HIV-gp120人源单链可变区抗体筛选与鉴定

目的 通过制备抗人类免疫缺陷病毒(HIV)gp120蛋白人源单链可变区抗体(seFv),探索新型抗人获得性免疫缺陷病毒(HIV)治疗方法.方法 采用噬菌体表面展示技术,将HIV-gp120蛋白固相包被于Nunc板,应用半合成的人源单链可变区抗体文库技术,从噬菌体单链可变区抗体库中经过5轮吸附-洗脱-扩增筛选过程,随机挑选96个克隆,利用酶联免疫吸附法(ELISA)、交叉反应和竞争抑制实验,进行免疫学检测和鉴定,获得与HIV-gp120蛋白结合活性较强的scFv阳性克隆,并对HIV-gp120蛋白ScFv的编码基因进行序列测定分析.结果 经过筛选96个克隆中有39株克隆ELISA的吸光度(A450 nm)值较高,将这些噬菌体上清与牛血清白蛋白(BSA)进行交叉反应,确定其中有7株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.提取质粒,进行DNA序列测定,DNA为699 bp.结论 用噬菌体抗体库技术成功地获得抗HIV-gp120蛋白的scFv,为抗gp120蛋白的ScFv防治艾滋病研究创造了条件.     

人源抗胃癌单链抗体的构建及初步鉴定

目的构建库容大、多样性好的噬菌体展示人源抗胃癌单链抗体。方法收集胃癌患者外周血分离淋巴细胞,利用噬菌体展示技术,构建人源单链抗体库并初步筛选抗胃癌单链抗体,用ELISA方法检验该抗体与胃癌细胞结合活性。结果成功构建了次级库容达4.0×109pfu/ml的噬菌体展示人源单链抗体库,并初步筛选到与胃癌细胞特异性结合的单链抗体。结论成功构建和筛选人源抗胃癌单链抗体,为进一步表达和应用特异性抗胃癌单链抗体奠定基础。     

人源抗HIV-1V3环噬菌体抗体筛选与鉴定

目的 以人类免疫缺陷病毒(HIV-1)gp120 V3环的合成环肽为抗原,从人的噬菌体抗体组合文库中筛选出与抗HIV-1 V3肽有结合活性的人源噬菌体抗体(Fab段).方法 用噬菌体抗体库技术筛选人源抗HIV-1V3环噬菌体抗体,ELISA测定其活性,竞争抑制实验证实了该噬菌体抗体的特异性.结果 序列分析表明.重链基因为IgG1亚类,可变区属VHI亚群.与胚系基因DP-88同源性最高,D区为D3-3、J区为JH5;轻链为k亚型.可变区属VL IV亚群,D区为DPK22,J链为Jk4胚系.结论 成功筛选了人源抗HIV-1 V3环噬菌体抗体.其具有很好的生物学活性和特异性.     

抗类风湿人源单链抗体库的制备及筛选

目的 通过噬菌体展示技术构建抗类风湿人源单链抗体库,为其进一步的临床应用研究奠定基础。方法提取患者外周淋巴细胞总RNA,逆转录合成cDNA,以人源免疫球蛋白H链和L链可变区的扩增引物,分别扩增出H链和L链可变区基因。采用SOE—PCR法将VH和比片段随机拼接成scFv片段,并克隆入噬菌粒载体pCANTAB5E中,构建噬菌体单链抗体库。结果初级库库容量为3×10^6,在大肠杆菌TG1中重组后得到1．2×10^6的次级抗体库。结论本研究成功构建类风湿人源单链抗体库,拟为类风湿病的预防、诊断、治疗奠定基础。     

鼠源抗烟曲霉单链抗体库制备及鉴定

目的 构建鼠源抗曲霉菌单链抗体（scFv）噬菌体展示文库并进行初步鉴定,为制备用于侵袭性曲霉感染实验室诊断的特异性抗体提供新的方法。方法 利用噬菌体展示技术构建鼠源抗烟曲霉半乳甘露聚糖（GM）ScFv库,经过4轮“吸附-洗脱-扩增”淘选富集,随机挑选克隆进行可变区基因测序确定文库多样性,并用phage-ELISA筛选烟曲霉特异性结合的克隆。结果 构建的scFv噬菌体表达文库,库容量约为1.8×10⁷;经4轮富集筛选,洗脱的表达scFv噬菌体滴度渐增;随机挑选出4轮筛选后的20个克隆,其可变区基因序列不同;随机挑选出4轮筛选后的90个克隆,其中6个克隆与烟曲霉GM特异性结合,而与对照的白假丝酵母菌无结合。结论 构建并初步鉴定了抗曲霉菌单链抗体库,库容量大,VH和VL区具备多样性;筛选的scFv与曲霉菌GM特异性结合,提供了快速制备、筛选GM高特异性抗体分子的新方法。     

乙肝病毒核心抗原人源单链可变区抗体的筛选

目的　筛选、鉴定乙型肝炎病毒 (HBV)核心抗原 (HBcAg)蛋白的人源单链可变区抗体 (ScFv)的编码基因 ,为细胞内表达小分子单链抗体的研究及抗HBV的基因治疗研究奠定基础。方法　采用噬菌体表面展示技术 ,以HBcAg蛋白为固相抗原 ,从噬菌体单链可变区抗体半合成库中经过 5轮“吸附 -洗脱 -扩增”筛选过程 ,获得抗原结合活性较强的HBcAg人源单链可变区抗体阳性克隆 ,并对其进行免疫检测及序列测定。结果　筛选得到的ScFv片段编码基因为 771nt,编码的产物由 2 5 7个氨基酸残基组成 ,具有典型的轻链和重链可变区结构特点以及与HBcAg结合的特异性。结论　利用噬菌体抗体库技术成功地获得了HBcAg人源单链可变区抗体的编码基因 ,并获得了可溶性单链抗体的表达。     

一株抗腮腺炎病毒Fab抗体克隆的筛选

[目的]获得抗腮腺炎病毒Fab段基因工程抗体。[方法]将腮腺炎病毒抗原（MUV-Ag）包被在固相ELISA板中,采用＂吸附-洗脱-扩增＂的方法,对已构建的抗腮腺炎病毒噬菌体抗体库进行富集。从富集后的抗体库中筛选抗腮腺炎病毒阳性克隆,并交叉筛选鉴定其特异性。[结果]在对176个克隆筛选后,再用麻疹病毒抗原（MEV-Ag）、混合副流感病毒抗原（mPIV-Ag）和呼吸道合胞病毒抗原（RSV-Ag）进行交叉筛选,最终获得特异性抗MUV-Ag阳性克隆2个（B206和D210）。对其中1株B206序列分析表明它的κ轻链V基因属于VκⅠ（O12）亚群,J基因属于Jκ1;γ链V基因属于VH1-69亚群,D基因来自D3-10,J基因则来自JH6。[结论]用腮腺炎病毒抗原成功地从抗体库中筛选到特异性阳性克隆,构建的噬菌体抗体库是有效的。     

天然人源性单链抗体文库的构建及鉴定

目的构建天然人源性单链抗体(scFv)文库,并对文库质量进行鉴定。方法采集220份未经主动免疫健康人外周血,分离单个核细胞后提取总RNA,逆转录成cDNA,混匀,以此为模板PCR扩增人抗体重链可变区(VH)和轻链可变区(VL,包括Vκ和Vλ),再经过overlap-PCR法,将VH基因和VL基因随机拼接成人scFv基因文库后,插入噬菌体载体pComb3XSS中,电转化大肠杆菌XL1-Blue制备scFv文库。通过菌落数量计算库容,并随机挑选100个单菌落验证scFv基因阳性克隆率,基因测序分析scFv文库基因的多样性。结果经过1次电转化得到容量为1.8×108的scFv文库,scFv基因阳性克隆率为96%,其中78%为正确插入,其scFv基因序列均不相同。结论成功构建了一种大容量、多样性高的天然人源性scFv文库,为进一步筛选特异性中和抗体提供了基础材料。     

噬菌体抗体库技术的发展及未来

噬菌体表面展示技术是近年发展起来的 ,将外源蛋白基因与噬菌体表面特定蛋白基因在其表面融合表达的新技术。应用PCR技术从生物体内扩增出整套抗体轻、重链可变区基因 ,克隆到λ噬菌体表达系统 ,构建噬菌体抗体库 ,通过抗原亲和吸附 -洗脱 -增殖 ,从中筛选富集多种目的克隆 ,制备相应单克隆抗体。     

全人源抗鼻咽癌噬菌体单链抗体的筛选与鉴定

目的从全人源抗鼻咽癌噬菌体抗体库中筛选特异性单链抗体（ScFv）,并对其特异性进行鉴定。方法通过噬菌体表面展示技术把ScFv表达在噬菌体表面,以鼻咽癌细胞作为抗原,用抗原递减法,通过“吸附-洗脱-扩增”过程筛选并富集特异性抗体,及ELISA筛选,获得特异阳性克隆进行免疫组化鉴定并测序。结果通过对抗体库进行三轮正负淘洗和富集后,随机挑选4212个克隆进行ELISA,发现3个克隆对CNE2呈强阳性反应,而与人正常细胞系HUVEC等呈弱阳性反应或不反应。对克隆HNSA033进一步进行免疫细胞化学验证,结果与ELISA反应一致;免疫组织化学鉴定表明克隆HNSA033与鼻咽癌组织和鼻咽组织阳性率的差别有统计学意义。结论通过淘选富集、ELISA和免疫化学鉴定获得特异性较强的噬菌体克隆,为鼻咽癌发病机制的研究和临床诊断以及治疗奠定了基础。     

抗氯菊酯人源scFv抗体筛选与鉴定

目的 筛选氯菊酯人源单链可变区（scFv）抗体,为研究快速检测试剂盒奠定基础.方法 采用噬菌体表面展示技术,以氯菊酯作为抗原,固相包被于Nunc板,应用半合成的人源单链可变区抗体文库技术,从噬菌体单链可变区抗体库中经过3轮＂吸附-洗脱-扩增＂筛选过程,随机挑选抗氯菊酯的100个克隆,利用酶联免疫吸附法（ELISA）、交叉反应及竞争抑制实验,对其进行免疫学检测和鉴定,获得与氯菊酯结合活性较强的scFv阳性克隆,从噬菌体抗体阳性克隆中提取质粒,经酶切鉴定Sfi Ⅰ/Not Ⅰ后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行酶切鉴定分析.结果 经过筛选100个克隆中有18株克隆ELISA的吸光度（A值）-490 nm波长（A490nm）值较高,将这些噬菌体上清与牛血清白蛋白（BSA）进行交叉反应,确定有5株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.亚克隆到pCANTAB5E载体;转化感受态细胞XL1-Blue.结论 提取质粒酶切片段与目的相符;为下一步其特异性亲和力的研究创造了条件.     

New approach for generation of neutralizing antibody against human T-cell leukaemia virus type-I (HTLV-I) using phage clones

We have screened a phage peptide library to address whether clones binding to a monoclonal antibody (mAb) could be isolated and if the selected phage particles would be able to elicit an in vivo immune response against the original antigen. A phage peptide library, consisting of seven random amino acids inserted in the minor coat protein (pIII), was screened for specific binding to a rat mAb LAT-27, which is capable of neutralizing human T-cell leukaemia virus type-I (HTLV-I) by binding to its envelope gp46 epitope, (amino acids LPHSNL). Total 37 clones were selected from the library and one clone named 4-2-22 was tested for its immunogenicity in three rabbits. The all rabbit immune sera showed strong binding activity to a gp46 peptide carrying the neutralization sequence, stained gp46-expressing cells and neutralized HTLV-I in vitro as determined by cell fusion inhibition assay. These results show that the selected phage clone was capable of mimicking the epitope recognized by a HTLV-I neutralizing mAb, and it can be used as an immunogen to induce protective immune response against HTLV-I. Thus, the present methodology could be one of the approaches to develop vaccines against infectious agents in a simple and inexpensive way.     

Engineering filamentous phage carriers to improve focusing of antibody responses against peptides

The filamentous bacteriophage are highly immunogenic particles that can be used as carrier proteins for peptides and presumably other haptens and antigens. Our previous work demonstrated that the antibody response was better focused against a synthetic peptide if it was conjugated to phage as compared to the classical carrier, ovalbumin. We speculated that this was due, in part, to the relatively low surface complexity of the phage. Here, we further investigate the phage as an immunogenic carrier, and the effect reducing its surface complexity has on the antibody response against peptides that are either displayed as recombinant fusions to the phage coat or are chemically conjugated to it. Immunodominant regions of the minor coat protein, pIII, were removed from the phage surface by excising its N1 and N2 domains (Δ3 phage variant), whereas immunodominant epitopes of the major coat protein, pVIII, were altered by reducing the charge of its surface-exposed N-terminal residues (Δ8 phage variant). Immunization of mice revealed that the Δ3 variant was less immunogenic than wild-type (WT) phage, whereas the Δ8 variant was more immunogenic. The immunogenicity of two different peptides was tested in the context of the WT and Δ3 phage in two different forms: (i) as recombinant peptides fused to pVIII, and (ii) as synthetic peptides conjugated to the phage surface. One peptide (MD10) in its recombinant form produced a stronger anti-peptide antibody response fused to the WT carrier compared to the Δ3 phage carrier, and did not elicit a detectable anti-peptide response in its synthetic form conjugated to either phage carrier. This trend was reversed for a different peptide (4E10_L), which did not produce a detectable anti-peptide antibody response as a recombinant fusion; yet, as a chemical conjugate to Δ3 phage, but not WT phage, it elicited a highly focused anti-peptide antibody response that exceeded the anti-carrier response by ∼65-fold. The results suggest that focusing of the antibody response against synthetic peptides can be improved by decreasing the antigenic complexity of the phage surface.     

Screening blood donors for human immunodeficiency virus antibody: cost-benefit analysis.

The costs and benefits of screening blood donors for antibody to human immunodeficiency virus (HIV) are assessed. Total costs, including testing, discarding processed blood, marginal donor recruiting, notifying and evaluating positive donors, are $36,234,000 annually for 10 million donors in 1986. Screening these donors will prevent 292 cases of transfusion-transmitted acquired immune deficiency syndrome (TT-AIDS), saving the costs of therapy and loss of earnings for total benefits of $43,490,480, a benefit:cost ratio of 1.2:1. Net economic benefits of $0.73 per donor will arise from the program. Calculated benefits will rise as increased numbers of infected recipients are diagnosed with longer follow-up or as partially effective therapy increases the cost of caring for patients with AIDS. Changes in test sensitivity, follow-up procedures, estimated value of life, and testing costs will also alter these projections, but none as dramatically as a change in the overall specificity of the screening process. The cost per case of TT-AIDS prevented, $124,089, and cost per year of life extended, $10,885, are comparable to costs of other screening programs.     

噬菌体抗体库技术及应用研究进展

噬菌体抗体库是近年发展起来的一项分子生物学新技术。构建容量大、特异性高和敏感性强的人源性抗体是此项技术的核心,也是其远大前景的基础。噬菌体抗体库的建立对生物学领域许多技术的进展起到很大的促进作用,本文对此项技术的进展情况作一综述     

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.     

以多表位融合抗原检测结核分枝杆菌抗体、TB-SA抗体检测和噬菌体裂解法快速检测在结核病诊断中应用价值研究

目的:评价以融合抗原检测结核分枝杆菌抗体、TB-SA抗体检测和噬菌体裂解法快速检测在结核病诊断中应用价值。方法:选择结核组病例134例与非结核组265例按说明书方法进行检测。结果:噬菌体裂解法灵敏度72.39%,特异度91.7%;TB-SA抗体灵敏度75.37%,特异度77.7%;融合抗原检测抗体灵敏度85.8%,特异度92.1%。结论:融合抗原检测抗体与噬菌体裂解法和TB-SA抗体试验相比敏感度更高,值得在各级医疗单位使用。