在肽库中筛选抗磷脂抗体的小肽抑制剂

β２糖蛋白Ⅰ（ｂｅｔａ－２－ｇｌｙｃｏｐｒｏｔｅｉｎ Ⅰ,β２ＧＰＩ）是一分子量为５０ ｋＤ的糖蛋白．从抗磷脂抗体综合症（Ａｎｔｉｐｈｏｓｐｈｏｌｉｐｉｄ ａｎｔｉｂｏｄｉｅｓ ｓｙｎｄｒｏｍ ,ＡＰＳ）患者身上获得的抗体可直接与β２ＧＰＩ作用．以β２ＧＰＩ为目标分子,在噬菌体十五肽库中筛选抗β２糖蛋白Ⅰ抗体的小肽抑制剂．通过四轮亲和性筛选,噬菌体的回收率从１．２６×１０－ ４％ 增加到３．１９×１０－ ２％ ．随机挑取噬菌体克隆测定其与β２ＧＰＩ的结合活性及其对β２ＧＰＩ与自身抗体结合的抑制活性,其中部分噬菌体克隆表现出４０％ 的抑制活性．经ＤＮＡ 序列测定,得到一组相关序列．该结果为研究β２ＧＰＩ的抗原表位奠定了良好的基础．     

麻蝇幼虫肠道蛋白酶BGP的分离纯化及性质

棕尾别麻蝇幼虫肠液经ＳＤＳ－ＰＡＧＥ后,Ｘ光片显影,呈现两条蛋白酶活性带．ＩＥＦ后,两条蛋白酶活性带的等电点分别为ｐＨ７．７和６．８．麻蝇幼虫肠液经５５％～７５％硫酸铵沉淀,以及连续两次制备等电聚焦,分离纯化出等电点约为ｐＨ７．７,分子量约为３５ｋＤ的蛋白酶ＢＧＰ．该酶能分解酪蛋白和类胰蛋白酶专一底物Ｂｚ－Ｐｈｅ－Ｖａｌ－ＡｒｇＮＡ,不能分解弹性蛋白酶专一底物ｅｌａｓｔｉｎ－ＣｏｎｇｏＲｅｄ和类胰凝乳蛋白酶专一底物Ｓｕｃ（Ａｌａ）２Ｐｒｏ－ＰｈｅＮＡ．ＳＢＢＩ,Ｌｅｕｐｅｐｔｉｎ和ＰＭＳＦ能强烈抑制其活性．专一底物和抑制剂的结果表明,ＢＧＰ是一种类胰蛋白酶．其最适反应温度为５０℃,最适作用ｐＨ为８．５．不耐高温,５０℃保温３０ｍｉｎ活性急剧下降．Ｈｇ２＋,Ｚｎ２＋和Ｃｕ２＋能抑制酶活性．Ｃａ２＋,Ｍｇ２＋对酶无激活作用,ＥＤＴＡ无抑制作用．     

抗人AFP抗体噬菌体呈现文库的构建筛选及基因序列测定

从人ＡＦＰ免疫小鼠脾细胞ｍＲＮＡ中扩增出全套抗体Ｖ区基因并随机拼接为ＳｃＦｖ基因,构建全套ＳｃＦｖ基因噬菌体呈现文库,经两轮ｐａｎｎｉｎｇ筛选富集,一次从９４个单个重组噬菌体克隆中筛选到１４个具有ＡＦＰ结合活性的克隆,测定两个阳性克隆中ＳｃＦｖ基因的核苷酸序列,获得一个Ｖ＿Ｈ基因和两个Ｖ＿Ｋ基因,其基因序列分别与鼠ＩｇＶ＿ＨＪ５５８、Ｖ＿ｋ－ＯＸ１和Ｖ＿ｋ４／５家族同源性最高;推导出的氨基酸序列中均含有抗体Ｖ区特征性的两个恒定的半胱氨酸残基、具有明确的三个ＣＤＲ和四个ＦＲ序列,表明这三个基因均系新发现的功能性鼠抗体Ｖ区基因序列。     

利用噬菌体肽库筛选胰凝乳蛋白酶的底物

利用脱水胰凝乳蛋白酶 (保持天然酶的完整结合部位 ,但没有催化活性 )作为靶蛋白 ,在噬菌体肽库中钓取一些有结合活力噬菌体 ,再将得的噬菌体同固定化的天然酶一起保温 ,能够被天然酶水解的那部分噬菌体即为底物噬菌体 .利用 DNA测序技术测出筛得的短肽序列 ,分析序列保守性 ,发现 WR和 YF的组合具有很强的保守性 .合成相应的几个短肽 ,与天然酶作用 ,发现芳香族氨基酸与碱性氨基酸的组合较易被胰凝乳蛋白酶切割 .而且 ,当 P2 、P3 位置为侧链较小的氨基酸或碱性氨基酸时 ,更有利于水解的发生 .精氨酸无论处在任何位置 ,对水解往往都有促进作用     

抗人AFP抗体噬菌体呈现文库的构建筛选及基因序列测定

从人ＡＦＰ免疫小鼠脾细胞ｍＲＮＡ中扩增出全套抗体Ｖ区基因并随机拼接为ＳｃＦｖ,构建全套ＳｃＦｖ基因噬菌体呈现文库,经两轮ｐａｎｎｉｎｇ筛选富集,一次从９４个单个重组噬菌体克隆中筛选到１４个具有ＡＦＰ结合活性的克隆,测定两个阳性克隆中ＳｃＦｖ基因的核苷酸序列,获得一个ＶＨ基因和两个ＶＫ基因,其基因序列分别与鼠Ｉｇ ＶＨｊＪ５５８,ＶＫ－ＯＸ１和ＶＫ４／５家族同源性最高,推导出的氨基酸序列中均含有抗体     

抗草鱼出血病病毒多肽的结构分析

将草鱼出血病病毒（ＧＣＨＶ８７３）颗粒与随机噬菌体九肽库在体外作用,三轮筛选后,从３００个转化的单菌落中获得１６个与病毒高亲和力的噬菌体克隆。接着经过抗病毒试验获得６个能强烈抑制病毒复制的阳性噬菌体克隆,能使病毒ＴＣＩＤ５０下降５个数量级。通过对阳性噬菌体克隆随机插入区域的核苷酸序列分析,推导出多肽的氨基酸序列。发现６个能强烈抑制病毒复制的阳性克隆中多肽的氨基酸序列完全一致（ＮＨ２ＬｅｕＴｒｐＶａｌＧｌｙＧｌｙＧｌｙＡｒｇＡｓｎＡｌａ）,该结果提示多肽的抗病毒能力与多肽特异性氨基酸组成及结构有关。因此,抑制ＧＣＨＶ８７３复制特异性多肽的氨基酸序列的确定及结构分析,不仅为人工合成抗草鱼出血病病毒的多肽奠定了基础,同时也为抗病毒多肽制剂的研制提供了依据。     

猪圆环病毒2型ORF2基因序列分析及真核表达载体的构建

根据ＧｅｎＢａｎｋ中猪圆环病毒２型（ＰＣＶ－２）ＯＲＦ２基因序列,设计一　对引物,应用ＰＣＲ从疑患断奶仔猪多系统消耗综合症（ＰＭＷＳ）的死亡仔猪组织病料中扩增出ＯＲＦ　２全基因（７０２ｂｐ）。将此片段克隆入ｐＧＥＭ－Ｔ　ｅａｓｙ载体,筛选获得重组质粒ｐＴＯＲＦ２,并对此质　粒中的插入序列进行了测序分析,结果表明本试验克隆的ＯＲＦ２与美国ＰＣＶ－２分离株ＡＦ２６４０３９　的核苷酸及氨基酸序列同源性均达到１００％,与其他ＰＣＶ－２毒株同源性分别为９２．３％～９８．　６％和　９２．３％～９６．６％。重组质粒ｐＴＯＲＦ２经　Ｂａｍ　Ｈ　Ｉ、Ｅｃｏ　Ｒ　Ｖ双酶切,回收ＯＲＦ２基因,转　移入真　核表达载体ｐＳｅｃＴａｇ２／ＨｙｇｒｏＢ的相应酶切位点之间,构建成重组质粒ｐＳｅｃＴａｇＯＲＦ２。此重组表　达载体的构建成功为进一步研究ＯＲＦ２编码蛋白的生物学活性及建立ＰＣＶ诊断试剂盒打下基础。     

小鼠4.5S RNA逆转座子对Luc基因表达的影响

利用ＲＴ－ＰＣＲ方法,从小鼠肝脏组织总ＲＮＡ中扩增出４．５ＳＲＮＡ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,酶切鉴定并测序。然后将该序列插入以Ｌｕｃ基因作为报道基因的表达载体ｐＳＶｌｕｃ２０的ＰｖｕⅡ位点,构建了含４．２ＳＲＮＡ逆转座子的表达载体ｐＳＶｌｕｃ２０－４．５Ｓ。脂质转染法将表达载体导入小鼠骨髓瘤细胞ＮＳ－１、ＳＰ２／０和人乳腺癌细胞Ｂｃａ６１。结果表明,小鼠４．５ＳＲＮ     

用逆转录病毒载体表达人粒细胞—巨噬细胞集落刺激因子

应用基因工程的方法,将含有巨细胞病毒（ＣＭＶ）启动子的基因片段和人粒细胞－茂噬细胞集落刺激因子（ｈＧＭ－ＧＳＦ）的ｃＤＮＡ,克隆进逆转录病毒载体Ｎ２Ａ,得到重组质粒Ｎ２ＱＡ／ＣＭＶ／ｈＧＭ－ＣＳＦ。经脂质体包装并转染包装细胞,通过Ｇ４１８药物筛选,得到抗生克隆。经ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ检测证实,ＧＭ－ＣＳＦ基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达１０＾４ＣＦＵ／ｍｌ,克     

用逆转录病毒载体表达人粒细胞－巨噬细胞集落刺激因子

应用基因工程的方法,将含有巨细胞病毒（ＣＭＶ）启动子的基因片段和人粒细胞－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）的ｃＤＮＡ,克隆进逆转录病毒载体Ｎ２Ａ,得到重组质粒Ｎ２Ａ／ＣＭＶ／ｈＧＭ－ＣＳＦ．经脂质体包装并转染包装细胞,通过Ｇ４１８药物筛选,得到抗性克隆。经ＰＣＲ和Ｓｏｕｔｈｅｍｂｌｏｔ检测证实,ＧＭ－ＣＳＦ基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达１０￣４ＣＦＵ／ｍｌ,克隆细胞培养上清用ＴＦ－１细胞可检测到ＧＭ－ＣＳＦ活性。     

直接用噬菌体肽进行功能性分析的研究

通过对胰蛋白酶抑制剂的筛选发现,噬菌体肽库不仅可以用于亲和性筛选,而且噬菌体肽（ｐｈａｇｅｐｅｐｔｉｄｅ——ｐｅｐｔｉｄｅｄｉｓｐｌａｙｉｎｇｏｎｐｈａｇｅ）可以直接用于对亲和筛选得到的克隆进行胰蛋白酶抑制实验,即所谓的功能性筛选．功能性筛选前后的测序结果表明用噬菌体肽直接进行功能性筛选确实能够筛去大部分的非抑制剂克隆．     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.     

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.     

Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.

A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.     

Identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase

Human immunodeficiency virus type 1 integrase catalyzes the integration of proviral DNA into the infected cell genome, so it is an important potential target for antiviral drug design. In an attempt to search for peptides that specifically interact with integrase (IN) and inhibit its function, we used an in vitro selection procedure, the phage display technique. A phage display library of random heptapeptides was used to screen for potential peptide ligands of HIV-1 IN. Several phage clones were identified that specifically bound IN. Two of the selected peptides (FHNHGKQ and HLEHLLF) exhibited a high affinity for IN and were chemically synthesized. High affinity was confirmed by a displacement assay which showed that these two synthetic peptides were able to compete with the phages expressing the corresponding peptide. These agents were assayed on the in vitro IN activities. While none of them inhibited the 3'-processing reaction, the FHNHGKQ peptide was found to be an inhibitor of the strand transfer reaction. Despite its high affinity for IN, the HLEHLLF peptide selected and assayed under the same conditions was unable to inhibit this reaction. We showed that the FHNHGKQ peptide inhibits specifically the strand transfer activity by competing with the target DNA for binding to IN. These IN-binding agents could be used as a base for developing new anti-integrase compounds as well as for structural studies of the still unknown three-dimensional structure of the entire integrase molecule.     

A polystyrene binding target-unrelated peptide isolated in the screening of phage display library

Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase.     

利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)颗粒筛选噬菌体肽库,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。【方法】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Peptide phage display in biotechnology and biomedicine

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors, etc.). Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nanomaterials.