噬菌体展示 protein A 及 protein L Ig 结合单结构域随机组合文库及 Ig 亲和筛选

Protein A 和 protein L 是细菌产生的两种结构和功能均不同的免疫球蛋白 (immunoglobulin , Ig) 结合分子,在细菌的致病中起重要作用 . 用含 SacⅠ位点的特定引物 PCR 分别扩增制备 protein A 的 A 、 B 、 C 、 D 抗体结合结构域和 protein L 的 B3 抗体结合结构域,各结构域 DNA 片段经 SacⅠ酶切后,再随机连接形成各种不同长度的分子组合文库,将该文库呈现在噬菌体表面构建了噬菌体展示 Ig 结合分子单结构域随机组合文库,所建组合文库容量为 2.3×10⁶个菌落形成单位,滴度为 4.1×10¹¹TU/ml , 包含各种单结构域片段,并以随机方式连接 . 用人 Ig 对该文库进行 4 轮亲和筛选,随机挑选 36 个代表性的阳性克隆进行序列测定分析表明,亲和筛选获得了多种非天然形式存在的新的 Ig 结合分子结构,其中 32 个克隆具有由 protein L 的单结构域和 protein A 的单结构域间隔重复排列而成的特征性 (MDPL-MDPA)n 结构 . 对噬菌体展示 Ig 结合分子单结构域随机组合文库的体外分子进化研究的尝试,为 Ig 结合分子的结构和功能研究提供了一新的途径,也为 Ig 结合分子的定向改造打下基础 .     

新型蛋白质配体-亲和体研究进展

亲和体(affibody)是一种衍生于葡萄球菌A蛋白B结构域的人工蛋白质分子．B结构域含58个氨基酸,形成3个α螺旋结构,分子质量约为6.5 ku．其中第一及第二螺旋中的13个特定位点的氨基酸对其结构无明显影响,这些位点可被随机突变形成理论上可与任何靶分子结合的亲和体文库．筛选该文库可获得能与某一靶分子特异结合的亲和体．亲和体与靶分子的结合特性与抗体相似,但与抗体相比具有一些独特的优势,如：通过体外筛选即可获得,以化学合成方法或原核表达即可大量制备,分子质量小、在生物体内组织穿透性强、血浆清除率高,理化稳定性好,可以通过交联或融合表达与标记分子(如荧光蛋白、生物素等)结合而不影响其与靶分子的结合能力．亲和体可作为抗体的替代品,用于蛋白质识别、分离及纯化、实验诊断、分子显像及靶向治疗．     

金黄色葡萄球菌蛋白A (SpA) Z结构域串联体的克隆、表达和筛选

筛选一种高效重组金黄色葡萄球菌蛋白A(SpA)用于制备抗体纯化亲和介质。首先通过基因操作获得金黄色葡萄球菌蛋白A(SpA)的Z结构域单体、二串体、三串体、四串体和五串体基因,将目的基因分别克隆至pET-22b表达载体并转化至大肠杆菌BL21(DE3)感受态细胞,获得不同串联个数的Z结构域基因工程菌,经诱导表达和Ni2+亲和层析纯化得到Z结构域单体和二-五串体蛋白。纯化后的目的蛋白偶联至琼脂糖凝胶作为亲和层析介质,对人免疫球蛋白G(IgG)进行分离纯化。分析比较Z结构域串联体蛋白产量及其偶联的亲和介质对抗体吸附载量的差异。结果表明,构建的Z结构域单体、二串体、三串体、四串体和五串体基因工程菌能有效表达目的蛋白,制备的凝胶亲和介质可特异性吸附人IgG。增加Z结构域串联数,重组蛋白产量和单位摩尔数多聚体蛋白吸附载量获得提高,其中,重组四串体蛋白产量大(160 mg/10 g湿菌体),对抗体的吸附载量高(34.4 mg人IgG/mL胶),更适合作为配基用于亲和层析介质的制备。     

金黄色葡萄球菌A蛋白基因的克隆及序列分析

 我们构建了金黄色葡萄球菌Cowan1株(CMCC26111)的染色体文库,转化大肠杆菌后筛选出一株protein A的阳性克隆。SDS-PAGE及Western-blot结果显示该克隆株表达的重组protein A的分子量为30 000,较天然protein A的小。该克隆中的protein A基因片段的序列分析表明,它含有天然protein A基因的启动子、信号肽以及至少四个与IgGFc段结合的结构域,而不含天然protein A的胞壁结合区,并发现其中有24个碱基对与Uhlen报告的protein A基因不同,由此导致三个编码的氨基酸发生变化,但这些差异并不影响该重组protein A与IgG Fc段的特异结合。     

利用噬菌体展示文库筛选纤维素结合结构基元

从编码随机１５肽序列的噬菌体表面展示文库中,筛选得到了与纤维素有特异亲和能力的噬菌体克隆。序列分析结果表明,这些噬菌体克隆编码的氨基酸序列中芒香族氨基酸残基高度保守,与天然纤维素结合结构域的结构有一定的类似性。     

噬菌体展示文库及其应用

近十几年来,噬菌体展示技术得到了迅速的发展。通过展示随机肽库可用来筛选与特殊靶分子相结合的配基;模拟非蛋白的配基;也可用作确定抗体表位的工具。展示蛋白;或其功能结构的文库为我们提供了分析结构与功能关系的体系,并能产生具有改变结合位点或新的催化活性的蛋白。展示短的抗原决定簇的融合噬菌体为开发新的疫苗提供了基础,而表达抗体片段的文库则提供了一种产生单克隆抗体的方法。     

日本血吸虫未成熟卯单链抗体库的构建、筛选及初步应用

运用噬菌体展示技术构建日本血吸虫未成熟虫卵可溶性抗原（SIEA）单链抗体（scFv）表达文库,以天然分子候选疫苗SIEA26～28ku为靶抗原筛选SIEA单链抗体库,获得特异性单链抗体．并将该scFv基因亚克隆至原核高效表达载体PET32a,诱导SIEA26-28ku特异性scFv大量表达．随后以此为探针筛选日本血吸虫尾蚴cDNA文库,以期获得SIEA2628ku天然分子候选疫苗相关的编码基因．结果显示,所获得的SIEA26-28ku特异性scFv,表达量高,采用该探针初步筛选出相关基因核糖体蛋白S4．SIEA26-28ku特异性scFv的获得,为进一步筛选、分析鉴定抗日本血吸虫病天然分子候选疫苗SIEA26-28ku的编码基因奠定了基础．     

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。     

双峰驼源天然噬菌体纳米抗体展示库的构建及抗GDH纳米抗体筛选

目的:构建噬菌体天然纳米抗体展示库,以期用于筛选不同抗原分子的纳米抗体筛选平台,并用艰难梭菌谷氨酸脱氢酶(GDH)抗原筛选靶向GDH的纳米抗体,对所构建的噬菌体天然纳米抗体展示库进行验证。方法:采用Oligo DT提取双峰骆驼脾脏总RNA进行反转录,通过巢氏PCR获取全套重链可变区基因,将其构建到噬菌粒pCANTAB5E载体,经多次电转化至E. coil TG1构建初级噬菌体抗体库,经辅助噬菌体拯救后构成噬菌体展示库,并对噬菌体展示库的库容及多样性进行分析和鉴定。同时以GDH为靶向抗原对文库进行淘筛,计算淘筛回收率,并对第三轮淘筛后平板的单克隆进行ELISA鉴定。结果:构建的天然噬菌体纳米抗体库的插入率为95%左右,随机挑取的9个克隆氨基酸同源性为66. 17%,经MEGA分析后具有较好的多样性,同时经辅助噬菌体拯救后,得到的噬菌体展示库滴度为4×10~(12)CFU/ml。在三轮淘筛过程中,回收率逐步升高,噬菌体得到了有效的富集,同时对阳性克隆进行测序及分析,最终得到2条抗GDH纳米抗体序列。结论:成功构建了双峰驼源天然噬菌体纳米抗体展示文库且多样性良好,为后续筛选其他的靶向抗原奠定了基础,同时筛选获得两条抗GDH纳米抗体序列,为制备艰难梭菌谷氨酸脱氢酶诊断抗体提供技术支撑。     

从噬菌体表面展示肽库中筛选衣原体单克隆抗体识别的抗原表位

利用抗体捕获法,经三轮淘洗,从表面展示随机肽序列的噬菌体文库中筛选到与衣原体单克隆抗体C17特异结合的噬菌体克隆,其一致序列为：（L／I）PGGS（P／W）,竞争抑制实验表明含特异序列的克隆能与天然抗原竞争。据此,我们认为此序列为衣原体的B细胞抗原表位。     

The YvfTU Two-component System is involved in plcR expression in Bacillus cereus

Background

Protein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting.

Results

We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively.

Conclusion

In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.     

Alternate arrangement of PpL B3 domain and SpA D domain creates synergistic double-site binding to VH3 and Vkappa regions of fab

In our previous study, a kind of novel hybrid immunoglobulin (Ig)-binding proteins (IBPs) was obtained with the characteristic structure of alternately arranged Finegoldia magna (formerly Peptostreptococcus magnus) protein L (P. magnus protein L, PpL) B3 domain (B3) and Staphylococcal protein A (SpA) D domain (D). In this study, two representative molecules of these novel proteins, LD3 (B3-D-B3) and LD5 (B3-D-B3-D-B3) (LD3/5), showed substantially higher affinity for IgG-F(ab')2, IgM, and IgA than 4L (B3-B3-B3-B3) or SpA, which were also demonstrated by surface plasmon resonance detection. Further, LD5 showed much stronger binding to single-chain Fv (scFv) KM38 (V(H)3-V(kappa)I) than to KM41 (V(H)1-V(kappa)III) or KM36 (V(H)3-V(kappa)III). Competitive inhibition studies showed that 4L alone or in combination with SpA (4L + SpA) was a weaker inhibitor than LD3/5 in inhibiting LD3/5's binding to IgG-F(ab')2, IgM, or IgA. The computer modeling suggested that the B3-D arrangement in LD3/5 could simultaneously bind to V(H)3 and V(kappa). Thus, our results indicated for the first time that alternate arrangement of B3 and D domains creates synergistic double-site binding to V(H)3 and V(kappa) regions of fragment of antigen binding. The different competitive inhibition pattern of binding of LD5 to scFv KM38 by 4L + SpA suggested strict use of antibody conformation for this simultaneous double-site binding. The demonstration of this novel binding property would promote to achieve the designed hybrid IBPs for useful immunological applications.     

Ig-binding bacterial proteins also bind proteinase inhibitors

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.     

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).     

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.     

Many group A streptococcal strains express two different immunoglobulin-binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M-type

Most group A streptococcal strains are able to bind immunoglobulin (Ig) in a non-immune manner, and the majority of these strains bind both IgA and IgG. Using molecular cloning and immunochemical techniques, we have purified and characterized the Ig Fc-receptors expressed by four such strains. Two of the strains express a novel type of receptor, designated protein Sir, which binds IgA and IgG of all subclasses, and therefore has broader reactivity than any Fc-receptor previously described. The other two strains express protein Arp, a receptor that binds IgA of both subclasses, and also binds polyclonal IgG weakly. Characterization of the weak IgG-binding ability of protein Arp shows that it binds only some monoclonal IgG proteins, in particular those of the IgG3 subclass. The four strains studied here were unexpectedly found to also express a second Ig-receptor, called protein Mrp, encoded by a gene closely linked to the gene for the first receptor. The Mrp protein does not bind IgA, but it binds IgG molecules of the IgG1, IgG2 and IgG4 subclasses, and it also binds fibrinogen. Binding of fibrinogen has been reported to be a characteristic property of streptococcal M proteins, which suggests that the Mrp protein may be an M protein that also binds Ig. Taken together, all available evidence now indicates that most strains of group A streptococci express two different Ig-binding proteins, encoded by closely linked genes.     

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.     

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.     

Protein L. A novel bacterial cell wall protein with affinity for Ig L chains

A novel Ig-binding protein has been isolated from the surface of bacteria belonging to the anaerobic species Peptococcus magnus. To solubilize the protein, peptococci were treated with different proteolytic enzymes (papain, pepsin, and trypsin) or with mutanolysin, a bacteriolytic agent known to digest the cell walls of streptococci. Papain, trypsin, and mutanolysin all solubilized peptides showing affinity for radiolabeled human IgG in Western blot analysis. Compared with papain and trypsin, mutanolysin liberated a more homogeneous material, which also had a higher m.w. This mutanolysin-solubilized protein (Mr 95 kDa) was obtained highly purified by a single isolation step on IgG-Sepharose, and the molecule was found to exhibit unique Ig-binding properties. Thus, in dot blots and in Western blots, human IgG, F(ab')2 and Fab fragments of IgG, and human kappa and lambda L chains all showed affinity for the protein. Moreover, the molecule also bound human IgM and IgA, whereas no binding was recorded for IgG-Fc fragments or IgG H chains. Finally, the protein bound to human polyclonal Ig L chains immobilized on polyacrylamide beads. These different data demonstrate that the isolated peptococcal protein binds Ig through L chain interaction. The name protein L is therefore suggested for this novel Ig-binding bacterial cell wall protein.     

Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.