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991.
利用DNA重组技术,将转录因子NF-κBp50基因片段插入pGEX-2T质粒载体,经DNA序列分析证明克隆的正确性.将重组的pGEX-2T/p50转化入大肠杆菌JM109,表达出pGEX-2T/p50融合蛋白.经WesternBlot检测证实表达产物对抗p50c的抗体呈特异性反应,表明克隆的正确性,并成功地应用纯化的p50蛋白制备了免疫血清.为进一步对NF-κB的功能进行研究打下了良好的基础 相似文献
992.
利用15肽随机肽库确定抗TNF单抗表位的研究 总被引:7,自引:0,他引:7
利用抗TNF的T5单抗作为筛选配基,对经DNA碱基组成分析证明具有良好随机性的15肽库进行亲和筛选.经过三轮筛选后,以硝酸纤维素膜斑点印迹法观察到良好的富集效果.由第三轮挑选出的31个克隆进行DNA测序,结果推出的优势克隆的短肽为CYRRPAGGLPGICSA等,竞争性ELISA实验证明带有以上短肽的噬菌体与TNF能竞争性地与T5单抗结合.该多肽可能是T5单抗所识别的模拟表位 相似文献
993.
恶性疟原虫多抗原表位基因表达载体的构建及其在大豆幼胚中的表达 总被引:2,自引:0,他引:2
疟疾是当今最需要研究有效疫苗的主要传染病之一。AWTE基因编码恶性疟原虫多种抗原表位基因 ,CTB基因编码霍乱毒素 B亚基 ,是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。把 AWTE- CTB融合基因构建到植物表达载体 p BVG- ny2上 ,通过基因枪导入法 ,转化大豆幼胚分生组织。 X- glu染色检测到 GUS基因的表达 ;抗原性分析实验结果表明 ,特异表达的融合蛋白可与 CTB和 AWTE抗体结合 ,具有 CTB抗原性。这个实验结果 ,表明疟疾多抗原表位基因首次在转基因大豆幼胚中得到瞬时表达 相似文献
994.
995.
Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies. 总被引:1,自引:0,他引:1
Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi. 相似文献
996.
Regional lymph node lymphocytes from five patients with primary lung cancer were analyzed for subset composition, and exposed
in vitro to the polyclonal human B cell mitogen Staphylococcus aureus Cowan I (SACI) or the murine B cell mitogen lipopolysaccharide
(LPS) and then fused with mouse myeloma cells for investigation at the clonal level of their antibody (Ab) production and
its statistical relation to the original subset composition. No correlation was found between the proportion of CD19+, CD23+,
or CD3+ cells in the lymphocyte sample prior to its exposure to either SACI or LPS, and the Ab production efficiency, defined
as the ratio of the number of Ab producing wells to the total number of proliferating wells. For lymphocytes exposed to LPS,
however, a strong correlation (r = 0.931, p = 0.02) was observed between the Ab production efficiency and the ratio of CD8+
to CD3+ cells (CD8/CD3) in the original sample at least within the ranges studied (CD8/CD3 = 0.216–0.288). For those exposed
to SACI, no correlation was found between the Ab production efficiency and the CD8/CD3 ratio (r = 0.881, p = 0.12) or the
proportion of CD8+ cells (r = 0.808, p = 0.19) in the original sample. These results suggest that the repertoire of B cells
responsive to LPS is different at least in part from the repertoire responsive to SACI and that the ratio CD8/CD3 could serve
as a practical predictor for Ab production by human lymphocytes stimulated with LPS.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
997.
H. Xiong C. Li E. Garami Y. Wang M. Ramjeesingh K. Galley C.E. Bear 《The Journal of membrane biology》1999,167(3):215-221
ClC-2 belongs to a large family of chloride channels and its expression in certain cell types is associated with the appearance
of swelling-activated chloride (Cl−) currents. In the present report, we examined the hypothesis that ClC-2 plays a role in regulatory volume decrease by expressing
ClC-2 in Sf9 cells using the baculovirus system. First, we showed that ClC-2 protein expression is associated with appearance
of a Cl− conductance which is activated by hypo-osmotic shock and can be distinguished from swelling-activated chloride currents endogenous
to Sf9 cells on the basis of its pharmacology and specific inhibition by an anti-ClC-2 antibody. Second, we show that the
rate of regulatory volume decrease is significantly enhanced in Sf9 cells expressing ClC-2 protein. Hence, our data support
the hypothesis that ClC-2 is capable of mediating regulatory volume decrease.
Received: 12 August/Revised: 23 October 1998 相似文献
998.
目的:构建及筛选高效表达原创性全人源抗人Ig E单克隆抗体的重组工程细胞株。方法:将采用核糖体展示技术筛选到的原创性全人源抗人Ig E单链抗体(sc Fv)基因改构设计为Ig G1κ型全长抗体,构建重组真核表达质粒并电转染CHO-S细胞,Dot-blot法选取多株高表达克隆进行40ml摇瓶批次培养,再据细胞生长特征及抗体表达量选取高表达克隆进行40ml摇瓶及3L摇瓶流加培养研究,选取候选细胞株并对改构前后抗体的生物学活性进行比较研究。结果:成功构建了p MH3-H、p MH3-L、p CApuro-H、p CApuro-L四种重组真核表达质粒并成功共转染CHO-S细胞。完成了4次电转染8轮细胞克隆筛选,获得两株表达量较高的候选克隆Mab1#和Mab2#,在3L摇瓶流加培养中抗体表达量分别达到470mg/L及499mg/L。生物膜光干涉技术(Bio-Layer Interferometry,BLI)亲和力结果显示Mab1#及Mab2#两株单抗亲和力均达到nmol/L级(10-9),与现有唯一上市的抗人Ig E单抗药物奥马珠单抗(Omalizumab)的亲和力相当。选取Mab1#全长抗体与其改构前的母本单链抗体的表面等离子共振技术(surface plasmon resonance,SPR)中和活性比较结果显示Mab1#抑制h Ig E与FcεRI结合的EC50为3nmol/L,EC90为9nmol/L,较改造前亲和力提高了4.3倍,中和活性(EC50)提高了23.7倍,中和活性(EC90)提高了41.3倍。结论:成功将表达原创性全人源抗人Ig E的单链抗体(约25k Da)改造为亲和力及中和活性均大幅提升的全长抗体(约150k Da),获得2个候选细胞株。 相似文献
999.
Kristyna Blakov Jana Beranov Martin Hradilek Libor Kostka Vladimír ubr Tom Etrych Pavel cha Jan Konvalinka 《The Journal of biological chemistry》2021,297(5)
Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design. 相似文献
1000.