应用RAD多肽展示体系制备抗hCG类抗体分子

应用基于激烈火球菌Pyrococcus furiosus重组酶RadA的ATP酶结构域(RAD骨架)的多肽展示体系,通过嫁接人绒毛膜促性腺激素(hCG)结合多肽,制备抗hCG类抗体分子。通过合成hCG结合多肽插入RAD多肽展示位点的类抗体基因,成功构建了pET30a-RAD/hCGBP-sfGFP原核表达载体,在大肠杆菌中诱导蛋白表达,分离、纯化获得类抗体蛋白,通过亲和吸附-GFP荧光检测方法测定类抗体对hCG的结合活性,并与应用单域抗体通用骨架制备的嫁接抗体比较活性差异。结果显示,RAD类抗体分子对hCG分子具有较高的亲和性和特异性,显著优于单域嫁接抗体,并与商业单克隆抗体的活性相当;同时,利用RAD多肽展示骨架制备的抗hCG类抗体,具有较高的生化稳定性,是一种具有应用潜力的抗体替代分子。     

结核分枝杆菌Rv0733 6His抗原羊驼单域重链抗体的筛选与鉴定

单域重链抗体是目前中和胞内病原体抗原的重要分子之一,研究以结核分枝杆菌Rv0733-6His融合抗原为靶标,对羊驼非免疫单域重链抗体库进行了3轮淘洗,通过ELISA和测序方法,从1024个克隆中筛选出10个独立单域重链抗体序列,继而用原核表达并鉴定了1株Rv0733-VHH-Fe-6His抗体。免疫印迹和免疫荧光结果均显示Rv0733-VHH—Fe-6His抗体可以特异性地结合Rv0733抗原。提示Rv0733-VHH抗体可能具备结合胞内菌相关抗原的潜力。     

抗脂多糖纳米抗体的筛选及其五聚体的制备

目的：筛选抗脂多糖（LPS）纳米单域抗体,并制备抗LPS纳米抗体五聚体。方法：以LPS为抗原,从驼源天然单域重链抗体库中筛选抗LPS纳米抗体,利用分子克隆技术将抗LPS单域抗体基因组装入志贺杆菌样毒素B亚基蛋白结构域（VTB）的五聚体特异性表达载体中进行可溶性表达,并用ELISA法鉴定所获抗体的抗原结合活性和特异性。结果：获得抗LPS纳米单域抗体及LPS纳米抗体五聚体;经鉴定,LPS纳米抗体五聚体的抗原结合活性优于抗LPS单域抗体。结论：利用驼源天然单域重链抗体库制备了抗LPS纳米单域抗体及抗LPS纳米抗体五聚体,为脓毒血症的分子诊断、预后判断及寻找生物治疗新靶点奠定了基础。     

EGFRvⅢ胞外区基因重组腺病毒的构建及单域抗体的制备北大核心CSCD

本研究旨在构建能够表达人表皮生长因子EGFRvⅢ胞外区基因的重组腺病毒,并通过免疫骆驼构建噬菌体单域抗体库,筛选和制备EGFRvⅢ胞外区特异性单域抗体并对其进行鉴定。从人前列腺癌细胞系PC-3细胞中提取总RNA,反转录为cDNA,以cDNA为模板扩增EGFRvⅢ胞外区基因,并连接pAdTrack-CMV质粒载体,转化含有pAdEasy-1的大肠杆菌(Escherichia coli)BJ5183感受态细胞,获得同源重组质粒转染HEK293A细胞,得到表达EGFRvⅢ胞外区蛋白的重组腺病毒。利用重组腺病毒免疫双峰驼,构建EGFRvⅢ胞外区特异性噬菌体单域抗体库,并以EGFRvⅢ蛋白为筛选抗原,对其进行筛选,对筛选得到的单域抗体进行诱导表达、纯化及鉴定。结果表明获得了表达EGFRvⅢ胞外区基因的重组腺病毒。构建得到的EGFRvⅢ特异性噬菌体单域抗体库的库容为1.4×109;经过3轮富集和筛选,通过噬菌体ELISA筛选出31个与EGFRvⅢ胞外区蛋白结合的阳性克隆,并对OD450值较高的重组单域抗体E14进行了表达和纯化。经ELISA鉴定,重组单域抗体E14可与EGFRvⅢ胞外区蛋白产生抗原抗体结合反应,具有较高的亲和力。说明制备的EGFRvⅢ特异性噬菌体单域抗体库具有较高的库容和多样性,且筛选得到的单域抗体具有较高的抗原活性和免疫学反应性,为今后以EGFRvⅢ为靶点的恶性肿瘤的诊断和治疗提供新的实验依据。     

基于纳米抗体CDR3区抗原表位展示制备生存素抗体及其鉴定

目的:通过纳米抗体CDR3区展示生存素N端表位的方式,探索纳米抗体在抗原表位展示中的作用。方法:通过基因合成方法将生存素N端起始表位(氨基酸序列1~15)插入纳米抗体CDR3区,再构建到原核表达载体pET24a中,IPTG诱导表达,用带His标签的填料纯化,获得高纯度的目的蛋白,免疫雌性BALB/c小鼠,间接ELISA检测5次免疫后的效价,用抗原偶联纯化介质纯化免疫多抗,Western印迹检测多抗特异性。结果:IPTG诱导后,目的蛋白主要以包涵体形式存在,亲和层析获得纯度大于96%的目的蛋白,包涵体经复性后免疫小鼠,效价可达1∶512000,West?ern印迹特异性检测显示免疫多抗能够特异性结合生存素。结论:纳米抗体CDR3区生存素抗原N端表位展示的方法可用于抗生存素抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。     

OGT 多克隆抗体的制备及特异性分析

目的:为了探讨O-GlcNAc糖基转移酶OGT的生理和病理作用,需制备能高效特异性检测OGT的抗体。方法:在NCBI数据库中,查找人源OGT基因序列,根据OGT的结构特点,选取OGT的C末端催化结构域中的一段多肽序列(464-949位点氨基酸)做抗原。首先,构建OGT的C末端催化结构域(464-949位点氨基酸)的重组表达载体pET30-a-OGT-C,转化至大肠杆菌BL21(DE3)感受态细胞中,IPTG诱导表达融合His标签的OGT-C蛋白,Ni+珠亲和层析法纯化提取OGT-C蛋白。再以OGT-C重组蛋白作为抗原,免疫Wistar大鼠制备多克隆抗体,并用间接ELISA法检测OGT抗体的效价,Western blotting鉴定抗体特异性。结果:多抗效价达1:80000;在免疫印迹实验中,此多抗可以高效的检测重组抗原,并可以特异性识别培养细胞内源表达的ncOGT和mOGT这2种OGT亚型。结论:实验结果表明,获得高效价、高特异性的OGT多克隆抗体,在OGT的生物学研究中可以用于检测ncOGT和mOGT的表达。     

重组骆驼源单域抗体的原核表达及纯化

目的:原核表达重组骆驼源单域抗体并纯化。方法:将雌二醇特异性的单域抗体基因片段克隆到pET32a载体中,经IPTG诱导表达后对形成的包涵体进行纯化,利用间接ELISA法检测获得的单域抗体的活性,并以孕酮、雌酮和雌三醇为对照,鉴定雌二醇单域抗体的特异性。结果:重组雌二醇单域抗体的相对分子质量约为34×103,通过大肠杆菌BL21(DE3)-p ET32a构建的原核表达体系实现了雌二醇单域抗体的高水平表达,并通过包涵体纯化获得了可溶性的高纯度单域抗体抗体,经间接ELISA检测,该雌二醇特异性抗体的50%抑制浓度(IC50)为20.03 ng/mL,对孕酮和雌酮2种化合物的交叉反应率分别为29.78%和1.63%,特异性较好。结论:通过构建原核表达体系,可以获得功能性的骆驼源单域抗体。     

单域抗体研究进展

近年来用基因工程方法从软骨鱼和骆驼科动物中克隆到的单域抗体（single—domain antibody,sdAb）具有无轻链、单一重链可变区保留了完整的抗原结合活性的特征。这类单域抗体具有分子小、稳定性高、体内组织渗透性好、可溶性好、易表达、抗原识别表位独特的特性,已引起生物技术研究与诊断治疗应用领域的广泛关注,取得了快速发展。综述了这类单域抗体发展历史、分子类别、结构特征、理化特征、分子演化及应用前景。     

抗CD133胞外区骆驼多克隆抗体的制备及鉴定

目的:制备高效价、高特异性的抗CD133胞外区新疆双峰驼来源的多克隆抗体,为制备高亲和力的抗CD133纳米抗体做准备。方法:将CD133胞外区基因序列构建到原核表达载体pET28a中,诱导表达及纯化CD133蛋白,免疫新疆双峰驼及新西兰兔。通过酶联免疫吸附实验(ELISA)和Western blot检测多克隆抗体的效价及与CD133特异性结合活性。结果:ELISA测定抗-CD133骆驼源抗体的效价可达到百万以上,通过Western blot检测多克隆抗体可特异性结合CD133蛋白。结论:重组人CD133蛋白可以在骆驼体内激发高滴度抗体反应,为今后构建骆驼免疫单域抗体噬菌体展示文库奠定基础。     

抗人CD3单链抗体与改形单域抗体的表达

设计并化学合成含有适当酶切位点及连接肽的寡核苷酸序列,与一定的背景载体连接并改造成适用于单链抗体表达的载体:外分泌型pWAI80和融合蛋白型pROH80从分泌抗人CD3单克隆抗体的杂交瘤细胞UCHT1中,经PCR扩增出轻、重链可变区基因V_H和V_K,并插入上述表达载体中构建成单链抗体基因.通过对鼠OKT3结合位点的结构模拟,并比较人、鼠抗体家族性保守序列,设计出改形OKT3的基因序列.化学法部分合成8个寡核苷酸片段,应用重叠PCR技术扩增出完整改形重链基因V_H,并克隆、酶切和测序鉴定.将所克隆V_H基因插入表达载体pCOMB3和 pGEX-4T-1中进行表达.经 IPTG诱导表达,对表达产物进行SDS-PAGE和 Western blot分析以及 ELISA检测,结果发现分泌型表达产物及 M13基因Ⅲ-V_H改形单域抗体融合蛋白具有与CD3单抗竞争抑制的活性;而融合型单链抗体及改形单域抗体表达产物主要以包涵体形式存在,占细菌总蛋白的 30％左右.     

VH-VL orientation prediction for antibody humanization candidate selection: A case study

Antibody humanization describes the procedure of grafting a non-human antibody's complementarity-determining regions, i.e., the variable loop regions that mediate specific interactions with the antigen, onto a β-sheet framework that is representative of the human variable region germline repertoire, thus reducing the number of potentially antigenic epitopes that might trigger an anti-antibody response. The selection criterion for the so-called acceptor frameworks (one for the heavy and one for the light chain variable region) is traditionally based on sequence similarity. Here, we propose a novel approach that selects acceptor frameworks such that the relative orientation of the 2 variable domains in 3D space, and thereby the geometry of the antigen-binding site, is conserved throughout the process of humanization. The methodology relies on a machine learning-based predictor of antibody variable domain orientation that has recently been shown to improve the quality of antibody homology models. Using data from 3 humanization campaigns, we demonstrate that preselecting humanization variants based on the predicted difference in variable domain orientation with regard to the original antibody leads to subsets of variants with a significant improvement in binding affinity.     

Preparation and immunological features of syngeneic monoclonal anti-idiotypic antibody using complementary determining region (CDR) peptide as the immunogen

A syngeneic monoclonal idiotypic antibody was prepared by immunizing the sequence peptide of complementary determining region-1 (CDRL-1) of 41S-2-L which is an antibody light chain capable of catalytically decomposing the antigen peptide (gp41 peptide:original antigen) as well as the intact gp41 molecule of HIV-1 envelope. The obtained idiotypic antibody, i41SL1-2, showed a high specificity to the CDRL-1 peptide. The intact i41SL1-2 and its heavy and light chains displayed apparent affinity constants to the CDRL-1 peptide of 3.6 × 10⁹, 2.7 × 10⁷, 1.8 × 10⁶/M, respectively. The i41SL1-2 recognized the artificial molecule CA2, which has a more complex steric conformation than the CDRL-1, while the i41SL1-2 showed very low affinity to the original monoclonal antibody 41S-2 and its light chain 41S-2-L. However, a homologous sequence, EGG-D, with the gp41 peptide was expressed in the complementary determining region-3 (CDRH-3) of the heavy chain of i41SL1-2. Furthermore, the consensus sequence EGG was located at the important position of the CDRH-3 loop of i41SL1-2. Although the sequence of CDRL-1 (16 mer) is quite shorter than that of whole light chain (112 mer), the CDRL-1 could induce the rearrangement of CDRH-3 gene of i41SL1-2 so as to express a homologous sequence with the original antigen.     

Switching antibody specificity through minimal mutation

Antibody 1E9, which was elicited with a hexachloronorbornene derivative and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, was successfully reengineered to bind a range of structurally diverse steroids with nanomolar affinities. Remarkably, two mutations (Leu^H47Trp/Arg^H100Trp) out of 36 total sequence differences suffice to switch the selectivity of 1E9 to that of the progesterone-binding antibody DB3. In contrast to the double mutant, which tightly binds multiple steroids with differently configured A-B ring junctions, the individual Leu^H47Trp and Arg^H100Trp single mutants both exhibit significantly greater specificity than DB3, preferentially binding 5α-pregnan-3β-ol-20-one (K_d ≈ 5 nM) over other steroids. These findings illustrate how easily differently shaped binding pockets can be created through subtle changes to the same primordial germ line template.     

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.     

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.     

Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.     

The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.     

Beta-edge interactions in a pentadecameric human antibody V kappa domain

Antibodies are the archetypal molecules of the Ig-fold superfamily. Their highly conserved beta-sheet architecture has evolved to avoid aggregation by protecting edge strands. However, the crystal structure of a human V kappa domain described here, reveals an exposed beta-edge strand which mediates assembly of a helical pentadecameric oligomer. This edge strand is highly conserved in V kappa domains but is both shortened and capped by the use of two sequential trans-proline residues in V lambda domains. We suggest that the exposure of this beta-edge in V kappa domains may explain why light-chain deposition disease is mediated predominantly by kappa antibodies.     

Modeling the anti-CEA antibody combining site by homology and conformational search.

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structures with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available to the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures. Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed.     

Improving the solubility of anti‐LINGO‐1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti‐LINGO‐1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.