Antiidiotypic antibodies against anti-cGMP polyclonal antibodies.

Affinity-purified polyclonal anti-cGMP antibodies were obtained from rabbit serum after immunization by succinyl derivative of cGMP coupled to bovine serum albumin. These antibodies were used to raise antiidiotypic antibodies in rats. Putative antiidiotypic serum inhibited the binding of [3H]cGMP to affinity-purified anti-cGMP antibodies. The influence of immunoglobulins isolated from antiidiotypic serum on the ion conductance of rod outer segment plasma membrane fragments from frog retina was studied in patch-clamp experiments. These immunoglobulins increased the conductance of ion channels acting like a natural agonist (cGMP). Preimmune immunoglobulins did not act. The data obtained suggest that antiidiotypic antibodies interact with regulatory cGMP-binding sites of the plasma membrane channels.     

Anti-idiotypic and natural catalytically active antibodies]

The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.     

研究报告：金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果。ELISA的本质是抗原与相应抗体之间的特异性相互作用。然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果。本课题组先前应用构象工程方法开发了一种基于金纳米粒子的人工抗体(简称金抗体)。金抗体可以像天然抗体一样特异性地与抗原相互作用,并且具备远优于天然抗体的稳定性。出色的稳定性使金抗体可能成为天然抗体更好的替代物,用于ELISA中。方法经过必要的优化并与辣根过氧化物酶(HRP)耦联后,制得酶标金抗体10HRP-(Au-400P1),然后用酶标金抗体代替天然酶标抗体用于ELISA检测中。结果通过一系列的实验证明,抗溶菌酶金抗体可用于ELISA特异性检测1～16 mg/L范围内的鸡蛋清溶菌酶(HEWL)样品。结论金抗体可以替代天然抗体用于ELISA检测,并具有优于传统ELISA法的检测准确性和一致性。     

Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies

Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.     

林下参内生生防细菌的分离鉴定及定殖能力

【背景】多年生林下参在自然环境下生长多年,其体内存在的内生菌具有更强的适应性和定殖性,可以提高植物自身抗性,抑制病原菌的生长,更好地发挥与植物的互作。【目的】筛选定殖能力强、繁殖能力快且对病原菌具有拮抗作用的优势菌株。【方法】采用常规组织分离方法,从健康林下参根部组织中分离内生菌,通过对峙试验筛选出对人参病原菌有拮抗作用的内生细菌并对其以传统的鉴定方法进行鉴定。【结果】在得到的6株内生细菌中,菌株LXS-N2对人参立枯病病原菌、人参猝倒病病原菌均有明显抑菌性,而且具有定殖性好、繁殖快的特点,通过破坏病原真菌细胞壁和细胞膜以及改变菌丝形态从而抑制病原真菌生长。【结论】经形态学观察、生理生化反应及16S rRNA基因序列分析鉴定内生菌LXS-N2为贝莱斯芽孢杆菌,具有良好的应用开发潜力。     

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.     

The clinical significance of HAMA in patients treated with mouse monoclonal antibodies

Twenty-four patients were analyzed for the development of HAMA (human antimouse antibodies) after being treated with repeated doses (200–500 mg) of the mouse monoclonal antibody (MAb) 17-1A. All patients developed anti-17-1A IgG antibodies, and most of them also developed IgM antibodies. In only two patients could immune complexes be demonstrated. Allergic reactions were rare (1.9%). In an extended study, a further 19 patient were analyzed for an idiotypic response. Forty-one out of 43 patients developed antiidiotypic antibodies (ab₂), and 20 of these also antianti-idiotypic antibodies (ab₃). Ab₃ ⁺ patients responded significantly better (p=0.01) and survived longer (p<0.001) compared to ab₃ ⁻ patients. In this study, we showed that MAb 17-1A could be repeatedly given on a safe basis. The development of high titers of HAMA did not cause significant clinical problems when further repeated infusions of MAb 17-1A were given. The development of an idiotypic response also indicate that the induction of HAMA might be beneficial and not harmful to the patient.     

Cardamonin inhibits LPS-induced inflammatory responses and prevents acute lung injury by targeting myeloid differentiation factor 2

BackgroundAcute lung injury (ALI) is a systemic inflammatory process, which has no pharmacological therapy in clinic. Accumulating evidence has demonstrated that natural compounds from herbs have potent anti-inflammatory efficacy in several disease models, which could be the potential candidates for the treatment of ALI.Hypothesis/PurposeAnti-inflammatory screening from natural product bank may provide new anti-inflammatory compounds for therapeutic target discovery and ALI treatment.Methods165 natural compounds were screened for their anti-inflammatory activity in LPS-stimulated macrophages. PCR array, SPR and ELISA were used to determine the potential target of the most active compound, Cardamonin (CAR). The pharmacological effect of CAR was further evaluated in both LPS-stimulated macrophages and ALI mice model.ResultsOut of the screened 165 compounds, CAR significantly inhibited LPS-induced inflammatory cytokine secretion in macrophages. We further showed that CAR significantly inhibited NF-κB and JNK signaling activation, and thereby inflammatory cytokine production via directly interacting with MD2 in vitro. In vivo, our data show that CAR treatment inhibited LPS-induced lung damage, systemic inflammatory cytokine production, and reduced macrophage infiltration in the lungs, accompanied with reduced TLR4/MD2 complex in lung tissues, Treatment with CAR also dose-dependently increased survival in the septic mice induced by DH5α bacterial infection.ConclusionWe demonstrate that a natural product, CAR, attenuates LPS-induced lung injury and sepsis by inhibiting inflammation via interacting with MD2, leading to the inactivation of the TLR4/MD2-MyD88-MAPK/NF-κB pathway.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

杆状病毒凋亡抑制基因IAP1促进家蚕CyclinB的核内积累

[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。     

Idiotypic network regulations of immune responses to HLA

The development of antiidiotypic autoimmunity with respect to HLA alloantigens provides an attractive explanation for the phenomenon of maternal tolerance to the fetus and for the tandem selection of two antagonistic traits, major histocompatibility complex polymorphism and alloreactivity. We have demonstrated that both T and B lymphocyte responses to allogeneic HLA antigens are subjected to feedback regulation by autologous antiidiotypic immunity. Idiotypic receptors for the alloantigen expressed by T lymphocytes induce antiidiotypic antibodies that are readily detectable in serum during pregnancy, and antiidiotypic T cells that can be revealed in the autologous mixed lymphocyte culture system. Such antiidiotypic T cells and antibodies inhibit specifically the alloimmune function of autologous T cell lines. Similarly, antiidiotypic antibodies (Ab2) to HLA antibody molecules (Ab1) block the binding of the latter to the immunizing HLA antigen. The prevalence of Ab2 over Ab1 during pregnancy may explain the maternal tolerance to the fetus.     

金黄色葡萄球菌黏附素ClfA等抗体抑制该菌黏附牛乳腺上皮细胞效果的比较

【目的】为从免疫的角度比较和分析黏附素分子细胞外纤维蛋白原结合蛋白(Extracellular fibrinogen-binding protein,EfB)和纤维连接蛋白结合蛋A(Fibronection binding protein,FnBP)对聚集因子A(Clumping factor A,ClfA)抑制牛源金黄色葡萄球菌(Staphylococcus aureus,SA)黏附牛乳腺原代上皮细胞作用的增强效果。【方法】本试验分离培养牛乳腺上皮细胞并鉴定;将真核重组质粒EfB和FnBPA分别与ClfA组合免疫新西兰大白兔,并利用免疫后抗体体外抑制2株SA分离株侵染牛乳腺上皮细胞,采用平板计数法定量检测与比较不同免疫组合诱导抗体对黏附的抑制作用;对SA和乳腺上皮细胞分别进行荧光染色,观察不同免疫组合诱导的抗体对黏附抑制效果的差异。【结果】成功分离培养了牛乳腺原代上皮细胞;证明了构建的ClfA、FnBPA和EfB真核重组表达质粒均可在细胞中表达,且在免疫实验兔后可诱导特异性抗体的产生;细菌平板计数和荧光染色观察的结果表明,黏附素分子单独和组合免疫的抗体对该菌不同菌株(GY278和GY309)的黏附抑制能力不同,ClfA抗体的黏附抑制能力最强,FnBPA分子A区的黏附抑制能力优于D区。FnBPA、EfB分别与ClfA联合免疫抗体对黏附的抑制程度高于黏附素分子单独免疫组,FnBPA分子A区对ClfA黏附抑制的增强作用优于D区,FnBPA-A区与Efb相比对ClfA的黏附抑制增强差异不显著。【结论】FnBPA-A、FnBPA-D和EfB分别与ClfA联合免疫可不同程度地影响ClfA的黏附抑制效果,该结果为以黏附素分子为靶点的牛乳腺炎疫苗的研究提供了实验数据。     

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.     

Stochastic prey-predator relationships: A random differential equation approach

A stochastic model describing two interacting populations is considered. The model involves a random differential equation of the form dX/dt=A(t)X+Y(t) where the random matrixA and vectorY represent the interactions and growth rates respectively andX is a (random) vector the components of which are the logarithms of the population's sizes. An expression for the solution of the above equation is obtained whence its statistical properties can be determined. Alternatively, a method based on Liouville's theorem is used to obtain the probability distribution of the solution. Application of both methods to simple cases indicates that the random solution is asymptotically stable in the mean even when the solution to the associated deterministic equation is not, viz. in the absence of self interactions.     

四川甘孜州高山葡萄酒产区酵母菌多样性分析

【背景】西南高山葡萄酒产区的甘孜州产区,具有生产优质葡萄酒的自然禀赋。【目的】研究四川甘孜州葡萄酒产区真核微生物种类多样性、本土酿酒酵母遗传多样性,以及商业酵母对本土酵母多样性的影响。【方法】利用ITS高通量测序技术对赤霞珠接种发酵和自然发酵过程中的微生物进行多样性分析,并利用Interdelta指纹图谱分析法,对经过26S rRNA基因鉴定的野生酿酒酵母基因型进行分类。【结果】ITS测序结果显示,接种发酵和自然发酵各时期均注释到7个科7个属的酵母,通过Interdelta指纹图谱分析发现甘孜州产区的酿酒酵母共有5种基因型。该产区酿酒酵母的6株代表菌株与我国其他产区109株酿酒酵母的进化树分析结果显示,均与来自北京产区的酿酒酵母菌株亲缘关系更近。【结论】甘孜州葡萄酒子产区酵母资源丰富,表现出较高的微生物多样性和中等程度的本土酿酒酵母基因型多样性,为后续优良本土酵母菌株的筛选奠定基础。     

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Objectives

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid.

Results

The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD.

Conclusions

These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

     

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans北大核心CSCD

［Objective］ Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata.Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses.Chemotaxis appears crucial for the growth of A.caulinodansin complicated environment and the construction of associative relationship with the plant.However, little is known about the chemotactic pathway of A.caulinodans.Thus, our study aimed to compare the chemotaxis-like genes of A.caulinodans with those of well-studied species.［Methods］ NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A.caulinodans.HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein （MCP）.［Results］ There was a major chemotaxis cluster in A.caulinodans and the CheR methylated MCPs independently of pentapeptide motif.There were 43 MCP homologs containing diverse signal-sensing architectures in A.caulinodans.In addition,cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.［Conclusion］ Despite the extremely high homology presented between the chemotactic system of A.caulinodans and those of well-studied species, A.caulinodans shows its own unique characteristics.The classification of these chemotactic pathways by comparative genomics enables us to better understand how A.caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.     

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

The repertoire of natural anti-glycan antibodies in naive chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.     

Synthesis and in vitro evaluation of fluorinated styryl benzazoles as amyloid-probes

The formation of proteinaceous aggregates is a pathognomonic hallmark of several neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. To date, the final diagnostic for these diseases can only be achieved by immunostaining of post-mortem brain tissues with the commonly used congo red and Thioflavin T/S amyloid-dyes. The interest in developing amyloid-avid radioprobes to be used for protein aggregates imaging by positron emission tomography has grown substantialy, due to the promise in assisting diagnosis of these disorders. To this purpose, the present work describes the synthesis and characterization of four novel fluorinated styryl benzazole derivatives 1–4 by means of the Wittig reaction, as well as their in vitro evaluation as amyloid-probing agents. All compounds were obtained as mixtures of geometric E and Z isomers, with the preferable formation of the E isomer. Photoisomerization reactions allowed for the maximization of the minor Z isomers. The authentic 1–4E/Z isomers were isolated after purification by column chromatography under dark conditions. Profiting from the fluorescence properties of the different geometric isomers of 1–4, their binding affinities towards amyloid fibrils of insulin, α-synuclein and β-amyloid peptide were also measured. These compounds share similarities with Thioflavin T, interacting specifically with fibrillary species with a red-shift in the excitation wavelengths along with an increase in the fluorescence emission intensity. Apparent binding constants were determined and ranged between 1.22 and 23.96 μM⁻¹. The present data suggest that the novel fluorinated styryl benzazole derivatives may prove useful for the design of ¹⁸F-labeled amyloid radioprobes.     

一种适用于链霉菌异源合成基因簇同框缺失的高效方法

【背景】对抗生素生物合成途径的阐明有助于提高目标化合物的产量并开发具有更高活性的新化合物。基因的同框缺失是天然产物生物合成研究的常规手段,通过分析突变菌株积累的中间产物,可以帮助推导天然产物的合成途径及相关基因的功能。天然产物生物合成基因簇的大小一般在20 kb以上,对每个基因进行同框缺失耗时耗力,因此,优化链霉菌来源的基因同框缺失的方法有重要的意义。【目的】基于PCR-targeting重新设计了一套在链霉菌柯斯文库质粒上进行基因同框缺失的方法,实现链霉菌基因在大肠杆菌中快速、高效的基因同框缺失的技术体系。【方法】使用氨苄青霉素抗性基因bla作为PCR-targeting DNA片段的筛选标记,同时使用体外的Pac I酶切和酶连系统代替体内的Flp/FRT系统来介导同框缺失的构建。【结果】利用这种方法,在6 d内完成了米多霉素生物合成基因簇中14个基因的同框缺失。【结论】此方法与传统的PCR-targeting方法相比,构建同框缺失载体的效率明显提高;Pac I识别序列在链霉菌基因组上的稀有性使得此方法在构建抗生素生物合成基因簇必需基因的同框缺失载体上具有普适性。