特异性吗啡抗原和单克隆抗体的制备

吗啡分子的不同部位与蛋白偶联诱导出的抗体的专一性差异很大。为减少交叉反应,提高抗体分子对游离吗啡的特异性识别,选择吗啡分子N位进行修饰,合成了半抗原降吗啡,并设计和合成不同的连接臂,将半抗原用不同的连接臂与不同的载体蛋白共价结合分别制备了免疫抗原和筛选抗原,经细胞融合和筛选,成功地获得了5株可分汔抗吗啡单克隆抗体的细胞株：28H10,29D5,36G3,42D5,43C4。     

Design and Synthesis of 6 α-Corticosteroid Haptens and Their Bovine Serum Albumin(BSA) Conjugates

The site of attachment of protein carrier to corticosteroids has great influence on the specificity of produced antibody.In order to obtain highly specific and accurate antibodies for bioimmunoassay determination of cortisol,different tether lengths of 60633－corticosteroid haptens and their BSA conjugates were designed and synthesized.     

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.     

Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification

In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.     

Chemical Modification of Chitosan for Gene Delivery

Abstract

Chitosan has been investigated as a non‐viral vector because it has several advantages such as biocompatibility, biodegradability, and low toxicity. However, low specificity and low transfection efficiency of chitosan as a DNA carrier need to be overcome prior to clinical trial. In this review paper, chemical modification of chitosan was tried in order to enhance cell specificity and transfection efficiency. Also, chemical modification of chitosan was performed to increase stability of chitosan/DNA complexes.     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

Mo-Co系加氢精制催化剂中TiO~2-Al~2O~3载体效应的PASCA研究

本文采用PASCA(化学分析正电子湮没谱)技术, 系统地研究了MoO~3/γ-Al~2O~3,CoO/γ-Al~2O~3, MoO~3/TiO~2-Al~2O~3, CoO/TiO~2-Al~2O~3等体系中活性组分的表面分散状态和化学状态及其变化规律, 揭示了用TiO~2改性Al~2O~3对Mo-Co系加氢精制催化剂所产生的载体效应的作用机理。Al~2O~3中引入TiO~2使得载体与MoO~3间的相互作用大大减弱, 导致分散态MoO~3易于还原和硫化。助剂CoO与TiO~2-Al~2O~3载体间存在极为显著的电子相互作用, 对于改善Co在活性相中的化学状态, 提高活性中心的内在活性具有重要的作用。PASCA研究的另一个重要结果是, 由于载体表面的不均匀性,使得上述负载型催化剂中氧化物活性组分的表面状态十分复杂, 存在与载体相互作用强弱程度不同的多类特征区, 这些特征区与内在活性不同的催化中心相关联. 通过TiO~2改性Al~2O~3载体, 有效地调节载体表面性质, 改变活性组分表面物种的分布及化学状态。     

Immobilized Laccase on a New Cryogel Carrier and Kinetics of Two Anthraquinone Derivatives Oxidation

A coordinatively immobilized laccase was prepared using a new cryogel type carrier. The support has a wide-pore texture facilitating diffusion of different substrates to the enzyme reaction center. The biocatalyst proved to be efficient in decolorization of two anthraquinone derivatives, namely Acid Blue 62 and bromaminic acid. After 24 h over 80% of the two substrates have been oxidated. The kinetic data (K (m) and V (max)) for the oxidation of the two anthraquinone derivatives, with the free and immobilized enzyme, have been determined and compared. Other parameters, like k (cat) and the specificity constant have been calculated and analyzed. The influence of substrate properties (hydrophobicity, polarity, etc.) has been discussed.     

Ag/TiO2聚丙烯负载膜的制备及其光催化与抗菌特性研究

以聚丙烯负载二氧化钛膜为载体,在一定浓度的AgNO3水溶液中,利用光还原在氧化钛膜表面还原Ag,制备载银二氧化钛负载膜,在室光和太阳光下,分别选择Ag/TiO2聚丙烯膜、TiO2聚丙烯膜和活性炭聚丙烯膜,研究膜的抗菌特性和光催化特性.结果表明:Ag/TiO2负载膜由于同时具有Ag和TiO2的双重效用,显示了良好的杀菌特性,无论是在室内还是太阳光下,都没有检测到活的微生物;在水溶液中检测Ag/TiO2负载膜和TiO2负载膜的光催化特性,发现由于银离子的催化活性中心的作用,光催化降解甲基橙的能力与TiO2负载膜相比,能够提高10%左右,显示了良好的光化学特性.     

One-pot chemo-enzymatic synthesis of reporter-modified proteins

To meet recent advancements in the covalent reporter labeling of proteins, we propose a flexible synthesis for reporter analogs. Here we demonstrate a one-pot chemo-enzymatic synthesis of reporter-labeled proteins that allows the covalent tethering of any amine-terminal fluorescent or affinity label to a carrier protein or fusion construct. This two-reaction sequence consists of activated panthothenate coupling, biosynthetic conversion to the coenzyme A (CoA) analog, and enzymatic carrier protein modification via phosphopantetheinyltransferase (PPTase). We also probe substrate specificity for CoAA, the first enzyme in the pathway. With this approach CoA analogs may be rapidly prepared, thus permitting the regiospecific attachment of reporter moieties from a variety of molecular species.     

Synthetic immunogen for the anti-relapse treatment of opioid dependence

A synthetic immunogen representing a complex of a conjugate of a macromolecular carrier (natural protein) and a hapten (morphine drug) covalently bound to poly(4-nitrophenyl acrylate) (PNPA) has been developed. The macromolecular carrier in the conjugate is a human serum protein (human gamma-globulin, HGG, or human serum albumin, HSA). The optimal design of the synthetic immunogen was developed. The epitope accessibility and specificity of the immunogen complexes were investigated by ELISA. It was established that antigenic determinants are not shielded upon binding to antibodies for complexes with the optimal (1: 10) ratio of the conjugate to the synthetic polymer. The accute toxicity of PNPA was estimated. The immunogenicity of synthetic complexes was studied in rat immunization models. An influence of the immunogen structure and vaccination dose on the ability to produce specific antibodies to morphine was found.     

Preparation of dry powder inhalation by surface treatment of lactose carrier particles

An attempt was made to produce carrier particles for dry powder inhalations by the surface treatment of lactose particles with aqueous ethanol solution. Drug/carrier powder mixtures were prepared consisting of lactose carriers with different particle surface properties and micronized salbutamol sulfate. These powder mixtures were aerosolized by Spinhaler, and in vitro deposition properties of salbutamol sulfate were evaluated by twin impinger. The degree of adhesion between drug particles and carrier particles was determined by the ultracentrifuge separation method. In addition, the air jet sieve method was used to evaluate characteristics of the separation of drug particles from carrier particles in airflow. The average adhesion force (F50) between the surface-treated lactose carrier and drug particles was significantly lower than that of powder mixed with the untreated lactose carrier, indicating that the degree of separation (T50) of drug particles from carrier particles was improved when surface-treated lactose carrier was used. This resulted in an improvement of in vitro inhalation properties.     

Application of micellar electrokinetic chromatography to pharmaceutical analysis

Electrokinetic chromatography is a new type of analytical separation method which belongs to the group of high performance capillary electrophoretic techniques but whose separation principle is based on that of chromatography. The solute distributes itself between a carrier and the surrounding medium. The carrier, which corresponds to the stationary phase in conventional chromatography, can be transported by electrophoresis with a different velocity from the surrounding medium. The separation is achieved by the differential solute distribution and the differential migration of the carrier. The charged molecules or charged molecular aggregates are employed as the carrier. Various kinds of carriers are available for electrokinetic chromatography along with different partition mechanisms. Among them, micellar electrokinetic chromatography, which employs an ionic micelle as a carrier, has become the most popular method because of its unique and attractive characteristics as well as the separating capability of electrically neutral or nonionic solutes in comparison with capillary zone electrophoresis. The present paper describes the principle, separation characteristics and its application to the analysis of pharmaceuticals.     

Biofunctionalization of polyelectrolyte microcapsules with biotinylated polyethylene glycol-grafted liposomes

Hollow polyelectrolyte microcapsules (PEMC) are prepared using layer-by-layer self-assembly of polyelectrolytes on melamine formaldehyde templates, followed by template dissolution, and subsequent coating with biotinylated polyethylene glycol-grafted liposomes. These potential site-specific carrier systems show a high specificity for NeutrAvidin binding and a strong resistance against unspecific protein binding. It is concluded that this design with NeutrAvidin as the outermost layer of such capsules provides an ideal platform for the biofunctionalization of PEMC as drug delivery systems or as artificial cell-like structures for biomimetic studies.     

Structure-based dissociation of a type I polyketide synthase module

Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.     

Selective Uptake of Cylindrical Poly(2‐Oxazoline) Brush‐AntiDEC205 Antibody‐OVA Antigen Conjugates into DEC‐Positive Dendritic Cells and Subsequent T‐Cell Activation

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well‐defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly‐2‐oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti‐DEC205 antibody to the periphery of the cylindrical brush polymer, antibody‐mediated specific binding and uptake into DEC205⁺‐positive mouse bone marrow‐derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205^? negative BMDC and non‐DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen‐specific T‐cell stimulatory capacity of pretreated BMDC than application of antigen or polymer–antigen conjugate.     

Highly sensitive detection of protein and small molecules based on aptamer-modified electrochemiluminescence nanoprobe

Amplified optical detection of biomolecules using nanoparticle as the carrier has attracted considerable interest in the scientific community. In this study, a promising aptasensor was developed for highly sensitive detection of protein and small molecules based on the construction of aptamer-modified electrochemiluminescence (ECL) nanoprobe. Specifically, thrombin and ATP serve as the examples for detection. By taking advantage of sandwich binding of two affinity aptamers for high specificity, tris-(2,2'-bipyridyl)ruthenium (TBR)-cysteamine loaded in gold nanoparticle (GNP) as barcodes for signal amplification, and micromagnetic particles (MMPs) based ECL technology for rapid detection, a novel assay for biomolecules quantification was developed. The sandwich complex containing targets could be selectively captured by MMPs and then quantified by ECL intensity. We have demonstrated that the detection limits of human thrombin and ATP are 1 pM and 10 pM, respectively, with high specificity. The proposed technology is expected to become a powerful tool for biomolecule analysis.     

Effect of surface covering of lactose carrier particles on dry powder inhalation properties of salbutamol sulfate

The effect of the surface covering of lactose carrier particles on the dry powder inhalation properties of salbutamol sulfate was investigated. Lactose carrier surfaces were covered with sucrose tristearate (J-1803F) by a high-speed elliptical-rotor-type powder mixer (Theta-Composer). In the present study, drug/carrier powder mixtures were prepared consisting of micronized salbutamol sulfate and lactose carriers with various particle surface conditions prepared by surface covering. These powder mixtures were aerosolized by a Jethaler), and the in vitro inhalation properties of salbutamol sulfate were evaluated by a twin impinger. Compared with the powder mixed with uncovered lactose carrier, the in vitro inhalation properties of the powder mixture prepared using the surface covering lactose carrier were significantly different, showing that the in vitro inhalation properties of salbutamol sulfate were improved. In vitro inhalation properties increased with the percentage of J-1803F added. Using this surface covering system would thus be valuable for increasing the inhalation properties of dry powder inhalation with lactose carrier particles.     

Rational Design of Biomolecules/Polymer Hybrids by Reversible Deactivation Radical Polymerization (RDRP) for Biomedical Applications

Hybrids, produced by hybridization of proteins, peptides, DNA, and other new biomolecules with polymers, often have unique functional properties. These properties, such as biocompatibility, stability and specificity, lead to various smart biomaterials. This review mainly introduces biomolecule-polymer hybrid materials by reversible deactivation radical polymerization(RDRP), emphasizing reverse addition-fragmentation chain transfer(RAFT) polymerization, and nitroxide mediated polymerization(NMP). It includes the methods of RDRP to improve the biocompatibility of biomedical materials and organisms by surface modification. The key to the current synthesis of biomolecule-polymer hybrids is to control polymerization. Besides, this review describes several different kinds of biomolecule-polymer hybrid materials and their applications in the biomedical field. These progresses provide ideas for the investigation of biodegradable and highly bioactive biomedical soft tissue materials. The research hotspots of nanotechnology in biomedical fields are controlled drug release materials and gene therapy carrier materials. Research showed that RDRP method could improve the therapeutic effect and reduce the dosage and side effects of the drug.Specifically, by means of RDRP, the original materials can be modified to develop intelligent polymer materials as membrane materials with selective permeability and surface modification.     

Effect of surface layering time of lactose carrier particles on dry powder inhalation properties of salbutamol sulfate

The effect of the surface layering time of lactose carrier particles on the dry powder inhalation properties of salbutamol sulfate was investigated. Lactose carrier particles were layered with vegetable magnesium stearate by physical mixing. In the present study, drug/carrier powder mixtures were designed consisting of micronized salbutamol sulfate and lactose carriers with various particle surface conditions prepared by surface layering. These powder mixtures were aerosolized by a Jethaler, and the in vitro deposition properties of salbutamol sulfate were evaluated by a twin impinger. Compared with the powder mixed with unlayered lactose carrier, the in vitro inhalation properties of the powder mixture prepared using the surface layering lactose carrier were significantly different, showing that the in vitro inhalation properties of the drug/carrier powder mixtures were improved. In vitro deposition properties (RP) increased with surface layering time. Using this surface layering system would thus be valuable for increasing the inhalation properties of dry powder inhalation.