噬菌体展示文库筛选技术的研究进展

噬菌体展示技术是将编码外源蛋白或多肽的基因片段定向插入到噬菌体的外壳蛋白基因区,使外源蛋白或多肽通过与噬菌体外壳蛋白融合而表达并展示于噬菌体表面,进而筛选表达特异蛋白或多肽的噬菌体,已发展成为生物学后基因组时代一个强有力的实验技术.噬菌体展示文库的筛选是其关键环节.为了提高筛选效率,许多研究者对传统的筛选技术进行了改进,如选择性感染噬菌体、迟延感染性噬菌体、以DNA为基础的筛选方法、亲合力捕获和反复筛选和封闭筛选法等,用于筛选的靶标也越来越具有多样性,使得这一技术有了更加广阔的发展前景.     

噬菌体展示文库及其应用

近十几年来,噬菌体展示技术得到了迅速的发展。通过展示随机肽库可用来筛选与特殊靶分子相结合的配基;模拟非蛋白的配基;也可用作确定抗体表位的工具。展示蛋白;或其功能结构的文库为我们提供了分析结构与功能关系的体系,并能产生具有改变结合位点或新的催化活性的蛋白。展示短的抗原决定簇的融合噬菌体为开发新的疫苗提供了基础,而表达抗体片段的文库则提供了一种产生单克隆抗体的方法。     

构建噬菌体展示的β转角多肽文库

可以形成I型 β转角构象的多肽CX2 GPX4 C融合表达于丝状噬菌体fd的次要衣壳蛋白 g3p的N端 ,从而展示在噬菌体的表面。构建的多肽文库容量达到 1.0 4× 10 8个。随机挑取了 19个克隆 ,序列分析表明 ,核苷酸和氨基酸的分布与预期的基本一致。19个多肽的疏水性和等电点的综合指标分布广泛。以单克隆抗体 12CA5为靶分子 ,经过 3轮筛选 ,出现明显富集。噬菌体酶联免疫吸附法 (ELISA)以及竞争性ELISA的结果表明 ,从第 3轮洗脱液中随机挑选的 15个噬菌体克隆都能结合于抗体的抗原结合位点。破坏多肽的构象 ,这种结合将丧失     

噬菌体cDNA文库筛选方法的改良

cDNA文库的构建和简便、快速的筛选是获得全长基因的重要途径,基于PCR的筛库方法具有快捷、灵敏的特点。研究改进了基于PCR的噬菌体cDNA文库筛选方法,用液体分装的方法,替代了文库筛选的关键步骤——涂板分区,省去了噬菌体文库铺平板、浸染、培养、划块洗脱的操作过程,使筛库的工作量减少,进一步提高了筛选速度和获得阳性克隆的效率。     

从噬菌体多肽文库中筛选α—葡萄糖苷酶的抑制剂

以黑曲霉葡糖淀粉酶为靶分子,筛选噬菌体展示的多肽文库,经过3轮生物淘选法筛选,得到3个特异性结合的噬菌体克隆,分别命名为GB-1、GB2和GB-3。其插入多肽中都含有一对二硫键,破坏二硫键将大大降低噬菌体与葡糖淀粉酶的结合。化学合成的环状多肽KCHFEECLAY,其序列对应于结合力最强的一个克隆GB-1的N端的10个氨基酸残基,该多肽可以竞争性地抑制黑曲霉葡糖淀粉酶（Ki=0.2mmol/L)以及大鼠小肠的α-葡萄糖苷酶（Ki=1.4mmol/L)。     

利用氨甲蝶呤－琼脂糖凝胶进行噬菌体展示人肝脏cDNA文库筛选的方法研究

目的：利用氨甲蝶呤（MTX）偶联琼脂糖凝胶吸附法从人肝脏细胞cDNA噬菌体展示文库中筛选与MTX相互作用的蛋白。方法：以偶联于琼脂糖凝胶表面的MTX为配基,通过"结合－洗脱－扩增"过程筛选与MTX相互作用的噬菌体。利用PCR对筛选结果进行监测,对筛选得到的噬菌体PCR产物进行序列测定和基因同源性分析。结果：通过五轮亲和筛选富集到特异噬菌体克隆,再通过PCR获得cDNA插入片段。通过BLAST程序搜索GenBank,证明筛选到的片段与人PI-3K相关蛋白激酶 SMG-1异构体1 蛋白同源性达100％。结论：利用偶联MTX的琼脂糖凝胶作为筛选基质,从T7噬菌体展示cDNA文库中富集特异噬菌体是一种方便、高效的MTX相互作用靶蛋白筛选方法。本方法可为探讨小分子药物的分子作用机制提供借鉴和参考.     

人抗体组合文库的构建和抗HBsAg噬菌体抗体的筛选

应用噬菌体表面递呈表达系统构筑建了人抗体组合文库,并同了结合乙肝表面抗原（ＨＢｓＡｇ）的人噬菌体抗体（Ｆａｂ片段）。以免疫球蛋白信号肽序列为引物进行半套式ＰＣＲ所得到的产物在质和量上优越于以可变区５末端保守库列为引物进行ＰＣＲ所得到的产物,经过３次亲和选择后,抗体阳性率为６９％,抑制实验表明,所筛选出来的噬菌体本具有抗ＨＢｓＡｇ的特异性,序更分析表明ＶＨ分别属于ＶＨＩ亚群和Ⅲ亚群,其轻链ＶＬ分别属     

红莲型水稻杂种F1花药噬菌体展示文库的构建

噬菌体展示是研究蛋白质相互作用的重要手段, 为深入研究红莲型水稻不育和育性恢复的分子机理, 以红莲型水稻杂种F1花药为材料, 分离纯化mRNA, 经反转录合成双链cDNA, 在双链cDNA 末端加上定向EcoRⅠ/HindⅢ接头, 再用EcoRⅠ和HindⅢ消化接头, 形成两端分别带有EcoRⅠ和HindⅢ粘性末端的双链cDNA。经Mini Column 纯化后, 收集300 bp 以上的双链cDNA 片段, 将其连接到带有EcoRⅠ和HindⅢ末端的T7 Select 10-3b 载体上, 经体外包装后, 以BL T5403 为受体菌构建了红莲水稻杂种F1花药的噬菌体展示文库。经测定显示, 该噬菌体展示文库容量为1.03×106 pfu/mL, 重组率为100%, 扩增后文库滴度为2.14×1012 pfu/mL。对随机挑取的100 个噬菌斑进行PCR 鉴定, 97%的插入片段大于300 bp。     

B3HM细胞免疫小鼠脾细胞scFv噬菌体展示文库的构建及表达

目的: 利用噬菌体展示技术构建B3HM细胞免疫小鼠的脾细胞表达scFv文库。方法: 用人骨髓细胞系B3HM细胞免疫小鼠,取其脾细胞采用RT-PCR方法扩增VH 和Vk基因并克隆入噬菌体展示表达载体,构建scFv文库,测定文库的库容量,BstNI酶切单克隆分析文库的多样性,对文库进行富集检测,鉴定单克隆噬菌体与B3HM细胞结合反应。结果:文库的库容为5×106cfu,单克隆的BstNI酶切图谱显示多样性,单克隆噬菌体抗体与B3HM细胞呈阳性反应。结论:噬菌体展示文库的成功构建为寻找新的致白血病相关基因,阐明白血病发病机理奠定了基础。     

人α型干扰素家族定向进化文库的构建及细胞筛选

Mixtures of twelve human interferon alpha genes were digested randomly with DNase I. DNA fragments of 30～50 bps were reassembled into a full length interferon gene with sexual PCR without primer (DNA Shuffling). This primerless PCR product was further amplified with additional PCR with primers containing restriction enzyme sites. Then these PCR products were cloned into the phagemid expression vector PcantAb5E to form an expression library displaying interferon molecules. Regarding WISH cells as a standard strain rich in IFN receptor complex, we developed a new and feasible panning method with WISH cells and performed a competitive washing selection strategy. Using this method in combination with antiviral activity assay, we got two clones showing higher antiviral activity compared with phage IFN α2b. Sequencing results showed that they are hybrids containing two and four interferon alpha genes. Now we are cloning them into prokaryotic expression vector and want to get purified sample to determine their biological activity and biochemistry characters.     

Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pill protein of MI 3 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.     

DNA改组的最新动态及应用前景

DNA改组(DNA shuffling)是目前最方便、有效的一种分子水平的体外定向进化技术,该技术同倾向错误PCR (Error-prone PCR) 相结合,通过对单基因或相关基因家族的靶序列进行多轮随机诱变、重组和高通量的筛选,可以有效富集正突变,去除负突变,提高突变文库的丰度,创造新基因和获得期望功能的蛋白质。DNA改组技术已在新药物等领域取得了广泛的应用,极大地推动了现代生物科学和生物技术的发展。该技术同计算机强大的数据分析系统相结合,将会为后基因组学的发展提供强有力的技术平台。     

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V_λ and 13/35 V_κ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V_H CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC₅₀ values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.     

利用DNA改组技术改造aacC1基因启动子活性的研究

从表达质粒pYPX251(GenBank 登陆号：AY178046)中获得aacC1基因启动子,采用DNA改组（DNA shuffling）技术在体外获得突变体。以lacZ作为报告基因,筛选获得活性明显改变的启动子。经过验证,对其中活性变化明显的7个启动子用邻硝基苯基β半乳糖苷(ONPG)作为底物进行表达活性测定。结果表明,获得的强启动子比原来的提高了3～8倍,而弱启动子则活性下降明显,其中3个几乎无活性。进一步对这7个启动子进行了序列分析。     

利用DNA家族重排提高青霉素G酰化酶合成活力

NA家族重排技术是酶定向进化的有力工具 ,已在实际应用中获得了巨大成功。来源于Providenciarettgeri、Escherichiacoli和Kluyveracitrophila的青霉素酰化酶基因序列同源性为 6 2 .5 %～ 96 .9%。在Providenciarettgeri青霉素酰化酶基因克隆和表达的基础上 ,利用DNA家族重排技术构建了上述基因的嵌合体突变库。通过平板初筛获得有活力的阳性克隆 ,表达提取突变酶测定其合成水解活力比。对突变酶进行随机测序的结果表明多基因嵌合体突变库显示出明显的多样性。通过一轮重排及筛选 ,获得了合成活力提高 4 0 %的突变酶 ,并且发现α亚基的重排对酶合成活力的提高更加有效。上述方法的应用有望获得合成活力进一步提高的青霉素G酰化酶。     

A novel BLyS antagonist peptide designed based on the 3-D complex structure of BCMA and BLyS

B lymphocyte stimulator (BLyS) is a member of tumor necrosis factor (TNF) family. Because of its roles in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS), BLyS antagonists have been tested to treat SLE- and RA-like symptoms in mice and obtained optimistic results. So far, reported BLyS antagonists were mostly decoyed BLyS receptors or anti-BLyS antibodies. In this study, a novel BLyS antagonist peptide, PT, was designed based on the modeling 3-D complex structure of BCMA and BLyS. The interaction mode of PT with BLyS was analyzed theoretically. The results of competitive ELISA demonstrated that PT could inhibit the binding of BCMA-Fc and anti-BLyS antibody to BLyS in vitro. In addition, PT could partly block the proliferating activity of BLyS on mice splenocytes. The BLyS antagonizing activity of PT was significant (p<0.05). This study highlights the possibility of using BLyS antagonist peptide to neutralize BLyS activity. Further optimization of PT with computer-guided molecular design method to enhance its biopotency may be useful in developing new BLyS antagonists to treat BLyS-related autoimmune diseases.     

D-泛解酸内酯水解酶的定向进化

易错PCR结合DNA改组方法向D-泛解酸内酯水解酶基因中引入突变,并构建突变体库。利用酶的催化特点和产物特性建立了基于平板初筛和高效液相复筛的两步法D-泛解酸内酯水解酶活性筛选系统。用该筛选系统以酶活力和pH稳定性为指标对突变体库进行筛选,最终获得一株酶活力高且在低pH条件下稳定性好的突变体Mut E-861。该突变体的酶活力是野生型酶的5.5倍。对突变体和野生型酶在pH 6.0和pH 5.0条件下的残余酶活进行对比,在这两种pH条件下,突变体酶的酶活残留分别为75%和50%,而野生型酶只能保持原来的40%和20%。通过软件对突变体Mut E-861酶基因和野生型酶基因进行分析对比,发现突变体Mut E-861酶基因发生了三处点突变,其中突变使两处氨基酸取代,另一处为沉默突变,未引起氨基酸的变化。     

家蚕核型多角体病毒遍在蛋白及结合肽的研究

从家蚕核型多角体病毒镇江株(Bombyx mori nuclear polyhedrosis virus Zhenjiang strain, BmNPV-ZJ)基因组DNA中克隆遍在蛋白（BmVUB）基因.序列分析结果显示,BmVUB基因长234 bp,编码77个氨基酸.BmVUB氨基酸序列内有一致HTH序列,遍在蛋白保守序列LRLRGG,参与遍在蛋白-蛋白酶复合体形成的4个保守性功能位点(Lys-29、Cys-48、Cys-63、Gly-76)及保守的Gly-Gly-X（X是疏水氨基酸残基）蛋白酶切信号序列,在遍在蛋白保守的Gly-Gly后多出由一个氨基酸组成的延伸肽.原核诱导表达表明BmVUB主要以可溶性形式存在,表达量占总菌蛋白的50%以上.纯化的遍在蛋白浓度为1.03～2.46 g/L,制备抗体,效价在3.2×10^-5以上.用噬菌体表面展示技术筛选遍在蛋白结合肽,所筛选的结合肽可与遍在蛋白特异性结合,其中结合肽VAPHHAYAPMRT对细胞增殖有明显的浓度调控作用,即低浓度促进细胞生长,高浓度强烈抑制细胞的生长.     

Discovery of high-affinity peptide binders to BLyS by phage display

B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.     

鼠源性人乳腺癌单链抗体库的构建与乳腺癌相关抗体的筛选

目的:获得乳腺癌的噬菌体呈现型单链抗体(scFv)库,筛选与乳腺癌细胞特异结合的抗体,为乳腺癌的诊断和治疗奠定基础。方法:用乳腺癌细胞系MCF-7、T47D、MDA-MB-435免疫BALB/c小鼠,取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区(VH和VL)基因,经Linker连接形成scFv基因片段。将scFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1。用辅助噬菌体M13KO7进行超感染,获得重组噬菌体抗体。选用乳腺癌细胞系MCF-7和人正常肝细胞系HL02做正负差异的筛选细胞,通过5轮筛选,随机挑取克隆,经phage-ELISA筛选特异性结合MCF-7细胞的scFv。结果:构建了1个库容为1.3×106的单链抗体库。筛选到2株与MCF-7细胞有较高结合活性的噬菌体-单链抗体scFv-873和scFv-874。数据库搜索表明这2株单链抗体基因是与以往抗体序列不同的新基因。用Westernblot检测了这2株单链抗体在琥珀密码子非抑制型菌株TOP10中的表达情况。结论:筛选到2个与乳腺癌细胞结合特异性较好的单链抗体,为乳腺癌的诊断和治疗研究奠定了基础。