链霉菌噬菌体φC31整合酶的性质和功能及其在哺乳动物细胞的应用研究

【作者】 张茂祥；

【导师】 薛京伦；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 利用基因转移技术有效地将目的基因位点特异性地整合到哺乳动物细胞的基因组中,从而避免外源基因随机插入基因组,这是基因治疗和转基因动物研究中未解决的重要问题。链霉菌噬菌体ΦC31整合酶可以将外源核苷酸序列定点整合到哺乳动物细胞基因组的特异性位点上,从而极大的降低了由于外源基因随机插入造成的基因突变风险,可以成为在哺乳动物细胞中进行转基因位点特异性重组操作和基因治疗的有用工具。现在常用的几种基因转移途径,病毒载体由于随机整合和免疫学性质,安全性一直受到人们的质疑。非病毒载体如裸DNA载体的转移效率受不同细胞类型的影响,也有潜在的随机整合外源基因到宿主基因组的可能性。近年来新出现的蛋白转导技术能够在几乎不受细胞类型限制的条件下,转移一个活性的蛋白进入哺乳动物细胞中,在体内可以通过小鼠的血脑屏障,提示该技术可以作为一种新的基因转移途径在基因治疗和转基因动物研究中得到应用。本文首先研究了ΦC31整合酶在体外的生物学特性。我们通过建立新的体外ΦC31酶的活性检测系统,表达纯化了23.8U/mg的重组ΦC31整合酶蛋白,它能够在pH6.5—11,温度9—37℃的条件下表现酶活性。在最适条件下（pH8.5,温度20℃）,ΦC31整合酶蛋白的Km和Vmax分别是6.261 nmol/L和0.8621 nM^-1min^-1。利用原子吸收质谱分析（ICP-mass）发现,ΦC31整合酶蛋白结合高浓度的K⁺离子,没有发现结合Ca²⁺,Mg²⁺和Mn²⁺等金属离子的特性。红外傅立叶光谱（FT-IR）和圆二色仪（CD）对该蛋白构象的研究表明,蛋白二级结构中的α—螺旋,β—折叠和无规则的卷曲的比例十分敏感地受pH值,温度,离子浓度和酶的DNA底物等条件的影响。在一些蛋白酶的抑止剂（如EDTA,EGTA,2-ME,PMSF,DTT）和低浓度的去垢剂（如0.1%Tween20和TritonX-100）的条件下,该整合酶活性基本不受影响。以上研究表明ΦC31整合酶的性质与已发现的λ重组酶家族成员（如Cre重组酶）完全不同,是一种新型的丝氨酸重组酶。ΦC31整合酶体外检测系统的建立和酶动力学性质的研究,将为后续的以该酶为代表的大分子量的丝氨酸整合酶家族的结构和功能研究奠定基础。在哺乳动物细胞中应用ΦC31整合酶在基因转移途径上仍然需要考虑其有效性和安全性。蛋白转导技术可以转移一个不受细胞类型限制的外源蛋白进入哺乳动物细胞中,转导后的蛋白在细胞中瞬时存在并表现生物活性。这一技术为有效地应用ΦC31整合酶介导的定点整合提示了新的方向。为了建立ΦC31整合酶的蛋白转导系统,我们构建了定量检测ΦC31整合酶功能的哺乳动物的293-PB[EGFP]报告细胞系。流式细胞仪分析（FACS）和细胞免疫组织化学的实验证实,我们制备的融合TAT-PTD（HIV-1的Tat蛋白的47—57aa的蛋白转导结构域）的ΦC31整合酶蛋白能够以密度依赖性的方式进入所有与之共孵育的哺乳动物细胞中,并且有效地介导特异性核苷酸序列位点的精确重组反应。至此,一个新的能够在哺乳动物细胞中应用蛋白转导技术,转移ΦC31整合酶的方法体系得到了初步的建立。我们从哺乳动物细胞中ΦC31整合酶的细胞定位与功能的研究入手,发现增加外源的ΦC31整合酶在细胞核内的分布,能够很好地增加该酶介导的细胞染色体水平的重组功能。为了更好在哺乳动物细胞中应用ΦC31整合酶,我们用NLS（SV40大T抗原的核定位信号）对ΦC31整合酶蛋白的C端进行了修饰。通过ΦC31整合酶分别以蛋白转导和质粒DNA转染的细胞学实验,证实C端融合NLS的ΦC31整合酶能够在哺乳动物细胞中有更好的重组效果。根据上述结果,我们利用TAT和NLS修饰的ΦC31整合酶（TAT-ΦC31-NLS）经蛋白转导介导人凝血因子Ⅸ（hFⅨ）基因特异性地整合到哺乳动物细胞基因组中,筛选得到的细胞系能够长期高效表达hFⅨ蛋白,并且表现出大约50%的外源hFⅨ基因位点特异性地整合在细胞基因组的psiA位点。在细胞中的瞬时存在的TAT-ΦC31-NLS能够介导外源基因定点整合到细胞基因组。活性TAT-ΦC31-NLS蛋白转导4天后的细胞,蛋白免疫印记（Western blot）实验已经不能检测到细胞中残存的整合酶蛋白。细胞周期和早期细胞凋亡分析实验表明,TAT-ΦC31-NLS蛋白转导对293和COS-1等哺乳动物细胞没有明显的影响。至此,我们应用TAT-ΦC31-NLS蛋白优化了ΦC31整合酶的蛋白转导技术,并且在哺乳动物细胞中有效介导外源hFⅨ基因定点整合在细胞基因组中,从而为更安全有效的转移ΦC31整合酶进入细胞的途径提供了新的例证。TAT-ΦC31-NLS整合酶蛋白能够从大量培养的细菌中经过一步快速的亲和纯化步骤制备,我们希望它能作为一个有效的工具应用在哺乳动物细胞的基因操作和基因治疗的领域。综上所述,本文说明了链霉菌噬菌体ΦC31整合酶的在体外催化重组反应的特性,为ΦC31整合酶在体外的基因重组操作和细胞中介导定点整合的应用提供了基础:通过对ΦC31整合酶的修饰和转移方法的优化,建立了在细胞中更有效和安全地利用ΦC31整合酶的蛋白转导方法,从而避免了利用的病毒和DNA载体可能造成的ΦC31整合酶基因随机整合和长期表达造成的毒性;最后以蛋白转导ΦC31整合酶在哺乳动物细胞中介导外源hFⅨ基因的位点特异性整合的应用研究,为ΦC31整合酶蛋白在哺乳动物细胞的基因操作和基因治疗领域的应用作了初步探索。更多还原

【Abstract】 Despite some success in the field of gene therapy and transgenic organisms,the research community is still facing an old problem,namely,adopting gene transfer approaches deliver a gene to target site-specific mammalian chromosome integration without adverse events and mutagenesis caused by random insertion.To minimize the potential disruption of genomic integrity,a target gene transfer should be inserted at a specific site proven to be a safe genomic location.Recently,Streptomyces phage F C31 integrase was reported to be able to mediate exogenous DNA containing wild att sequences site-specific integration into pseu-att sites on mammalian cellular genome.It is possible that the F C31 integrase system has great potential for a tool of gene manipulation and gene therapy.There have been several gene transfer approaches to deliver corrective genetic materials into target cells,some reported incident have been raising concerns about the safety of gene delivery viral vectors.Non-viral gene delivery approaches,such as naked DNA vector was advancing to gene delivery with poor transfection efficiency in some cell types and in vivo,and the potential for random integrating into the host genome resulting in continued expression.A new protein transduction technique was developing in several past years,that can deliver a biologically active enzyme to all cells and tissue of mice,thereby,overcoming limitations of DNA transfection and viral infection.It suggests that this new technique can be used as a tool of delivery F C31 integrase to mammalian cells for the sake of efficiency and safe.The catalytic activity and biological characterization of Streptomyces phage F C31 integrase was be studied by our established an in vitro linear intramolecular assay system and successfully expressed a C-terminal 6His-taggedφC31 integrase fusion protein in Escherichia coli BL21（DE3）.φC31 integrase purified in a single Ni-NTA affinity chromatographic step to a specific activity of 23.8U/mg protein at optimum pH 8.5 and 20℃.The partial purified enzyme was highly active in the pH and temperature ranges of pH 6.5 to pH 11,and temperature from 9℃to 37℃,respectively.Its catalytic activity was enhanced in the presence of K⁺,corresponding to the results of the purified protein contained more K⁺ other than Ca²⁺,Mg²⁺ or Mn²⁺ detected by the inductively coupled plasma mass spectroscopy（ICP-mass）.The K_m and V_max of the recombinantφC31 integrase were 6.261 nmol/L and 0.8621 nM^-1 min^-1,respectively.The stability of the purified protein showed that the protein second structural changes are sensitive to the conditions of pH,temperature,metal ions and DNA substrates as well,investigated by circular dichroism（CD） and Fourier-transform infrared（FF-IR） spectroscopy.All results indicated thatφC31 integrase was a member of serine recombinase family,but not characteristically and mechanistically,analogous toλ.recombinases,such as Cre recombinase.The catalytic and conformational characterization ofφC31 integrase will provide a base to catch on emzymic structure and function relationship of the large serine recombinase subgroupIn order to new application ofφC31 integrase protein transduction,fusion 11 amino acids peptide of HIV-1 Tat protein toφC31 integrase was expressed and purified,which can enter into mammalian cells and perform recombination function in the manners of dose-dependency in our established mammalian cellular quantitative assay system.The results were demonstrated using a fluorescence-activated cell sorter analytical flow cytometer and indirect immunofluorescence.The study of relationship betweenφC31 integrase activity and its sub-cellular localization in mammalian cells,indicates that nuclear translocation is a limiting factor to F C31-mediated recombination at genome level in mammalian cells.A nuclear localization signal（NLS） of SV40 large T antigen modified C-terminalφC31 integrase was designed to transferφC31 integrase into cells by protein transduction and DNA transfection.Consequently,a TAT-F C3-NLS protein transduction was used to mediate human coagulating factorⅨ（hFⅨ） gene site-specific integration into mammalian genome,those isolated and expanded cell lines can express hFⅨand have about 50%integration in cellular genomic psiA sites. Transient TAT-F C31-NLS displayed in transduced cells by immunoblotting analysis and had no influence on the cell cycle and early apoptosis in human embryonic kidney 293-cell and Monkey kidney COS-1 cell.TAT-F C31-NLS turned out to have efficient transducible ability and high recombination function in mammalian cell,and can be produced readily from bacteria expression on a large scale and purified rapidly by a single affinity chromatographic step under native conditions.We expect that TAT-F C31-NLS protein transduction can apply to serve as a useful tool for genetic manipulation and gene therapy.In conclusion,the results of catalytic characterization of Streptomyces phage F C31 integrase in vitro provide a base for its application in gene manipulation and gene therapy. We introduce F C31 integrase protein transduction into mammalian cells supplying the catalytic F C31 integrase with non-viral vector system in the form of DNA,which has low transfection efficiency in particular cell types and the potential for integrating into the host genome resulting in continued expression.Internalized F C31 integrase protein mediated hFⅨgene site-specific integration in mammalian genome represents an effective site-specific integration system for cells and may be of value in gene therapy and other chromosome engineering strategies.更多还原