HIV-1整合酶抑制剂的筛选及其活性的测定
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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。
Integration is a critical step in the retroviral life cycle. HIV-1 integrase is involved in the integration of HIV-1 DNA into host chromosomal DNA. It has become an attractive and rational target for selective anti-AIDS therapy. A random heptapeptide phage displayed library was panned on the recombinant HIV-1 integrase. After five rounds of panning, 13 positive phage clones were selected and sequenced. Two consensus peptides (TPSHSSR and HPERATL) were chemically synthesized. The non-radioactive ELISA-based HIV-1 integrase assay showed that the synthetic peptides TPSHSSR and HPERATL were able to inhibit the 3'cleavage or strand transfer activity of HIV-1 integrase to some extent (IC_(50)=54.56±5.18 μM, IC_(50)=28.29±1.32 μM, respectively). These heptapeptides could be used for developing new anti-HIV drug candidates, as well as for mechanism studies of the integration.
1. Expression, purification and activity of recombinant HIV-1
integrase
A culture of Escherichia coli BL21 (DE3) cells, which contained the plasmid pT7-7-His-TX-WT-IN was used to expressed His-tagged HIV-1 integrase. The
culture was grown at 37 ℃ until OD_(600) reached 0.6-0.8. Expression was induced by 1 mM IPTG, collected the cells after 5 h incubation at 30℃ by centrifugation. Cells were resuspended and the lysate was sonicated and centrifuged. The supernant was loaded onto a NTA resin column. The protein was eluted with buffer A containing 200 mM imidazole. Purified integrase filtered on membranes (0.22 μm) and was kept at -20 ℃. Both the integration and disintegration activity was assayed. The integration assay was performed by a non-radioactive ELISA-based method. And the disintegration products were analyzed on a 20% polyacrylamide gel.
2. Panning of a peptide library and analysis of the selected peptides
for integrase by ELISA
In our experiment, a phage display library of randomized linear heptamer peptides was used. The 96-well microtiter plates were coated with the integrase, and then blocked with 5% BSA. After five rounds of binding, elution, neutralization, titer and amplification, the selected phage was enriched approximate 700-fold. In order to identify the positive clones, we amplified the individual clones randomly from the fifth round of panning products. Assaying the different interaction of individual phage clones with the integrase and BSA, we determined the phage clones which specifically band to the integrase but not BSA as the positive ones. Using this method, we identified 13 phage clones from the linear heptamer peptides library which displayed peptides specifically band to the integrase.
3. Sequence analysis of the positive clones
Sequencing the single stranded DNA encoding the peptides displayed on the positive phages surface, we deduced the amino acid sequence of the selected heptapeptides. Scanning the amino acid sequence, two of the seven sequences have the same amino acid residues Thr, Pro and Ser at position one to three; another two sequences have the same amino acid residues Ser, His and Gly at position one, two and four. Besides, most of the seven amino acid sequences contain His, Arg, Pro and Ser.
4. Competitive ELISA experiments
The relative affinity of the two synthetic peptides was measured by their ability to compete for the integrase binding with their corresponding peptide-displaying phage using a competitive ELISA assay. This experiment was carried out on the 96-well immunosorbent plates coated with the integrase. The coated integrase was incubated with a solution containing a serially diluted synthetic peptide solution and a constant concentration of the corresponding phage. The phage binding rate was calculated by the formula: Phage binding rate = [ 1-(A_(450_-A'_(450))/A_(450)]× 100%, where A_(450) and A '_(450) represent without and with the corresponding peptide, respectively. During the concentration of the synthetic peptides increased, the phage binding rate dropped. Thiese results further support the specific nature of the interaction between HIV-1 integrase and the selected peptides.
5. Integration and disintegration inhibition experiments
The inhibit effect of the synthetic peptides was investigated on the two in vitro activities of integrase (integration and disintegration) corresponding to different substrates. Inhibition experiment was performed as integrase activity assay expect that increasing concentrations of synthetic peptides were preincubated with the integrase. In our experiments, we found that the peptides TPSHSSR and HPERATL have the ability to inhibit both integration and disintegration reactions. Interestingly, the catalytic domain (or core domain) alone is capable of performing disintegration reaction while all three integrase domains (the core domain, N-terminal domain and C-terminal domain) are required for integration activity. These results indicate that the peptides TPSHSSR and HPERATL may act on the central catalytic domain. Of course, they also may have an effect on the DNA substrates.
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。
Integration is a critical step in the retroviral life cycle. HIV-1 integrase is involved in the integration of HIV-1 DNA into host chromosomal DNA. It has become an attractive and rational target for selective anti-AIDS therapy. A random heptapeptide phage displayed library was panned on the recombinant HIV-1 integrase. After five rounds of panning, 13 positive phage clones were selected and sequenced. Two consensus peptides (TPSHSSR and HPERATL) were chemically synthesized. The non-radioactive ELISA-based HIV-1 integrase assay showed that the synthetic peptides TPSHSSR and HPERATL were able to inhibit the 3'cleavage or strand transfer activity of HIV-1 integrase to some extent (IC_(50)=54.56±5.18 μM, IC_(50)=28.29±1.32 μM, respectively). These heptapeptides could be used for developing new anti-HIV drug candidates, as well as for mechanism studies of the integration.
1. Expression, purification and activity of recombinant HIV-1
integrase
A culture of Escherichia coli BL21 (DE3) cells, which contained the plasmid pT7-7-His-TX-WT-IN was used to expressed His-tagged HIV-1 integrase. The
culture was grown at 37 ℃ until OD_(600) reached 0.6-0.8. Expression was induced by 1 mM IPTG, collected the cells after 5 h incubation at 30℃ by centrifugation. Cells were resuspended and the lysate was sonicated and centrifuged. The supernant was loaded onto a NTA resin column. The protein was eluted with buffer A containing 200 mM imidazole. Purified integrase filtered on membranes (0.22 μm) and was kept at -20 ℃. Both the integration and disintegration activity was assayed. The integration assay was performed by a non-radioactive ELISA-based method. And the disintegration products were analyzed on a 20% polyacrylamide gel.
2. Panning of a peptide library and analysis of the selected peptides
for integrase by ELISA
In our experiment, a phage display library of randomized linear heptamer peptides was used. The 96-well microtiter plates were coated with the integrase, and then blocked with 5% BSA. After five rounds of binding, elution, neutralization, titer and amplification, the selected phage was enriched approximate 700-fold. In order to identify the positive clones, we amplified the individual clones randomly from the fifth round of panning products. Assaying the different interaction of individual phage clones with the integrase and BSA, we determined the phage clones which specifically band to the integrase but not BSA as the positive ones. Using this method, we identified 13 phage clones from the linear heptamer peptides library which displayed peptides specifically band to the integrase.
3. Sequence analysis of the positive clones
Sequencing the single stranded DNA encoding the peptides displayed on the positive phages surface, we deduced the amino acid sequence of the selected heptapeptides. Scanning the amino acid sequence, two of the seven sequences have the same amino acid residues Thr, Pro and Ser at position one to three; another two sequences have the same amino acid residues Ser, His and Gly at position one, two and four. Besides, most of the seven amino acid sequences contain His, Arg, Pro and Ser.
4. Competitive ELISA experiments
The relative affinity of the two synthetic peptides was measured by their ability to compete for the integrase binding with their corresponding peptide-displaying phage using a competitive ELISA assay. This experiment was carried out on the 96-well immunosorbent plates coated with the integrase. The coated integrase was incubated with a solution containing a serially diluted synthetic peptide solution and a constant concentration of the corresponding phage. The phage binding rate was calculated by the formula: Phage binding rate = [ 1-(A_(450_-A'_(450))/A_(450)]× 100%, where A_(450) and A '_(450) represent without and with the corresponding peptide, respectively. During the concentration of the synthetic peptides increased, the phage binding rate dropped. Thiese results further support the specific nature of the interaction between HIV-1 integrase and the selected peptides.
5. Integration and disintegration inhibition experiments
The inhibit effect of the synthetic peptides was investigated on the two in vitro activities of integrase (integration and disintegration) corresponding to different substrates. Inhibition experiment was performed as integrase activity assay expect that increasing concentrations of synthetic peptides were preincubated with the integrase. In our experiments, we found that the peptides TPSHSSR and HPERATL have the ability to inhibit both integration and disintegration reactions. Interestingly, the catalytic domain (or core domain) alone is capable of performing disintegration reaction while all three integrase domains (the core domain, N-terminal domain and C-terminal domain) are required for integration activity. These results indicate that the peptides TPSHSSR and HPERATL may act on the central catalytic domain. Of course, they also may have an effect on the DNA substrates.
引文
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[20] Bae W, Mulchandani A, Chen W. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. J Inorg Biochem, 2002, 88: 223
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[22] Shibasaki S, Ueda M, Ye K, et al. Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration. Appl Microbiol Biotechnol, 2001, 57: 528
[23] Aoki T, Tahara T, Fujino H, et al. "GFP-display," an easy detection method for single amino acid changes in a target polypeptide: application to random mutagenesis. Anal Biochem, 2002,300: 103
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[29] Irving RA, Coia G, Roberts A, et al. Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics. J Immunol Methods, 2001, 248: 31
[30] Ihara H, Mie M, Funabashi H, et al. In vitro selection of zinc finger DNA -binding proteins through ribosome display. Biochem Biophys Res Commun, 2006,345: 1149
[31] Xu L, Aha P, Gu K, et al. Directed evolution of high-affinity antibody mimics using mRNA display. Chem Biol, 2002, 9: 933
[32] Weng S, Gu K, Hammond PW, et al. Generating addressable protein microarrays with PROfusione covalent mRNA-protein fusion technology. Proteomics, 2002, 2: 48
[33] Yan X, Xu Z. Ribosome — display technology: applications for directed evolution of functional proteins. Drug Discov Today, 2006,11: 911
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[36] Bernath K, Magdassi S, Tawfik DS. Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery. J Mol Biol, 2005, 345: 1015
[37] Griffiths AD, Tawfik DS. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J, 2003,22: 24