DC-SIGN胞外区基因的克隆、蛋白表达及抗体制备

旨在构建DC-SIGN胞外区基因原核表达质粒,诱导蛋白表达并制备多克隆抗体。用PCR的方法扩增编码DC-SIGN胞外区的基因序列,将其克隆到原核表达载体pET-28a(+)中,利用大肠杆菌表达系统表达DC-SIGN胞外区蛋白,用H is抗体做W estern Blotting鉴定目的蛋白的免疫原性。用纯化的DC-SIGN胞外区蛋白免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,免疫荧光法检测其特异性。结果显示,原核表达载体pET-28 a(+)-DC-SIGN胞外区基因成功构建,可在大肠杆菌BL21(DE3)中高效表达,获得相对分子质量约20 kD的DC-SIGN胞外区蛋白,经Westernb lotting鉴定为正确。纯化后的蛋白免疫大耳白兔,制备的多克隆抗体具有较强免疫原性和特异性。本研究得到了纯化的DC-SIGN胞外区蛋白,并制备了具有特异性和高效价的抗体,为研究DC-SIGN生物学功能提供试验基础。     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a(+) 中,从而构建了原核表达质粒pET30-VP1.将pET30-VP1转化至感受态细胞E.coli BL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达.该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清.以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上.以Western blot 检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性.VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础.     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a( )中,从而构建了原核表达质粒pET30-VP1。将pET30-VP1转化至感受态细胞E.coliBL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达。该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清。以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上。以Westernblot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性。VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础。     

鹅细小病毒vp2基因片段在原核系统中的表达及多克隆抗体的制备

根据鹅细小病毒(Gooseparvovirus,GPV)中国分离株HG5/82基因序列,设计引物,利用PCR技术扩增出HG5/82株vp2基因,将其克隆到pMD18-T载体后,转化入感受态细胞TG1中增殖。筛选阳性质粒,将其与原核表达载体pPROEXTMHTb分别用NcoI酶切后回收目的片断,进行定向连接,产物转化入感受态DH5α,重组质粒经酶切和测序证实目的基因正确克隆到表达载体的预期位点且插入方向正确,构建了含有HG5/82主要结构基因vp25’端969bp片段的原核表达载体。经IPTG诱导后表达出与预期大小相符的约36kDa的融合蛋白,表达形式为包涵体。薄层扫描结果表明表达产物约占菌体总蛋白的21.4%。包涵体通过6mol/L盐酸胍裂解后,利用镍离子亲和树脂进行纯化,用纯化的分子量为36kDa的融合蛋白免疫新西兰白兔,制备兔抗鹅细小病毒部分结构蛋白多克隆抗体。Westernblot分析表明该多克隆抗体与HG5/82毒株具有反应性,说明该融合蛋白具有抗原性。     

Ataxin-3融合蛋白表达、纯化及抗血清的制备

目的:表达GST-ataxin-3-N融合蛋白并制备GST-ataxin-3特异性抗体,为深入研究其功能及其在SCA3发病机制中的作用提供重要的技术和材料保障.方法:将人ataxin-3氨基端基因克隆入原核表达载体pGEX-4T-2,在大肠杆菌(E.coli)BL21中表达,用Glutathione sepharose4B凝胶亲和柱纯化目的蛋白.利用纯化的GST-ataxin-3-N蛋白制备多克隆抗体.结果:成功构建了原核表达载体,得到高表达量的融合蛋白,经亲和层析柱纯化获得较高纯度的GST-ataxin-3-N融合蛋白.以融合蛋白免疫新西兰兔得到Ataxin-3-N多克隆抗体,Western Blotting及免疫荧光均证实该抗体能够识别Ataxin-3-myc蛋白,具有较高特异性.结论:利用原核表达人GST-ataxin-3-N融合蛋白制备的Ataxin-3多克隆抗体具有较好的特异性,可用于该蛋白的相关研究.     

A novel peptide tag for detection and purification of recombinant expressed proteins

Peptide tags have proven useful for the detection and purification of recombinant proteins. However cross reactions of antibodies raised to the tag are frequently observed due to the presence of host proteins containing all or parts of the tag. In this report we have identified a unique viral peptide sequence, R-tag, that by blast searches is absent from the commonly expression hosts Arabidopsis thaliana, Escherichia coli, Pichia pastoris and mouse myeloma cell NSO. We have prepared monoclonal antibodies to this peptide and confirmed the absence of this peptide sequence from the above genomes by Western blotting. We have also modified protein expression vectors to incorporate this sequence as a fusion tag in expressed proteins and shown its use to successfully purify recombinant proteins by immunoaffinity procedures.     

Sequential deposition of latent TGF-β binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts

Latent TGF-beta binding proteins (LTBPs) mediate the targeting of latent TGF-beta complexes into ECM structures, which is important for TGF-beta activation and functions. LTBPs-1, -3 and -4 associate with and regulate the bioavailability of TGF-betas. We investigated whether LTBP-3 and -4 are associated with pericellular fibrillar structures of human lung fibroblast ECM, and which of their domains are important for this function. Immunoblotting analyses of isolated insoluble matrices as well as immunofluorescence analyses and confocal microscopy indicated that both LTBP-3 and -4 get assembled into the ECM. Interestingly, LTBP-4 was not detected until 7-10 days of culture and LTBP-3 until 14 days of culture. This was a major difference from the deposition kinetics of LTBP-1, which was detected already within 2 days of culture. Expression analyses by real time RT-PCR indicated that the slow appearance of LTBP-3 and -4 was due to the low expression levels soon after subculture. Recombinant N-terminal fragments of LTBP-3 and -4 bound readily to fibroblast ECM. The C-terminal domain of LTBP-4, but not of LTBP-3, also associated with the matrix structures. The levels of ECM-associated latent complexes of TGF-beta1 increased in parallel with the increased production and deposition of the LTBPs. The amount of active TGF-beta in the conditioned medium decreased during extended culture. Our results suggest that ECM is an important site of deposition also for LTBP-3 and -4 and that the temporal and spatial targeting of the TGF-beta complexes are associated with ECM maturation.     

Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.     

Expression and subcellular localization of Spred proteins in mouse and human tissues

Spred-1 and Spred-2 (Sprouty-related protein with an EVH1 domain) are recently described members of the EVH1 (Ena/VASP-homology domain 1) family. Both Spred-1 and Spred-2 are membrane-associated substrates of receptor tyrosine kinases and they act as negative regulators of the Ras pathway upon growth factor stimulation. Since the Spred family members seem to exert overlapping molecular functions, the isotype-specific function of each member remains enigmatic. To date, no comprehensive expression profiling of Spred proteins has been shown. Therefore, we compared mRNA and protein expression patterns of Spred-1 and Spred-2 systematically in mouse organs. Furthermore, we focused on the tissue-specific expression of Spred-2 in adult human tissues, the subcellular localization, and the potential role of Spred-2 in the organism. Our studies show that expression patterns of Spred-1 and Spred-2 differ markedly among various tissues and cell types. In mouse, Spred-1 and Spred-2 were found to be expressed predominantly in brain, whereas Spred-2 was found to be more widely expressed in various adult tissues than Spred-1. In humans, Spred-2 was found to be strongly expressed in glandular epithelia and, at the subcellular level, its immunoreactivity was associated with secretory vesicles. Using confocal microscopy we found Spred-2 to be strongly colocalized with Rab11 and, to a lesser extent, with Rab5a GTPase, an observation that was not made for Spred-1. We conclude that the two members of the recently discovered Spred protein family, Spred-1 and Spred-2, show a highly specific expression pattern in various tissues reflecting a specific physiological role for the individual Spred isoforms in these tissues. Furthermore, it becomes most likely that Spred-2 is involved in the regulation of secretory pathways.     

Expression of overlapping PreS1 fragment recombinant proteins for the determination of immunogenic domains in HBsAg PreS1 region

The virion of the hepatitis B virus (HBV) is a sphericalparticle of 42-nm diameter whose envelope contains threerelated surface glycoproteins called the large (L), middle(M) and small (S) proteins.All these proteins are expressedfrom one open reading frame using three in-frame startsites [1]. The L protein is the translation product of thewhole open reading frame. The M protein lacks the N-terminal amino acid residue 108–119 of Lprotein (the preS1sequence), and the S protein lacks the N…     

HIV-1型核衣壳蛋白P24截短体在大肠杆菌中的表达,纯化和鉴定

用聚合酶链式反应（ＰＣＲ）从ＨＩＶ １ｇａｇ基因中扩增出衣壳蛋白Ｐ２４截短体（ｔＰ２４）基因,插入质粒ｐＧＥＸ ４Ｔ３中构成重组表达质粒ｐＧＥＸ ｔｐ２４,将ｐＧＥＸ ｔｐ２４转化大肠杆菌ＢＬ２１后获得了高效表达,表达量占菌体总蛋白量的３４３８％。经Ｇｌｕｔａｔｈｉｏｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化的截短体Ｐ２４纯度为９２７７％。纯化的截短体Ｐ２４在间接ＥＬＩＳＡ和免疫印迹检测ＨＩＶ抗体阳性血清和正常人血清中,具有很高的抗原特异性和免疫反应性。     

Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH(2)), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH(2)), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH(2)). The very low antimicrobial potency (MIC>200microM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.     

Expression, purification, and biochemical characterization of a recombinant Iectin of Sarcocystis muris (Apicomplexa) cyst merozoites

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia coli as a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.     

Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.     

In vivo functional dissection of human inner kinetochore protein CENP-C

CENP-C is a fundamental component of the inner kinetochore plate and contributes to the formation of functional centromeres in eukaryotic organisms. Recruitment of CENP-C to kinetochore requires other centromere proteins, particularly CENP-A, CENP-H, and CENP-I. However, how CENP-C is correctly localized at the kinetochore is not clearly determined, mainly due to the functional variety of its domains, which hints at a complex recruitment mechanism. Here, by both immunofluorescent labeling and chromatin/immunoprecipitation we could show that human CENP-C contains two distinct domains, one in the central region, between amino acids 426 and 537, and the second one in the carboxyl terminal region, between amino acids 638 and 943, which are both capable of localizing at centromeres and binding alpha-satellite DNA. The presence of two domains that iterate the same function despite being significantly different in their amino acid sequence and structure suggests that CENP-C may target the centromere by establishing multiple contacts with both the DNA and protein constituents of the kinetochore.     

Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates

The expression of the gene encoding a thermostable α-amylase (EC 3.2.1.1) (optimal activity at 100°C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant α-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.