特异性抗体致敏SPA菌体协同凝集反应的研究

金黄色葡萄球菌 A 蛋白能够与许多哺乳动物 IgG 的 FC 段发生非特异的结合,这一特性已广泛应用于免疫研究中。作者等用含 A 蛋白葡萄球菌(简称 SPA 菌体),以细菌、钩体和流感病毒的抗体制成致敏试剂,对 SPA 菌体协同凝集反应进行了实验和应用研究。现将所得结果报道如下。     

酶标葡萄球菌A蛋白组化法检测腺病毒抗原的研究

葡萄球菌 A 蛋白(SPA)具有与人和许多哺乳动物血清 IgC 的 Fc 段结合的特性。目前已成为一种适用范围很广的免疫试剂。近年来曾将它与辣根过氧化物酶(HRP)结合替代人工免疫制备的第二抗体(抗 IgC),用于 ELISA 法测定病毒抗体的研究,而用 HRP 标记 SPA(HRP—SPA)组化法检测病毒抗原的研究,国内尚无报导。本文是在徐氏用 HRP—SPA 法快速测定炭疽病人英膜抗体的实验研究的启发下,为了解 HRP—SPA 组化法在小儿腺病毒肺炎早期诊断上的意义。我们首先用腺病毒(AdV)模拟标本接种人胚肾(HK)单层细胞,然后涂片检测 AdV 抗原,与酶标抗体、荧光抗体法进行了特异性、敏感性比较,在实验中对 AdV 的复制做了动态观察。并于1981年冬1982年春应用本法对34例小儿肺炎患儿进行了临床诊断,现将初步结果报告如下:     

金黄色葡萄球菌蛋白A串联Z结构域的表达、纯化及其亲和填料制备

目的构建金黄色葡萄球菌蛋白A(Staphylococcal Protein A,简称SPA或蛋白A),串联Z结构域的重组表达克隆,并实现在大肠杆菌中诱导表达,随后纯化获得重组蛋白,制备亲和填料.方法对目的基因进行元和表达的密码子优化,利用PCR扩增方法获得SPA的3个串联Z结构域,并在蛋白的N端加入组氨酸标签.将优化后的基因序列克隆到p ET43.1a(+)表达载体上,获得p ET43.1a(+)-SPA-3Z重组质粒;测序正确的重组质粒转化大肠杆菌感受态细胞BL21(DE3),IPTG诱导表达,通过亲和层析方法纯化表达的重组蛋白,并使用SDS-PAGE鉴定表达蛋白.将纯化获得的SPA纯品偶联到经环氧氯丙烷活化的Sepharose 6B Fast Flow凝胶上.使用兔多抗和腹水单抗对填料进行验证.结果成功构建p ET43.1a(+)-SPA-3Z克隆,纯化的SPA蛋白纯度高.成功将重组SPA偶联到经环氧氯丙烷活化的Sepharose 6B Fast Flow凝胶上.制备的填料在0.1mol/L的Na OH溶液里浸泡30小时,填料亲和性能未见明显变化,这为填料的长期使用提供了可能.将该填料用于抗体纯化,湿胶的多抗结合量可达22.7mg/ml,纯化的腹水单抗纯度在98%以上.结论本研究为SPA的大规模制备提供了基础.     

酶标SPA染色法快速检出感染组织中炭疽杆菌的研究

利用炭疽杆菌毒株在动物机体内可形成荚膜的特性,本文采用酶标葡萄球菌A蛋白染色法(酶标SPA法),检测人工感染动物组织中的炭疽杆菌,并与荧光抗体法及常规培养法作了比较。     

酶标SPA免疫酶法检测宫颈癌患者血清中单纯疱疹病毒抗体

免疫酶法用于检测病人体液中病毒特异性抗体与其它方法相比。是一种简便易行、敏感性、特异性较好的方法,应用日广。用它检测病毒IgG抗体,通常以酶标抗人IgG(简称酶标抗体)作为第二抗体。本文将辣根过氧化物酶标记葡萄球菌A蛋白(简称酶标SPA)用于免疫酶法中检测宫颈癌患者血清中单纯疱疹病毒     

狂犬病病毒抗体胶体金检测试纸的制备

通过胶体金免疫层析技术建立一种特异、便捷、快速的狂犬病病毒抗体检测方法,对犬等动物免疫狂犬病疫苗后的抗体水平监测提供参考。用醋酸锌沉淀法沉淀狂犬病病毒CVS11,Sepharose 4FF进行层析纯化。用柠檬酸三钠还原法制备的胶体金,标记纯化的狂犬病病毒,喷于试纸的结合释放垫 (金标垫),将SPA (葡萄球菌表面A蛋白) 和纯化的兔抗狂犬病病毒IgG分别喷于试纸的T (检测线) 处和C (对照线) 处,组装试纸条。用制备的试纸条对261份犬血清进行检测,与快速荧光灶抑制试验 (RFFIT) 检测的结果一致;对已知效价的犬狂犬病毒中和抗体 (VNA) 大于0.5 IU,结果为阳性;对狂犬病病毒中和抗体 (VNA) 小于0.5 IU/mL的血清,结果为阴性。制备的狂犬病病毒抗体胶体金检测试纸检测犬血清抗体,具有特异、便捷、快速的特点,能够检测出狂犬病病毒中和抗体大于0.5 IU的血清,适用于临床犬血清抗体水平监测,具有良好的应用前景。     

脱落酸抗体的抗独特型抗体的制备与鉴定

用ABA多克隆抗体(Ab_1)以金黄色葡萄球菌菌体(SPA)作为载体,免疫家兔,制备的抗独特型抗体(Ab_2)初步纯化后用ELISA试验鉴定其特异性的结果表明,该抗独特型抗体具有良好的特异性,能阻断ABA抗体对ABA的结合反应,并与ABA结合蛋白结合,暗示其有模拟抗原的作用。 Jerne的免疫网络学说认为,特异性抗体(Ab_1)可变区中的独特型决定簇(Id)可诱导抗独特型抗体(Ab_2)的产生,Ab_2中的一部分可以模拟抗原的作用。Sege和Peterson提出,     

Protein A磁性纳米颗粒载体的制备及应用

本研究采用本课题组合成的表面氨基化磁性纳米微球,首先通过化学共价交联制备了葡萄球菌Protein A磁性纳米微球载体（SPA-MP）,并探讨了载体制备的优化条件。然后根据生物分子特异性亲合作用原理,在外加磁场的定向控制下,通过亲和吸附、清洗和解吸附等操作,探讨了SPA-MP载体在抗体分离纯化领域的应用可行性。载体制备优化实验结果显示,通过改变蛋白质浓度、交联剂浓度和交联剂活化时间可以制备不同表面密度的SPA-MP载体。300 μg SPA,2.5% (V/V)戊二醛浓度和3小时的活化时间可以获取表面密度高达35 mg SPA/g磁性纳米微球的载体。此外,应用结果显示每克SPA-MP磁性微球载体可以结合高达14 mg 的CD25抗体,同时可有效地分离纯化人抗血清样品中的IgG抗体。     

SPA-IgG直接免疫荧光菌团法应用于志贺氏菌的检定

本文利用金黄色葡萄球菌A蛋白(SPA)所具有的与人和多种哺乳动物IgG的Fc段非特异结合的特性,和免疫荧光技术相结合,对SPA结合志贺氏菌抗血清的IgG,以及异硫氰酸荧     

牛源金黄色葡萄球菌粘附因子Efb与ClfA的免疫生物学特性比较

为评价与比较金黄色葡萄球菌的粘附因子Efb(Extracellular fibrinogen-binding protein)和Clf A(Clumping factor A)的抗原性、粘附特性及其抗血清对金黄色葡萄球菌菌体的识别能力、抗粘附能力及免疫保护力等免疫生物学特性,分别构建Efb和Clf A的重组表达载体,并用纯化的重组蛋白免疫实验动物,分别检测家兔抗血清对菌体的识别能力、抗粘附能力以及小鼠抗血清的抗体效价和免疫保护作用。结果显示,Efb和Clf A均有与纤维蛋白原(Fibrinogen,Fg)结合的能力,且Efb蛋白对纤连蛋白(Fibronectin,Fn)的结合能力优于Clf A(P0.01),Clf A免疫组抗血清对全细菌的识别能力优于Efb免疫组(P0.01)。与对照组相比Efb和Clf A免疫组的抗血清均能显著抑制金黄色葡萄球菌对Fg和Fn的粘附(P0.01),Efb免疫组对金黄色葡萄球菌的粘附抑制优于Clf A,两种粘附因子免疫小鼠后产生的血清抗体效价与对照组比较有显著的升高,抗体滴度可达1∶40 500,攻毒结果显示Efb与Clf A免疫组均对小鼠具有良好的保护效果。该研究结果对Efb在金黄色葡萄球菌的亚单位疫苗的应用奠定了基础。     

LamB as a carrier molecule for the functional exposition of IgG-binding domains of the Staphylococcus aureus Protein A at the surface of Escherichia coli K12

Summary One, two or four IgG-binding domains of the Staphylococcus aureus Protein A (SPA) were inserted into the LamB protein which was expressed under control of the tac promoter. The chimeric proteins were shown to be exposed at the cell surface by analysis of isolated outer membranes and also by testing their functional interaction with IgG molecules. We hereby show that the LamB protein can accept as many as 232 amino acids (four SPA domains) and still be incorporated into the Escherichia coli outer membrane, while maintaining the functional conformation of the inserted SPA polypeptides.     

Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2

The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.     

Staphylococcal protein A promotes osteoclastogenesis through MAPK signaling during bone infection

Bone infection is a common and serious complication in the orthopedics field, which often leads to excessive bone destruction and non‐union. Osteoclast is the only type of cells which have the function of bone resorption. Its over activation is closely related to excessive bone loss. Staphylococcus aureus (S. aureus) is a major pathogen causing bone infection, which can produce a large number of strong pathogenic substances staphylococcal protein A (SPA). However, few studies were reported about the effects of SPA on osteoclastogenesis. In our study, we observed that S. aureus activated osteoclasts and promoted bone loss in bone infection specimens. Then, we investigated the effects of SPA on RANKL‐induced osteoclastogenesis in vitro, the results revealed that SPA promoted osteoclastic differentiation and fusion, and enhanced osteoclastic bone resorption. In addition, we also showed that SPA upregulated the expression of NFATc1 and c‐FOS through the activation of MAPK signaling to promote osteoclastogenesis. Our findings might help us better understand the pathogenic role of S. aureus in bone infection and develop new therapeutic strategies for infectious bone diseases.     

Secretion of recombinant proteins into the culture medium by Escherichia coli and Staphylococcus aureus

The ability of staphylococcal protein A (SPA) to bind to the Fc part of IgG has been used for the purification of a number of heterologous gene products as fusion proteins. Both the SPA promoter and signal sequence function in Escherichia coli, as well as in a number of Gram-positive bacteria, which facilitates comparisons of the expressed specific products in different hosts. The expression system developed for E. coli yields excretion of the fusion protein to the growth medium, which makes E. coli a competitive alternative to Gram-positive bacteria for the expression of secreted products. The human peptide hormones insulin-like growth factors (IGF) I and II were expressed using the protein A system in E. coli and Staphylococcus aureus. Despite a high degree of structural homology, large differences in the yields were observed in the two hosts. This underlines the importance of investigating different bacterial hosts for a particular protein product.     

Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.     

Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli

Nucleotide sequence analysis of the gene (spa) for staphylococcal protein A (SPA) from Staphylococcus aureus strain Cowan 1 (NCTC8530) shows that the sequence differs from previously reported SPA nucleotide sequences, especially in the number of repeat units in the cell-wall-binding region of the gene. Dot matrix comparison with streptococcal protein G and the macrophage receptor for the constant fragment (Fc) of immunoglobulins shows a limited but significant homology. The homology to the latter probably identifies the Fc-binding region in the immunoglobulin-binding domains of SPA. Enhanced production of SPA in Escherichia coli was achieved using the lac promoter immediately upstream from the spa gene.     

Cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli.

A gene bank of Staphylococcus aureus strain Cowan I was established using an E. coli HB101/pBR327 host-vector system. Recombinants expressing staphylococcal protein A (SPA) were detected using an IgG-binding assay. A 3.2 Kb DNA fragment directing the synthesis of SPA in E. coli was identified. SPA produced by E. coli was characterised in minicells and by Western blotting and double diffusion experiments.     

Suppression of DNA synthesis in mitogen stimulated leukocytes with partial trypsin digests of Staphylococcus aureus protein A

Staphylococcus aureus protein A (SPA) acts as a mitogen stimulating DNA synthesis and is not antagonistic to the mitogenic activity of other mitogens. The mitogenicity of SPA is due to the protein's multivalent Fc antibody receptors. Partial trypsin digestion of SPA yields monovalent Fc antibody receptors which are not mitogenic. Using a thymidine incorporation assay, results indicated that tryptic digests of SPA added to mouse splenic leukocytes 12 hours after stimulation with either concanavalin A, lipopolysaccaride or SPA suppressed the DNA synthesis normally elicited by these mitogens. SPA was digested with 1% trypsin at pH 8 for varying periods of time. As the digestion time increased from 0 to 60 minutes the mitogenicity of the SPA-trypsin digests decreased indicating cleavage from multivalent to monovalent SPA Fc antibody receptors. The decrease in mitogenicity of the SPA digests was directly associated with increased ability to inhibit DNA synthesis in mitogen stimulated leukocytes. A proposed mechanism for the inhibition of DNA synthesis suggests that SPA-monovalent Fc antibody receptors mimic cellular Fc receptor function thereby influencing cellular gene expression.     

Protein A chromatography for antibody purification

Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.     

Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureus and protein A

The aim of this study was to establish an IMS-MS/SPR technique for the detection of Staphylococcus aureus (S. aureus) and Staphylococcus protein A (SPA) at the same time, which consists of isolating S. aureus and trapping-enrichmenting its SPA by IMS, and the end point is determined by using either MS or SPR measurements. Magnetic bead (MB) containing aldehyde group was synthesized with latex-polymerization and immunomagnetic bead (IMB) was fabricated by modifying its surface with an oriented layer of human IgG in covalent linkage. As soon as sample of pulverator-treated bacterial cell lysate (10(8) cfu/mL) was incubated with IMB at 4 degrees C for 30 min, SPA was captured and separated from the mixed solution in a few minutes by the IMB and then detected with mass spectrometry after washing. SPR was used to detect S. aureus quantitatively in situ at the end-detection procedure. All in all, this technique can be employed to detect rapidly SPA and S. aureus within 2h and also be applied to detect other cells or their membrane proteins with changed modified antibodies.