Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

重组鼠疫菌F1抗原在大肠杆菌中的表达及免疫原性分析

利用基因重组技术,用pET42(b+)质粒在大肠杆菌DE3中表达鼠疫菌F1抗原。经分析rF1抗原基因序列与天然F1抗原结构基因序列完全一致,电泳扫描测其表达量为25%:W estern B lot结果表明,rF1抗原可与F1特异性抗体相互作用,具有天然F1抗原的活性。用镍离子亲和层析纯化rF1抗原免疫BALB/c小鼠,在其血清中可检测到高滴度的抗F1抗体。     

结核杆菌抗原HSP65基因在大肠杆菌中的表达及纯化

目的 :为研制预防结核病疫苗 ,选取结核杆菌HSP65蛋白为免疫抗原 ,将结核分枝杆菌HSP65抗原基因在大肠杆菌中表达、纯化。方法 :将HSP65的全长cDNA插入到原核表达载体pGEX5T中 ,构建成pGEX5T HSP65重组质粒。将质粒转化到E .coli.K80 2细菌 ,用IPTG诱导HSP65表达 ,然后用亲和层析的方法进行纯化 ,最后用Western blot方法确认表达蛋白的特异性。结果 :获得了pGEX5T HSP65重组子 ,HSP65蛋白在k80 2菌中获得了表达 ,表达的蛋白条带大小约 86kDa ,与预期的结果相符。表达产物经亲和层析后获得了较单一的蛋白条带 ;表达及纯化的蛋白在纯化前后均可被结核病患者血清特异地识别。为进一步研究其在结核病诊断和防治中的应用打下了基础。     

The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties

We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.     

重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段,直接克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定后,再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间,构成重组质粒ｐＫＧ－ｈＦＡＳ,将此质粒导入大肠杆菌,经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达,用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白,经凝血酶酶切和二次亲合层析去除ＧＳＴ部分,得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体,经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡     

白喉毒素A片段的表达纯化与单克隆抗体制备

白喉毒素 (Diphtheriatoxin ,DT)A片段 (DTA)是白喉毒素的酶活性区 ,也是DT类免疫毒素的关键结构域。DTA蛋白及其单克隆抗体在免疫毒素的毒性机理、检测与纯化研究等方面具有重要价值。通过在E .coli中表达了DTA ,经Q SepharoseFF和Ni²⁺ Sepharose两步层析纯化 ,得到纯度约为 90 %的融合蛋白。以DTA为抗原免疫BalB c小鼠 ,获得了分泌抗DTA特异单抗的杂交瘤细胞株 3B6和 3B9。单抗为IgG1亚型 ,滴度达 1∶10⁶ 以上 ,与DTA的结合可被抗DT马血清竞争抑制。抗DTA单抗用于免疫印迹试验 ,或制备成免疫亲和柱纯化基于DT的重组免疫毒素 ,均获得较好效果 ,为免疫毒素的研究奠定了良好基础     

猪流感病毒抗原表位基因表达载体构建与免疫原性分析

根据猪流感病毒血凝素蛋白基因(Heamuglutinine, HA)的核苷酸序列, 设计、筛选HA蛋白氨基酸序列的主要表位多肽4个, 将4个片段以柔性连接串联成模拟蛋白, 核苷酸约为300 bp, 体外扩增该模拟蛋白基因, 插入到原核表达载体pET30a(+)中, 转染宿主菌诱导表达, 结果获得分子量为20 kD的表达蛋白, 该蛋白可与抗His-tag抗体、抗猪流感病毒H1N1、H3N2亚型高免血清发生免疫学反应。纯化后免疫小鼠, ELISA及血凝抑制(Heamuglutinine inhibitor, HI)试验检测, 小鼠产生针对多肽抗原的血清抗体, 同时还可检测到H1N1、H3N2亚型SIV血凝抗体。流氏细胞仪检测免疫组外周血淋巴细胞高于对照组, 说明该模拟蛋白具有与H1N1、H3N2亚型猪流感病毒相似的免疫原性及反应原性, 为H1N1、H3N2血清亚型猪流感病毒疫苗研制提供了新手段。     

猪流感病毒抗原表位基因表达载体构建与免疫原性分析

根据猪流感病毒血凝素蛋白基因(Heamuglutinine, HA)的核苷酸序列, 设计、筛选HA蛋白氨基酸序列的主要表位多肽4个, 将4个片段以柔性连接串联成模拟蛋白, 核苷酸约为300 bp, 体外扩增该模拟蛋白基因, 插入到原核表达载体pET30a(+)中, 转染宿主菌诱导表达, 结果获得分子量为20 kD的表达蛋白, 该蛋白可与抗His-tag抗体、抗猪流感病毒H1N1、H3N2亚型高免血清发生免疫学反应。纯化后免疫小鼠, ELISA及血凝抑制(Heamuglutinine inhibitor, HI)试验检测, 小鼠产生针对多肽抗原的血清抗体, 同时还可检测到H1N1、H3N2亚型SIV血凝抗体。流氏细胞仪检测免疫组外周血淋巴细胞高于对照组, 说明该模拟蛋白具有与H1N1、H3N2亚型猪流感病毒相似的免疫原性及反应原性, 为H1N1、H3N2血清亚型猪流感病毒疫苗研制提供了新手段。     

应用细菌鞭毛递呈的随机肽库研究丙型肝炎病毒核心蛋白B细胞抗原表位

设计了利用大扬杆菌鞭毛蛋白递呈的随机十二肽库研究ＨＣＶ核心蛋白Ｂ细胞抗原位的实验程序：１利用大肠杆有达质粒ｐＱＥ－３０有达并纯化ＨＣＶ核心蛋白Ｐ１９;２利用Ｐ１９蛋白亲和层析纯化ＨＣＶ感染者血清的抗ＨＣＶ核心蛋白多克隆抗体;     

单核增生李斯特菌Internalin A的克隆表达与抗体制备

目的克隆表达单核增生李斯特菌Internalin A（InlA）,并以之为抗原制备检测单核增生李斯特菌的抗体。方法通过PCR技术从单核细胞增生李斯特菌4b中扩增出inlA基因,克隆筛选和测序鉴定后,最终构建该基因的原核表达质粒pGEX-4T-InlA,谷胱甘肽树脂亲和层析纯化表达产物后,免疫小鼠分别制备相应的多抗和单抗。结果在大肠埃希菌中成功表达了InlA,并对其进行了纯化,融合表达产物分子量约为110 kD;免疫小鼠获得的抗血清效价达到1∶1600;得到了3株抗InlA的单克隆抗体杂交瘤细胞株,腹水单抗效价为1∶1×10^5-1∶3×10^5。2种抗体与其他病原菌均无交叉反应。结论通过表达单核增生李斯特菌的特异性蛋白制备的抗体,能有效地消除交叉反应,提高检测的特异性。     

Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3’s high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3’s stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3’s framework and where A3’s CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3’s binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability.     

炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

EB病毒早期蛋白P54的原核表达及其免疫原性的研究

PCR法获得编码EB病毒早期蛋白P54的基因BMRFl,序列分析后亚克隆入原核表达载体pET30a。表达质粒pET30a-BMRF1在大肠杆菌BL21(DE3)菌株中经IPTG诱导后表达了P54抗原,SDS—PAGE表明其相对分子质量为51000;采用镍离子亲和柱纯化重组蛋白。Western印迹结果表明纯化蛋白免疫BALB／c小鼠后产生了P54特异性抗体。间接免疫荧光表明免疫血清可以识别激活的Raji细胞中表达的P54蛋白。以上结果表明构建了原核表达质粒pET30a-BMRF1并在大肠杆菌细胞中成功表达EB病毒早期蛋白P54,表达蛋白具有很好的抗原性和免疫原性。     

HIV-1p24蛋白在大肠肝菌中的高效可溶性表达、一步纯化及抗原性分析

合成引物扩增HIV 1p2 4基因 ,并将其克隆到 pQE 30质粒中 ,使其在大肠杆菌E .coliM 15中以IPTG诱导高效表达 ,经SDS PAGE分析 ,该表达产物约占菌体总蛋白 2 0 % ,并且以可溶蛋白的形式存在于细菌裂解液上清之中。经镍离子柱亲和层析一步纯化 ,洗脱产物中 p2 4蛋白纯度达95 %。ELISA分析表明 ,该蛋白可与HIV感染者血清发生特异性免疫反应。以此蛋白交联Sepharose 4B ,亲和层析纯化HIV感染者血清中的抗体 ,用所得抗体与HIV确认试剂反应 ,发现该纯化抗体仅与确认试剂中的 p2 4蛋白反应。上述结果表明在大肠杆菌中已经高效表达了可溶性HIV 1p2 4蛋白 ,该蛋白具有良好的抗原性     

Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

Expression, purification and refolding of the phosphatase domain of protein phosphatase 1 (Ppt1) from Saccharomyces cerevisiae

Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein.     

人免疫缺陷病毒早期蛋白Nef的表达、纯化及免疫原性研究

目的：通过原核细胞表达人免疫缺陷病毒（HIV）Nef抗原,制备特异抗血清,为Nef抗原检测提供技术方法。方法：以HIVBotswana毒株基因组为模板,用PCR法获得Nef蛋白编码基因,将其克隆到pET30a载体中,在大肠杆菌中表达Nef融合蛋白;用纯化的融合蛋白免疫BALB／c小鼠获得抗血清,用真核表达的Nef抗原对其特异性进行分析。结果：构建的Nef融合基因在大肠杆菌中获得表达,相对分子质量约为36x103,免疫BALB／c小鼠获得针对融合蛋白的高效价抗血清,ELISA抗体滴度为1：6400;免疫荧光和Westemblot检测表明,该抗血清能特异地与重组痘苗病毒表达的Nef抗原反应。结论：在大肠杆菌中表达了HIVNef融合蛋白,制备了Nef融合蛋白的高效价小鼠免疫血清,该血清能特异性识别HIVNef抗原,为HlVNef抗原检测提供了技术方法。     

乙肝病毒前S1抗原突变体在大肠杆菌中的可溶性表达及鉴定

乙肝病毒前S1抗原含多个免疫优势表位及乙肝病毒肝细胞受体结合位点,具有重要的生物学功能。为使其高效、可溶性表达,在DnaStar软件辅助分析下,将前S1基因5′端复杂二级结构突变后,克隆入原核表达载体pQE-30a,转化大肠杆菌M15感受态细胞,经IPTG诱导后,获得了高水平、可溶性表达;并对其进行了纯化和鉴定,为进一步研究乙肝病毒前S1抗原的结构功能特点奠定了基础。     

人偏肺病毒融合蛋白F的原核表达及初步应用

为了探讨人偏肺病毒(hMPV)融合蛋白(F)在原核表达系统中的表达效果及其应用价值,用大肠杆菌表达系统表达hMPV F1亚单位蛋白并用镍柱(Ni-NTA)进行亲和层析纯化,并以其为抗原,用Western Blot方法进行人血清抗体检测的探索。根据F蛋白的疏水性、抗原位点和表面可及性的预测结果选择F1亚单位为目标区域,在大肠杆菌BL21中分别得到了带有6个组氨酸(His)标记的不同基因型hMPV的F1亚单位蛋白的高效表达,目的蛋白大小约37.0 kD,主要以包涵体形式存在。Western Blot检测显示表达的目的蛋白可以特异性地结合抗hMPV的豚鼠多克隆抗体以及确定为hMPV急性感染患者的血清,而且与副粘病毒科肺病毒属的呼吸道合胞病毒(RSV)和副流感病毒(PIV)(2和3型)之间没有交叉反应,显示表达的F1蛋白的特异性和抗原性良好。应用该表达蛋白进行部分人群血清抗体检测的探索,结果显示人群血清抗hMPV-F蛋白的IgG抗体总阳性率约为66%~67%,0~2岁组的血清抗体阳性率随年龄增长呈现逐渐下降趋势,新生儿的抗体阳性率最高,达85%左右,>1岁~2岁组幼儿阳性率最低,提示母传抗体的存在。随后各年龄组人群随年龄的增长血清抗体阳性率逐渐增高,>60岁年龄组人群抗体水平最高,上述结果提示<2岁儿童为hMPV的易感人群。