A蛋白亲和色谱法纯化动物血清抗体的效果比较

比较Hitrap A蛋白琼脂糖凝胶亲和色谱系统,对不同动物血清或抗血清抗体的纯化效率,提供选择抗体纯化方法的依据。该方法简使快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。色谱柱反复使用40多次,纯化抗体效率不变。但A蛋白对不同动物血清的IgG吸附能力不同,对兔和豚鼠的血清抗体吸附能力强,而对小鼠、山羊和驴的血清抗体吸附能力较弱。说明虽然该方法纯化的抗体纯度高,但它对动物品种有选择。因此,应根据动物品种,选择适合的纯化方法。     

采用免疫亲和色谱方法从骆驼血清中纯化抗CD47特异性抗体

本研究从骆驼血清中制备抗CD47特异性抗体并分析其与重组蛋白CD47和EC9706细胞膜表面CD47结合情况。从骆驼血清中纯化出总Ig G,再利用偶联上CD47的CNBr琼脂糖树脂免疫亲和纯化出抗CD47特异性抗体,通过ELISA和Western blotting验证其结合情况,通过细胞ELISA鉴定抗CD47特异性抗体与食管癌细胞膜表面CD47结合。结果显示通过两步免疫亲和色谱成功获得抗CD47特异性抗体,ELISA和Western blotting说明该抗体与重组蛋白CD47有强的结合活性和特异性,细胞ELISA检测抗CD47抗体浓度在20μg/mL时与EC9706细胞表面CD47有较好的结合。本研究首次利用免疫亲和色谱方法从骆驼血清中获得抗CD47特异性抗体,并与食管癌细胞有一定的结合活性,为肿瘤靶向治疗提供了可能。     

重组蛋白G Fc结合区单结构域的纯化及理化性质

蛋白 G 是存在于链球菌 C 群和 G 群胞膜上的一种分子量为65kD 的膜蛋白,完整的蛋白 G 分子主要由四部分组成,其中第三部分能与许多动物的 IgG Fc段特异结合。近年来,蛋白 G 的应用研究已展示出广阔前景,它一方面可与多种标记物(如酶、放射性同位素、胶体金等)连接,用于免疫分析;另一方面,可用作纯化单克隆抗体及多克隆抗体的亲和配基。但是有关蛋白 G 的空间结构及其与 IgG Fc 的结合机制     

ANKRD17蛋白抗体的制备、纯化及检测

目的：制备ANKRD17（P260）蛋白的兔多克隆抗体,以与抗原相结合的方法进行抗体的纯化,并利用纯化的抗体对该蛋白进行细胞内免疫荧光检测。方法：构建表达GST—ANKRD17C端融合蛋白的质粒,在大肠杆菌中诱导表达;制备GST—ANKRD17C端抗原融合蛋白后免疫家兔,对获得的兔多克隆抗血清进行亲和纯化;纯化后的抗体经过Western blot鉴定,用于细胞免疫荧光染色检测。结果：获得较高效价的血清抗体,并对血清抗体进行了纯化;利用纯化的抗体对ANKRD17蛋白进行了细胞内免疫荧光检测,发现改蛋白定位于细胞质中。结论：制备得到的纯化抗体为研究ANKRD17蛋白的功能打下了必要的基础。     

A蛋白亲和层析法纯化动物血清抗体的效果比较

用ＨｉｔｒａｐＰｒｏｔｅｉｎ－ＡＳｅｐｈａｒｏｓｅ亲和层析系统,对不同动物血清或抗血清抗体的纯化效率进行了比较,为选择抗体纯化方法提供依据。该方法简便快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。层析柱反复使用４０多次,纯化抗体效率不变。但Ｐｒｏｔｅｉｎ－Ａ对不同动物血清的ＩｇＧ吸附能力不同：对兔和豚鼠的血清抗体吸附能力强     

PTD融和蛋白的原核表达及其抗体制备

目的:为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原核表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础.方法:应用重叠延伸的基因融合技术将LHRH(黄体生成激素释放激素)基因、TAT(HIV-1反式转录激活因子)基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28a-LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni-NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体.结果:表达产物经聚丙烯酰胺凝胶电泳检测,LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12.6%.LTA蛋白经Ni-NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清.结果表明抗体的效价为1: 12800.结论:应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体.     

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。     

PTD融合蛋白的原核表达及其抗体制备

目的：为制备凋亡素重组蛋白抗体,首先获得凋亡素重组蛋白融合基因LTA,而且通过原恢表达系统表达重组蛋白并制备其抗体,为进一步利用凋亡素重组蛋白导向治疗肿瘤的检测奠定基础。方法：应用重叠延伸的基因融合技术将LHRH（黄体生成激素释放激素）基因、TAT（HW—1反式转录激活因子）基因和凋亡素基因重组,构建成凋亡素重组蛋白原核表达载体pET-28α～LTA,随后将表达质粒转入BL21菌株,经IPTG诱导表达重组融合蛋白,将已表达的重组蛋白通过Ni—NTA亲和色谱柱进行纯化,并制备LTA凋亡素重组蛋白抗体。结果：表达产物经聚丙烯酰胺凝胶电泳检测．LTA蛋白融合基因获得高效表达,凝胶薄层扫描分析表明表达蛋白占菌体蛋白12．6％。LTA蛋白经Ni—NTA亲和色谱柱柱纯化,以纯化蛋白为抗原免疫獭兔制备凋亡素重组蛋白抗血清。结果表明抗体的效价为1：12800。结论：应用重叠延伸的基因融合技术获得凋亡素重组蛋白融合基因LTA,通过原核表达系统表达重组蛋白并制备其抗体。     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

口蹄疫病毒非结构蛋白3AB 的原核表达及应用

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA方法。克隆、表达了口蹄疫病毒非结构蛋白3AB基因,原核表达的重组蛋白经亲和层析法纯化及Western blotting鉴定后作为包被抗原,建立检测口蹄疫病毒非结构蛋白抗体的3AB间接ELISA方法,通过与商品化试剂盒3ABC-ELISA的比对试验对其进行评价。结果显示,重组蛋白3AB以包涵体形式表达;能与口蹄疫病毒感染血清发生特异性反应,而不能与疫苗免疫动物血清发生反应;在检测田间样品时,与3ABC-ELISA具有同样的特异性和敏感性 (P＞     

Production, characterization and applications of mouse anti-grass carp (Ctenopharyngodon idellus) growth hormone monoclonal antibodies

Mouse anti-grass carp growth hormone (gcGH) monoclonal antibody (MAb) secretors were produced by PEG-mediated fusion of NS-1 myeloma cells and splenic B-lymphocytes of gcGH hyper-immunized mice. Positive secretors were screened by direct ELISA and cloned by limiting dilution. Three positive secretors, 21D3, 22G5 and 23B3, were obtained in a single fusion trial. Anti-gcGH MAbs were produced by growing hybridomas in the peritoneal cavity of pristane-primed mouse. The three MAbs were isotyped to be IgG2a, IgG2b and IgM, respectively. IgG MAbs were purified from ascitic fluid by Hitrap protein G column and IgM MAb was purified by gel filtration chromatography. The purified MAbs were highly specific and had moderate binding affinity. The MAbs were successfully used for the purification of native gcGH from mature grass carp pituitary extract by one-step immunoaffinity chromatography, for the quantification of gcGH by competitive sandwich ELISA, and for the probing of somatotropes in grass carp pituitary by immunohistochemistry.     

两种果子狸血清IgG纯化方法 的比较

目的 探讨一种具有简便快捷、高纯度、活性好的果子狸血清IgG纯化方法。方法 比较Hitrap Protein A亲和层析和PAGE电泳两种纯化法对果子狸血清IgG的纯化,用PAGE还原电泳和Western-Blot法对IgG作纯度鉴定。结果 对Hitrap Protein A纯化的果子狸血清IgG,其活性虽好,但纯度不高;而非还原PAGE电泳所纯化的果子狸血清IgG不但纯度高（〉95%）、并具有较强的免疫活性。结论 非还原PAGE电泳法纯化果子狸血清IgG,是一种具有高纯度、免疫活性强的纯化方法 。     

长爪沙鼠IgG的纯化及抗血清的制备

目的 纯化长爪沙鼠血清IgG,制备兔抗长爪沙鼠IgG抗血清。方法 采用Hitrap Protein G亲和层析预装柱来纯化长爪沙鼠血清IgG;通过SDS-PAGE电泳和Western-Blotting免疫印迹法对长爪沙鼠血清IgG进行纯度鉴定,免疫兔子制备抗血清。结果 7 mL长爪沙鼠血清纯化得到11 mg IgG;电泳和免疫印迹测定,IgG纯度大于95%;用纯化的IgG作抗原制备了兔抗血清,免疫双扩散测定效价达1∶32。结论 建立了长爪沙鼠血清IgG的纯化方法,制备了长爪沙鼠IgG抗血清,证实长爪沙鼠血清IgG和Protein G具有较高的亲和性。     

A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties

Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.     

貉血清IgG纯化及抗血清的制备

目的 制备高纯度貉血清IgG和兔抗貉IgG抗血清,作为建立多种动物抗体检测技术的储备。方法 采用Hitrap Protein A亲和层析及盐析再沉淀法纯化貉血清IgG,通过PAGE电泳和Western-Blot免疫印迹法对IgG作纯度及免疫活性检测;常规免疫法制备兔抗貉IgG血清。结果 貉血清IgG与Protein G虽有较强的结合力,但同时也结合血清中其他杂蛋白;用二步纯化法可从5 mL貉血清中纯化IgG约7 mg,电泳和免疫印迹测定显示,IgG纯度大于95%,常规免疫法制备抗血清免疫双扩散效价达1∶32。结论 建立了可行的貉血清IgG的纯化方法和高效价的兔抗貉血清IgG抗血清,为貉血清IgG二级抗体酶联物的制备储备了资源。     

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.     

IgG PURIFICATION USING AFFINITY FILTRATION WITH SULFAMETHAZINE-AFFINITY CARRIERS

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 μmol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography.