共查询到20条相似文献，搜索用时 15 毫秒
1.
Escherichia coli secretion of an active chimeric antibody fragment 总被引：28，自引：0，他引：28
A chimeric mouse-human Fab protein that binds specifically to the human carcinoma cell line C3347 has been expressed and secreted from Escherichia coli. This molecule, which contains functionally assembled kappa and Fd proteins, binds as effectively to sites on the surface of C3347 cells as Fab fragments prepared proteolytically from whole chimeric or mouse antibody. The production in Escherichia coli of foreign heterodimeric protein reagents, such as Fab, should prove useful in the management of human disease. 相似文献
2.
The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure. 相似文献
3.
van der Neut Kolfschoten M Schuurman J Losen M Bleeker WK Martínez-Martínez P Vermeulen E den Bleker TH Wiegman L Vink T Aarden LA De Baets MH van de Winkel JG Aalberse RC Parren PW 《Science (New York, N.Y.)》2007,317(5844):1554-1557
Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies. 相似文献
4.
ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion. 相似文献
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【背景】非洲猪瘟（ASF）于2018年8月在中国首次出现,对养猪业造成了巨大危害,损失惨重。目前尚无安全有效的疫苗用来预防ASF,于是建立快速特异的检测方法对于防控ASF提供了有效的手段。【目的】制备非洲猪瘟病毒（ASFV）特异性单克隆抗体,建立ASF快速特异性的检测方法。为ASF的检测和防控提供借鉴技术手段。【方法】构建表达载体pET-28a-P30,通过原核表达系统获得ASFV P30重组蛋白,以纯化的P30蛋白为抗原免疫BALB/c小鼠,经过细胞融合和细胞亚克隆制备出ASFV P30蛋白特异性杂交瘤细胞株;对P30蛋白进行截短表达,利用Western Blot和间接酶联免疫吸附试验（iELISA）鉴定单克隆抗体所对应的抗原表位;并利用制备的单克隆抗体建立非洲猪瘟阻断ELISA抗体检测方法。【结果】通过双酶切和PCR验证,结果显示构建出重组载体pET-28a-P30,经测序其序列未发生突变;IPTG诱导后,P30重组蛋白主要表达在包涵体中,分子量约为33 kD。纯化的P30蛋白与弗氏佐剂1﹕1混合免疫小鼠,3次免疫后,小鼠血清效价达到1﹕102 400,说明表达的蛋白具有良好的免疫原性。经细胞融合和亚克隆,获得8株P30蛋白特异性杂交瘤细胞,Western Blot和间接免疫荧光试验（IFA）检测获得的8株单抗均具有良好的反应性。叠加试验显示8株单克隆抗体均针对相同的抗原位点;截短表达P30蛋白不同片段,选取制备的2-12B单克隆抗体与不同的截短P30蛋白反应,显示单克隆抗体的抗原表位区为187—194aa。利用2-12B单克隆抗体并经过条件优化,成功建立了ASF阻断ELISA抗体检测方法,检测了190份临床样品,并与商品化非洲猪瘟ELISA抗体检测试剂盒进行对比,两方法阳性符合率为90.91%,总符合率为96.32%。【结论】本研究成功获得ASFV P30蛋白,经过iELISA、Western Blot和IFA筛选出反应性良好的特异性单克隆抗体8株,抗原识别表位区为187—194aa。并利用制备的单克隆抗体建立了特异性高,敏感性好的ASFV阻断ELISA抗体检测方法,为ASF的检测及其防控提供了手段和支撑。 相似文献
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引物原位标记技术及其应用 总被引：1，自引：1，他引：1
顾丰 《南京农业大学学报》1997,20(3):106-109
本文综述了近几个来引物原位标记技术的发展和应用，并以检测猪染色体端位序列为例，介绍了这项技术的基本原理和实验程序。现已表明，引物原位标记技术不仅可以应用于检测染色体特定的ＤＮＡ序列，而且可以应用于在细胞和组织切片上检测特定的ＲＮＡ序列。 相似文献
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猪圆环病毒2型Cap蛋白核定位信号区抗原表位的鉴定 总被引：1，自引：0，他引：1
【目的】猪圆环病毒2型(PCV2)是引起断奶仔猪多系统衰竭综合症(PMWS)的重要病原,其基因组ORF2编码的Cap蛋白为病毒的主要结构蛋白,起保护性抗原作用,对其进行抗原表位鉴定十分必要。【方法】本研究采用杆状病毒表达的重组Cap蛋白作为免疫原制备5株单克隆抗体,通过原核表达系统对ORF2基因进行了截短表达,先行对Cap蛋白抗原表位进行宽幅定位,然后利用合成多肽对Cap抗原表位做精确扫描定位。【结果】5株单抗中有4株针对同一抗原表位,坐落在Cap蛋白N末端核定位信号区,经多肽扫描证实,核心序列为26RPWLVHPRHRY36;另1株单抗(1D2)仅与重组Cap蛋白产生免疫活性反应,对4个分段截短表达的Cap蛋白无免疫活性反应,鉴于该单抗具有中和病毒活性,针对的可能是构象表位。【结论】本研究首次鉴定出位于Cap蛋白核定位信号区的一个抗原表位,为以后Cap蛋白功能及核定位机理的研究奠定基础。 相似文献
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[目的]为C型FMDV型特异性多克隆抗体、单克隆抗体的制备及FMDV定型提供理论依据。[方法]以含有C型口蹄疫病毒（FMDV）结构蛋白基因VP1的重组质粒pGEM—CP1为模板,设计特异性表达引物,扩增VP1及其C端编码区。对C型口蹄疫病毒VP1及其C端进行原核表达,并测定反应原性。利用纯化的C型VP1及其C端融合蛋白建立间接ELISA,分别对O、A、C、Asia14型豚鼠阳性血清进行检测,确定C型VP1及其C端与其他3型FMDV抗体的型间交叉反应性。[结果]构建了pPRO-CVP1、pPRBO-CVP1c重组原核表达质粒,实现了C型口蹄疫病毒VP1及其C端的高效表达,目的蛋白的分子量大小分别为33和20kD。Westernblot显示。VPl及其C端融合蛋白均可与对应血清型的豚鼠阳性血清反应。C型VP1及其C端与其他血清型的FMDv阳性血清均未发生交叉反应。且以VP1c端的型特异性最好。[结论]获得了C型FMDV特异性抗原。 相似文献
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Ekiert DC Friesen RH Bhabha G Kwaks T Jongeneelen M Yu W Ophorst C Cox F Korse HJ Brandenburg B Vogels R Brakenhoff JP Kompier R Koldijk MH Cornelissen LA Poon LL Peiris M Koudstaal W Wilson IA Goudsmit J 《Science (New York, N.Y.)》2011,333(6044):843-850
Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the V(H)1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies. 相似文献
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The molecular basis for the anti-inflammatory property of intravenous gamma globulin (IVIG) was investigated in a murine model of immune thrombocytopenia. Administration of clinically protective doses of intact antibody or monomeric Fc fragments to wild-type or Fcgamma receptor-humanized mice prevented platelet consumption triggered by a pathogenic autoantibody. The inhibitory Fc receptor, FcgammaRIIB, was required for protection, because disruption either by genetic deletion or with a blocking monoclonal antibody reversed the therapeutic effect of IVIG. Protection was associated with the ability of IVIG administration to induce surface expression of FcgammaRIIB on splenic macrophages. Modulation of inhibitory signaling is thus a potent therapeutic strategy for attenuating autoantibody-triggered inflammatory diseases. 相似文献
12.
Pejchal R Doores KJ Walker LM Khayat R Huang PS Wang SK Stanfield RL Julien JP Ramos A Crispin M Depetris R Katpally U Marozsan A Cupo A Maloveste S Liu Y McBride R Ito Y Sanders RW Ogohara C Paulson JC Feizi T Scanlan CN Wong CH Moore JP Olson WC Ward AB Poignard P Schief WR Burton DR Wilson IA 《Science (New York, N.Y.)》2011,334(6059):1097-1103
The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface. 相似文献
13.
本文测定了麦长管蚜及其天敌七星瓢虫对六种菊酯类杀虫剂的敏感性。测定结果表明,山西晋中地区麦长管蚜对菊酯类药剂的敏感性次序为,溴氰菊酯>氯氰菊酯>氰戊菊酯>氟氰戊菊酯>甲氰菊酯>戊菊酯。七星瓢虫幼虫对这六种菊酯类药剂的敏感性次序与麦长管蚜相同。在麦长管蚜和七星瓢虫之间,六种菊酯类药剂的选择性指数以氯氰菊酯最高,依次为溴氰(?)酯、氰戊菊酯、氟氰戊菊酯、甲氰菊酯和戊菊酯。 相似文献
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[目的]制备鸡MHCⅡ类分子的单克隆抗体。[方法]在分析鸡MHCII类分子蛋白序列基础上,选取鸡MHCⅡα链基因第2～6外显子和β链基因第3～6外显子进行原核表达,以纯化的融合蛋白免疫Balb/c小鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,经克隆和间接ELISA筛选阳性细胞株。[结果]共得到1株分泌鸡MHCⅡα链抗体和2株MHCⅡβ链抗体的杂交瘤细胞,分别命名为MHCⅡα-4、Ⅱβ6-2和Ⅱβ6-31,其腹水效价分别为1∶256000、1∶256000和1∶1280000。Western blotting分析表明其特异性强,能与相应蛋白结合。[结论]成功获得了3株稳定分泌抗鸡MHCⅡ类分子抗体的杂交瘤细胞。 相似文献
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S Paul D J Volle C M Beach D R Johnson M J Powell R J Massey 《Science (New York, N.Y.)》1989,244(4909):1158-1162
Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody. 相似文献
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[目的]探讨14%马联苯乳油对经济作物茶树小绿叶蝉的防治效果及最佳施用量。[方法]设置14%马联苯乳油1 000、1 500、2 000倍液,以50%马拉硫磷乳油600倍液和2.5%天王星菊脂4 000倍液为对照,研究各处理对茶树小绿叶蝉的防治效果。[结果]14%马联苯乳油1 000、1 500、2 000倍液药后1 d平均防效分别为73.03%、52.65%和36.48%,药后3 d平均防效分别为94.94%、81.18%和52.66%,药后7 d分别为97.73%、88.38%和63.31%。[结论]14%马联苯乳油1000倍液为最佳施用量。 相似文献
17.
猪瘟病毒石门株E2基因4个抗原结构域的原核表达 总被引：1，自引：1，他引：1
【目的】对猪瘟病毒石门株E2基因进行原核表达，以期获得可溶性表达产物，为检测猪瘟抗体的ELISA试剂盒的研制奠定基础。【方法】用PCR技术扩增了重组S21质粒载体上的猪瘟病毒石门株E2基因的4个主要抗原结构域ABCD，A1A2，B和C。分别将4个片段克隆于pMAL－p2X载体中，经PCR、双酶切和测序鉴定，E2基因的4个主要抗原结构域片段的位置、大小和读码框均正确。将4个片段分别转化到表达菌TB1、BL21、BL21－CodonPlus(DE3)-RP和BL21(DE3)中，共得到16株重组表达菌，用IPTG进行诱导表达，对表达产物进行SDS-PAGE电泳和免疫印迹分析。【结果】E2基因的4个主要抗原结构域均可在这4种表达菌中表达，但以BL21－CodonPlus(DE3)-RP的表达效果最好，可表达出可溶性并且产量较高的目的蛋白。免疫印迹结果表明，表达的目的蛋白可以被猪瘟阳性血清和针对E2蛋白的单克隆抗体所识别。【结论】只表达目的基因的抗原结构域，可以缩短表达片段的长度，有利于获得可溶性目的蛋白，并且具有良好的血清学反应的特异性。 相似文献
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[目的]检测猪圆环病毒2型（PCV2）核衣壳蛋白（Cap）的表达,为进一步研制PCV2单克隆抗体检测抗原提供参考依据。[方法]根据已经发表的PCV2的衣壳蛋白基因（Cap）设计1对引物,利用PCR方法从已知PCV2病毒中扩增到1条DNA片段。将PCR产物按预定的阅读框架插入表达载体质粒pET28a（＋）中,获得重组质粒pCAP-PCV2,并转化到大肠杆菌BL21（DE3）中。[结果]序列测定表明,所克隆的序列大小为579 bp,与DQ104422的Cap基因序列一致;通过对菌体裂解物的SDS-PAGE分析证明,携带重组质粒pET-PCV2-Cap的大肠杆菌可以表达可溶性的衣壳蛋白。[结论]PCV2衣壳蛋白能通过表达载体质粒pET28a（＋）进行可溶性表达。 相似文献