波形蛋白-核酸复合物诱导的多特异性单抗

本文首次采用中间纤维波形蛋白(vimentin)-核酸复合物免疫Balb/c小鼠,通过杂交瘤技术制备了一组多特异性单克隆抗体(polyspecific McAb),这些抗体能够分别与波形蛋白、DNA、磷脂以及其它生物化学结构不同的生物大分子结合,其抗原特异性与病人血清中的自身抗体(autoantibody)极为相似。抗原表位分析结果表明,抗体所识别的波形蛋白NH_2末端恰是波形蛋白结合DNA的位点。多特异性单抗的研制将为研究抗体交叉反应机制、自身抗体来源以及核酸与蛋白质的相互关系提供了一条新的途径。     

免疫亲和质谱法研究／β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法,对β2-微球蛋白抗原表位进行了系统研究.完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后,用Endoproteinase Glu-C,Trypsin,AminopeptidaseM和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分了,并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究.结果表明:β2-微球蛋白抗原表位位于整个蛋白分了氨基酸序列的61～67位,即为SFYLLYY.通过合成肽段的分析,证明了SFYLLYY即为抗原表位,与亲和质谱方法分析结果一致.     

免疫亲和质谱法研究β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法, 对β2-微球蛋白抗原表位进行了系统研究. 完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后, 用Endoproteinase Glu-C, Trypsin, Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子, 并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究. 结果表明: β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位, 即为SFYLLYY. 通过合成肽段的分析, 证明了SFYLLYY即为抗原表位, 与亲和质谱方法分析结果一致.     

氧氟沙星噬菌体库的构建、筛选及抗体结构模拟

应用噬菌体展示和重组抗体技术制备抗氧氟沙星单链抗体(scFv)库,筛选获得氧氟沙星特异性噬菌体scFv以及同源模拟其三维结构。将从氧氟沙星杂交瘤细胞提取的总RNA,用RT-PCR反转录合成cDNA,以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的兼并引物,扩增获得VH和VL可变区基因,通过SOE-PCR法将VH基因和VL基因通过柔性多肽Linker(Gly4Ser)3拼接成全长scFv基因片段,将双酶切后的scFv基因片段插入T7噬菌体,经体外包装后转化宿主菌BLT5403,成功构建库容量为3×105pfu/mL的抗体库,经4轮吸附-洗脱-扩增的富集,采用直接竞争ELISA筛选到4个特异性噬菌体scFv,运用Expasy软件模拟特异性scFv的三维结构。为进一步大量表达氧氟沙星单链抗体奠定了基础。     

虾过敏原Pen a1抗原表位的关键氨基酸分析

建立了一种利用表位抗体筛选虾过敏原Pen a1抗原表位中关键氨基酸的方法。利用生物信息学软件MEGA5计算Pen a1中表位的氨基酸组成和出现频率,并采用DNAMAN软件对SDAP数据库中致敏食物原肌球蛋白的氨基酸保守性分析,综合两种方法筛选出各表位可能的关键氨基酸,Epitope 1:K,E,N;Epitope2:K,L,E;Epitope 3:E,R,D,L,Q;Epitope 5:K,L,Q。用丙氨酸取代并合成突变多肽。利用表位抗体,通过竞争性免疫斑点法及间接ELISA方法检测突变多肽的抗体结合能力,筛选出各表位的关键氨基酸。分别为:Epitope1:谷氨酰胺;Epitope2:亮氨酸和谷氨酸;Epitope3:亮氨酸和天冬氨酸;Epitope5:亮氨酸。实验结果证明了此种筛选关键氨基酸的方法的可行性,为Pen a1致敏机理的研究及利用基因修饰脱敏提供了一定的理论依据。     

γ-羟基膦酸酯诱导梭曼水解单链抗体酶

新型半抗原３－羟基－１－对硝基苯基甲磷酸单频哪基酸酯（Ｐ_６），与血蓝蛋白结合后（Ｐ_６－ＬＰＨ）免疫小鼠，从小鼠脾细胞出发构建单链抗体ｃＤＮＡ文库和噬菌体表面呈现文库．用固相化抗原 Ｐ_６－ＢＳＡ对噬菌体抗体库进行了 ４轮淘选，从中筛选到１１株对梭曼水解有催化作用的单链抗体.对其中一株（ＥＰ_６）进行可溶性抗体的制备，经凝胶过滤和离子交换层析纯化后，ＳＤＳ－ＰＡＧＥ为 ３１ｋｕ的单一条带．酶动力学常数测定其对梭曼水解的催化常数为ｋ_（ｃａｔ）＝１９８ｍｉｎ~(－１)，Ｋ_ｃａｔ／Ｋ_ｕｎｃａｔ＝１２２ ４１９．单链抗体酶ＥＰ６_０．１６ｍｇ·ｍＬ~(－１)与梭曼 ０． １３２ ｍｍｏｌ· Ｌ~（－１）在 ２０℃共温１ｈ后给小鼠背部皮下注射相当于１． １个致死剂量的梭曼， １９只小鼠全部存活，而对照组 １４只小鼠 ３０ ｍｉｎ内全部死亡．小鼠体内抗毒实验显示惊厥潜伏期及死亡时间延长．     

汉坦病毒嵌合基因G2S0.7与CTL多表位重组腺病毒的构建及免疫学分析

目的:构建含有汉坦病毒M基因(编码糖蛋白G2)和部分S基因(编码核蛋白主要抗原区段)以及S基因的多个CTL表位的多种蕈组腺病毒表达载体,并对这些重组腺病毒的免疫学特性进行研究.方法:构建含目的基因的重组腺病毒载体,并在VeroE6细胞中进行表达,利用纯化后的重组腺病毒免疫BALB/c小鼠,通过ELISA、微量细胞中和试验、T淋巴细胞增殖实验及CTL杀伤实验等方法检测免疫反应.结果:成功表达出可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单克隆抗体(mAb)所识别的融合蛋白;重组的腺病毒可以有效诱导针对汉坦病毒NP及GP的体液免疫应答和细胞免疫应答;同时我们发现含有CTL表位的重组腺病毒比不含CTL表位的重组腺病毒可以诱导小鼠产生更为有效的细胞免疫反应.结论:构建的含CTL多表位的重组腺病毒可以有效的提高嵌合基因刺激细胞免疫应答的能力.     

可溶性人源含硒抗体酶GPx的研究

对噬菌体展示人单链抗体库进行筛选,得到与半抗原S-二硝基苯取代的谷胱甘肽二丁酯特异结合的单链抗体3B10。用计算机模拟分析了单链抗体的空间结构,发现抗原结合的CDR3区位于抗体的表面,推测其可能进一步参加硒化反。利用突变引物,在大肠杆菌中表达了可溶性抗体蛋白,并用化学方法将催化必需基团硒代半胱氨酸（Sec）组装到3B10抗原结合部位,获得了具有谷胱甘肽过氧化酶活力的人源抗体酶。动力学研究结果表明,抗体酶和天然酶一样,符合乒乓反应机制。     

HIV-1嵌合抗原的纯化及免疫原性分析

为获得高效的HIV诊断试剂,选定HIV-1的外膜蛋白env中469-511aa,538-674aa和700-734aa3处包含较多抗原位点的区域作为免疫抗原,用PCR的方法从HIV-1全基因扩增编码这3处片段的基因序列,将它们克隆到原核表达载体中,利用大肠杆菌表达系统表达嵌合蛋白.结果发现,3段嵌合基因能在大肠杆菌BL21(Star)中表达,通过Ni-sepharose4B金属Ni螯合层析柱分离纯化目的产物,酶联免疫检测结果表明,纯化抗原有较强的抗原特异性.     

以烟草花叶病毒为半抗原载体的活体免疫反应

以烟草花叶病毒的赖氨酸突变体(TMV-EPMK)为半抗原载体,利用“点击化学”反应,将半抗原小分子雌三醇(E3)经由两个不同亲/疏水性的连接臂修饰在TMV-EPMK上,成功制备了TMV-EPMK/E3完全抗原。 通过免疫实验发现,以亲水性连接臂制备的TMV-EPMK/E3偶联体可在小鼠体内诱导更高水平的E3特异性免疫反应,所产生的IgG抗体的滴度达21800。 该结果表明,以烟草花叶病毒为载体制备的完全抗原,其免疫原性受连接臂性质的影响,亲水性的连接臂更有利于抗体反应的发生。     

M13噬菌体在分析传感中的应用进展

为生物传感分析选择合适的目标识别分子是提高检测灵敏度和准确度的关键.为此,人们开发了一系列诸如小分子、抗体、多肽和适配体等亲和配体.基于噬菌体展示技术筛选出数千种特异性配体,可以噬菌体为组合元件构建生物传感探针.该方法具有优异的靶向能力,并具有较强的环境耐受性.常见的M13噬菌体表面由多达2700个拷贝的主要衣壳蛋白p...     

不同生长时期盐藻无机元素分析

测定了对数生长期和稳定期盐藻中10种无机元素K、Na、Ca、Mg、P、Cu、Zn、Fe、Mn、Se的含量。结果表明，稳定期盐藻中K、Ca、Mg、Cu、Zn、Fe、Mn、Se的含量比对数生长期都有不同程度的增加，Na、P的含量反而降低。     

Efficiency of MS2 phage and Qβ phage removal by membrane filtration in water treatment: Applicability of real-time RT-PCR method

MS2 phage is currently used as a surrogate to evaluate pathogenic virus removal efficiency by filtration membrane during water treatment. Phage removal is commonly defined upon comparing number of infectious units in permeate with that in feed solution by PFU method. This method may lead to overestimation of the virus removal because of possible occurrence of viruses aggregation.     

Phage display of enzymes and in vitro selection for catalytic activity

Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes. The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography. The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.     

Y-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitro-phenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcatt= 198 min-1 and the rate enhancement kcatkuncat = 122 419. When 0.16 mg · mL-1     

γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcat = 198 minγ1 and the rate enhancement kcat/kuncat = 122 419. When 0.16 mg·mLγ1 EP6 was preincubated in vitro with 0.132 mmol·Lγ1 (220 g·kgγ1 = 1.1×LD95) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP6 could prolong the latent time of spasm and death when mice were passively immunized with EP6 intravenously 15 min before 1×LD95 of soman challenge. These results demonstrate that EP6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.     

Nucleophilic proteolytic antibodies

Proteolytic antibodies appear to utilizecatalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity ofactive-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.     

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.     

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals—The Etiological Factor of Chondrocalcinosis

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg²⁺-dependent and also works against soluble species of pyrophosphate.