麦迪霉素3-O-酰基转移酶在螺旋霉素链霉菌F21中酰化特性

螺旋霉素(SP)与麦迪霉素(MD)均为16元环大环内酯类抗生素, 并且结构非常相似。螺旋霉素含有3个组分,其结构差异表现在16元内酯环C3上的一个取代基的差异, SP I组分为羟基、SP II组分羟基乙酰化、SP III组分羟基丙酰化; 麦迪霉素是以麦迪霉素A1为主要组分的多组分抗生素, 麦迪霉素16元内酯环C3上连接的均为丙酰化羟基。已知这类抗生素16元内酯环C3羟基酰化是由一种称为3-O-酰基转移酶的蛋白催化完成。本研究将螺旋霉素产生菌—Streptomyces spiramyceticus F21中的螺旋霉素3-O-酰基转移酶基因用Streptomyces mycarofaciens ATCC 21454中的麦迪霉素3-O-酰基转移酶基因原位替换后, 发现所产生的螺旋霉素仍然含有3个组分, 并且螺旋霉素III组分也不是主要组分, 说明麦迪霉素3-O-酰基转移酶在螺旋霉素产生菌—S. spiramyceticus F21中不具有16元内酯环C3羟基丙酰化特异性以及酰化高效性, 也提示其在麦迪霉素产生菌中的丙酰化特异性和高效性可能与该菌株(种)的特性有关。     

麦迪霉素3-O-酰基转移酶在螺旋霉素链霉菌F21中的酰化特性

螺旋霉素(SP)与麦迪霉素(MD)均为16元大环内酯类抗生素,并且结构非常相似.螺旋霉素含有3个组分,其结构差异表现在16元内酯环C<,3>上的一个取代基的差异,SPⅠ组分为羟基、SPⅡ组分羟基乙酰化、APⅢ组分羟基丙酰化;麦迪霉素是以麦迪霉素A1为主要组分的多组分抗生素,麦迪霉素16元内酯环C3上连接的均为丙酰化羟基.已知这类抗生素16元内酯环C3羟基酰化是由一种称为3-O-酰基转移酶的蛋白催化完成.本研究将螺旋霉素产生菌-Streptomycesspiramyceticus F21中的螺旋霉素3-O-酰基转移酶基因用Streptomyces mycarofaciens ATCC 21454中的麦迪霉素3-O-酰基转移酶基因原位替换后,发现所产生的螺旋霉素仍然含有3个组分,并且螺旋霉素Ⅲ组分也不是主要组分,说明麦迪霉素3-O-酰基转移酶在螺旋霉素产生菌-S.spiramyceticus F21中不具有16元内酯环C3羟基丙酰化特异性以及酰化高效性,也提示其在麦迪霉素产生菌中的丙酰化特异性和高效性可能与该菌株(种)的特性有关.     

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒（HTNV）核衣壳蛋白（NP）B细胞抗原表位。以抗HTNV NP单克隆抗体(mAb）5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库。阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV76－118株S基因进行同源性分析。结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制。10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76－118株NP氨基端的aa25-33一致。证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定。     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoVN蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Westernblotting和间接免疫荧光方法分别检测了4株抗SARS-CoVN蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoVN蛋白对上述4种单克隆抗体的识别表位进行了初步定位。结果显示:(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoVN蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoVN蛋白的aa30-60,1-1C2针对的抗原表位则位于SARS-CoVN蛋白的aa170-184。这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料。     

抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoV N蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Western blotting和间接免疫荧光方法分别检测了4株抗SARS-CoV N蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoV N蛋白对上述4种单克隆抗体的识别表位进行了初步定位.结果显示(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoV N蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoV N蛋白的aa 30-60,1-1C2针对的抗原表位则位于SARS-CoV N蛋白的aa 170-184.这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料.     

应用噬菌体展示随机肽库淘筛mAb5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒(HTNV)核衣壳蛋白(NP)B细胞抗原表位.以抗HTNV NP单克隆抗体(mAb)5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库.阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV 76～118株S基因进行同源性分析.结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制.10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76～118株NP氨基端的aa25～33一致.证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定.     

Molecular analysis of cross-reactive human monoclonal antibody AE6F4 generated by in vitro immunization: Epitope mapping of AE6F4 antibody on 14-3-3 family proteins and cytokeratin 8

We reported previously that adenocarcinoma-reactive human monoclonal antibody AE6F4, which had been generated by in vitro immunization method, recognizes both 14-3-3protein and cytokeratin 8 (CK8). In this study, to analyze the cross-reactivity of AE6F4 antibody, epitopes of AE6F4 antibody on 14-3-3 proteins and CK8 were studied by using synthetic linear peptide scanning technology. To determine the locations of B cell epitope, 48 and 95 of decapeptides covering the entire 14-3-3 proteins and CK8, respectively,were synthesized and binding to AE6F4 antibody was examined by ELISA. The AE6F4 antibody was strongly reactive to peptides containing amino acid sequences TLWTSDTQGD in 14-3-3 proteins and INFLRQLYEE in CK8. These results indicate that AE6F4 antibody can recognize the different peptide sequences in 14-3-3 proteins and CK8. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Mapping Epitope Structure and Activity: From One-Dimensional Prediction to Four-Dimensional Description of Antigenic Specificity

Our knowledge of antigenic specificity has greatly increased in recent years mainly through X-ray crystallographic studies of proteins and peptides complexed with monoclonal antibodies. However, our ability to predict the location of antigenic sites in proteins remains limited partly because prediction algorithms reduce the complexity of epitopes to one-dimensional, linear peptide models. Epitopes and paratopes are relational entities definable by their mutual complementarity and adaptation potential as well as by their activity. A complete account of antigenic specificity demands the integration of both structural and binding activity data that can be achieved only through a spatiotemporal four-dimensional analysis. Failure to include the fourth dimension, i.e., time, in the analysis of antigen–antibody complementarity amounts to considering proteins as rigid bodies and ignores the mutual adaptation that occurs when the two partners interact. Reducing four-dimensional protein systems to three-dimensional or two-dimensional representations inevitably distorts our perception of the dynamic nature of epitopes.     

Epitope Specificity of a Monoclonal Antibody Generated against the Dissociated CFA/I Fimbriae of Enterotoxigenic Escherichia coli

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide ³⁹TFESY⁴³, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The ³⁹TFESY⁴³ sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.     

马传染性贫血发病过程中细胞毒性T细胞的作用及其表位研究进展

马传染性贫血(Equine infectious anemia,EIA)是马属动物感染的一种主要经由虫媒传播的传染性疾病。该病的临床表征为反复发热、贫血、出血、水肿以及消瘦等,病理生理变化以血小板减少为主[1]。EIA的致病原为马传染性贫血病毒(Equine infectious anemia virus,EIAV),该病毒与人     

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.     

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2

Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3].¹ Troisi EM, Rockman ME, Nguyen PP, Oliver EE, Hines JK. Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE3-1]. Mol Microbiol 2015; 97:926-41; PMID:26031938; https://doi.org/10.1111/mmi.13076[Crossref], [PubMed], [Web of Science ®] [Google Scholar] [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation.     

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.     

脊髓灰质炎病毒C3表位和C—myc＋肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性,化学合成脊髓灰质炎病毒C3表位和C-myc十肽有位的基因片段,并插入到上述载体的相应位点间,采用全菌PCR筛选到正向插入的重组子,全细胞ELISA和电镜观察显示,重组蛋白以杂和菌毛的形式得到了表达,并保持CS3和外源位的抗原性,表明脊髓灰质炎病毒C3表位和C-myc十肽捕位在细菌表面得到了表达,证实载体pCSB136和pCSX72可用于外源表位的呈现,为构建基因工程活苗疫苗奠定了基础。     

脊髓灰质炎病毒C3表位和C-myc十肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性 ,化学合成脊髓灰质炎病毒C3表位和C myc十肽表位的基因片段 ,并插入到上述载体的相应位点间 ,采用全菌PCR筛选到正向插入的重组子 ,全细胞ELISA和电镜观察显示 ,重组蛋白以杂和菌毛的形式得到了表达 ,并保持CS3和外源表位的抗原性 ,表明脊髓灰质炎病毒C3表位和C myc十肽表位在细菌表面得到了表达 ,证实载体pCSB136和pCSX72可用于外源表位的呈现 ,为构建基因工程活菌疫苗奠定了基础     

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non‐human primate models

Antibody based manipulation of the CD137 (4‐1BB) co‐signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti‐human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti‐CD137 mAb (4B4‐1), recognized activated PBMCs from both human and non‐human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species (～95% identical), with the extracellular domain differing by only 9–10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co‐stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes. Copyright © 2009 John Wiley & Sons, Ltd.     

Plasmid Specificity of The Origin of Transfer of Sex Factor F

The ability of F-like plasmids to promote transfer from the F origin of transfer was determined. Chromosome transfer was measured from plasmid derivatives of RecA(-) Hfr deletion strains which had lost all the F transfer genes but which in some cases retained, and in others had also lost, the origin sequence. ColV2 and ColVBtrp could initiate transfer from the F origin, but R100-1, R1-19, and R538-1 drd could not. These results can be correlated with the plasmid specificity of the traI components of the different plasmid transfer systems, supporting the hypothesis that the origin of transfer is the site of action of the traI product. Most F-like plasmids, including R1-19 and R538-1 drd, could transfer ColE1, consistent with previous findings that the (plasmid-specific) traI product is not necessary for ColE1 transfer by Flac; ColE1 transfer may be initiated by a ColE1-or host-determined product. R100-1 and R136fin(-) could not transfer ColE1 efficiently, apparently because of differences residing in their pilus-forming genes.     

利用rBE3和rBE4系统进行水稻基因单碱基编辑的特异性和遗传稳定性分析

对基于rBE3(Rice base editor)和rBE4碱基编辑系统创制获得Os SERK1(D428N)和pi-ta(S918F)等基因的单碱基编辑突变体材料进行编辑特异性和遗传稳定性分析,旨在全面了解和更好地利用该碱基编辑系统。首先对Os SERK1(D428N)和pi-ta(S918F)sg RNA的潜在脱靶位点进行预测,并对T0代材料中的各脱靶位点进行PCR扩增和Sanger测序检测;同时对该两个基因的突变体材料自交获得的T1代植株的靶位点序列和外源T-DNA分离进行检测。结果显示各T0代材料均未检测到潜在脱靶位点发生单碱基编辑;此外,Os SERK1(D428N)和Os08g07774含有相同的sg RNA位点,且两个位点均能发生单碱基编辑;rBE3或rBE4系统介导产生的碱基编辑可稳定遗传至T1代,并在T1代可获得无外源T-DNA的纯合突变体。上述结果表明由rBE3或rBE4介导的碱基编辑具有较高的特异性,可进行多位点编辑,引入的碱基替换可稳定遗传至后代。