极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌(Archaea)是一类与细菌及真核生物显著不同的生命的第三种形式[1],大多生活在极端或特殊环境,主要包括产甲烷古菌(Methanogenic Achaea)、极端嗜盐古菌(Extremely Halophilic Archaea)和极端嗜热古菌(Extremely Thermophilic Archaea)等三大类.极端古菌是极端环境微生物的重要成员,也是极端环境微生物资源开发的重要领域.其中,嗜盐古菌可产生一类蛋白类抗生素,称为嗜盐菌素(halocin).     

极端嗜盐古菌中CRISPR结构的生物信息学分析

摘要：【目的】利用生物信息学方法了解目前拥有全基因组序列的极端嗜盐古菌中CRISPR结构的特征。【方法】通过比对,保守性分析,GC含量分析,RNA结构预测等方法对已有全基因组序列的嗜盐古菌基因组进行研究。【结果】在5株嗜盐古菌基因组中发现CRISPR结构,在leader序列内得到具有回文性质的保守motif。发现在大CRISPR结构内repeat序列具有很强的保守性。同时根据第四位碱基的不同,repeat序列可形成两类不同的RNA二级结构。【结论】leader序列中回文结构的发现对其可能为蛋白结合位点的假     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌 (Ａｒｃｈａｅａ)是一类与细菌及真核生物显著不同的生命的第三种形式[1] ,大多生活在极端或特殊环境 ,主要包括产甲烷古菌 (ＭｅｔｈａｎｏｇｅｎｉｃＡｃｈａｅａ)、极端嗜盐古菌 (ＥｘｔｒｅｍｅｌｙＨａｌｏｐｈｉｌｉｃＡｒｃｈａｅａ)和极端嗜热古菌 (ＥｘｔｒｅｍｅｌｙＴｈｅｒｍｏｐｈｉｌｉｃＡｒｃｈａｅａ)等三大类。极端古菌是极端环境微生物的重要成员 ,也是极端环境微生物资源开发的重要领域。其中 ,嗜盐古菌可产生一类蛋白类抗生素 ,称为嗜盐菌素 (ｈａｌｏｃｉｎ)。与细菌素相似[2 ] ,嗜盐菌素是由质粒编码、核糖体合…     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

嗜盐古菌噬菌体研究进展

嗜盐古菌噬菌体是噬菌体的一个重要分支,在群体生态学以及生命的起源与进化历程中扮演重要的角色.综述了嗜盐古菌噬菌体的形态多样性、研究方法、起源与进化几方面的研究报道,指出目前研究中存在的问题,对未来研究进行了展望.     

极端嗜盐古菌遗传转化系统的研究

对极端嗜盐古菌遗传转化系统的研究进展进行了综述,内容包括抗性标记基因的选择,基因克隆和表达载体系统的发展以及受体系统的改造。     

嗜盐古菌分类学研究进展

嗜盐古菌是一类需要高盐维持生长的古菌。到目前为止,已发现的嗜盐古菌都属于古菌域的广古菌门,主要包括:嗜盐甲烷古菌类群、嗜盐古菌纲的全部成员以及尚不能培养的纳米嗜盐古菌类群。嗜盐古菌是盐环境的土著类群,驱动着盐环境生态系统的生物地球化学循环。作为极端微生物,嗜盐古菌在理论研究和应用领域具有重要的研究价值。本文从嗜盐古菌分类学地位的变迁、分类学方法、分类学研究现状及我国的嗜盐古菌分类学研究等方面综述了嗜盐古菌分类学的最新研究进展。     

嗜盐古菌启动子的研究及应用

嗜盐古菌是古菌域的一个重要代表类群,在遗传与代谢、进化与适应、前沿生物技术及合成生物学领域都显示了其重要的研究价值。嗜盐古菌启动子的认识和利用,可以为嗜盐古菌的基础和应用研究提供必要的条件。本文从古菌启动子的结构与功能出发,就启动子的研究方法、嗜盐古菌启动子的特征及嗜盐古菌启动子的应用3个方面综述了嗜盐古菌启动子的研究现状,并对嗜盐古菌启动子未来研究的重点和方向进行了展望。     

基于PHA颗粒-PhaP标签和内含肽元件的极端嗜盐古菌蛋白的表达纯化系统

【目的】建立一个基于PHA颗粒-PhaP标签的简便实用的极端嗜盐古菌蛋白表达纯化系统,并探讨嗜盐古菌内含肽在该系统优化中的应用。【方法】以嗜盐古菌-大肠杆菌穿梭载体pWL502为基本骨架,构建带有嗜盐古菌强启动子和PhaP融合标签的表达载体;在地中海富盐菌phaP缺陷株(ΔphaP)中融合表达目的基因,通过蔗糖密度梯度离心分离纯化结合于PHA颗粒上的PhaP融合蛋白;在phaP基因与多克隆位点之间引入特定的嗜盐古菌内含肽元件,尝试通过定点突变改变该内含肽的剪切活性。【结果】成功构建了以PhaP作为N端融合标签的表达载体pPM以及作为C端融合标签的载体pIP;在phaP基因簇强启动子控制下,二者均实现了目标蛋白的高效表达;通过PHA颗粒介导的蛋白分离纯化策略,实现了以PhaP为融合标签的目标蛋白的分离纯化;发现内含肽序列Hbt21在地中海富盐菌中保持了高效的剪接活性,通过定点突变其C端末位氨基酸天冬酰胺(N182)及邻位的丝氨酸(S183)失活了该内含肽的C端剪接活性。【结论】首次建立了一个基于PHA颗粒-PhaP标签的简便节约的极端嗜盐古菌蛋白表达纯化系统,并确定了嗜盐古菌型内含肽C端剪接的活性位点,为该内含肽将来应用于PhaP融合蛋白的标签去除奠定了基础。     

Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

大肠杆菌-链霉菌穿梭表达载体pMF的构建及其应用

【目的】构建能定点整合到链霉菌(Streptomyces)染色体上的高效表达载体。【方法】以链霉菌自杀型表达载体pLSB2为基础,通过插入链霉菌噬菌体ΦC31整合酶基因int和attP位点(Phage attachment site),构建了能在大肠杆菌和链霉菌之间进行接合转移并定点整合到链霉菌染色体上的表达载体pMF。将pMF转化大肠杆菌ET12567(pUZ8002),并分别接合转移天蓝色链霉菌(Streptomyces coelicolorM145)、变铅青链霉菌(Streptomyces lividansTK24)和红色糖多孢菌(Saccharopolyspora erythraea2338),挑取接合子进行PCR和Southern杂交检测。将来自刺糖多孢菌S08-4的S-腺苷甲硫氨酸合成酶基因(SAM-s)克隆到载体pMF的启动子下游,接合转移到天蓝色链霉菌中。【结果】表明pMF成功整入链霉菌染色体,并且检测到目的蛋白的表达。【结论】构建的pMF载体可作为外源基因定点整合表达的有效工具,为后续的基因功能研究以及链霉菌的遗传改造奠定了基础。     

Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea

The pNB101 is the first plasmid to be isolated from an haloalkaliphilic archaea. With insertion of the ColE1 replicon of Escherichia coli, as well as two antibiotic resistance genes at its unique Hin dIII site, a novel shuttle vector between haloarchaea and E. coli was developed. This vector, named pNB102, was successfully transformed into two non-alkaliphilic haloarchaea, Halobacterium salinarum SNOB and Haloarcula hispanica ATCC33960. The presence and stability of pNB102 in the transformants were confirmed by PCR identification, Southern blotting and restriction endonuclease digestion. Results also indicated that the presence of restriction-modification (R-M) systems in some Halobacterium species prevented this transformation. It is the first report that the replicon of pNB101 has such a wide host range, and has taken the first step for construction of the vector/host system in haloalkaliphilic archaea.     

Construction of a new high-copy number shuttle vector of Bacillus thuringiensis

AIMS: Construction and characterization of a new cloning shuttle vector for gene transfer and expression in Bacillus thuringiensis. METHODS AND RESULTS: A novel short and high-copy number shuttle vector called pHBLBIV, was constructed for gene transfer and expression in Bacillus thuringiensis. A 1.6-kbp replicon of a relatively high-copy number endogenous plasmid of a selected B. thuringiensis strain was ligated to Escherichia coli pUC18 replicon containing the ampicillin and the erythromycin resistance genes used for the selection of respectively E. coli and B. thuringiensis transformants. The constructed vector was shown to have a high copy number compared with the conventional B. thuringiensis vectors, and used successfully for the transfer of vegetative insecticidal protein-encoding gene (vip) in between B. thuringiensis strains. CONCLUSIONS: A new shuttle vector of B. thuringiensis-E. coli named pHBLBIV was constructed. It was characterized by its high copy number, small size and segregational stability. This vector was successfully used for vip gene cloning and transfer in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel shuttle vector has been constructed, which has demonstrated potential for the cloning and expression of genes in B. thuringiensis.     

大肠杆菌-鸭疫里默氏杆菌高效穿梭质粒pFY02的构建和应用

鸭疫里默氏杆菌(Riemerella anatipestifer,RA)是引起鸭、鹅、火鸡等家禽传染性败血症及浆膜炎的主要病原。目前主要通过基因缺失及基因回补的方法对鸭疫里默氏杆菌的基因功能进行研究。然而,目前使用的穿梭质粒pLMF03存在结合转移效率低、酶切位点少等缺陷,不能用于所有鸭疫里默氏杆菌基因的回补。为解决这一问题,文中将结合转移位点oriT、鸭疫里默氏杆菌复制起始基因pRA0726ori、高表达启动子基因及多种酶切位点逐一克隆至质粒pPM5,构建了新的穿梭质粒pFY02。结果表明,该质粒能够稳定存在于鸭疫里默氏杆菌,且具有较高的结合转移效率。通过回补鸭疫里默氏杆菌tonB2基因缺失株表明,该质粒可用于鸭疫里默氏杆菌基因的回补。总之,文中构建的穿梭质粒pFY02更加完善了用于鸭疫里默氏杆菌基因回补的材料。     

谷氨酸棒杆菌/大肠杆菌穿梭型启动子探测载体构建

从质粒pXZ10145和pUC19出发,构建了一个谷氨酸棒杆菌/大肠杆菌穿梭载体pAK6。pAK6的大小为5684bp,带有卡那霉素和氨苄青霉素抗性选择标记,以及多克隆位点。在pAK6基础上,构建了以氯霉素乙酰转移酶为报告基因的启动子探测载体pAKC6,pAKC6的大小为6474bp。采用鸟枪法,将经Sau3AI消化的谷氨酸棒杆菌基因组片段连入pAKC6;根据谷氨酸棒杆菌对氯霉素的抗性,从中分离出两个具有启动子功能的插入片段。通过测定报告基因氯霉素乙酰转移酶的活性,对两个启动子片段在谷氨酸棒杆菌中的强度进行了初步的判断;测序后,用启动子预测软件对其结构进行了预测,证实了启动子序列的存在。     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.