Simultaneous Expression of Vitreoscilla Globin Gene and Lytic Genes of Phage A in a Novel Recombinant Escherichia Coli Used for Production of PHB

Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli cell, simultaneously or respectively. Six novel strains containing phbCAB and vgb with or without lytic genes were constructed. Strain VG1 (pTU14), in which vgb, phbCAB and S-RRz could all be successfully expressed, has superior characteristics in cell growth and PHB accumulation, while the results of strains containing vgb and phbCAB without S- RRz were not better than that of strains harbored ph&CAB only. The simultaneous expression of vgb and S- RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.

Study on Genetic Inactivation Bacterial Ghosts of Pasteurella multocida based PhiX174 Gene E Lysis Cassette Mediated

[Objective] To study on genetic inactivation bacterial ghosts of Pasteurella multocida based PhiX174 gene E lysis cassette mediated. [ Method ] Recombinant pPBA1100-e was constructed by which the gene E of bacteriophage Phix174 and temperature sensitivity expressing control system hybridized with plasmid pPBA1100 by genetic engineering method. Recombinant was transformed to Pasteurella multocida and lysis gene E expressed by temperature induction. Recombinant was detected by restriction endonuclease. Cell morphology of bacterial ghost of Pasteurella mul- tocida was observed by scanning electron microscopy and inactivation ratio was estimated by CFU analysis. I Result~ The results indicated that the recombination plasmid presented three bands by restriction endonuclease and agarose electrophoresis and that molecular weight of every band ac- corded with theoretical value. The result of SEM observing showed that recombination plasmid expressed successfully in P. multocida and produced bacterial ghost. The result of CFU detecting demonstrated that inactivation ratio of P. multocida reached 99 per cent. ~Conclusion~ This study pro- vided technical bases for the preparation of antigen vaccine of natural bacterial outer membrane protein.

THE THERAPEUTIC EFFECT OF INTRATUMORAL INJECTION OF GM-CSF GENE-MODIFIED ALLOGENIC MACROPHAGES ON TUMOR-BEARING MICE

Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GMCSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GMCSF genemodified allogenic macrophages on tumorbearing mice observed. The peritoneal macrophages of C57BL/6 mice were transfected with GMCSF gene mediated by recombinant adenovirus and the subcutaneous CT26 colon adenocarcinomabearing BALB/c mice were treated by intratumoral injection of the above macrophages. The survival time of the tumorbearing mice were prolonged significantly and some tumor mass disappeared completely. The necroses of the tumor cells and massive infiltration of inflammatory cells were observed 6 days after treatment. 30 days after treatment, only the leftover of tumor cells and the inflammatory cells remained. The data indicated that introtumoral injection of GMCSF genemodified allogenic macrophages displayed more potent therapeutic effect on the preestablished tumorbearing mice.

Overexpressions of Lambda Phage Lysis Genes and Biosynthetic Genes of Poly-β-hydroxybutyrate in Recombinant E.coli

A plasmid (pTU9) containing the lambda (λ) phage lysis genes S( )RRz and the biosynthetic genes phb CAB of poly β hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.

Bacteriophage secondary infection

Phages are credited with having been first described in what we now, officially, are commemorating as the 100 th anniversary of their discovery. Those one-hundred years of phage history have not been lacking in excitement, controversy, and occasional convolution. One such complication is the concept of secondary infection, which can take on multiple forms with myriad consequences. The terms secondary infection and secondary adsorption, for example, can be used almost synonymously to describe virion interaction with already phage-infected bacteria, and which can result in what are described as superinfection exclusion or superinfection immunity. The phrase secondary infection also may be used equivalently to superinfection or coinfection, with each of these terms borrowed from medical microbiology, and can result in genetic exchange between phages, phage-on-phage parasitism, and various partial reductions in phage productivity that have been termed mutual exclusion, partial exclusion, or the depressor effect. Alternatively, and drawing from epidemiology, secondary infection has been used to describe phage population growth as that can occur during active phage therapy as well as upon phage contamination of industrial ferments. Here primary infections represent initial bacterial population exposure to phages while consequent phage replication can lead to additional, that is, secondary infections of what otherwise are not yet phage-infected bacteria. Here I explore the varying meanings and resultant ambiguity that has been associated with the term secondary infection. I suggest in particular that secondary infection, as distinctly different phenomena, can in multiple ways influence the success of phage-mediated biocontrol of bacteria, also known as, phage therapy.

Screening Peptide Inhibitors Using Phage Peptide Library with Isocitrate Lyase in Mycobacterium tuberculosis as Target

When devoured by macrophages,Mycobacterium tuberculosis remains persistent in macrophages and gains energy through the glyoxylate bypass to maintain its long-term existence in host cells.Therefore it is possible to stop persistent infections by interdicting the glyoxylate bypass in which the isocitrate lyase（ICL） is the key rate-limiting enzyme and a persistence factor.ICL is the target of anti-TB（TB：tubercular） drugs,which could screen ICL out and effectively inhibit the activity of ICL in Mycobacterium tuberculosis,and because of this,anti-TB drugs can be used to kill persistent Mycobacterium tuberculosis.In this study,the ICL gene of the Mycobacterium tuberculosis H37Rv was cloned successfully and recombinant protein with bioactivity was obtained through the enzyme characteristic appraisal.The specific activity of the recombined ICL is 24μmol·mg-1·min-1.The recombined ICL protein was used as the target,and phages which can specifically combine to ICL were screened in the phage 7 peptide library.According to the results of the ELISA and DNA sequence detection,eventually three 7-peptide chains were synthesized.Then the peptide chains were reacted with ICL,respectively,to detect their inhibitory effects on ICL.The results show that all the three 7-peptide chains possessed varying inhibitory effects on the activity of ICL.This study provided lead compounds for the research and development of new peptide anti-TB drugs.

Release the iron: does the infection of magnetotactic bacteria by phages play a role in making iron available in aquatic environments?

Magnetotactic bacteria(MTB)are ubiquitous prokaryotes that orient along magnetic field lines due to magnetosomes’biomineralization within the cell.These structures are ferrimagnetic organelles that impart a magnetic moment to the cell.To succeed in producing magnetosomes,MTB accumulate iron in(i)cytoplasm;(ⅱ)magnetosomes;and(ⅲ)nearby the organelle.It has already been estimated that a single MTB has an iron content of 10 to 100-fold higher than Escherichia coli.Phages are the most abundant entity in oceans and are known for controlling nutrient flow such as carbon and nitrogen by viral shunt and pump.The current work addresses the putative role of phages that infect MTB on the iron biogeochemical cycle.Can phage infection in MTB hosts cause a biogenic iron fertilization-like event in localized microenvironments?Are phages critical players in driving magnetosome biomineralization genes(BGs)horizontal transfer?Further investigation of those events,including frequency of occurrence,is necessary to fully comprehend MTB’s effect on iron cycling in aqueous environments.

Phage display creates innovative applications to combat hepatitis B virus

Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.

Identification of a gene engineering antibody against cystic echinococcosis in liver

OBJECTIVE: To identify a gene engineering antibody against cystic echinococcosis in liver. METHODS: A single chain of variable fragment of human antibodies (ScFvs) was selected from the library by using affinity selection technique with the recombinant antigen on solid surface. The positive clones were demonstrated by ELISA and their DNA sequences were also determined. RESULTS: The DNA sequence data showed that the antibody gene is composed of 768bp. In addition, a specific combination capacity with recombinant Echinococcus granulosus antigen B (r-EgB) was demonstrated by ELISA. CONCLUSION: The obtained gene engineering antibody against r-EgB may have potential implications in immunological treatment and drug targeting delivery.

The Structural Proteins of Bdellovibrio bacteriovorus Bacteriophage MAC-1

In the present investigation the structural proteins associated with MAC-1 bacteriophage have been characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE);tandem mass spectrometry of protein bands from SDS-PAGE gel;from the open reading frames (ORFs) deduced from MAC-1 genome sequence and amino acid sequence homology searches from the Uniprot database (up000002418). Results have led to the identification of at least three structural proteins associated with MAC-1 phage genome. They are: capsid protein (~55,000-daltons);spike protein (~22,000-daltons) and a low molecular weight DNA binding protein (~4000-dal- tons). In addition, two other minor proteins were tentatively identified as replicative and scaffold proteins based on two to three unique peptides from mass spectrometry data. However, other proteins coded (ORFs) by phage genome remain to be identified.

一株具有多种毒力基因的粪肠球菌噬菌体的研究分析

以之前分离出的奶牛乳房炎源粪肠球菌SL22为宿主菌,从临床型乳房炎乳样中分离纯化噬菌体,对其进行生物学特性分析、小鼠治疗试验及毒力基因与耐药基因检测。结果分离到1株噬菌体,该噬菌体形成的噬菌斑如针尖状,电镜下发现该噬菌体无尾,头部呈对称多面体,直径约66 nm,将其命名为vB_Eco T_PSL22(简称PSL22);只裂解其宿主菌,核酸类型为dsDNA;最佳感染复数为0.1,裂解量约为52 PFU/cell;在p H4.0~12.0时活性稳定,能耐受60℃左右高温。对小鼠治愈率为33.33%(2/6),含有2个耐药基因(Acc和aac(3)-Ⅳ)和4种毒力基因(gelE、cylA、ace和efaA)。通过本次试验,可为临床型乳房炎奶牛乳房中噬菌体的多样性研究提供参考,也可为研究奶牛乳房中的微生态系统提供依据。

解淀粉欧文氏菌噬菌体Kuerle的分离、基因组测定及其裂解功能分析

【背景】解淀粉欧文氏菌(Erwiniaamylovora)是仁果类果树火疫病的病原体,对全球苹果和梨的生产构成严重威胁。随着抗生素耐药菌株的出现,火疫病的防治面临巨大的挑战。【目的】分离一种新的裂解解淀粉欧文氏菌的噬菌体,并分析该噬菌体裂解相关基因的功能,为火疫病的防治提供新的选择。【方法】以解淀粉欧文氏菌Ea102为宿主菌,采用双层平板法从流行火疫病的果园土壤中分离噬菌体。通过噬菌斑和透射电镜观察其形态。用PacBio测序技术测定噬菌体基因组,SPAdes组装序列,RAST注释基因组。通过大肠杆菌原核表达系统分析噬菌体Kuerle的裂解机制。【结果】分离纯化出一株裂解解淀粉欧文氏菌的噬菌体,命名为Kuerle。Kuerle由二十面体衣壳的头部和短尾组成,潜伏期约为50 min,裂解量约为240 pfu/cell。噬菌体基因组全长75599 bp,GC含量48.0%,末端有393 bp的重复序列,未发现与溶原调控相关的基因。共预测到85个开放阅读框(open reading frame,ORF),其中33个已知功能蛋白中包含一个由3550 aa组成的巨大病毒颗粒相关的RNA聚合酶(virion-associated RNA polymerase,vRNAP),该vRNAP是Schitoviridae科噬菌体的主要特征之一。病毒粒子和基因结构表明噬菌体Kuerle属于有尾噬菌体类的Schitoviridae科病毒。聚集在DNA晚期表达区的3个裂解相关基因holin、endolysin和spanin组成了“裂解盒”。在大肠杆菌中Kuerle-holin的表达会抑制细胞的生长,而Kuerle-endolysin的表达可裂解细胞。二者共表达时Kuerle-holin会加速Kuerle-endolysin引起的细胞裂解。用叠氮化钠抑制细菌的一般分泌系统(Sec)或Kuerle-endolysin N端信号序列的缺失均会导致endolysin裂解功能丧失。上述结果表明噬菌体Kuerle具有pinholin-SAR endolysin裂解系统。Kuerle-holin(pinholin)使细胞质膜去极化以激活分泌的Kuerle-endolysin(SAR endolysin)降解肽聚糖层。【结论】Kuerle是一株烈性噬菌体并且Kuerle-endolysin抑菌效果显著,为后续火疫病生防试剂的制备提供了理论依据和研究材料。

猕猴桃类胡萝卜素裂解双加氧酶基因AcCCD的克隆与表达分析

【目的】对猕猴桃类胡萝卜素裂解双加氧酶(CCD)基因进行克隆,并进行理化和表达分析。【方法】从猕猴桃基因数据库中鉴定CCD候选基因,克隆3条目的基因全长,并利用荧光定量PCR技术分析所选基因在猕猴桃果实发育期及不同组织的表达量。【结果】克隆得到3个类胡萝卜素裂解双加氧酶(CCD)基因,命名为AcCCD1.1、AcCCD1.2和AcCCD7。其开放阅读框(ORF)分别为1 890、1 632和1 818 bp,编码629、543和605个氨基酸。经预测,3种AcCCD蛋白均定位于细胞质,不具有信号肽,不属于分泌蛋白。经分析,AcCCD1.1、AcCCD1.2蛋白属于CCD1蛋白家族,AcCCD7蛋白属于CCD7家族。系统进化树显示,3个基因编码的蛋白与野茶树等植物蛋白相似性高。对AcCCD基因qRT-PCR,结果表明AcCCD1.1在根中表达量高,叶中最低;AcCCD1.2在花、果实发育后期表达量高。AcCCD7表达量总体较低,根中表达量高于在其茎、叶、花和果的表达量。【结论】推测AcCCD1基因对于猕猴桃果实颜色以及香味的形成有重要联系。AcCCD7基因对于猕猴桃根系生长上具有重要调控作用。

一株副溶血弧菌噬菌体VPp1的分离鉴定及裂解性能

为探究副溶血弧菌(Vibrio parahaemolyticus)的生物防治方法,从水产品市场处污水中分离出一株副溶血弧菌噬菌体VPp1。并借助噬菌斑形态、电镜、酶切等技术对其进行了分类鉴定,同时测定了其裂解谱、最佳感染复数、一步生长曲线以研究其裂解性能。分类鉴定结果表明,其核酸是线型双链DNA,大小在15 kb左右。具有一个正二十面体的头部,头部直径大约为44 nm,无尾,属盖噬菌体科(Tectivirus);裂解性能研究结果表明,其在双层平板上培养12 h可形成中心清亮的噬菌斑,周围有大而明显的晕环。最佳感染复数为0.0001,潜伏期为10 min,裂解量为90.3,是符合条件的潜伏期短裂解量大的理想噬菌体,可用作进一步的应用。

致病性鸡大肠杆菌噬菌体的分离与效价测定

鉴于临床分离的致病性大肠杆菌的耐药谱很广,常规抗生素的治疗效果较差,死疫苗往往存在毒力返祖的问题.实验从污水中分离得到了5株噬菌体,采用自行分离和鉴定的大肠杆菌为宿主菌,经纯化增殖提高效价后进行裂解试验,取得了比较满意的效果.

裂解基因E和核酸酶基因串联表达载体的构建及大肠杆菌菌影的制备

目的构建噬菌体裂解基因E和核酸酶基因串联表达载体,制备高质量的大肠杆菌菌影。方法自行设计一对引物,PCR扩增PhiX174噬菌体裂解基因E,将该基因亚克隆至原核表达载体pGEX-6P-1,构建大肠杆菌菌影的表达载体pGEX-E。在E基因基础上与辅助基因葡萄球菌核酸酶A(SN)基因串联,并插入pGEX-6P-1载体,构建双基因串联高效表达载体pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,采用CaCl2法将其转入大肠杆菌BL21(DE3),经IPTG诱导,制备大肠杆菌菌影。结果裂解基因E单基因及串联基因已成功插入融合表达载体pGEX-6P-1,所构建的大肠杆菌菌影表达载体pGEX-E、pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,诱导后经透射电镜及活菌计数显示大肠杆菌已发生不同程度裂解,制成了大肠杆菌菌影。电镜下菌影形态完整,内容物已释放到胞外。结论已成功构建了噬菌体裂解基因E单基因及裂解基因和核酸酶基因串联基因表达载体,并制成了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗及佐剂形式奠定了基础。

两株副溶血弧菌噬菌体的分离鉴定及裂解性能

为探究副溶血弧菌的生物防治方法,自水产品市场污水排放处及青岛岸边海水中分离出两株副溶血弧菌噬菌体VPp2、VPp3。借助噬菌斑形态、电镜、酶切等技术对其进行了分类鉴定,测定了其裂解谱、最佳感染复数、一步生长曲线并研究其裂解性能。分类鉴定结果表明,VPp2、VPp3分别属于2株不同的噬菌体。其核酸均是线型双链DNA,均具有正二十面体的头部及较长的尾部,且均属肌尾科噬菌体。VPp2头部直径约64nm,尾长约114nm。VPp3头部直径约89nm,尾长约106nm。裂解性能研究结果表明,VPp2、VPp3在双层平板上均能形成清晰透亮的噬菌斑。其裂解谱相同。最佳感染复数均较小,潜伏期均较短,VPp2的裂解量只有11.1,而VPp3的裂解量可达45.3,是符合条件的潜伏期短裂解量大的噬菌体,可进一步应用于噬菌体治疗及检测等。

副猪嗜血杆菌菌影的制备

为研制副猪嗜血杆菌(H.parasuis)菌影疫苗,本实验通过PCR扩增噬菌体PhiX174的裂解基因E,将其连接到含有λPL/PR-cI857启动阻遏系统的pBV220载体中,使裂解基因E与λPL/PR-cI857串联成为温度敏感的裂解盒,构建重组质粒pBV-E。并将含有E基因的裂解盒插入pMD18-T载体中,构建H.parasuis打孔质粒(pMD-E)。采用电击穿孔法将其转入E.coliβ2155中,利用接合的方式将打孔质粒转入H.parasuis 5型菌株中。将含有打孔质粒的H.parasuis在28℃培养至对数生长期,升温到42℃诱导E基因表达,制备H.parasuis菌影。电镜观察菌影形态完整,内容物全部被释放到胞外。本实验为进一步研究菌影疫苗奠定了基础。

猪传染性胸膜肺炎放线杆菌“菌影”的制备

本试验通过PCR扩增噬菌体PhiX174的裂解基因E,将该基因连接到含有λPL/PR-cI857启动阻遏系统的pBV220载体中,从而使裂解基因E和启动阻遏系统λPL/PR-cI857串联成为温度敏感的裂解盒,构建重组质粒pBV-E。再将含有E基因的裂解盒插入到App-E.coli穿梭载体pGZRS-18中,构建胸膜肺炎放线杆菌打孔质粒。采用电击穿孔法将其转入胸膜肺炎放线杆菌中,含有打孔质粒的胸膜肺炎放线杆菌在28℃条件下生长到对数生长期,升温42℃诱导E基因的表达,制备了胸膜肺炎放线杆菌菌影。电镜观察菌影形态完整,内容物全部被释放到胞外。本试验为进一步研究菌影这一新型菌苗及佐剂奠定了基础。

土壤病毒的研究进展与挑战

病毒是由蛋白质和核酸组成的结构简单的非细胞型生物实体,全球病毒数量估算为4.80Í1031,远高于具有细胞型结构的生物数量。土壤是病毒重要的储藏库,土壤中病毒主要以侵染原核生物的噬菌体为主,其数量与寄主细胞数目和土壤理化指标密切相关。虽然学术界已意识到土壤病毒在调控微生物群落结构组成、影响土壤元素循环利用、促进生物进化、以及影响动植物病害,乃至人类健康等方面可能起到重要的作用,但由于受到土壤病毒颗粒的吸附性、土壤异质性,以及研究手段和分析平台的制约,我们对土壤病毒的认知和重视程度远落后于流动的海洋等水体环境。土壤病毒宏基因组中大量的基因序列无法判断其来源及功能,被划分为未知基因,所以病毒也被喻为生物“暗物质”和最大的未知基因宝库。本文在简要介绍了病毒的形态,在土壤中数量、存在形式和多样性的基础上,着重介绍了利用分子标记基因、RAPD和宏病毒组研究技术解析土壤病毒多样性的方法及其局限性,阐述土壤病毒已证实和潜在生态功能。最后,文章提出未来土壤病毒研究应加强的研究方向,强调土壤病毒与土壤微生物耦合研究在揭示土壤生态功能研究中的重要性。