseudovirion, we proved that the protect humoral response could be elicited in Balb/c mice immunized with the HPV16 VLP constructed in our lab. In this study, HPV16 pseudovirion was first tried to infect the insect cells, and eGFP expression was found in some infected insect cells.Numerous research and application works on the recombinant baculovinis expression system have showed that it was very important to know the exact titer of a recombinant baculovinis stock in time for the stability of experiments and protein productions. In this study, utilizing the centrifugal transduction method and VV-T7RP coinfection, a new strategy of determining the recombinant baculovinis titer in mammalian cells was developed. Recombinant baculoviruses BacDGCG and BacT7G were constructed and capable of expressing EGFP in mammalian cells and insect cells. Firstly the virus titers were determined in insect cells. Then the same virus samples were used to transduce the CV1 cells by centrifugal method, followed by coinfected with VV-T7RP. 12～24h post transduction, the numbers of EGFP-positive wells were counted by fluorescence microscopy, and the transducing titer was calculated using TCID₅₀ method. Results showed that the CMV-T7-EGFP expression cassette in the baculovirus genome has the highest detection sensitivity than single CMV or T7 promoter with presence of T7 RNA polymerase and the obtained virus titer was similar to that determined in insect cells. In this study, we first suggested that the CMV-T7-EGFP expression cassette could be used as an excellent element for virus titer detection in mammalian cells, with no interfere with the protein or virus productions in insect cells. It was a new way could be chosen by researchers for the detection of recombinant baculovirus titers.更多还原