猪瘟兔化弱毒E2蛋白A/D区的高效表达和蛋白纯化及其应用

本试验对表达猪瘟E2囊膜糖蛋白的原核表达载体pET-E2的表达条件进行了优化,在此基础上,对表达的蛋白质进行了纯化.在6 mol/L盐酸胍存在的条件下,将包涵体溶解在Tris-HCI中,并直接用于His Band亲和层析.收集过柱产物并透析,在2%SDS条件下,对所得的重组蛋白质进行了定量.重组蛋白质被用来包被96孔ELISA板并应用于免疫猪瘟抗体水平的测定.     

抗对虾白斑综合症病毒的单链抗体A1的表达和生物化学特性

用噬菌体展示技术制备了抗对虾白斑综合症病毒(WSSV)的单链抗体A1。该抗体在30℃培养条件下诱导表达20h后,其蛋白表达量可达总菌体蛋白的3.67%。用亲和层析柱和SephadexG-100层析柱可将单链抗体A1纯化为一条单电泳条带,其分子量约为31.5kD。用等电聚焦电泳测定,其等电点为pH5.8。ELISA测定表明冻干的单链抗体A1在室温储藏4年后与WSSV结合仍具有较高的活力。       

萤火虫荧光素酶单克隆抗体的制备与抗原决定簇的研究

以大肠杆菌表达的萤火虫荧光素酶 (fireflyluciferase)为抗原 ,免疫小鼠并进一步筛选与克隆 ,共得到 6株单克隆抗体 .制备腹水并纯化获得抗体后 ,对这 6株抗体与天然态和热变性态蛋白质以及蛋白酶解片段的结合性质进行了鉴定 .认为这 6株抗体的抗原决定簇都是顺序决定簇 .发现其中有 2株单抗与热变性态蛋白质及酶解片段的结合能力较强 ,而不与天然态蛋白质结合 ,其抗原决定簇可能是位于蛋白质内部的肽段 .另外 4株抗体既可与热变性态蛋白质以及酶解片段结合 ,也可与天然态蛋白质结合 ,其抗原决定簇可能位于蛋白质分子表面 .     

猪瘟兔化弱毒E2蛋白A／D区的高效表达和蛋白纯化及其应用

本试验对表达猪瘟E2囊膜糖蛋白的原核表达载体pET—E2的表达条件进行了优化,在此基础上,对表达的蛋白质进行了纯化。在6mol/L盐酸胍存在的条件下,将包涵体溶解在Tris-HCl中,并直接用于His Band亲和层析。收集过柱产物并透析,在2％SDS条件下,对所得的重组蛋白质进行了定量。重组蛋白质被用来包被96孔ELISA板并应用于免疫猪瘟抗体水平的测定。     

基于抗体的水稻蛋白质组学——开端与展望

为了深入开展水稻基因功能研究,借鉴人类蛋白质研究中的相关理念,提出了基于抗体的水稻蛋白质组学的策略.AbRP的要点在于,建立针对水稻蛋白质的抗体资源库,利用大规模免疫分析技术,揭示水稻蛋白质在不同时空条件下的表达状态并了解其相关功能.用AbRP策略可以对特定的基因家族或途径相关蛋白质进行研究,可以检测蛋白质的表达量及修饰的变化等.AbRP的实施将为水稻基础和应用研究创建一个重要的支撑平台,基于抗体的蛋白质表达数据与基因组、转录组数据相整合,可望进一步丰富水稻乃至其他植物研究的公共数据资源.     

利用基因免疫进行日本血吸虫Sj22蛋白抗体制备的研究

应用基因免疫方法制备日本血吸虫Sj22蛋白的抗体,并研究CpG佐剂和蛋白加强策略在增强基因免疫效果中的作用。采用肌肉注射的方法免疫小鼠,实验动物分为四组：A组注射pVAX1-sj22质粒100μg /只;B组注射pVAX1-sj22质粒50μg /只,同时注射CpG佐剂20μg /只;C、D组注射pcDNA3.1-sj22质粒100μg /只。分别在0,3,6周进行免疫,第9周A、B、C组用30μg重组sj22蛋白加强免疫,D组则用pcDNA3.1-sj22质粒100μg加强免疫。第0,9,10周割尾采血,使用ELISA方法进行抗体效价测定,Western blot验证抗体的特异性。结果表明：使用基因免疫的方法获得了针对Sj22的特异性抗血清,CpG佐剂能够有效降低质粒用量,基因免疫-蛋白加强的策略使得抗体的滴度提高了40～1280倍。     

抗体的性质及生成机制

若有一种异种蛋白质,不经消化吸收途径而直接被注射进入动物体内,则此异种蛋白质可以影响体内的蛋白质生成机制,使其生成一种新的蛋白质。此新的蛋白质若在体内或体外遇到上述异种蛋白质,则可与之特异结合,并起沉淀或其他反应。此新的蛋白质称为抗体;影响机体以生成抗体的异种蛋白质称为抗原。抗原与抗体的特异结合称为抗原     

细胞工程

860752全色印迹(Golden blot):用蛋白质A一金黄色复合体测定在硝化纤维上与抗原结合的多克隆和单克隆抗体〔英〕/Brada,D.…IAnal.Bioehem一1984,142(1)一79~83〔译自DBA,1985,4(1),85一00444] 一种用蛋白质A一gold"西方印迹"测定免疫复合体方法得到了发展。用电泳分离蛋白质后,将抗原固定在硝化纤维薄板上,然后将硝化纤维带与亲和纯化     

用免疫亲和层析法纯化萝卜 PHGPx 天然蛋白

萝卜磷脂氢谷胱甘肽过氧化物酶 (RsPHGPx) 是一个定位于线粒体的蛋白质 . 为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的 RsPHGPx. 用重组 RsPHGPx 蛋白免疫兔子获得了抗 RsPHGPx 的多克隆抗血清,以重组 RsPHGPx 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗 RsPHGPx 的抗体 . 将纯化好的抗体偶联到一个 N- 羟基琥珀酰亚胺 (NHS) 预先激活的琼脂糖柱子上,装配成一个以单特异性的抗 RsPHGPx 抗体为配体的免疫亲和层析柱 . 经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案 . 按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质 . 免疫印迹分析表明,该蛋白质被抗 RsPHGPx 的抗血清特异识别 . 酶活性分析表明,该蛋白质具有显著的 PHGPx 活性 . 这些结果表明,纯化到的特异蛋白质是萝卜的 RsPHGPx 天然蛋白 . 这是首个关于定位于植物细胞器的 PHGPx 蛋白纯化的报道 . 这一结果为准确测定 RsPHGPx 信号肽的切割位点奠定了基础,并将有助于对植物 PHGPx 的亚细胞定位机制及其生理功能的深入研究 .     

中枢注射抗Orexin抗体对禁食大鼠摄食的抑制作用

目的:探讨中枢注射抗Orexin抗体对禁食大鼠摄食的抑制作用。方法:采用免疫组织化学法和蛋白质免疫印迹分析法,对Orexin抗体的特异性进行了检测,分析Orexin阳性神经元和阳性神经纤维在大脑中的分布。给予24 h禁食大鼠中枢注射抗Orexin抗体,计算其对大鼠食物摄入量的影响。结果:蛋白质免疫印迹分析显示,Orexin抗体能够检测到合成的Orexin-A。免疫组织化学分析显示,Orexin阳性神经元存在于外侧下丘脑区域和穹窿周核,Orexin阳性神经纤维大量投射至弓状核、下丘脑室周核和下丘脑室旁核、菱形丘脑核、丘脑室旁核、缰内侧核和纹质丘脑、中脑的中央灰质区、蓝斑核和中缝核、脑桥和髓质的网状结构、对疑核和迷走神经复合体。与注射羊血清相比,给予45μg/10μL抗Orexin抗体侧脑室注射则抑制了大鼠的食物摄入量(P0.05)。高剂量抗Orexin抗体能显著地抑制大鼠摄食,并且呈剂量依赖关系(P0.05)。结论:中枢注射抗Orexin抗体对禁食大鼠摄食具有抑制作用,并呈剂量依赖关系。     

Dynamic analysis of irreversible adsorption of protein on porous polymer resins as studied by pulse injection method

Irreversible adsorption of bovine serum albumin onto porous polymer resins is examined by a pulse injection method. The experimental results are theoretically analyzed by a model based on the assumption that there exist three kinds of binding sites with different binding rates, which are considered to exist in the vicinity of the outer periphery surface, the inner surface of macropore, and the micropore, or center part, of particles. The three kinds of adsorption rates are also evaluated by a batch method and are nearly equal to the corresponding kinetic parameters by this method. The amount of BSA bound irreversibly to the resins is independent of the protein concentration and the flow rate examined, which suggests that protein molecules penetrate into pores of resins and occupy almost all the effective binding sites in the resins. The total amount of bound BSA shows a pH dependence with a maximum near the isoelectric point of BSA, and the amount of BSA bound at the slowest rate is most largely influenced by pH.     

用硝酸纤维素膜亲和法纯化抗体

介绍一种改进的纯化抗体方法。将抗原蛋白固定在硝酸纤维素膜上,然后对抗体进行亲和纯化,能快速有效获得高特异性抗体。     

Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.     

Antibodies against the human cellular 3,3′,5-triiodo-L-thyronine-binding protein (p58)

High-titer antibodies against a cellular thyroid hormone-binding protein (M_r 58 000, p58) were developed by a special immunization method. To enhance immune responses, this method uses a boosting protocol in which repeated injections of small amounts of antigen are administered at 2-day intervals. Antibodies were detected 1 week after the last injection of antigen by ELISA, Western dot blotting and immunoprecipitation. The anti-p58 antibodies recognize p58 which is bound to the thyroid hormone. With the availability of anti-p58 antibodies, it has become possible to study cellular localization and function.     

Inactivation of viruses using novel protein A wash buffers

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc‐fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc‐fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:406–413, 2015     

Benzoxazinone — kanamycin derivative: a new fluorescent probe for flow cytometry analysis of bacteria (Agrobacterium tumefaciens)

Abstract A new polycation fluorescent dye (BVC-kinamycin-conjugate) has been synthesized and used to detect alive bacteria by flow cytometry. This fluorescent chromophore has the noteworthy property of being excited at the same wavelength as fluoresceinylated conjugates (488 nm) and to show a much longer emission wavelength (616 nm) than fluoresceinylated derivates (520 nm); furthermore its fluorescence intensity is not quenched at low pH in contrast with fluorescein. In such conditions, bacteria can easily be detected with cytofluorimeter equipped with a single excitation wavelength beam. The binding of fluoresceinylated lectins and antibodies onto a strain of Agrobacterium tumefaciens has been studied by this method.     

Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.     

The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues.

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.     

Isolation and characterization of a low-molecular-weight immunoglobulin-binding protein from Yersinia pseudotuberculosis

A low-molecular-weight immunoglobulin-binding protein (IBP) bound with the cell envelope has been isolated from Yersinia pseudotuberculosis cells and partially characterized. This IBP is a hydrophilic protein with a high polarity index of 55.3%. The molecular weight of the protein has been determined by MALDI-TOF mass spectrometry as 14.3 kD. CD spectroscopy showed that the IBP has high contents of the beta-structure and random coil structure. The IBP contains glycine as the N-terminal amino acid. The protein can be stored for a long time at acidic pH values but aggregates and loses activity at alkaline and neutral pH. The IBP binds rabbit IgG with optimum at pH of 6.0-7.5. The IBP interacts with IgG molecule in the Fc-fragment region. The protein retains activity after heating at 100 degrees C in the presence of SDS.     

Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.