【作者】 王春艳；

【导师】 张玉静；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 作为一类新型的非病毒基因转运工具,蛋白或多肽介导的基因转运越来越表现出强大的基因转运效率。在当前的研究中,一些研究人员发现,组蛋白能有效的介导基因转运（组蛋白转染）。小麦组蛋白H4是构成核小体的核心组蛋白,由103个氨基酸组成,它的基因序列和蛋白的空间结构最初是由Tabata等人1983年发表的。它与人的组蛋白H4及其它真核生物的组蛋白H4有着高度的同源性,基因内部均不含内含子序列。在氨基酸组成上来讲,与人的组蛋白H4氨基酸序列相比,只有第61和78两个位点的氨基酸差异。这个分子量大约在11.0 kDa小分子蛋白,由于它的低毒性和低免疫原性,是一个较好的基因转运工具的候选分子。基于此,我们构建了组蛋白H4融合蛋白基因转运载体。蛋白跨膜结构转导域（PTDs）是一类能穿越哺乳动物细胞质膜的小分子多肽类物质,这类物质已经被广泛应用于蛋白质治疗的各个领域。研究人员可以通过基因工程的方法将PTDs的基因与细胞毒作用的蛋白进行融合与表达,借此将异源目的蛋白携入细胞内。PTD-tat是研究比较透彻的一个穿膜肽。它是来源于HIV-1病毒的TAT蛋白的一个多肽结构域,由11个氨基酸组成。它的组成序列,YGRKKRRQRRR,由几个强碱性的氨基酸序列组成的,并且是完成穿膜作用的关键肽段。考虑到PTD-tat在药物运输中起到的重要作用,很多人利用此分子作为治疗性分子或粒子进入哺乳动物细胞的重要工具。尽管有报导称,四种核心组蛋白都有蛋白跨膜转运的能力,但是在研究表明,与单纯的组蛋白相比较,引入PTD-tat融合组蛋白作为DNA转运工具,在很大程度上能够提高组蛋白转染细胞的效率。正是因为此多肽能够高效的介导小分子和大分子的细胞内摄,在本研究中,我们将此肽整合进入小麦组蛋白H4的C末端,以保证小麦组蛋白H4能通过PTD-tat的途径高效的将治疗基因携入细胞内。除此之外,靶向性药物专一地用于治疗癌症有着许多优点,例如可以维持一个较低的给药浓度,就能将药物从血清中运送至靶细胞,还可以减少药物的副作用,并且可以提高抗肿瘤的效率。一个成功的靶向性药物传输系统必须能够获得很高的细胞穿膜效率并能停滞于特异性的细胞类群上。这种特异性是通过将靶向性的基序与药物传输系统相结合,而这些基序也必须能够精确的与癌症细胞表面的受体相结合。这样才能有效的降低药物对正常的健康组织的副作用。促黄体素释放素（LHRH）的受体在人乳腺癌、卵巢和子宫内膜肿瘤以及前列腺癌细胞等肿瘤细胞表面过量表达,而在正常内脏组织细胞表面没有表达,因此,我们利用LHRH作为靶向性的基序转运抗肿瘤药物至特定的癌症细胞,起到了很好的效果。本研究构建了一个异源性的、有组氨酸标签的重组小麦组蛋白H4,命名为H4TL。在小麦根尖组织中提取了小麦基因组,目标基因H4TL包含小麦组蛋白H4基因,蛋白跨膜转导肽TAT基因以及靶向性配体分子LHRH的基因序列。通过PCR方法在小麦基因组中扩增H4基因,利用重叠延伸PCR方法对基因进行改造。经过6次PCR,每次PCR片段均克隆进pMD18T载体中进行鉴定,鉴定正确后的片段进入下一次延伸反应,最后得到了编码完全正确的基因片段,将此片段插入改造过的原核表达载体pET28a （++）中,得到重组pET28a（++）-H4TL质粒。将鉴定正确的质粒转入大肠杆菌E.coli BL21（DE3）中,利用IPTG诱导表达。此目的蛋白在大肠杆菌中获得高效表达。目的蛋白是一个含有141个氨基酸,分子量为15.5 kDa小分子蛋白。利用抗His标签的单克隆抗体对目的蛋白进行了Western blot鉴定。将表达阳性的细胞通过摇瓶进行发酵培养,通过改变诱导条件,优化了最佳表达条件,培养基PH为6.8-7.6,诱导剂IPTG浓度范围0.6-1.0mM,诱导温度25-30℃,诱导时间>10h,得到了高效可溶性表达,表达量占菌体总蛋白的35%以上。收集发酵瓶中细胞,用超声波破碎后离心,上清过滤后冷冻保存。用质量分数为30%的硫酸铵溶液盐析沉淀目的蛋白,随后用6M盐酸胍变性结合亲和层析分离和纯化融合蛋白H4TL,亲和纯化的介质为Ni⁺-葡聚糖。100-200mM的咪唑进行梯度洗脱,收集到峰期的洗脱液,用凝胶层析脱盐柱快速脱盐,纯化后的样品经超滤柱或PEG20000进行浓缩,于超净工作台用0.22μm滤膜过滤后冷冻保存。最终建立了一种有效的纯化方法,对重组蛋白进行了高效的分离和纯化,高效液相色谱分析其纯度达到了95%以上。用SmartSpecTM plus分光光度法测定纯化后蛋白浓度,分别按蛋白/质粒DNA质量比0-8进行凝胶电泳阻滞试验,凝胶电泳阻滞结果证明重组蛋白H4TL可以有效的结合质粒DNA。在凝胶电泳阻滞试验的基础上,我们对结合后的蛋白/DNA复合物进行了降解试验。核酸降解试验结果证明了重组蛋白与DNA形成的复合物有很强的稳定性,并且可以有效的防止DNA酶的降解。更重要的是,此蛋白成功地将报告基因pEGFP/C1转染进入人乳腺癌细胞MCF-7,人卵巢癌细胞HO8910,人前列腺癌细胞LNCap,人肺癌细胞A549以及人宫颈癌细胞HeLa中,转染效率与细胞类型、复合物浓度、蛋白与质粒DNA的配比、细胞密度及Ca²⁺浓度有关。我们确立的最佳的转染条件为蛋白与质粒DNA质量比4:1,转染前细胞为60-70%丰度,Ca²⁺浓度为2-4mM。凋亡素,是一个来源于鸡贫血病毒的小分子蛋白质,之所以受到人们关注,是因为它可以选择性的杀死肿瘤细胞而对正常细胞无毒性作用。凋亡素的亚细胞定位是肿瘤特异性的关键因素,在正常细胞中,它位于细胞质中,而有肿瘤细胞中它可以转运进入细胞核中。凋亡素的这种核定位机制主要是由自身的磷酸化水平控制的。在肿瘤细胞中,凋亡素引起活细胞内包括Akt等激酶物质的核聚集,并且可以被CDK2磷酸化。因此,凋亡素可以间接地在活细胞内传递细胞凋亡信号。此外,凋亡素也会以多聚物的形式与DNA分子相结合,通过与几个核靶分子相互作用,例如与促细胞后期复合物相互作用,导致G2/M期逃逸。这种促凋亡信号由信号分子Nur77经核传递给细胞质,激活非p53依赖的线粒体途径,诱导肿瘤细胞凋亡。我们利用靶向性的蛋白H4TL作为载体,进行凋亡素基因的转染研究,来评价H4TL的转染效率及其凋亡素转入细胞后的释控及表达,同时检测凋亡素基因对人乳腺癌细胞MCF-7及人卵巢癌HO8910两种肿瘤细胞的凋亡作用。我们构建了凋亡素真核表达载体pcDNA3.1（+）/apoptin,利用重组蛋白H4TL将其转染进入受试细胞中。利用经典的RT-PCR和western blot分析其在受试细胞中的表达情况;MTT法检测了转染凋亡素后细胞的增殖抑制情况,利用Annexin V–FITC /PI双荧光染色法对转染后的细胞进行细胞凋亡的检测,得到了较高凋亡率,对阴性的对照细胞系-正常人肺胚二倍体细胞2BS的凋亡率不超过20%,MCF-7细胞的凋亡率达到了42%（对照组为9.18%）; HO8910细胞的凋亡率为44%（对照组为3.31%）。此外,我们利用DNA ladder法检测了凋亡后细胞中的基因组DNA的片段化,利用罗丹明123作为荧光染料检测了处于凋亡状态的细胞的线粒体膜电位的缺失情况,这些试验帮助我们进一步验证了重组蛋白H4TL作为基因转运载体和凋亡素作为治疗基因的有效性。同时,这些结果说明了靶向于肿瘤特异性受体的非病毒载体为基因转运提供了一个廉价的、简单高效的工具。更多还原

【Abstract】 Protein/peptide-mediated gene delivery has recently emerged as a powerful approach in non-viral gene transfer. In previous studies, several groups found that histones can efficiently mediate gene transfer （histonefection）. Wheat histone H4 is a nucleosome core histone whose gene was cloned and structure determined by Tabata et al. It is relatively conservative in eukaryotic core histones. There are only two amino acid differences at sites 61 and 78 between human histone H4 and that of wheat, which allows for a good candidate for gene transfer. For these reasons, we have constructed recombinant wheat histone H4 as a new gene delivery carrier.Protein transduction domain TAT （PTD-tat） is one of the most well-studied and efficient cell-penetrating peptides. It contains an 11-amino acid peptide of the human immunodeficiency viruses type 1 （HIV-1） Tat Protein. The amino acids of PTD-tat are highly basic, YGRKKRRQRRR, which is crucial for penetrating the plasma membrane. Because of the potential of PTD-tat for mediating cellular uptake of small and large molecules, in this study we genetically incorporated PTD-tat into the C-terminus of wheat histone H4 to allow H4 to enhance penetration of the plasma membrane via the PTD-tat pathway. In addition, a targeted drug delivery system must achieve high cell permeability and retention by the specific cell population. Target cell specificity is achieved by attaching molecular targeting moieties to the drug delivery system that interact precisely with receptors of cancer cell surface. This ensures that adverse side effects on healthy tissues will be minimized. Receptors for the luteinizing hormone-releasing hormone （LHRH） are overexpressed in the plasma membrane of cancer cells, and expression is not detected in normal visceral organs. Therefore, LHRH can potentially be used as the targeting moiety to deliver anticancer drugs specifically to cancer cells and facilitate their cellular uptake.Here, we report the generation of plasmid constructs that overproduce H4-TAT-LHRH （H4TL） as heterologous, His-tagged fusion proteins. The wheat genome was extracted from wheat root tissue. The putative fusion H4TL gene containing the wheat histone H4 gene with PDT-tat （YGRKKRRQRRR） and LHRH （QHWSYGLRPG） gene sequences was cloned from the Triticum aestivum genome and amplified by overlap extention polymerase chain reaction （OE-PCR）. Every PCR fragment was determined by cloning it into pMD18-T vector and sequenced. The sequence of modified wheat histone H4 gene was in alignment with the GenBank sequence. The last amplified sequence was digested using EcoR I-Not I, cloned into pET28a （++）, transferred E.coli JM109 cells, and identified by PCR and restriction digest. Correct constructs were identified by sequencing. E.coli BL21（DE3） containing the pET28a（++）-H4TL recombinant plasmid were induced by isopropyl-β-D-thiogalactopyranoside （IPTG） .The expression positive cells were induced using shake-flask fermentation and collected by centrifugation （7000xg at 4 oC for 15 min）, washed twice with PBS and lysed by sonication. The clear supernatant was collected by centrifugation （10000xg at 4oC for 15 min）, then obtained by filtration through a 0.22μm Millipore membrane. The supernatant proteins were denaturated by 6 M guanidine hydrochloride and loaded onto a nickel column, （0.7x2.5 mm, GE Healthcare）. Chromatography operation was performed on an AKTA^TM Explorer Fast Protein Liquid Chromatography system （GE Healthcare）. Ni⁺ -metal affinity chromatography was used to purify His-tagged H4TL fusion proteins from supernatant proteins by guanidine hydrochloride denaturation.Purified proteins were desalted using a HiTrap desalt column （GE Healthcare） according to the manufacturer’s instructions, concentrated using an Amicon Ultra-15 Centrifugal Filter Units （Milipore, UK）, and buffer-exchanged with 5% dextrose in 20 mM Tris-HCl pH 7.4. The H4TL fraction was freeze-dried and stored at -70oC for further use.The modified H4TL gene encoded a recombinant protein of 141 amino acids with an approximate molecular weight of 15.5 kDa. Gel electrophoresis mobility shift assays demonstrated that the purified modified H4 protein had high affinity for DNA. Most significantly, complexing plasmid pEGFP/C1 with H4TL was transfected with high efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. The transfection efficiency mediated by histones is dependent on the relative concentration （DNA: histone ratio）, type of cells and the incubation time.The chicken anaemia virus-derived protein apoptin is a tumour-specific cell-killing agent. This 14 kDa protein selectively induces apoptosis in a wide variety of transformed cells but not in primary cells. The cancer-selective toxicity of apoptin correlates with its cellular localization. Apoptin enters the nuclei of cancerous cells, whereas in nontransformed cells it remains in the cytoplasm. The current hypothesis is that nuclear trafficking and tumor specific phosphorylation of apoptin at Thr-108 are essential for the induction of apoptosis. Recently, Rohn and colleagues reported that phosphorylation of apoptin is required for apoptosis, but that abolishing the phosphorylation site of apoptin does not significantly disrupt its nuclear input into tumor cells.In the present study, we attempted to use H4TL with naked plasmid DNA, which contains a apoptin tumor suppressor gene, to evaluate the transfection efficiency and antitumor activity in human ovarian carcinoma （HO8910） and breast cancer （MCF-7） cells in vitro. We observed that apoptin delivery resulted in a more differential growth inhibition pattern in cancer cells in vitro. In this study, the wild-type apoptin gene was transfected into cancer cells using the H4TL as a vector, and the expression level and the activity of apoptin gene were evaluated in vitro. Gene expression was determined by classic reverse transcriptase-PCR and western blotting analysis. The cellular growth inhibition of H4TL-mediated apoptin transfection were assessed by MTT （[3-（4,5-dimethylthiazol-2-yl） -2,5-diphenyltetrasolium bromide] benzene sulfonic acid hydrate） assay. The apoptosis induced by apoptin were measured by Annexin V–FITC （fluorescein isothiocyanate） /PI staining, DNA ladder and lost of mitochondrial membrane potential. The results showed that remarkable apoptotic characteristics such as nuclear shrinkage appeared in tumor cells, but few apoptotic characteristics were observed in control groups. The apoptosis present were higher than control, the cell cycle were exchanged also detected by FACS. It was suggested that the recombination protein H4TL could mediate apoptin’s transfection and could promote them expression and induce apoptosis in different tested tumor cells effectively. Our data suggest that H4TL can mediate apoptin gene transfer in vitro with high efficiency and is a promising new strategy for apoptin gene therapy. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provide a cheap, simple, highly efficient tool for gene delivery.更多还原