HIV-1整合酶抑制剂的筛选及其活性的测定

发布时间：2018-01-15 02:30

本文关键词：HIV-1整合酶抑制剂的筛选及其活性的测定　出处：《浙江大学》2007年硕士论文　论文类型：学位论文

更多相关文章： HIV-1 整合酶 抑制剂 多肽 噬菌体展示

【摘要】： 病毒DNA整合进宿主DNA的过程是某些反转录病毒在宿主细胞中增殖的关键步骤。由于在正常人类细胞中不存在相似的功能蛋白，其抑制剂对人体的副作用可能很小。相对于经典AIDS治疗药物的众多毒副作用，整合酶抑制剂理论上要具有优势，因此成为受人瞩目的治疗艾滋病的新靶点。以重组HIV-1整合酶为目标蛋白，采用噬菌体展示技术，在随机线性七肽库中筛选与整合酶有特异结合作用的噬菌体展示肽，得到13个具有特异结合能力的阳性克隆；经DNA测序推断出七条氨基酸序列。选取同源性较高的TPSHSSR和HPERATL两条肽，它们可以竞争性地抑制展示相应肽段的噬菌体与整合酶的结合。通过体外整合酶活性实验显示，它们对整合酶的整合活性(3’加工和链转移过程)有抑制作用，TPSHSSR和HPERATL的半数抑制率即IC_(50)分别为54.56±5.18μM和28.292±1.32μM。同时，实验证明这两条多肽还有抑制去整合的作用。我们的研究表明：用噬菌体展示技术可以筛选到有效的HIV-1整合酶的抑制剂，可用于整合酶机理的研究，并有潜在的新药开发前景。 1．HIV-1整合酶的表达与纯化及活性鉴定将转化有质粒PT7-7-His-TX-WT-IN的E.coli BL21(DE3)菌接种于含有Amp(终浓度为100μg／ml)LB培养基中培养，加IPTG诱导表达后6 000 rpm，4℃离心收集菌体。超声破碎后，15 000 rpm，4℃离心收集上清。上清经Ni—NTA柱分离纯化，得到较纯的整合酶。整合酶活性的鉴定分别检测了整合酶的整合与去整合两个作用。整合作用采用的是一种非放射性的类似于ELISA的方法，而去整合活性则是将整合酶与相应底物反应，经电泳分析底物的变化来定性鉴别。 2．噬菌体肽库的筛选及阳性克隆的鉴定本实验筛选的肽库为随机线性七肽库。将纯化得到的具有活性的HIV-1整合酶蛋白包被在酶标板上，经5％BSA封闭，噬菌体的结合，酸洗脱、中和，测滴度、扩增等基本步骤的循环筛选后，噬菌体得到了富集，程度达到了700倍左右。将第五轮洗脱下来的噬菌体感染E.coli ER2738，挑取平板中的清晰菌斑，制备单克隆噬菌体溶液。在96孔板上包被整合酶蛋白，同时包被BSA作为阴性对照。经封闭后加入单克隆噬菌体溶液。洗涤各孔后加入辣根过氧化物酶标记的M13抗体，而后用TMB进行显色。比较各个克隆与整合酶和BSA的结合效果，筛选出特异结合整合酶蛋白而非BSA的阳性克隆。本实验筛选得到了13个阳性克隆。 3．DNA测序及多肽序列的推断提取阳性克隆的单链DNA，送交上海生工进行测序，测序以—96通用引物(5'-GCCCTCATAGTTAGCGTAACG-3’)为引物。13个阳性克隆共得到7条序列。根据DNA测序结果，推测出这七条序列的氨基酸序列，分析他们之间的氨基酸组成特点。其中有两条多肽的前三个氨基酸一致，另两条多肽的第1、2和第4个氨基酸一致，，并且七条肽序列中，大部分都含有组氨酸，精氨酸，脯氨酸和丝氨酸。 4．合成七肽的竞争抑制ELISA实验两条合成的七肽与展示相应肽段的噬菌体竞争性地结合整合酶的能力反映了这两条肽的相对亲和力。在96孔免疫吸附板中包被整合酶蛋白，加入不同浓度的合成多肽及一定量展示相应肽序列的噬菌体溶液，使两者竞争结合整合酶。通过辣根过氧化物酶标记的抗M13抗体结合，TMB显色后，计算出噬菌体结合率：结合率％=[1-(A_(450)-A’_(450))／A_(450)]×100％；其中A_(450)为未加抑制剂时450nm波长下的吸光度，A’_(450)为加入抑制剂后450 nm波长下的吸光度。随着合成多肽浓度的增加，与整合酶结合的展示相应肽序列的噬菌体逐渐减少，结合率下降。这些结果充分证明了筛选出来的两条多肽与整合酶蛋白特异性结合的特点，亲和力较好。 5．多肽对整合酶活性抑制实验针对整合酶的整合与去整合作用，用合成的多肽分别对这两个反应的抑制作用做了分析。实验方法与整合酶活性检测相似，只是整合酶需要和合成的多肽先进行预反应。实验发现合成的两条多肽TPSHSSR和HPERATL对整合酶的整合、去整合作用都有抑制作用。整合酶的去整合作用对整合酶的序列及结构完整性的要求不高，只要有核心结构域存在即可；而整合作用必须要求整合酶既有核心结构域，又有N末端和C末端结构域。所以，这两条多肽对整合酶的作用位点很可能就在核心结构域上。
[Abstract]:The integration of viral DNA into the host DNA process is the key step of some retroviruses in human cells. The function of protein similar does not exist in normal human cells, the side effects of inhibitors on human may be small. Compared to the side effects of classic AIDS drugs, integrase inhibitors have theory the advantage, therefore become a new target for treatment of AIDS attention. The recombinant HIV-1 integrase for target protein using phage display technology, in linear random seven peptide library screening specific binding phage display peptide and integrase, 13 with specific binding ability of positive clones by DNA; sequencing seven deduced amino acid sequence. Selected homologous TPSHSSR and HPERATL two peptide, which can competitively inhibit phage display peptide and the corresponding integrase by. In vitro experiments showed the integration of enzyme activity, enzyme activity of integration integration (3 'processing and chain transfer process) are inhibited, and the inhibition rate of half of the TPSHSSR HPERATL IC_ (50) were 54.56 + 5.18 and 28.292 + M 1.32 M. at the same time, effect of the experiment shows that the two polypeptides have inhibition to integration. Our research shows that: display technology can be screened effective inhibitors of HIV-1 integrase with phage integrase can be used to study the mechanism, and new drug development potential.
Expression and purification of 1.HIV-1 integrase and identification of its activity
The transformation of plasmid PT7-7-His-TX-WT-IN E.coli BL21 (DE3) in bacteria containing Amp (final concentration of 100 g / ml) LB medium, and after the induction of IPTG, 6000 rpm, 4 C centrifugal collection cell. After ultrasonication, 15000 rpm, 4 DEG C collected by centrifugation the supernatant by Ni - NTA. Column separation, integration of pure enzyme. Identification of integrase activity were detected by enzyme and integration to integrate two. Integration uses a non radioactive method similar to ELISA, and to integrate the activity is to integrate with the corresponding reaction enzyme substrate, by electrophoresis substrate change analysis to qualitative identification.
Screening of 2. phage peptide library and identification of positive clones
The peptide library screening experiments for linear random seven peptide library. The purified active HIV-1 integrase protein was coated on the ELISA plate, the 5%BSA closed, binding phage, acid washing, neutralization, titration, amplification and other basic steps of the cycle after screening, phage enriched, reached the level of about 700 times. The fifth round of the eluted phage infection of E.coli ER2738, were clear plaque in the plate, the preparation of monoclonal phage integrase protein coated solution. In the 96 hole plate, and coated by BSA as negative control. After being closed after adding single clone phage solution. M13 antibody of each hole after washing horseradish peroxidase labeled, then use the TMB color. Comparing the combined effect of cloning and integrase and BSA, screened the specific binding integrase protein and non BSA positive clones. This experiment screened 13 gram positive Long.
3.DNA sequencing and peptide sequence inference
The extraction of single stranded DNA positive clones, sent to Shanghai SANGON sequencing, sequencing with 96 universal primers (5'-GCCCTCATAGTTAGCGTAACG-3) primers and.13 clones were obtained 7 sequences. According to the results of DNA sequencing, deduced amino acid sequences of the seven sequences, analysis of amino acid composition between them. There are three characteristics two amino acid polypeptide, another two polypeptide 1,2 and fourth amino acids, and seven peptide sequences, most of them contain histidine, arginine, proline and serine.
Competitive inhibition ELISA experiment of 4. synthetic seven peptide
Two of seven synthetic peptides and peptide phage display corresponding competitive binding ability of integrase reflects the relative affinity of these two peptides. Coated integrase protein in 96 hole immune adsorption plate, and a certain amount of synthetic peptides with different concentrations of phage display peptide sequences of the corresponding solution. The competitive binding of integrase. By combining horseradish peroxidase labeled anti M13 antibody, TMB staining, calculate the phage binding rate: binding rate%=[1- (A_ (450) -A (450) _) / A_ (450)] * 100%; A_ (450) for the absorbance at 450nm under with the inhibitor, A (450) _ absorbance wavelength of 450 nm after adding inhibitors. With the increase of the concentration of synthetic peptides, show the corresponding peptide sequence combined with phage integrase gradually reduced, the binding rate of two. These results demonstrate that the polypeptides were screened out with the whole The specific binding of the synthase protein is good, and the affinity is good.
Inhibition of 5. polypeptide on the activity of integrase
According to the integration and integration of the role of the enzyme, with synthetic peptides of the two reaction inhibition were analyzed. Test method and integration of enzyme activity is similar, just need integrase and synthetic peptide pre reaction. The experimental results showed that the synthesis of two polypeptides TPSHSSR and HPERATL integration of the integrase to integrate, have inhibitory effect. Sequence and structural integrity to integration of the role of the integrase enzyme on the integration of the requirements is not high, as long as the core domain can; and integration must require the integration of both the core enzyme domain, and at the end of the N terminal and the C domain. Therefore, the two polypeptide action sites for integrase is likely in the core domain.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

【参考文献】