利用噬菌体肽库筛选IL-2Rα模拟表位肽的研究

目的 筛选IL-2Rα模拟表位肽,为研制高效、特异性强的小分子肽类免疫抑制剂奠定基础.方法 应用离子交换层析法从小鼠腹水中纯化抗人IL-2Rα单克隆抗体5G1,细胞ELISA方法检测其特异性.用5G1单抗筛选噬菌体环七肽库,并对噬菌体克隆进行抗原性鉴定.根据阳性克隆的DNA序列合成环肽并鉴定其生物活性.结果 经5轮筛选、鉴定,获得5个与5G1有较强结合特性的噬菌体阳性克隆.DNA测序并推导氨基酸序列,得到Lys-X-{X}-Lys-Gly保守序列.依此序列合成环七肽CP,小鼠淋巴细胞增殖试验证实其有免疫抑制活性.结论 环肽CP为IL-2Rα模拟表位肽,可作为IL-2Rα的拮抗剂发挥免疫抑制效应.     

人源性抗乳腺癌单链抗体库的筛选与初步鉴定

目的:本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定。方法:以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况。结果:成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白。结论:本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库。研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础。     

用噬菌体抗体库分离NK细胞抑制受体特异性的ScFv片段

目的探讨从噬菌体抗体库分离杀伤细胞抑制受体（ＫＩＲｓ）特异性ＳｃＦｖ（ｓｉｎｇｌｅｃｈａｉｎＦｖ）的可行性及其特征。方法所用ＫＩＲ为ＮＫＡＴ２,其可溶性形式为ＮＫＡＴ２－ＩｇＧ１。噬菌体抗体库为Ｎｉｓｓｉｍ等报道的半合成抗体库。抗体库经包被免疫管的ＮＫＡＴ２－ＩｇＧ１５次筛选后,可溶性ＳｃＦｖ由ＩＰＴＧ诱导细菌而获得,筛选效果由测定噬菌体的菌落形成单位及ＤＮＡ酶切图谱分析。ＥＬＩＳＡ、聚丙烯酰胺凝胶电泳及免疫印迹分析ＳｃＦｖ特异性及免疫学特征,并进行ＤＮＡ的序列测定。结果５次筛选后,噬菌体得到２００倍的富集,酶切图谱也显示某种构型噬菌体的富集。４０个克隆的细菌上清经测试,３７个显示与ＮＫＡＴ２有较强的反应。ＳｃＦｖ经聚丙烯酰胺凝胶电泳及免疫印迹显示相对分子质量为３０×１０３,其ＤＮＡ序列完全相同,重链ＣＤＲ３区编码氨基酸为ＥＳＮＬＶＴＣ,重链其它部分与ＤＰ３５一致。结论研究初步结果表明,用噬菌体抗体库可以快速、简便地产生用于ＫＩＲ研究的较高特异性的试剂。     

The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library

Molecular cloning techniques and V gene phage display have revolutionised the production of human monoclonal antibodies. Antibodies of a defined specificity can be obtained by selecting phage display libraries on antigen in a process known as panning. We have applied these techniques to the isolation of three HLA-A2-specific single chain variable domain fragments (scFv) from a patient alloimmunised by blood transfusion. Analysis of specificity with cells of HLA genotyped donors revealed the following: i) in addition to the major reactivity with HLA-A2, cross-reactivity with the HLA-A28 epitope; and ii) inhibition of scFv binding to the antigen by the patients' antibodies. The heavy chain variable genes of all three were derived from the germline gene Cos-3, carry the hallmarks of somatic hypermutation, and are most likely derived from clonally related B cells. The light chain variable domains were encoded by DPK1 and DPK8 from the VkappaI family. These data show that phage display can be used to clone HLA-specific alloantibodies that recognise the native antigen from alloimmunised patients.     

从半合成噬菌体抗体库筛选基质金属蛋白酶—2人抗体

目的：从半合成噬菌抗体库中筛选抗金属蛋白酶－2（MMP－2）人源抗体。方法：将重组人MMP－2固相化,通过多轮“吸附－洗脱－扩增”从半合成抗体库筛选特异性抗体,用ELISA方法对所获克隆进行特异性分析及MMP－2中和活性。结果：经过4轮筛选,得到多个可与MMP－2结合的克隆,但这些克隆也可与其它无关抗原结合,选1个克隆AD20进行竞争抑制性ELISA分析,发现其与MMP－2的结合可被游离MMP－2特异性抑制,不被无关抗原抑制,而与无关抗原的结合不能被所测的任何游离抗原所抑制,进一步检测AD20可中和MMP－2的酶解活性,DNA序列测定证明其为人抗体。结论：从半合成抗体库中获得一株具有中和活性的抗MMP－2人抗体,该抗体对无关抗原显示的非特异性结合是由不同模式介导,属于多反应性。     

人源性抗HER2胞外段Fab噬菌体抗体库的构建及筛选

目的:构建全人源抗HER2胞外段(HER2 ECD)噬菌体Fab抗体库,从中筛选出特异性的抗体,并对其进行鉴定。方法:体外致敏并用EB病毒(EBV)转化HER2高表达乳腺癌患者的外周血单核细胞(PBMC),用PCR分别扩增重链Fd和轻链κ/λ基因。经SacI/XbaI和XhoI/SpeI双酶切,依次克隆入噬菌体载体pComb3中,并电转化大肠杆菌XL1-Blue,以辅助噬菌体VCSM13进行超感染,构建抗HER2 ECD人源化Fab噬菌体抗体库。以纯化HER2 ECD蛋白为抗原进行3轮固相淘选,富集抗HER2 ECD的抗体,并随机挑选克隆进行ELISA,获得的阳性克隆进一步以Western blot鉴定其抗原结合活性,对其中活性最高的克隆进行DNA测序。结果:构建了容量为2.5×107的抗HER2 ECD的Fab噬菌体抗体库,并筛选获得了4株与HE2 ECD特异性结合的阳性克隆,Western blot分析显示其与HER2 ECD能较好的结合。选取结合活性最高的阳性克隆进行DNA序列分析,结果显示其重链可变区、轻链可变区分别与人胚系免疫球蛋白基因有高度的同源性。结论:成功构建了全人源抗HER2 ECD噬菌体抗体库,并筛选出抗HER2 ECD特异性较强的噬菌体克隆,为获得新的有临床应用价值的HER2 ECD抗体提供了实验基础。     

The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library

A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K_d of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.     

Identification of peptide motifs recognized by a human IgG autoanti-IgE antibody using a phage display library

BACKGROUND: The potential of murine monoclonal anti-IgE antibodies as long-term therapy for atopic diseases will have to rely, for the time being, on passive antibody administration. There is therefore considerable interest in developing a peptide-based vaccine for active immunization to elicit long-term protective anti-IgE antibodies in the patient. It has been shown that some human IgG autoanti-IgE antibodies have the ability to partially block the binding of IgE to Fc receptors such as Fc epsilonRI. Therefore, the epitopes recognized by such antibodies could have vaccine potential. OBJECTIVE: To determine the epitope specificity of one such human IgG anti-IgE antibody. METHODS: A 15-mer phage-peptide library was used to establish the epitope specificity of an IgG anti-IgE antibody isolated from the serum of an asthma patient. RESULTS: The SRPSP sequence, or part of it (i.e. RPS, RPSP, SPS or PSP), was present in all 18 phage-peptides that have been sequenced. This common motif was found to be within the human epsilon chain sequence Ser341-Thr355 near the N-terminus of the C epsilon3 domain. According to the human Fc epsilon model, the most accessible residues in this sequence are Arg342, Ile350, Arg351, Lys352 and Ser353. CONCLUSIONS: The present data should provide the molecular basis for the rational design of a suitable peptide immunogen (vaccine) for boosting the production of protective autoanti-IgE antibodies.     

Isolation of the Taenia crassiceps antigens from a phage display cDNA library and evaluation of their use for diagnosis of neurocysticercosis

A novel cDNA cloning strategy consisting in elimination of non-coding DNA sequences from 3' regions of cDNAs was applied to construct the Taenia crassiceps phage displayed cDNA expression library. After biopanning using immune sera, three phage clones expressing T. crassiceps-derived antigens specifically recognizing antibodies present in cerebrospinal fluid and plasma samples from neuroimaging-confirmed neurocysticercosis patients were selected. This novel cloning strategy may be applied to other pathogens allowing rapid identification of peptides/proteins for immunodiagnostic tests.     

抗KGla细胞噬菌体展示ScFv的筛选及鉴定

目的 应用ＫＧ１ａ细胞（髓系白血病细胞系）筛选噬菌体抗体库,获得能以一定特异性结合ＫＧ１ａ细胞的单链抗体（ＳｃＦｖ）,克隆重其基因并研究其对应抗原在不同细胞表面的分布。方法 用完整的ＫＧ１ａ细胞筛选一个经ＫＧ１ａ细胞免疫小鼠脾细胞的ＳｃＦｖ噬菌体抗体库,重复筛选４轮;细胞ＥＬＩＳＡ鉴定了其中９６个克隆重与ＫＧ１ａ细胞的结合反应;从２６个ＫＧ１ａ细胞ＥＬＩＳＡ阳性克隆中挑选了３个克隆重,进一步用免疫     

绒毛和蜕膜中的Th1,Th2细胞因子水平与反复流产的关系

目的　了解Th1,Th2细胞因子在反复流产患者绒毛和蜕膜中的浓度以及与反复流产的关系。方法　采用放射免疫的方法 (RIA)对 80例反复流产患者和 2 8例正常妇女的绒毛和蜕膜中Th1,Th2细胞因子进行检测。结果　反复流产患者中绒毛、蜕膜Th1,Th2细胞因子的浓度增高 ,与正常对照组相比 (P <0 0 5 ,0 0 1) ;Th1/Th2偏移。结论　Th1、Th2型细胞因子的异常分布与妊娠丢失有关     

Identification of a human heavy chain antibody fragment directed against human platelet alloantigen la by phage display library

Abstract: The human platelet alloantigen HPA-la (Pl^A1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombo-cytopenia in the Caucasian population. HPA-la and HPA-lb are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recom-binant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-la and HPA-lb. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-la homozygous donors, the HPA-la form of recombinant N-terminal GPIIIa and intact HPA-la platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-la form of platelet GPIIIa or other platelet glycoproteins. This HPA-la specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-lb homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.     

一种能与Fas结合的融合型九肽生物学活性分析

目的鉴定从噬菌体随机肽库筛选所获得的融合型多肽3A8的生物学活性。方法制备融合型多肽3A8,经纯化,采用ELISA检测融合型多肽3A8对Fas．Fc的特异性结合;细胞ELISA检测3A8能否与Jurkat细胞表面天然Fas受体结合;^3H-TdR掺入法检测3A8对Jurkat细胞增殖的影响;流式细胞仪检测细胞凋亡。结果ELISA结果显示融合型多肽能与Fas．Fc特异性结合,呈浓度剂量依赖关系。细胞ELISA实验证实3A8能与Jurkat细胞表面Fas受体结合。^3H—TdR掺入法检测发现3A8可抑制Jurkat细胞增殖,抑制率为50％。经3A8处理细胞经PI染色流式细胞仪分析,发现25．41％的Jurkat细胞发生凋亡,明显高于未经处理的对照组。结论从噬菌体随机肽库获得的融合型多肽3A8能与Jurkat细胞表面受体Fas结合,诱导细胞凋亡。     

用细胞筛选噬菌体抗体库分离自然杀伤细胞抑制受体的单链抗体

目的与方法：用Ｎｉｓｉｍ等噬菌体抗体库经细胞筛选，分离自然杀伤细胞抑制受体（ＮＫＡＴ１）的单链抗体。结果：８次筛选循环后，分离到的单链抗体不但可以特异性识别到表达在细胞表面的ＮＫＡＴ１分子，而且也能与纯化的ＮＫＡＴ１蛋白结合，这些单链抗体为３１ｋＤａ的分子。结论：用噬菌体抗体库可以简便、快速产生自然杀伤细胞抑制受体特异性的单链抗体     

Selection and characterization of single chain Fv fragments against murine recombinant prion protein from a synthetic human antibody phage display library

We describe the selection of single chain Fv fragments (scFv) against recombinant murine prion protein (mPrP) from a synthetic human antibody phage display library. Six different antibodies were isolated after three rounds of panning against full-length mPrP. All antibodies recognized a truncated form of mPrP containing residues (121-231). The amino acid sequence of the CDR3 of the scFv fragments has been determined. Five of the antibodies have been over-expressed, purified and their affinity for full-length mPrP determined by ELISA. The observed binding affinities vary from 30 nM to 2.7 microM.     

膜表达的HLA-G1对同种反应性PBMC分泌Th1/Th2型细胞因子的影响

目的研究人脐静脉内皮细胞系(ECV304)表达的HLA-G1对同种异体外周血单个核细胞(PBMC)Th1/Th2型细胞因子分泌的影响。方法采用脂质体介导的基因转染技术将pcDNA3-HLA-G1转入ECV304,以间接免疫荧光技术在蛋白质水平上检测HLA-G1分子在ECV304上的表达;以表达HLA-G1的ECV304作为刺激细胞,灭活后,与健康人PBMC共同培养,用ELISA检测上清液中Th1/Th2型细胞因子的浓度,观察HLA-G1对同种异体抗原激活的PBMC分泌Th1/Th2型细胞因子的影响。结果与转染pcDNA3空质粒的对照组相比,HLA-G1能使PBMC的IL-10分泌增加(P<0.05),而IL-2,IFN-γI、L-4分泌无明显影响。结论HLA-G1能引起由同种异体抗原激活的PBMC分泌IL-10增加,提示HLA-G1有可能使Th1/Th2型细胞因子向Th2型偏移。     

Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library

BACKGROUND: Phl p 5 represents a major allergen of timothy grass pollen (Phleum pratense). Detailed knowledge about the structures responsible for IgE binding would allow the design of a novel generation of allergy vaccines. OBJECTIVE: We aimed to characterize the IgE epitopes of Phl p 5a using phage display combined with a molecular modeling approach. METHODS: Phl p 5a-specific IgE from sera of patients with grass pollen allergy was used for screening of a random peptide phage library displaying constrained decamers. RESULTS: Fifteen phage clones that shared sequence motifs and could be grouped into families were selected by using Phl p 5a-specific IgE. Peptide alignment with the solvent-accessible amino acids of Phl p 5a revealed 3 sequence sections with frequent hits of identical or similar amino acids. On the surface of Phl p 5a, these sections assembled in compact patches, most likely representing conformational IgE epitopes, whereas no matching clusters were found on the back sides of the 2 Phl p 5a halves. In surface plasmon resonance experiments, the high-affinity interaction between IgE and Phl p 5 could be competed by phage-displayed peptides up to 24%, indicating that they represent true epitope mimics (ie, mimotopes). Allergen-specific immunogenicity of the mimotopes was proved in Biozzi mice. CONCLUSION: The selected mimotopes facilitated the localization of conformational IgE epitopes of Phl p 5. We suggest them to be suitable candidates for the development of an epitope-specific immunotherapy.     

人源腺病毒伴随病毒Ⅱ型单克隆抗体Fab段及其全抗体的研制

目的 运用噬菌体表面表达技术，获得基因工程抗腺病毒伴随病毒抗体Fab、IgG。方法 从酶联免疫吸附试验阳性的人外周血中分离淋巴细胞。提取总RNA，逆转录cDNA，聚合酶链反应扩增人IgG Fab轻、重链基因，利用pComb3载体构建噬菌体抗体库。用纯化的腺病毒伴随病毒为固相抗原筛选抗体Fab段，并在E.coli中进行分泌性表达。通过ELISA和间接免疫荧光试验、筛选和鉴定Fab抗体，并进行序列测定。然后将其重链和轻链基因克隆到全抗体表达载体pAC-L-Fc上。转染昆虫sf-9细胞，利用杆状病毒，昆虫细胞系统实现全抗体的分泌型表达，免疫沉淀试验鉴定它的抗蛋白。结果 分离到一株Fab克隆AAVs-31，腺病毒伴随病毒抗原和抗Fab抗体直接ELISA检测阳性，间接免疫荧光试验呈阳性，序列分析结果表明是一新的序列，所获得的基因为人源IgG Fab基因。由Gamma链和Kappa链组成。表达的全抗体ELISA反应、免疫荧光反应和间接免疫荧光试验均为阳性，免疫沉淀试验结果显示它结合病毒颗粒，不结合任一VP1、VP2、VP3单独衣鞘蛋白。结论 用噬菌体表面表达技术首次获得了抗腺病毒伴随病毒Ⅱ型单克隆抗体Fab段，并在真核系统中表达了其全抗体，它们识别的可能是衣鞘蛋白组装时形成的表位。     

重组人G—CSF免疫小鼠全套单链抗体噬菌体表面展示文库的构建

利用全套噬菌体抗体表面展示技术,绕过杂交瘤技术,从重组人G-CSF免疫的小鼠脾淋巴细胞中提取总RNA,反转录成cDNA后,用抗体可变区PCR混合引物进行全套抗体重、轻链可变区（VH和VL）基因的扩增。经重叠延伸反应,在体外随机装配成单链抗体（ScFv）。将其克隆至噬菌粒载体pCANTAB5E中,电转化含SupE的E.Coli菌株,以辅助噬菌体M13K07超感染噬菌粒文库,构建成全套ScFv表面展示文库。为利用亲和富集筛选技术,获得具有G-CSF结合活性的完整重组噬菌粒克隆奠定了基础。