Lysogeny in Lactobacillus delbrueckii strains and characterization of two new temperate prolate-headed bacteriophages

Aims: Frequency of lysogeny in Lactobacillus delbrueckii strains (from commercial and natural starters) and preliminary characterization of temperate bacteriophages isolated from them. Methods and Results: Induction of strains (a total of 16) was made using mitomycin C (MC) (0·5 μg ml⁻¹). For 37% of the MC-treated supernatants, it was possible to detect phage particles or presence of killing activity, but only two active bacteriophages were isolated. The two temperate phages isolated were prolate-headed phages which belonged to group c of Lact. delbrueckii bacteriophages classification. Different DNA restriction patterns were obtained for each phage, while the structural protein profiles and packaging sites were identical. Distinctive one-step growth curves were exhibited by each phage. An influence of calcium ions was observed for their lysis in broth but not on the adsorption levels. Conclusions: Our study showed that lysogeny is also present in Lact. delbrueckii strains, including commercial strains. Significance and Impact of the Study: Commercial strains could be lysogenic and this fact has a great practical importance since they could contribute to the dissemination of active-phage particles in industrial environments.     

Phage display: practicalities and prospects

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variant nucleotide sequences may be converted into populations of variant peptides and proteins which may be screened for desired properties. It is now some seventeen years since the first demonstration of the feasibility of this technology and the intervening years have seen an explosion in its applications. This review discusses the major uses of phage display including its use for elucidating protein interactions, molecular evolution and for the production of recombinant antibodies.     

胃溃疡患者血清多肽类激素及胃粘膜中单胺类神经递质的水平及其临床意义

目的:分析胃溃疡患者血清多肽类激素及单胺类神经递质的水平变化及其临床意义。方法:选取2014年8月-2015年8月在我院经胃镜检查确诊为胃溃疡的患者103例作为研究组,另选取54例健康志愿者作为对照组。检测两组血清中胃动素(MTL)、肾上腺髓质素(AM)、胃肠激素胃泌素(Gas)、生长抑素(SS)及降钙素基因相关肽水平(CGRP),以及胃黏膜中血管活性肠肽(VIP)、5-羟色胺(5-HT)、去甲肾上腺素(NE)、P物质(SP)水平。结果:胃溃疡患者血清Gas,AM及MTL明显高于对照组,而SS及CGRP明显低于对照组,差异具有统计学意义(P0.05)。胃溃疡活动期患者血清Gas,AM及MTL明显高于愈合期及瘢痕期患者,而SS及CGRP低于愈合期及瘢痕期患者,差异具有统计学意义(P0.05);胃溃疡愈合期患者血清Gas,AM及MTL高于瘢痕期患者,而SS及CGRP低于瘢痕期患者,差异具有统计学意义(P0.05)。胃溃疡患者胃粘膜内5-HT,SP及NE明显低于对照组,而VIP明显高于对照组,差异具有统计学意义(P0.05)。胃溃疡活动期患者胃粘膜5-HT,SP及NE明显低于愈合期及瘢痕期患者,而VIP明显高于愈合期及瘢痕期患者,差异具有统计学意义(P0.05);胃溃疡愈合期患者胃粘膜5-HT,SP及NE明显低于瘢痕期患者,而VIP明显高于瘢痕期患者,差异具有统计学意义(P0.05)。结论:胃溃疡患者血清多肽类激素及胃黏膜中单胺类神经递质的水平变化与疾病进展密切相关,对胃溃疡的诊断及疗效评价具有指导意义。     

Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene and lysis genes of phage λ in a recombinant E. coli

Glutaryl-7-aminocephalosporanic acid acylase (GLA), recommended for use in the form of immobilized-enzyme, is one of the two key enzymes in the two-step synthesis of 7-aminocephalosporanic acid. For simplifying the process of cell disruption and immobilization, the lysis genes of phage λ (S⁻RRz) with the S amber mutation were designed to introduce into the over-expression system of GLA. A novel recombinant strain, E. coli TB1/pMKC-AS, simultaneously containing the maltose binding protein gene (malE), the lysis genes (S⁻RRz) and the target GLA gene (Acy) in a same operon, was successfully constructed. Under neutral pH conditions, cell growth and GLA activity of TB1/pMKC-AS was not affected by the presence of the lysis genes, however, autolysis phenomenon was observed under weak alkaline conditions. Through pH control and fed-batch culture, the GLA activity of TB1/pMKC-AS reached as high as 6810 U/L with 24.8 g/L dry cell density (OD₆₀₀ = 67.9) in a 5 L fermentor. In contrast to the cells of E. coli TB1/pMKC-Acy without the lysis genes, the mild EDTA/Tris buffer (pH 8.0) can cause the lysis of the cells of TB1/pMKC-AS containing the lysis genes. Correspondingly, a mild pH 9.0/42 °C incubation method was developed for conveniently degrading the recombinant cells of TB1/pMKC-AS, based on the expression of the lysis genes. Further experiments showed that the cell lysate after the mild incubation disruption can be directly immobilized by 10% polyacrylamide to make the immobilized enzymes. In comparison with the immobilized GLA from TB1/pMKC-Acy, the immobilized cell lysate of TB1/pMKC-AS has the similar characteristics of catalysis stability, implying a great potential for industrial application of the lysis genes-assisted cell disruption.     

玉米对二氧化硫胁迫响应的相关cDNA克隆与分析

应用mRNA差异显示技术,对高耐SO2玉米自交系Q9进行了SO2胁迫下的基因差异表达分析.结果显示,30对引物组合进行的PCR扩增中,获得了13条差异表达的cDNA片段,其中3条诱导表达,2条增强表达,6条减量表达,2条抑制表达.序列分析和数据库比对表明,GenBank中只搜寻到5条cDNA序列,其中2条增强表达的cDNA片段D1和D5分别与氨基酸结合蛋白ABP、锌指结构转录因子蛋白DOF部分序列高度同源,其他诱导表达的3条cDNA(D3、D6、D11)功能未知,可能是新的cDNA片段.经半定量RT-PCR分析验证,D1和D5转录水平受SO2胁迫表达显著增强,推测D1和D5可能参与了玉米对SO2胁迫的抗性反应.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.     

Stepwise prediction of conformational discontinuous B-cell epitopes using the Mapitope algorithm

Mapping the epitope of an antibody is of great interest, since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. Here we present Mapitope, a computer algorithm for epitope mapping. The algorithm input is a set of affinity isolated peptides obtained by screening phage display peptide-libraries with the antibody of interest. The output is usually 1-3 epitope candidates on the surface of the atomic structure of the antigen. We have systematically tested the performance of Mapitope by assessing the effect of the algorithm parameters on the final prediction. Thus, we have examined the effect of the statistical threshold (ST) parameter, relating to the frequency distribution and enrichment of amino acid pairs from the isolated peptides and the D (distance) and E (exposure) parameters which relate to the physical parameters of the antigen. Two model systems were analyzed in which the antibody of interest had previously been co-crystallized with the antigen and thus the epitope is a given. The Mapitope algorithm successfully predicted the epitopes in both models. Accordingly, we formulated a stepwise paradigm for the prediction of discontinuous conformational epitopes using peptides obtained from screening phage display libraries. We applied this paradigm to successfully predict the epitope of the Trastuzumab antibody on the surface of the Her-2/neu receptor in a third model system.     

水产动物副溶血弧菌病及其噬菌体防治研究进展

副溶血弧菌是水产动物弧菌病的重要病原微生物之一,又是食源性致病菌,摄入被其污染的水产品后可引发肠胃炎、败血症和坏死性筋膜炎等疾病,对水产养殖业及公共卫生安全均具有较大威胁。抗生素大量使用甚至滥用,不可避免地会带来水产品药物残留和细菌耐药等问题,开发安全有效的抗生素替代品迫在眉睫。作为细菌病毒,噬菌体具有宿主特异性强、易筛选、易保存、高效直接等优点,在水产养殖病害防控和食品安全领域受到广泛关注。本文概述了水产动物的副溶血弧菌病及该菌噬菌体防治的研究进展,为副溶血弧菌噬菌体及制剂应用于水产养殖病害生物防控提供参考。     

荧光假单胞菌抗噬菌体菌株的选育

本实验从荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓ）ＡＳ—３菌株的不正常发酵液中分离到一种噬菌体,将其命名为ＰＦＡＳ。ＡＳ—３菌株能利用葡萄糖发酵产生Ｄ－异维生素Ｃ的前体物质２－酮基－Ｄ－葡萄糖酸。电镜观察表明ＰＦＡＳ噬菌体呈蝌蚪形,具有直径为６６ｎｍ的六角形头部及长１１７ｎｍ的尾部。通过紫外线诱变及自然选育两种途径,配合简便有效的初筛方法,经多次分离、纯化、复筛最终在摇并发酵试验中获得６株产量稳定地高于对照敏感菌的抗噬菌体菌株,可望用于生产。     

一种基于配体肽的CRP荧光检测探针的研制

C反应蛋白（C-reaction protein,CRP）是反映机体炎症的有效标志物,早期检测是判断炎症相关疾病的关键,因此,研制CRP新型检测制剂具有重要意义。利用噬菌体表面展示技术对CRP特异性亲和配体进行了筛选,采用固相肽合成技术对目标配体进行了合成,并经生物标记、高效液相色谱法（high performance liquid chromatography,HPLC）分析及质谱（mass spectrometry,MS）鉴定,成功制得检测CRP的荧光探针。经3轮筛选、ELISA检测、重组噬菌体测序及序列比对后得到1个目标配体肽：S-P-H-N-R-S-N-L-V-Q-E-L;经肽合成及生物标记获得1种CRP荧光探针：FITC-(Acp)-S-P-H-N-R-SN-L-V-Q-E-L。研究结果为CRP的有效检测提供了一种新型制剂。