淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性

【目的】构建融合基因原核表达载体pET-30a/ltB-porB,并表达重组融合蛋白LTB-PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据。【方法】构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平。【结果】在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性sIgA水平随免疫时间呈上升趋势,第42天A450值达0.66,明显高于对照组(P0.01),效价高达1∶1280;血清中产生的PorB特异性IgG第28天达最高,A450值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A450:0.57)无明显差异(P0.05)。LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和SolutionBuffer对照组(P0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P0.05)。【结论】重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫。首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫。     

Evolution of cell division in bacteria

Molecular evolution in bacteria is examined with an emphasis on cell division. For a bacterial cell to assemble and then divide required an immense amount of integrated cell and molecular biology structures/functions to be present, such as a stable cellular structure, enzyme catalysis, minimal genome, septum formation at mid-cell and mechanisms to take up nutrients and produce and use energy, as well as store it. The first bacterial cell(s) capable of division must have had complex cell and molecular biology functions. At this stage of evolution, they would not have been primitive cells but would have reached a threshold in evolution where cell division occurred in a regulated manner.     

衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E．coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

Characterization of the NgoAXP: phase-variable type III restriction–modification system in Neisseria gonorrhoeae

Methyltransferases associated with type III restriction–modification (RM) systems are phase-variably expressed in a variety of pathogenic bacteria. NgoAXP, the type III RM system encoded by Neisseria gonorrhoeae , was characterized in this study. The cloned resngoAXP and ngoAXPmod genes were expressed in Escherichia coli strains. The restriction and modification activities of NgoAXP were confirmed in vivo by the λ phage restriction and modification test and in vitro by the methylation of DNA substrates in the presence of [ methyl -³H]AdoMet. As in all known type III systems, the restriction activity needed the presence of both genes, while the presence of the ngoAXPmod gene was sufficient for DNA methylation. Following its overexpression, the DNA methyltransferase M.NgoAXP was purified to apparent homogeneity using metal affinity chromatography. The specific sequence recognized by this enzyme was determined as a nonpalindromic sequence: 5'-CCACC-3', in which the adenine residue is methylated. We observed that in E. coli cells, the expression of the restriction phenotype associated with NgoAXP switched randomly. This phase variation was associated with the change in the number of pentanucleotide repeats (5'-CCAAC/G-3') present at the 5'-end of the coding region of the ngoAXPmod gene.     

Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli

A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila

Summary The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8⁺. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8⁺ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.     

Nucleotide sequence and expression in Escherichia coli of the recA gene of Neisseria gonorrhoeae

The nucleotide sequence of the recA gene of Neisseria gonorrhoeae MS11 has been determined. The product of this gene can act as a recombinase in Escherichia coli, but does so with a decreased efficiency, probably because of the formation of mixed multimers with the equivalent E. coli protein.     

Activation of the Escherichia coli cell division protein FtsZ by a low-affinity interaction with monovalent cations

We have investigated the activation of FtsZ by monovalent cations. FtsZ polymerization was dependent on the concentrations of protein and monovalent salts, and was accompanied by the uptake of a single ion per monomer added. The affinity and the specificity for the cation were low. Potassium, ammonium, rubidium or sodium activated FtsZ to different extents. Electron microscopy showed that polymers formed with either rubidium, or potassium, were very similar, as were their nucleotide turnover rates. The GTPase activity was lower with rubidium than with potassium, indicating that nucleotide exchange is independent of nucleotide hydrolysis. Control of polymerization by binding of a low affinity cation might govern the dynamic behavior of the FtsZ polymers.     

Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

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Highlights

• •Quantitative proteomes of the 2016 WHO Neisseria gonorrhoeae reference strains.
• •Novel gonorrhea vaccine candidates and potential global proteomic AMR markers.
• •First large-scale proteomic profiling of gonorrhea vaccine candidates and AMR.
• •A reference proteomics databank for gonococcal vaccine and AMR research endeavors.

     

Cloning and expression in Escherichia coli of tryptophan genes from Streptomyces griseus IMRU 3570

Two Sau3A fragments of Streptomyces grisues IMRU 3570 were cloned in pBR322 as a vector. One of these clones contained the genetic information needed to complement trpA and trpB mutations in Escherichia coli. The other complements trpA, trpB and trpC mutations in E. coli. Both fragments originated in the same region of the chromosome but the latter is 1 kilobase (kb) longer in the region nearest the tetracycline promoter.     

Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum

Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.     

Abnormal cell division caused by inclusion bodies in E. coli; increased resistance against external stress

Inclusion body formation occurs naturally in prokaryotic cells, but is particularly common when heterologous foreign proteins are overexpressed in bacterial systems. The plant disease virus protein CMV 3a (cucumber mosaic virus movement protein) and the 56 kDa Orientia tsutsugamushi (OT56) protein (an outer membrane protein), which causes tsutsugamushi disease, were expressed in Escherichia coli, and found to form inclusion bodies. Confocal laser scanning microscopy revealed that these inclusion bodies are localized at the cellular poles within E. coli. Cells expressing inclusion bodies appeared to be interconnected, and divided abnormally. The clustered cells exhibited biofilm-like characteristics in that the interior cells of the community were protected by the antibiotic resistance of the outer cells. We compared the number of colony-forming units in inclusion body-forming versus non-forming E. coli to demonstrate the effects of lysozyme, sonication or antibiotic treatment. E. coli clustering provided significantly improved protection against cell disruption/lysis by physical and biochemical stress. This is the first report that shows that abnormal cell division caused by inclusion body formation can cause cellular clustering, resulting in improved resistance to stress in vitro.