极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用

摘要：【目的】利用有自主知识产权的嗜盐古菌θ型复制质粒和启动子,构建在极端嗜盐古菌模式菌株西班牙盐盒菌（Haloarcula hispanica）中方便使用、功能完善的基因表达载体。【方法】以pSCM201的最小复制子为基础,通过引入莫维诺林抗性基因,大肠杆菌质粒复制子以及氨苄抗性基因,构建了一个新的嗜盐古菌-大肠杆菌穿梭载体。利用定点突变和末端补平法依次将其中多余的酶切位点去除后,再添加hsp5启动子核心序列、人工合成的多克隆位点以及蛋白纯化标签His?Tag等重要元件成功构建了嗜盐古菌表达载体pSCM307。将报告基因bgaH插入到该载体的多克隆位点中并转化H. hispanica AS2049,通过X-gal平板筛选和β-半乳糖苷酶酶活实验检测pSCM307的表达能力。【结果】pSCM307具有独立的自主复制能力,其多克隆位点方便实用,报告基因bgaH在hsp5启动子控制下实现了高效表达。【结论】成功构建了嗜盐古菌领域中第一个方便使用的基因表达载体。     

大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达

目的:构建大肠杆菌-长双歧杆菌穿梭表达载体,并通过此载体使人内皮抑素基因在大肠杆菌和长双歧杆菌中得到表达。方法:以质粒pDG7、pBCSK( )、pET-9C为基础,构建大肠杆菌-长双歧杆菌穿梭表达载体pET-1128,并将人内皮抑素基因插入到新构建的表达载体中,分别转化大肠杆菌BL21(DE3)和长双歧杆菌NQ-1501。诱导表达,表达产物经SDS-PAGE和WesternBlot鉴定。结果:成功构建了大肠杆菌-双歧杆菌穿梭载体,人内皮抑素基因在大肠杆菌和长双歧杆菌中均可表达。结论:构建的穿梭载体为今后用双歧杆菌作为生理菌载体进行肿瘤的基因治疗奠定了基础。     

深海来源低温诱导表达载体pSW4的构建及其应用

【目的】在深海来源穿梭载体p SW2的基础上构建低温诱导高效表达载体,为最终获得环境污染物高效降解菌提供基础和工具。【方法】通过最小化敲除实验确定载体必需基因,以绿色荧光蛋白GFP为报告基因检测载体的表达能力变化,进一步添加纯化标签、多克隆位点及替换启动子等改造获得低温诱导表达载体pSW4。【结果】敲除实验结果显示编码单链结合蛋白的基因fps B敲除后载体的表达效率显著提高。以GFP为报告基因检测发现,pSW4的表达效率较pSW2有显著提升(4°C条件下提高10.7倍)。以深海细菌Shewanella piezotolerans WP3为宿主菌,应用pSW4为表达载体表达深海细菌Shewanella psychrophila WP2的聚合酶亚基,检测显示其在Mg~(2+)条件下具有切割单链DNA的核酸酶活性,而在Mg~(2+)或Mn~(2+)条件下具有切割双链DNA的核酸酶活性。【结论】构建了低温诱导表达载体pSW4,并证实了其适用性,有助于今后构建环境修复菌及相关的应用性研究。     

链霉菌和大肠杆菌穿梭质粒载体的构建

本文报道了链霉菌和大肠杆菌穿梭质粒载体pSE-3的构建;把具有双启动子的大肠杆菌的质粒pGEM-3与新霉素抗性基因启动子缺失的链霉菌的探针质粒pIJ486分别用BamHI和BglⅡ酶切,T4 DNA连接酶连接后转化到E.coli HB101(Amp(?),Neo(?)),所得重组质粒能强启动pIJ486质粒上的氨基糖苷磷酸转移酶基因(aph),并使新霉素抗性基因在大肠杆菌中得到强表达。此重组质粒被命名为pSE-3,当其转化到变青链霉菌TK54(Tsr(?),Neo(?))的原生质体前,新霉素抗性基因亦能得到强表达。酶切结果表明,构建的具有两个启动子的穿梭质粒载体pSE-3上有HindⅢ和EcoRI的单酶位点,拷贝数约为39。经再转化和传代50代等研究表明,穿梭质粒载体pSE-3在链霉菌和大肠杆菌中均是稳定的。为某些有应用价值的目的基因在大肠杆菌和链霉菌中的克隆与表达提供了一个有价值的穿梭质粒载体。     

圆红冬孢酵母磷酸盐饥饿诱导表达载体的构建

摘要:【目的】建立一种适用于圆红冬孢酵母代谢工程的磷酸盐饥饿诱导表达系统。【方法】对圆红冬孢酵母pho89基因5'侧翼序列进行生物信息学分析,设计相应引物,PCR扩增pho89基因启动子(pPHO89)和hsp70基因终止子(tHSP),利用RF克隆方法置换出发载体上的pPGK组成型启动子和tNOS终止子,以潮霉素磷酸转移酶基因hyg为报告基因,得到响应磷酸盐饥饿诱导的单表达盒载体pZPK-pPHO89-hyg-tHSP,利用ATMT方法转化圆红冬孢酵母,通过转化子潮霉素抗性表型鉴定pPHO89和tHSP的启动子和终止子活 性。在此基础上,构建了适合外源基因表达的双表达盒诱导表达载体pZPK-HYG-pPHO89-MCS-tHSP,并利用该载体构建了苹果酸酶重组表达菌株。【结果】成功构建了响应磷酸盐饥饿的圆红冬孢酵母诱导性表达载体,该载体在圆红冬孢酵母中可表现出启动子和终止子活性。【结论】该启动子受磷酸盐浓度的严谨调节,响应度高,操作简单,无需额外诱导剂,经济便捷,为后续圆红冬孢酵母代谢工程研究提供了基本材料。     

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。     

谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析

摘要：【目的】获得谷氨酸棒杆菌10147基因组中具有启动子活性片段的结构序列,为构建表达载体做准备。【方法】利用启动子探测载体pAKC6,采用鸟枪法克隆经过限制性内切酶Sau3A I完全酶切的谷氨酸棒杆菌10147染色体DNA片段,并测定pAKC6上报告基因编码的氯霉素乙酰转移酶（CAT）的比活力,以筛选有启动子功能的片段。【结果】共克隆到30个具有启动子功能的片段。其中有三个插入片段起动的氯霉素乙酰转移酶比活力大于24 U/mg,插入片段F57起动的CAT比活力为32.50 U/mg;而插入有启动子Ptrc的阳性对照的CAT比活力为26.33 U/mg。【结论】获得三个DNA插入片段具有与已知启动子Ptrc相当的启动活性,这些片段可以用于构建谷氨酸棒杆菌表达载体。     

大肠杆菌-链霉菌穿梭载体的构建及应用

pIJ6021和pIJ4123是链霉菌的高拷贝表达载体,它们携带有受硫链丝菌素诱导的强启动子P_tipA。分别在它们的合适位点插入大肠杆菌质粒的复制子和在大肠杆菌中选择用的抗性标记基因(bla),得到了两个能在大肠杆菌和链霉菌中穿梭复制、并保持结构稳定的链霉菌表达载体：pHZ1271和pHZ1272。将透明颤菌(Vitreoscillia sp.)血红蛋白基因(vhb)克隆到pHZ1272中,用它转化变铅青链霉菌(Streptomyces lividans),经Western blotting分析和CO结合实验表明,在变铅青链霉菌中表达出了有生物活性的透明颤菌血红蛋白,从而证明所构建的pHZ1272载体具有在链霉菌中表达外源基因的功能。     

一个热诱导穿梭表达载体的构建及其在链霉菌中的应用

为探索大肠杆菌λ噬菌体表达调控元件在链霉菌中的应用,构建了一个链霉菌大肠杆菌穿梭表达载体pHZ1080,并将来自链霉菌FR-008的聚酮合酶(PKS)基因置于其中的λ噬菌体启动子P_R下游,得到表达PKS的穿梭质粒pHZ1067。与在大肠杆菌中一样,该质粒在变铅青链霉菌中也受热诱导表达100kD的PKS蛋白;表达的PKS蛋白可由SDSPAGE和Western-blot实验检测到。PKS在链霉菌中的热诱导表达表明,构建的载体也能用于链霉菌诱导表达外源基因。       

变形链球菌F-ATPase启动子荧光蛋白载体的构建及表达

目的构建由质子移位膜ATP酶(membrane-bound proton-translocating ATPase,F-ATPase)启动子启动的绿色荧光蛋白报告基因穿梭表达载体,观察其在大肠埃希菌中的表达同时鉴定表达产物。方法以变形链球菌(UA159)基因组为模板,扩增F-ATPase启动子片段,构建由F-ATPase启动子启动的绿色荧光表达载体pFgfp,酶切F-ATPase启动子及绿色荧光蛋白编码基因,连接到穿梭质粒pDL276,构建重组载体pLFgfp。结果重组质粒pLFgfp酶切及基因序列分析证实目的片段成功插入,重组载体转化后的大肠埃希菌有绿色荧光蛋白的表达,并能随着细菌传代继续表达。结论 F-ATPase启动子启动的绿色荧光蛋白穿梭表达载体pLFgfp构建成功,为研究生物膜环境中耐酸菌F-ATPase毒力因子的表达奠定基础。     

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-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

A plasmid vector for an extreme thermophile, Thermus thermophilus

The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis. A Thermus-E. coli shuttle vector pYK109 was constructed. pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E. coli plasmid vector pUC13. pYK109 transformed T. thermophilus HB27 trpB5 to Trp+ at a frequency of 10(6) transformants per microgram DNA.     

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。     

Construction of a green-fluorescent protein-based, insertion-inactivation shuttle vector for lactic acid bacteria and Escherichia coli

A shuttle vector, p5aGFP2201a, for lactic acid bacteria and E. coli was constructed by using the gene of a jellyfish green fluorescent protein ( gfp) as a selection marker. The plasmid was shown to function as a shuttle vector by its ability to carry and express a staphylococcal chloramphenicol acetyltransferase ( cat) gene into targeted hosts.     

糖多孢红霉菌多拷贝表达载体pZM的构建

对糖多孢红霉菌染色体上红霉素生物合成基因进行改造 ,已经合成了多种红霉素类似物。在糖多孢红霉菌中对红霉素类似物进行结构修饰 ,以pWOR1 0 9质粒为基础构建糖多孢红霉菌多拷贝表达载体pZM。pZM载体带有PermE启动子、fd终止子、多克隆位点、硫链丝菌肽和氨苄青霉素抗性基因、以及在大肠杆菌和糖多孢红霉菌中复制的ColE1ori和pJV1ori复制子 ,系可在大肠杆菌和糖多孢红霉菌中扩增的穿梭质粒。在糖多孢红霉菌中 ,pZM可以表达氨普霉素抗性基因和绿色荧光蛋白基因 ,从糖多孢红霉菌中提取的表达质粒酶切图谱与转化前一致 ,表明pZM是糖多孢红霉菌中多拷贝、稳定的表达载体。