C-terminal fragment of tetanus toxin: its use in neuronal network analysis and its potential as non-viral vector

The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical conjugation. Thus, these tracers have been used to study and analyse the synaptic connections of a neural network. In this article, we shortly review the various methods used with this aim including: injection of the fusion protein, adenovirus in vivo expression and transgenesis. Since neural activity is essential for neuronal TTC binding and internalization, the functionality of connections can be also evaluated. Moreover, modifications of the retrograde transport can be detected by using this fragment. Thus, TTC fragment is an excellent tracer to analyse the connectivity and functionality of a neural network. The TTC fragment was also soon proposed as potential therapeutic vector to transport and to deliver a biological activity or gene in a neural network. With this aim, the efficiency of a translocation domain to induce the cytosolic release of the associated activity has been evaluated. The use of the TTC fragment to target specifically a neurotrophic factor to neurons and thus avoid secondary effects has been tested with interesting results.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

信号肽序列对禽流感病毒H5N1 HA 蛋白GFP融合分子在真核细胞中表达的影响

[目的]构建绿色荧光蛋白(GFP)与禽流感病毒(AIV)HA基因的融合表达载体,观察信号肽序列的有无及位置对HA-GFP在293T细胞中的表达影响.[方法]应用PCR方法从H5N1亚型AIV质粒DNA中扩增出完整的或除去信号肽序列的HA基因片段,PCR产物经Xho Ⅰ和SmaⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的不同位点,将得到的重组体M1,M2和M3分别转化宿主菌DH5a,经双酶切及.DNA测序鉴定分析后,采用脂质体转染法将pEGFF-C1,M1,M2和M3转染人胚肾细胞系293T细胞,荧光显微镜下观察HA-GFP的表达,流式细胞仪检测表达HA蛋白的细胞百分数.[结果]经酶切及测序鉴定成功构建了HA-GFP重组表达载体M1,M2和M3,荧光显微镜及流式细胞仪检测到重组质粒转染的293T细胞表达强的荧光蛋白,信号肽的有无对HA-GFP融合蛋白在293T细胞中的表达有显著影响,信号肽序列使HA-GFP融合蛋白在293T细胞中的表达减少,而信号肽的位置对融合蛋白表达量的影响不显著.[结论]信号肽的有无对HA-GFP融合蛋白在细胞中的表达有显著影响.     

庚型肝炎病毒E2区cDNA在毕赤酵母中的表达及抗原性鉴定

从含有庚型肝炎病毒(GBVC/HGV)包膜蛋白E2 cDNA(559bp)的质粒pGEX\|E2中,扩增得到能够编码日本血吸虫谷胱甘肽硫转移酶(GST)和GBVC/HGV包膜蛋白E2的融合基因片段。将此长度为1324bp的DNA片段插入到酵母表达载体pPIC9K中,使之位于α因子信号肽下游,且与之同框。通过电激转化将构建的重组表达质粒pPIC9K\|GST\|E2插入到Pichia pastoris GS115菌株染色体中。筛选His\++Mut\+s表型的转化子,震荡培养,用05%甲醇诱导表达5d后,在培养液中得到表达的GSTE2融合蛋白。经过表达条件的优化,GSTE2蛋白可占培养液中总蛋白的50%。通过谷胱甘肽亲和层析柱纯化,GSTE2融合蛋白的纯度可达95%左右。以庚型肝炎病人血清为探针,进行免疫印迹及ELISA实验,结果表明该融合蛋白具有能被庚型肝炎病人血清特异性识别的抗原性。     

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.     

PDGF受体结合域与乙肝病毒核心抗原的融合表达

化学合成血小板源性生长因子受体结合域１３肽基因,并与乙肝病毒核心抗原基因５′端融合,序列分析表明化学合成的１３肽基因及融合后基因的阅读框架正确．将融合基因亚克隆于ｔａｃ启动子控制的ｐＥＴ３ａ表达质粒中并于大肠杆菌中表达．表达产物经ＥＬＩＳＡ、ＷｅｓｔｒｅｎＢｌｏｔ鉴定表明,融合蛋白已被表达,其单位分子量与推算值一致．电镜观察证明所表达的融合蛋白能形成颗粒．     

Transneuronal tracing of neural pathways influencing both diaphragm and genioglossal muscle activity in the ferret.

In prior experiments that employed the transneuronal transport of isogenic recombinants of pseudorabies virus (PRV), we demonstrated that neurons located ventrally in the medial medullary reticular formation (MRF) of the ferret provide collateralized projections to both diaphragm and abdominal muscle motoneurons as well as to multiple abdominal muscle motoneuron pools. The goal of the present study was to determine whether single MRF neurons also furnish inputs to diaphragm motoneurons and those innervating an airway muscle with inspiratory-related activity: the tongue protruder genioglossus. For this purpose, PRV recombinants expressing unique reporters (beta-galactosidase or enhanced green fluorescent protein) were injected into either the diaphragm or the genioglossal muscle. The virus injections produced transneuronal infection of overlapping populations of MRF neurons. A small proportion of these neurons (<15%) was infected by both PRV recombinants, which indicated that they provide collateralized inputs to genioglossal and diaphragm motoneurons. These findings show that, whereas some MRF neurons simultaneously influence the activity of upper airway and respiratory pump muscles, other cells in this brain stem region independently contribute to diaphragm and genioglossal muscle contraction regulation.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase.

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display

A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.     

带有蛋白转导结构域的Bcr/Abl癌蛋白片段的表达及其跨膜转运

HIV 1编码的反式激活蛋白TAT具有将细胞外蛋白转导进入细胞的基序 ,称为蛋白转导结构域 (PTD) .为研究PTD介导的PTD Bcr Abl融合蛋白的跨膜转运 ,合成了编码PTD的基因片段 ,并与PCR扩增的慢性粒细胞白血病癌蛋白bcr abl基因片段融合 .在大肠杆菌中表达纯化了融合蛋白 ,将纯化的融合蛋白加入培养的HL60细胞和C2C12细胞后 ,发现PTD基序可以介导Bcr Abl蛋白自由从细胞外跨膜转导进入细胞内 .研究结果可能为用外源蛋白负载 (loading)免疫活性细胞如抗原提呈细胞提供新的途径 .     

Identification of the motoneurons innervating the stapedius muscle in Gallus gallus: a horseradish peroxidase study

The location of the stapedial motoneurons in Gallus gallus was investigated by means of the retrograde transport of HRP, injected into the stapedius muscle. The labeled neurons are located in both the ventral and dorsal divisions of the VII nerve nucleus, in a lateral and ventral position respectively, facing the superior olivary nucleus. The neurons are distributed in two size classes. The functional implications of these findings are discussed, in relation both to the absence of the acoustic stapedial reflex in birds and to the functional properties of the stapedius muscle.     

信号肽对短杆菌胆固醇氧化酶基因在大肠杆菌中表达的影响

从Brevibacterium sp．DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a（＋）中,构建重组质粒pET28a—COD（s＋）。以pET28a-COD（s＋）为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD（s-）。两种重组载体转化大肠杆菌BL21（DE3）通过IPTG诱导均获得活性表达,SDS-PAGE分析,目的产物表达量都占到了细胞总蛋白的50％以上,Brevibacterium sp．DCR2DC-82胆固醇氧化酶的信号肽对重组酶的空间构象和表达量并没有太大的影响。     

Misidentification of cardiac vagal pre-ganglionic neurons after injections of retrograde tracer into the pericardial space in the rat

We evaluated whether pericardial injections of the retrograde tracers cholera toxin subunit B (CTb) or Fast Blue (FB) reliably labelled cardiac vagal pre-ganglionic neurons. Injections of CTb into the pericardial space of the rat labelled neurons in both the external and compact formations of the nucleus ambiguus. Most labelled neurons were found in the compact formation of the nucleus ambiguus, and the majority of these, and only these, expressed immunoreactivity for calcitonin gene-related peptide. This distribution of labelled neurons and their immunohistochemical properties is characteristic of oesophageal motoneurons. Examination of the oesophagus following intra-pericardial CTb applications revealed strong labelling of motor end plates within the skeletal muscle of the thoracic but not the abdominal oesophagus. When a second retrograde tracer, FB, was injected into the abdominal oesophagus, labelled somata were found adjacent to CTb-labelled neurons in the compact formation of the nucleus ambiguus. No co-localisation of tracers was found, but identical proportions of calcitonin gene-related peptide (CGRP) immunoreactivity were observed in both groups of neurons. FB injected into the pericardial space labelled intra-cardiac neurons but not brainstem neurons. We conclude that intra-pericardial, and perhaps sub-epicardial, injections of some retrograde tracers are likely to label a subset of oesophageal, as well as cardiac, vagal motor neurons in the brainstem.This work was supported in part by grant No. G 00 M 0670 from the National Heart Foundation of Australia.     

In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system

We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.     

手掌参γ-硫素的原核表达及纯化

目的：在原核系统中高效表达手掌参γ-硫素,并对其进行纯化。方法：通过筛选手掌参cDNA文库获得γ-硫素基因（gcthionin）,分别对其全长及信号肽编码序列缺失的cDNA片段进行PCR扩增,克隆入原核表达载体pET-32（a）,构建重组质粒pET-32（a）/gcthionin和pET-32（a）/Δgcthionin;测序鉴定后,转化大肠杆菌BL21（DE3）,经IPTG诱导表达融合蛋白;SDS-PAGE分析后,采用Ni-NTA亲和层析柱及凝胶柱对可溶性蛋白进行纯化,Western blotting鉴定。结果：gcthionin基因开放式阅读框全长225nt,编码一个由74个氨基酸残基组成的蛋白;带有信号肽的重组质粒在大肠杆菌BL21（DE3）中以包涵体形式表达;信号肽缺失可以极大地提高外源蛋白的可溶性,该可溶性产物经Ni-NTA柱及凝胶过滤后可获得纯度较高的蛋白,经Western blotting分析,相对分子质量约21.9×10^3处有明显的蛋白条带,与预期蛋白分子大小一致。结论：信号肽编码序列缺失的Δgcthionin可在大肠杆菌中可溶、高效表达。     

Overproduction of Biologically-active Human Nerve Growth Factor in Escherichia coli

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

新型真核表达质粒pcDNA6/myc-his-EGFP B 的构建及其在重组基因表达中的应用

增强型绿色荧光蛋白（EGFP, enhanced green fluorescent protein）、myc抗原和6×His已在众多真核表达载体中用作重组蛋白的表达标记,EGFP能发出的绿色荧光,myc抗原能用相应的抗体检测,6×His能被相应的树脂特异吸附。但目前为止,没有一个质粒表达载体能够同时整合三者的功能。本研究构建了一个能够同时整合EGFP、myc抗原和6×His功能的新型真核质粒表达载体,我们将其命名为pcDNA6/myc-his-EGFP B。值得注意的是,为确保目的基因与EGFP基因融合表达后,融合表达产物各组成部分能够保持原有的生物活性,我们运用LINKER程序在EGFP基因的5'端设计了一段编码八肽的连接DNA序列。将一段含有人白细胞介素2（IL-2, human interleukin 2）信号肽编码序列的基因亚克隆进pcDNA6/myc-his-EGFP B的多克隆位点中,使之与EGFP、myc抗原和6×His融合表达,构建成质粒pMHES。用pcDNA6/myc-his-EGFP B和pMHES转染2.2.15细胞,48 h后成功观察到绿色荧光;用pcDNA6/myc-his-EGFP B尾静脉注射Balb/c小鼠,8 h后在小鼠肝脏冰冻切片中同样观察到绿色荧光。用同源建模软件Modeller8V2模拟IL-2与EGFP、myc抗原和6×His融合表达产物的三维结构,结果表明：IL-2、EGFP、myc和6×His各部分互不干扰,连接八肽具有一定的柔性。以上结果表明pcDNA6/myc-his-EGFP B可望作为外源基因在哺乳动物细胞中表达研究和基因治疗的新型载体。