【作者】 何俊；

【导师】 邓松华； 潘卫；

【作者基本信息】 安徽医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 当前,AIDS对人类的生存和发展提出了严峻的挑战,已成为全球面临的严重公共卫生问题和社会问题。目前针对AIDS的高效抗逆转录病毒疗法(highly active anti-retroviral therapy, HAART),临床作用显示可以明显减少AIDS相关发病率和死亡率。HAART主要使用一种HIV-1蛋白酶(Protease, PR)抑制剂(HIV-1 Protease inhibitor, PI)药物结合两种HIV反转录酶抑制剂药物治疗。由于HIV逆转录酶易发生复制错误使得感染人群中的HIV基因序列差异很大,以及抗HIV药物的广泛应用导致HIV的耐药突变株不断出现,影响治疗效果。因而迫切要求更多﹑更新的抗HIV药物以对抗不同序列的HIV和不断出现的HIV耐药突变株。目前常用的PI类药物筛选模型其原理如下:在HIV PR对其靶序列切割作用时,加入待筛药物并检测其对于切割的抑制作用,从而鉴定该待筛药物的实际应用。然而,由于HIV PR对不同的靶序列具有不同的切割活性,针对不同的耐药突变PR,需要选择与之适应的PR靶位点来进行筛选,而目前出现的各种耐药突变PR,其最适应的PR靶序列尚未明确,因而现有方法难以应对各种耐药突变PR进行有效的PI类药物筛选。因此对抗HIV药物的体外筛选模型提出了更高的要求:迅速建立新的﹑针对不同序列HIV及耐药突变HIV的体外药物筛选模型,使之既与现有的模型相互补充,又能提高抗HIV药物的筛选效率。我们的设想是应用噬菌体展示技术,构建展示HIV蛋白酶PR靶序列随机突变的噬菌体文库,从中筛选出对耐药突变PR高敏感性的PR靶序列,从而应用于针对耐药突变PR的PI类药物筛选。同时,利用展示耐药突变PR敏感的PR靶序列的噬菌体,结合耐药突变PR,可以直接针对耐药突变PR进行PI类药物筛选。本研究在HIV PR的靶序列CAP2和NC之间的切割位点部分进行了随机化,将随机化序列展示在噬菌体载体上,构建噬菌体展示文库。用HIV PR对噬菌体展示文库进行切割筛选。本研究分以下三个部分进行:一.HIV-1型HXB2株PR在大肠杆菌中的表达、纯化及鉴定根据HIV-1 HXB2株PR及CAP2NC DNA序列设计引物,用重叠延伸PCR方法合成使用大肠杆菌偏好密码子的HIV-1 PR的DNA编码序列,并在PR序列上游添加自切割位点GTVSFNF7个氨基酸的编码序列,用T/A克隆法将其插入pMD18-T载体进行测序。将PR DNA克隆至原核表达载体pET-32a,在大肠杆菌BL-21 DE3诱导表达,表达出相对分子质量为30 000的HIV-1 PR融合蛋白。用Ni-NTA亲和柱对表达蛋白进行纯化,纯化的目的蛋白浓度为2.54mg/ml。将纯化的PR蛋白与复性缓冲液进行1:20比例混合进行稀释复性。此部分工作由本研究合作者第二军医大学陈铭硕士完成。二.HIV-1蛋白酶靶位点CAP2NC序列的随机化文库的构建借助噬菌体载体,构建适合筛选用的大容量突变体文库是进行体外分子进化研究的重要部分。我们对PR靶序列区域的随机突变设计为:应用含有随机核苷酸序列的引物,使用重叠延伸PCR(overlapping PCR)的方法,将HIV PR作用的靶序列CAP2和NC之间引入随机突变。为了能更好的研究突变靶序列和增大PR靶序列中被筛选的氨基酸范围,我们分别随机化了三组不同的氨基酸,将其连接于自主构建的噬菌体载体pCANTAB5S-F7上,构建了三个噬菌体展示文库。所得到的三个原代噬菌体文库,库容量分别为9.7×10~6﹑6.3×10~6﹑1.2×10~7;噬菌体文库滴度均达1013TU/ml数量级;PCR鉴定为阳性克隆的阳性插入率分别占50%﹑62.5%﹑41.67%;重组质粒的双酶消化可以得到大小正确的目的片段;测序分析表明切割位点中随机化的核苷酸与氨基酸随机分布均匀;各文库中有60%以上的单克隆噬菌体与IgG有良好的结合活性。文库在库容量、多样性、随机性及IgG结合活性评价良好,满足后续体外HIV蛋白酶切割筛选的要求。三.HIV-1 PR对蛋白酶靶位点CAP2NC序列的随机化文库的筛选模型和切割筛选噬菌体载体pCANTAB5S-F7是第二军医大学微生物教研室在关于免疫球蛋白结合分子的体外分子进化的研究中筛选得到的一种与IgG结合活性很好的噬菌体载体。我们将人IgG包被在ELISA酶标板中,可以有效吸引结合噬菌体,将其固相化于ELISA酶标板上。加入HIV SF2 PR进行切割筛选,以期获得对PR敏感的PR靶序列。但前期筛选并未收取到良好的成效。实验中加入了验证HIV-1 PR的切割活性的部分。我们制备出展示有PR原靶位点(即未经过突变改造的)CAP2NC序列的噬菌体pCANTAB5S-F7-CAP2NC,和不展示任何序列的空载体pCANTAB5S-F7分别按照1:1,1:10,1:100,1:1000,1:10000的比例混和后,加入HIV SF2 PR进行切割作用,HIV SF2 PR作用的灵敏度可达1:1000的混和浓度。在这个灵敏度,一次酶切筛选即能将阳性噬菌体检出,证明了HIV SF2 PR能够有效的切割固相化在酶标板上的展示于噬菌体表面的HIV PR靶序列,并且能被有效检测。证实了我们利用HIV SF2 PR能够对PR靶序列随机突变文库进行切割筛选以获得PR敏感的PR靶序列的可行性之后,在后续筛选中,对NC段缺失的噬菌体展示文库的筛选得到了一些对HIV SF2 PR较敏感的序列,后续研究仍在进行中。本研究成功对HIV-1 PR靶蛋白CAP2NC间的酶作用位点进行了随机化,并构建了展示包含有随机突变序列的噬菌体文库,文库的库容量、多样性、随机性及IgG结合活性评价良好,满足后续体外HIV蛋白酶切割筛选的要求。在第二部分的筛选工作中,证实了HIV SF2 PR能够有效的切割固相化于酶标板上的展示噬菌体表面的HIV PR靶序列,并在突变体文库中筛选得到一些对HIV SF2 PR较为敏感的序列结构。本研究为目前所使用的PI类药物体外筛选模型提供了一种新的思路和方法,也为酶与底物相互作用的研究提供了一个新平台。更多还原

【Abstract】 Nowadays, we are faced with the difficult challenge to battle with acquired immunodeficiency syndrome (AIDS). It is a serious global puzzle of public health and society.Clinical data showed that the highly active anti-retroviral therapy (HAART), which aims to AIDS, could reduce the rates of disease and death significantly. It uses a kind of HIV-1 protease inhibitor (PI). But, the emergence of mutated protease was hindering the application of HAART. More and better PIs are demanded for clinical usage.Different protease target peptide was susceptive to different protease, so during the screening of PIs that against to the resistant mutated protease, adapted protease target peptides should be meet the demands of the resistant mutated protease. Whereas, the most adapted PR target peptides to many kinds of HIV PR resistant mutants are not confirmed. The current screening mode cannot satisfy the demands of facing all manner of resistant mutated protease.We attempt to construct phagemid libraries, which displaying the protease target peptide randomized mutants, in order to find highly sensitive ones that fed on mutated protease. And these highly sensitive protease target peptides should be screened from the libraries by enzyme action of the resistant mutated HIV protease. We choose the CAP2NC sequence; randomize three amino acids of it, and conjunct to the phagemid vector. The protease target sequences were displayed on the surface of phage. The phagemid could be fixed on the surface that IgG were coating on. Three parts:1. Expression, purification and identification for protease of HIV-1 HXB2 subtype in E.coliThe primers were designed according to the PR amino acid sequence of HIV-1 HXB2 subtype and the E.coli preferred codon. The PR DNA fragment with E.coli preferred codon was synthesized in vitro by overlapping PCR and sequenced after inserted into pMD18-T vector by T/A method. Then the PR DNA sequence was cloned into pET-32a vector which is used as expression vector in E.coli. Expression of HIV PR was induced by IPTG in E.coli BL21 and the expressed PR protein was purified by the Ni-NTA affinity column. The purified PR protein was analyzed by SDS-PAGE. The HIV PR DNA fragment with E.coli preferred codon was successfully synthesized and its corresponding amino acid sequence is identical with the primary amino acid sequence of HIV-1 HXB2 subtype. The expression vector pET-32a -PR was constructed successfully, The HIV-1 PR was expressed in E.coli BL21 after the induction by IPTG with a relative molecular weight of 30000. The purified PR protein has a concentration of 2.54mg/ml. The protein was then diluted with 20 volumes of refolding buffer and allowed to stand at 4°C for 12 hours.The part of work was completed by Chen Ming.2. Construction of phagemid libraries to display randomization sites on CAP2NC sequence of HIV-1 gag proteinConstruction of high capacity libraries was essential for molecular evolution study in vitro. We designed primers to implement the mutation of protease target peptides.Methods:Introduce nine randomized bases into 3′terminal of CAP2 fragment by PCR, and obtain CAP2NC fragment that contained these randomized sites and had StuⅠrestriction site on 5′terminal and SalⅠrestriction site on 3′terminal by overlapping PCR. Clone the three different CAP2NC fragment into pMD18-T, then into phage display vector F7-pCANTAB5S.The three phagemid libraries contain 9.7×106﹑6.3×106﹑1.2×107 clones respectively. Each of the titer of the three libraries goes beyond 1013TU/mL. The positive clones reach to 50%﹑62.5%﹑41.67% in the libraries. The analysis of sequences shows that the randomicity and variety of these libraries can meet the demand. About 60% clones can bind IgG protein specifically. So, three CAP2NC fragment libraries, each of which contains nine randomized bases, were displayed by pCANTAB5S-F7 phage successfully. The capacity, titer, rate of positive clone, and randomicity could fit the requests of construction a model for in vitro screening the phages that carried susceptible cleavage sites or sequences.3. Establishment of cleavage model of HIV protease and screening workThe phage vector pCANTAB5S-F7 was able to bind to IgG validly. We fix IgG on solid surface, adds HIV SF2 PR to cleave the phage libraries, and hope to obtain significant ones. There are no significant phages initially. So, we do some research to check the activity of the HIV-1 PR we using. Serials of experiments were designed and enforce: we mixed the positive and negative phages in proportion at 1:1, 1:10, 1:100, 1:1000, and 1:10000. At the rate of 1:1000, HIV-1 SF2 PR could recognize and cleave the target peptides effectively. Meanwhile, we adjust and optimize the condition of the enzyme action. Some sensitive target peptides were obtained. The research is carrying on.We randomized nine bases in CAP2NC-- one of the target peptides of HIV-1 protease, connected it into phage vector pCANTAB5S-F7, and construct three phagemid libraries. The capacity, titer, rate of positive clone, and randomicity could fit the requests of construction a model for in vitro screening the phages that carried susceptible cleavage sites or sequences. The screening system was feasible and some sensitive target peptides were obtained from the library. The new screening system is to bring new ideas of screening drugs in vitro and provide a platform of research for co-action of enzymes and their substrates.更多还原