【作者】 何红秋；

【导师】 王存新；

【作者基本信息】 北京工业大学 ， 生物医学工程， 2010， 博士

【摘要】 人类免疫缺陷病毒(HIV)引起的获得性免疫缺陷综合症(艾滋病)是人类历史上最严重、最致命的疾病之一。艾滋病目前仍然不能治愈。HIV编码的整合酶(IN)介导病毒DNA与宿主细胞基因组整合,是病毒复制所必需的关键酶之一。抑制IN的功能能够有效阻断病毒在宿主细胞内的复制。同时,由于正常人体细胞中没有IN的功能类似物,特异性作用于IN的抑制剂对人体的副作用可能很小。因此,IN被认为是抗HIV药物研发的理想靶点。IN在体内主要通过催化3′加工和链转移两步反应实现整合过程。以上两步反应能够在体外使用重组IN蛋白、寡核苷酸DNA底物以及二价金属辅助离子实现。此外,IN还能在体外催化链转移的逆反应——去整合,将链转移形成的DNA产物重新还原生成病毒DNA和靶DNA。建立高效的IN活性检测方法,并据此开展抑制剂的筛选,是当前IN抑制剂体外筛选的主要手段,也是以IN为靶点的抗病毒药物研发的热点之一。本研究表达纯化了重组IN蛋白,建立了IN活性检测的一系列高通量方法,并应用这些方法进行了IN的性质、抑制剂筛选等方面的研究。论文内容主要包括以下几个方面:(1)整合酶蛋白表达纯化及3′加工活性高通量检测方法的建立使用PCR获取了HXB2CG病毒株IN基因,通过序列分析,设计突变引物,使用重叠PCR方法将HXB2CG IN基因突变为HIV NL4-3 IN基因,并引入了F185K/C280S突变以提高蛋白溶解性。将目的基因连接到pET表达载体中,在大肠杆菌BL21中实现了高效且可溶性表达。通过亲和层析方法,从细胞破碎上清液中纯化到纯度高且具有3′加工和链转移功能活性的重组IN蛋白。根据分子信标的原理,设计了荧光基团和淬灭基团标记的模拟病毒DNA序列的3′加工DNA底物,提出了一种检测3′加工反应活性的新型荧光分析法。实验表明,该方法不但灵敏度、特异性高,操作简单,方便快捷,为全液相反应环境,而且能够实时定量监测IN 3′加工反应。该方法能够用于IN 3′加工反应性质研究和以3′加工为靶标的IN抑制剂高通量筛选。本研究建立的3′加工反应荧光检测方法有潜力应用到其他蛋白质与DNA相互作用的研究中,具有重要的学术和应用价值。(2)整合酶链转移活性高通量检测方法的建立和应用抑制链转移反应被认为是体内抑制IN功能活性的关键,当前IN抑制剂的体外筛选主要以链转移反应为靶标。本研究设计合成了分别用生物素和地高辛修饰的供体DNA和靶DNA,引入了链霉亲和素磁珠捕获反应DNA产物,提出了检测IN链转移反应活性或者检测3′加工与链转移反应整体活性的高通量酶联免疫吸附测定法(ELISA)。与之前的各种方法相比,我们提出的高通量ELISA具有明显的改进:(i)无需使用放射性底物,实现了高通量;(ii)反应时所有的试剂均自由悬浮在液体环境中,磁珠–DNA产物、磁珠–抗体接触更容易更充分,显著提高灵敏度,还能灵活应用到研究反应中各试剂的相互作用,有助于研究抑制剂的作用机理;(iii)无需包被、封闭微孔板,省时省力,检测前转移磁珠至新的微孔板能够几乎完全消除微孔板对试剂的非特异性吸附,有效降低了背景值,提高了特异性;(iv) ELISA和荧光免疫吸附测定(FLISA)两种检测策略并用,增加了方法的应用范围。成功应用高通量ELISA研究了金属离子对链转移反应的影响,得出了一些有意义的结果。使用已知IN抑制剂证明了高通量ELISA应用到IN抑制剂筛选中的有效性,且从天然产物提取物和合成化合物中成功筛选了数个具有初步活性的IN抑制剂。(3)整合酶去整合活性高通量检测方法的建立IN在体外能通过去整合反应实现对整合过程的逆转,且整合酶核心区(IN-CCD)具有独立催化去整合功能。建立去整合活性高通量检测方法对研究IN功能及筛选IN抑制剂都具有重要意义。本研究表达纯化了具有功能活性的野生型IN及IN-CCD蛋白,设计合成了生物素和地高辛双标记的去整合DNA底物,基于链霉亲和素磁珠捕获生物素标记DNA,建立了一种检测IN和IN-CCD体外去整合活性的高通量ELISA。研究了金属离子对去整合高通量ELISA的影响,结果表明与3′加工和链转移反应相似,IN去整合反应也更偏好于使用Mn2+而不是Mg2+为金属辅助离子。进一步使用NaCl滴定实验分析表明,IN去整合对Mn2+的选择偏好性与Mn2+比Mg2+更能稳定IN–DNA复合物有关。使用了已知IN抑制剂baicalein验证去整合高通量ELISA筛选IN抑制剂的有效性,证明了baicalein是明确的以IN-CCD为靶标的IN抑制剂。本研究建立的去整合高通量ELISA具有高通量、高灵敏度、高特异性、低背景、省时省力等优点,能够应用到去整合反应、抑制剂作用机理等研究中,且具有应用到以IN为靶标,特别是以IN-CCD为靶标的抑制剂高通量筛选的潜力。(4)整合酶核心区野生型和F185K可溶性突变型蛋白活性和溶解性研究野生型(WT) IN和IN-CCD溶解性较差,在大肠杆菌中表达时容易形成包涵体,给后期纯化及蛋白质功能研究带来不便,而F185K突变后IN和IN-CCD能实现可溶性表达且活性不受影响。通过构建、表达、纯化得到WT和F185K突变型IN-CCD,比较了其溶解性和活性差别。分析并比较了WT和F185K/C280S突变型IN蛋白溶解性和活性差别。结果表明,F185K和F185K/C280S突变后IN和IN-CCD的溶解性显著提高,能够实现可溶性表达;同时,突变后IN-CCD的去整合活性有一定程度的降低,IN的3′加工和链转移活性有一定程度降低。进一步通过同源模建,构建了WT和F185K突变型IN-CCD的蛋白结构,并在水溶液中进行了1800 ps的分子动力学(MD)模拟,得出了一些有意义的结果:(i) F185K突变后,体系的功能loop区柔性有一定程度的降低,整体运动性略有减小,催化核心DDE基序残基之间距离无显著变化,因此,突变后蛋白催化活性降低,但活性受影响程度不大;(ii) F185K突变对IN-CCD内部静电相互作用网络的改变驱动了蛋白构象的变化,特别是loop区构象变化明显,引起蛋白表面的部分疏水残基被包埋,亲水残基暴露,造成IN-CCD相对的极性溶剂可接近面积增大。同时,F185K突变后蛋白与水之间形成的氢键数量有明显的增加,这些变化使IN-CCD的溶解性显著提高。MD模拟与实验结果相吻合。该工作为理解蛋白质溶解性和蛋白质工程中蛋白质可溶性改造提供了一些有价值的信息和理论依据。更多还原

【Abstract】 Acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), is one of the most serious and deadly diseases in human history. Right now, there is still no cure for AIDS. HIV-encoded integrase (IN) catalyzes the integration of the viral DNA into the host genome, and IN is one of the vital enzymes necessary for viral replication. The inhibition of IN activities can effectively block the viral replication cycle of HIV in host cells. Moreover, there is no recognized counterpart of IN in normal human cells, and inhibitors specifically targeting IN may thus have little side effect on human body. Therefore, IN is considered to be an ideal target for the research and development of anti-HIV drugs.IN catalyzes two successive reactions, termed 3′-processing and strand transfer, to facilitate the integration process in vivo. Both the above reactions can be modeled in vitro using purified recombinant IN protein, oligonucleotide DNA substrates, and divalent cationic cofactor. Besides, in vitro, IN can also carry out an apparent reversal of strand transfer which has been termed disintegration, in which the DNA product of strand transfer is resolved into viral and target DNA segments. To develop high efficient assays for IN activities and apply these assays to screen inhibitors is the main method for in vitro IN inhibitor screening, and it is also a focus in the research field of antiviral drug development targeting IN. In this thesis, different types of recombinant IN proteins are expressed and purified, and a series of high-throughput assays for IN activities have been developed. These assays have also been applied in the studies of the properties of IN and IN inhibitor screening. The main content of the thesis consists of the following major aspects:(1) Expression, purification of integrase protein and development of a high-throughput assay for the 3′-processing reaction of integraseThe IN gene of HIV HXB2CG strain was acquired by PCR. After sequence analysis, primers for gene mutation were designed and site-directed mutagenesis of the HXB2CG IN gene was done by overlapping PCR to construct the IN gene of HIV NL4-3 strain. Site-directed mutagenesis was also employed to bring the F185K/C280S mutations to the constructed NL4-3 IN gene for the purpose of enhancing the protein solubility. The correctly constructed IN gene was ligated to a pET expression plasmid vector, and IN was highly expressed as a soluble protein in Escherichia coli strain BL21. After purification from the supernatant of cell suspension using affinity chromatography, the highly purified recombinant IN protein was active in 3′-processing and strand transfer reactions.Based on the principle of molecular beacons, we designed a fluorophore and a quencher labeled 3′-processing DNA substrate mimicking the viral DNA and developed a novel fluorescent assay for the detection of IN 3′-processing activity. The results obtained show that this assay is an overall liquid assay with high sensitivity, high specificity, easy and simple assay procedure, as well as real-time monitoring of the 3′-processing reaction of IN. The assay is also applicable in the 3′-processing reaction character study of IN and high-throughput screening of inhibitors targeting the 3′-processing reaction of IN. The fluorescent assay for IN 3′-processing reaction proposed in this thesis has the potential to be applied in the studies of interactions between other proteins and DNA. This work has important academic significance and application value.(2) Development and application of a high-throughput assay for the strand transfer reaction of integraseThe inhibition of strand transfer reaction is reported to be the primary key to block the biological functions of IN in vivo. Nowadays, In vitro assays for IN inhibitor screening are generally based on the strand transfer reaction. In this work, biotin and digoxin modified donor DNA and target DNA were designed and composed, and streptavidin-coated magnetic beads were involved to capture the reaction product DNA strand, and a novel high-throughput enzyme-linked immunosorbent assay (ELISA) was developed to measure the IN-catalyzed strand transfer reaction activity or 3′-processing and strand transfer reaction activities altogether. Compared to previous assays, the high-throughput ELISA proposed in this thesis has notable improvements: (i) It is a high-throughput format assay with no need of radioactive substrate; (ii) All the reagents are freely suspended in solution, makes the magnetic beads–DNA product contact and subsequent magnetic beads–antibody contact much easier and more sufficient, the sensitivity of this assay is thus enhanced. In addition, the assay is flexible to investigate the interactions among all reagents and it is easy to study the mechanism of IN inhibitors; (iii) Neither the precoating nor the blocking of microplate is required, it is less laborious and time consuming. The easy transfer of magnetic beads into fresh microplate before detection helps to eliminate almost all the non-specific binding of reagents on the microplate, the background readings are effectively reduced and the higher specificity of this assay is thus achieved; (iv) Both the ELISA and the fluorescence-linked immunosorbent assay (FLISA) associated detection strategy are used, and the applicable range of this assay is expanded.The high-throughput ELISA was successfully applied to study the effects of divalent cations on the strand transfer reaction, and several meaningful results were obtained. The assay was also proved to be effective in inhibitor identification of IN by the employment of known IN inhibitors. Several samples from natural product extracts and composed compounds were screened out to be active IN inhibitors by using this high-throughput ELISA.(3) Development of a high-throughput assay for the disintegration reaction of integraseIN can carry out the disintegration reaction in vitro, a reversal of the integration process. The central catalytic domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction alone. To develop high-throughput assays for disintegration reaction is of great significance for investigating the functions of IN and IN inhibitor screening. In this work, wild type IN and IN-CCD proteins with functional activities were expressed and purified. Biotin and digoxin-labeled disintegration DNA substrate was designed and composed. Based on the application of streptavidin-coated magnetic beads to capture the biotin-labeled DNA, we proposed a high-throughput ELISA for detecting the disintegration activity of IN and IN-CCD in vitro.We studied the effects of metal ions on the disintegration reaction using this high-throughput disintegration ELISA. The results showed that as in 3′-processing and strand transfer, IN displayed dramatic preference for Mn2+ over Mg2+ to be the cationic cofactor in disintegration. Further NaCl titration study indicates that the preference for Mn2+ over Mg2+ in disintegration reaction is ascribed to a higher effect of Mn2+ than Mg2+ in stabilizing the IN–DNA complex. Baicalein, a known IN inhibitor, was involved to test the efficiency of this high-throughput disintegration ELISA in IN inhibitor screening. The results proved that baicalein clearly inhibit IN activities by targeting IN-CCD protein. The high-throughput disintegration ELISA presented in this work has the advantages of high-throughput, high sensitivity, high specificity, low background, as well as less laborious and time consuming. The assay is capable to be applied to study the disintegration reaction character and pharmacology of IN inhibitors. In addition, the assay has the potential to be applied for the high-throughput identification of drug candidates targeting IN, especially targeting IN-CCD.(4) Study on the activity and solubility of the wild type and F185K soluble mutant type integrase central catalytic domainDue to their poor solubility, the wild type (WT) IN and IN-CCD proteins form insoluble inclusion bodies when expressed in Escherichia coli, which brings difficulty in subsequent purification and functional studies. The introduction of F185K mutation into IN gene enhances the solubility of IN, and both IN and IN-CCD can be expressed as soluble protein, whereas the activities of IN and IN-CCD are not affected. In this work, the WT and F185K mutant type IN-CCD proteins were expressed and purified, and their solubility and activity were compared. The solubility and activities of WT and F185K/C280S full IN proteins were also compared. The results show that after F185K and F185K/C280S mutations, the solubility of IN-CCD and full IN proteins were both dramatically increased, both proteins were expressed as soluble proteins. In the meantime, the disintegration activity of mutant type IN-CCD, and the 3′-processing and strand transfer activities of mutant type full IN were reduced to some extent.We further constructed the WT and F185K mutant type IN-CCD structures by homology modeling, and 1800 ps of molecular dynamics (MD) simulations for these two types of IN-CCD proteins in water were performed. Some meaningful results were obtained: (i) After the F185K mutation, the flexibility of the catalytic loop region and the total mobility of IN-CCD was reduced, whereas the distances between the residues of the catalytic site (DDE motif) had no notable change. Therefore, the activities of mutated proteins were decreased, but were not significantly affected. (ii) After the F185K mutation, changes of the electrostatic interaction network drove the conformational change of IN-CCD, especially changed the conformation of the loop regions, and resulted in the burying of some hydrophobic residues and exposure of some other hydrophilic residues on the protein surface. The relative hydrophilic solvent accessible surface area of IN-CCD was increased. Moreover, the F185K mutation notably increased the hydrogen bonds between the IN-CCD protein and water molecules. These above changes contribute to the solubility increase of IN-CCD. It is found that the results obtained from MD simulations are in good agreement with the experiment data. This work supplies useful information and provides valuable insight for understanding the protein solubility and will be helpful in protein engineering for increasing the solubility of proteins.更多还原