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Background: Even though PD-1/PD-L1 is an identified key "don't find me" signal to active adaptive immune system for cancer treatment, the overall response rate (ORR) for all cancer patients is still limited. Other effective therapeutic modalities to bridge the innate and adaptive immunity to improve ORR are urgently needed. Recently, CD47/SIRPα interaction is confirmed as a critical "don't eat me" signal to active innate immunity. However, the red blood cell (RBC) toxicity is the big concern for the development of CD47-based anti-cancer therapeutics. Methods: Here, we report the development of a CD47/PD-L1 bispecific antibody 6MW3211 to block both PD-1/PD-L1 and CD47/SIRPα signals, and studied the effects of 6MW3211 on anti-tumor immune functions in vitro and in vivo. The pharmacokinetic and toxicity profiles of 6MW3211 were evaluated in GLP non-human primate (NHP) studies. Results: The dual immune checkpoint inhibitory signaling blocker 6MW3211 shows high binding affinity to PD-L1 and low binding affinity to CD47. This inequivalent binding affinity design makes 6MW3211 preferentially bound to PD-L1 on tumor cells followed by disrupting the interaction of CD47/SIRPα. Complex structure determination and flow cytometry assay demonstrated that 6MW3211 has no binding to either human or rhesus monkey RBCs. 6MW3211 effectively blocked both PD-1/DP-L1 and CD47/SIRPα signaling and promoted macrophage phagocytosis of tumor cells. Potent therapeutic efficacies of 6MW3211 in three different mouse models were further observed. Moreover, 6MW3211 was demonstrated to have a fairly good safety profile in a GLP NHP study. In addition, multiplex fluorescent immunohistochemistry (mIHC) staining shows that PD-L1 and CD47 co-express on several different types of human tumor tissues. Conclusions: These results support the development of 6MW3211 for the treatment of PD-L1 and CD47 double positive cancers.
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Anticorpos Biespecíficos , Neoplasias , Animais , Camundongos , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Antígeno CD47/metabolismo , Antígeno B7-H1 , Receptor de Morte Celular Programada 1/uso terapêutico , Fagocitose , Neoplasias/patologia , Imunoterapia/métodos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêuticoRESUMO
Residues of neonicotinoid pesticides have potential risks to food, environmental and biological safety. In this study, the hapten toward imidacloprid was adopted to gain antibodies. After molecular modeling, the electrostatic potentials of eight commonly-used neonicotinoid pesticides were individually calculated to analyze the structural similarity. Two representative compounds (imidacloprid and acetamiprid) with moderate similarity were rationally selected for hybridoma screening. Using this strategy, four clones of broad-specific monoclonal antibodies (mAbs) against multiple neonicotinoids were obtained, and the clone 6F11 exhibited the broadest spectrum to six neonicotinoid pesticides and two metabolites, with half-maximal inhibitory concentrations (IC50) ranging from 0.20 to 5.92 ng/mL. Then, the novel antibody gene was sequenced and successfully expressed in full-length IgG form using mammalian cells. Based on the sensitive recombinant antibody, a gold lateral-flow immunosensing strip assay was developed and it was qualified for rapid detection of imidacloprid, clothianidin or imidaclothiz residues in food samples.
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Inseticidas , Animais , Anticorpos Monoclonais , Ouro , Haptenos , Imunoglobulina G , Inseticidas/análise , Mamíferos , Neonicotinoides , NitrocompostosRESUMO
The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Arabidopsis/química , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestrutura , Transporte Biológico/efeitos dos fármacos , Microscopia Crioeletrônica , Ácidos Indolacéticos/química , Ácidos Indolacéticos/metabolismo , Ftalimidas/química , Ftalimidas/farmacologia , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, several variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and have consistently replaced the previous dominant variant. Therapeutics against variants of SARS-CoV-2 are urgently needed. Ideal SARS-CoV-2 therapeutic antibodies would have high potency in viral neutralization against several emerging variants. Neutralization antibodies targeting SARS-CoV-2 could provide immediate protection after SARS-CoV-2 infection, especially for the most vulnerable populations. In this work, we comprehensively characterize the breadth and efficacy of SARS-CoV-2 RBD-targeting fully human monoclonal antibody (mAb) MW3321. MW3321 retains full neutralization activity to all tested 12 variants that have arisen in the human population, which are assigned as VOC (Variants of Concern) and VOI (Variants of Interest) due to their impacts on public health. Escape mutation experiments using replicating SARS-CoV-2 pseudovirus show that escape mutants were not generated until passage 6 for MW3321, which is much more resistant to escape mutation compared with another clinical staged SARS-CoV-2 neutralizing mAb MW3311. MW3321 could effectively reduce viral burden in hACE2-transgenic mice challenged with either wild-type or Delta SARS-CoV-2 strains through viral neutralization and Fc-mediated effector functions. Moreover, MW3321 exhibits a typical hIgG1 pharmacokinetic and safety profile in cynomolgus monkeys. These data support the development of MW3321 as a monotherapy or cocktail against SARS-CoV-2-related diseases.
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The toxicity of clothianidin to non-target organisms has gradually attracted world-wide attention. It is essential to develop reliable methods for the on-site detection of clothianidin residue. In this study, analogue-based heterologous ic-ELISAs were designed to rapidly screen desirable hybridomas, which could be used for the construction of recombinant antibodies (RAbs) against clothianidin. Based on the antibody variable region genes, two full-length IgG RAbs (1F7-RAb and 5C3-RAb) were produced by the mammalian cell expression system. The performance of the two RAbs was characterized and compared by heterologous ic-ELISAs and non-competitive surface plasmon resonance (SPR) assays. Using heterologous ic-ELISAs, the 1F7-RAb exhibited highly specific and sensitive recognition to clothianidin with an IC50 of 4.62 µg/L, whereas the 5C3-RAb could bind to both clothianidin and dinotefuran. The results of the non-competitive SPR assay further verified that the 1F7-RAb had a higher specificity and affinity to clothianidin than the 5C3-RAb. Finally, a gold immunochromatographic assay based on the novel antibody, 1F7-RAb, was developed for rapid detection of clothianidin with high sensitivity (visual detection limit of 2.5 µg/L), specificity, and good reproducibility, which can be used as an effective supervision tool for clothianidin residue in agricultural and environmental samples.
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Imunoglobulina G , Tiazóis , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Guanidinas , Imunoensaio/métodos , Mamíferos , Neonicotinoides , Reprodutibilidade dos Testes , Tiazóis/análiseRESUMO
Understanding the underlying molecular mechanisms behind ADE of SARS-CoV-2 is critical for development of safe and effective therapies. Here, we report that two neutralizing mAbs, MW01 and MW05, could enhance the infection of SARS-CoV-2 pseudovirus on FcγRIIB-expressing B cells. X-ray crystal structure determination and S trimer-binding modeling showed that MW01 and MW05 could bind to RBDs in S trimer with both "up" and "down" states. While, the neutralizing mAb MW07, which has no ADE activity only binds to RBD in S trimer with "up" state. Monovalent MW01 and MW05 completely diminished the ADE activity compared with their bivalent counterparts. Moreover, both macropinocytosis and endocytosis are confirmed involving in ADE of SARS-CoV-2 pseudoviral infection. Blocking endosome transportation and lysosome acidification could inhibit the ADE activity mediated by MW05. Together, our results identified a novel ADE mechanism of SARS-CoV-2 pseudovirus in vitro, FcγRIIB-mediated uptake of SARS-CoV-2/mAb complex with bivalent interaction.
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Anticorpos Facilitadores , COVID-19 , Anticorpos Antivirais , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
Ionic polymer-metal composites (IPMC), one of the most popular materials in the field of artificial muscle research, have attracted much attention because of their high flexibility, low drive voltage (<10 V), high force density, large deformation, and so forth. However, the results show that the serious electrode fatigue crack and water loss of traditional IPMC greatly decrease its fatigue life and limit the practical application. In this study, we developed a novel liquid metal composite electrode. A layer of eutectic gallium-indium alloy (EGaIn) liquid metal was applied to the surface of the platinum electrode of the IPMC using a mask. Because of the good self-healing performance of the liquid metal, it is expected to solve the above problems of resistance increase and water loss caused by cracks. It turns out that the prepared EGaIn/Pt-IPMC exhibits a driving force up to 120 mN and maximum fatigue life of about 25,000 s at a driving voltage of 3 V. Compared with the best work reported, the fatigue strength of EGaIn/Pt-IPMC was increased by about 210%, and the maximum driving force of EGaIn/Pt-IPMC prepared by a single-layer basement membrane was between the IPMC prepared by 4-6 layer basement membrane. The electromechanical properties were significantly improved, and it is expected to realize a series of bionic applications.
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The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Anticorpos Facilitadores , COVID-19/terapia , Sequência Conservada , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Pandemias , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Tratamento Farmacológico da COVID-19RESUMO
Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, with SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with FcγRIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity is eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Potent prophylactic and therapeutic effects against SARS-CoV-2 are observed in rhesus monkeys. A single dose of MW05/LALA blocks infection of SARS-CoV-2 in prophylactic treatment and clears SARS-CoV-2 in three days in a therapeutic treatment setting. These results pave the way for the development of MW05/LALA as an antiviral strategy for COVID-19.
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Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Antivirais/imunologia , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Pandemias/prevenção & controle , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/prevenção & controle , Receptores de IgG/genética , Receptores de IgG/imunologia , Receptores Virais/metabolismo , SARS-CoV-2 , Células Vero , Ligação ViralRESUMO
Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 µg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/química , Região Variável de Imunoglobulina/genética , Inseticidas/imunologia , Neonicotinoides/imunologia , Tiazinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Cromatografia Líquida , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridomas/metabolismo , Ligação de Hidrogênio , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Espectrometria de Massas em TandemRESUMO
Programmed cell death 1 (PD-1) is inhibitory receptor and immune checkpoint protein. Blocking the interaction of PD-1 and its ligands PD-L1/ L2 is able to active T-cell-mediated antitumor response. Monoclonal antibody-based drugs targeting PD-1 pathway have exhibited great promise in cancer therapy. Here we show that MW11-h317, an anti-PD-1 monoclonal antibody, displays high affinity for PD-1 and blocks PD-1 interactions with PD-L1/L2. MW11-h317 can effectively induce T-cell-mediated immune response and inhibit tumor growth in mouse model. Crystal structure of PD-1/MW11-h317 Fab complex reveals that both the loops and glycosylation of PD-1 are involved in recognition and binding, in which Asn58 glycosylation plays a critical role. The unique glycan epitope in PD-1 to MW11-h317 is different from the first two approved clinical PD-1 antibodies, nivolumab and pembrolizumab. These results suggest MW11-h317 as a therapeutic monoclonal antibody of PD-1 glycosylation-targeting which may become efficient alternative for cancer therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/química , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Antineoplásicos Imunológicos/química , Asparagina/metabolismo , Antígeno B7-H1/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Epitopos/química , Feminino , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/tratamento farmacológico , Nivolumabe/química , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Domínios e Motivos de Interação entre ProteínasRESUMO
A flexible filter paper based composite electrode was prepared via the convenient one-step synthesis of silver doped graphene for the first time, followed by in-situ polymerization of aniline monomers. Using L-ascorbic acid for simultaneous reduction of grapheme oxide and silver nitrate, we provided a new and green method to prepare graphene hybrid sheets without toxicity. It was found that the as-fabricated hybrid electrode formed a three-dimensional porous architecture, which not only increased the specific surface area of composite, but also facilitated the ion diffusion of the electrolyte. In addition, according to the tests of electrochemical performances, the flexible hybrid electrode subsequently exhibited exceptional specific capacitance of 437.3 F/g, energy density of 1133.5 W·h/kg and power density of 88.8 kW/kg, respectively. Meanwhile, the as-prepared hybrid demonstrated a good cycling stability with only 10.99% specific capacitance deterioration after 5000 times of cycling. This preparation technology presented here shows great potential for the development and application of wearable and portable energy storage devices, particularly for flexible supercapacitors. Moreover, this study puts forward a general, simple and low-cost route of fabricating a novel flexible electrode on a large scale, eventually for environmental protection.
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An asymmetric supercapacitor was prepared on a sheet of filter paper with two modified surfaces acting as electrodes in 1 M potassium hydroxide aqueous solution. By choosing carbon nanotubes and two different kinds of metal oxides (zinc oxide and ferro ferric oxide) as electrode materials, the asymmetric supercapacitor was successfully fabricated. The results showed that this device exhibited a wide potential window of 1.8 V and significantly improved electrochemical performances of its counterparts. Particularly, the one-sheet asymmetric supercapacitor demonstrated high energy density of 116.11 W h/kg and power density 27.48 kW/kg, which was attributed to the combined action and shortened distance between the two electrodes, respectively. Besides, it showed superior electrochemical cycling stability with 87.1% capacitance retention under room temperature. These outstanding results can not only give researchers new insights into compact energy storage systems, but they also provide a good prospect for flexible asymmetric supercapacitors.
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In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71 ng mL-1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10-9 M and 4.26 × 10-9 M for parathion, 6.75 × 10-9 M and 4.17 × 10-9 M for methyl-parathion, and 2.44 × 10-8 M and 1.19 × 10-8 M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. Graphical abstract á .
Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos , Técnicas Biossensoriais/métodos , Compostos Organofosforados/química , Praguicidas/química , Ressonância de Plasmônio de Superfície/métodos , Animais , Líquido Ascítico , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Haptenos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Água/químicaRESUMO
Neonicotinoid insecticides are commonly used for pest control on tea plantations as a result of their broad-spectrum activity. However, neonicotinoid residues released from tea leaves into tea infusions pose a dietary risk to consumers. Therefore, a rapid, sensitive, and reliable on-site detection method for neonicotinoids is needed. We developed a quantum-dot-based fluorescent lateral flow immunochromatographic strip (LFICS) combined with a broad-specific antibody for detection of typical neonicotinoids (imidacloprid, imidaclothiz, and clothianidin), with sensitivities [50% inhibitory concentration (IC50)] of 0.104-0.33 ng/mL and visual detection limits of 0.5-1 ng/mL. The strip assay could be completed in less than 30 min. Using the LFICS to analyze spiked tea samples (green tea, black tea, and oolong tea), the average recovery of the three neonicotinoids ranged between 71 and 111%, with the coefficient of variation below 12%. The results from the LFICS tests for field samples were consistent with results from ultraperformance liquid chromatography-tandem mass spectrometry. The newly developed strip is a useful tool for the on-site detection of neonicotinoid residues in tea.
Assuntos
Imunoensaio/métodos , Inseticidas/análise , Neonicotinoides/análise , Resíduos de Praguicidas/análise , Pontos Quânticos/química , Chá/química , Camellia sinensis/química , Imunoensaio/instrumentação , Limite de DetecçãoRESUMO
BACKGROUND: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution. RESULTS: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826. CONCLUSIONS: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.
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Cromossomos Artificiais Bacterianos/genética , Genes MHC da Classe II/genética , Mapeamento Físico do Cromossomo/métodos , Animais , Bovinos , OvinosRESUMO
Previous DNA sequencing of BAC clones covering entire ovine MHC (OLA) region resulted in identification of approximately 130 functional genes in the region, of which 8 were predicted by computer software to be exclusively existed in sheep, but not in any other species known to date. In the present study, we successfully identified and cloned cDNA sequence of OaN2, OaN5, and OaN6 from representative sheep tissues, confirmed their existence in reality. The sequences obtained experimentally exactly identical to those predicted previously. The length of cDNA fragments for OaN2, OaN5, and OaN6 was 270 bp, 309 bp, and 205 bp, respectively, with GenBank accession number assigned as JF330782 (OaN2), JF330783 (OaN5), and JF330784 (OaN6). Northern analyses indicated that the mRNA transcripts of OaN2 were mainly seen in ovine mesenteric lymph nodes and spleen, while OaN5 was observed in only in mesenteric lymph nodes. In contrast, OaN6 transcripts were detected in all tissues except for liver and heart. Western blot showed that OaN2 protein expression level was detected in mesenteric lymph nodes, spleen, and liver, essentially consistent with that of mRNA transcripts. Immunohistochemistry analysis showed that OaN2 protein was highly expressed in ovine mesenteric lymph nodes, moderately expressed in, and not expressed in heart, liver, and pancreas, consistent with the results of Western blotting. The cloning and expression analysis of 3 novel genes provide a basis for revealing their specificities and would be helpful to further study of their expression profile and their potential functions.