Red/ET重组AHBA生物合成基因簇打靶载体的构建与鉴定

为确定3-氨基-5-羟基-苯甲酸(AHBA)生物合成基因簇在链霉菌中与次生代谢产物的关系,运用PCR技术,从33株AHBA合酶基因阳性菌株扩增与AHBA生物合成基因簇中编码AHBA合酶(A)、氧化还原酶(O)、磷酸化酶(P)基因,获得24株AOP基因阳性菌株.根据靶基因A基因下游和P基因上游同源序列设计50 bp引物,中间插入卡那霉素抗性基因的DNA片段,进行PCR,获得外源DNA片段.经过电转化,将外源DNA片段和pKC1139-AOP重组质粒共转入含重组酶质粒大肠杆菌HS996/ pSC101-BAD-gba-(Tet).在Red重组酶的作用下,外源DNA片段与重组质粒pKC1139-AOP上的AHBA基因簇的同源区域重组,构建了AHBA基因簇打靶载体.研究显示了Red/ET重组工作效率高、操作简单、精确的优点,可大大缩短构建打靶载体的时间.     

PCR扩增乙酰乳酸合酶基因片段的研究

实验对大肠杆菌ＰＰＡＷ－１质粒的ＤＮＡ（含乙酰乳酸合酶基因）进行了提取分离与纯化,并以此质粒ＤＮＡ为模板,用乙酰乳酸合酶基因的３个引物５′－Ｂ．ｎ．、３′－ＭＷ－１和３′－Ｂ．ｎ．,通过ＰＣＲ扩增得到了乙酰乳酸合酶基因的大片段,克服了用２个引物不能扩增合成乙酰乳合酶基因的困难．     

大肠杆菌硫氧还蛋白thioredoxin基因的克隆及表达

以大肠杆菌XL1blue为模板，通过PCR技术扩增大肠杆菌硫氧还蛋白基因，并将目的基因分别连接到克隆载体pUC18和表达载体pTrcHisC上，构建重组质粒．重组质粒pTrcHisC-TRX在大肠杆菌中高效表达，最后利用固定化金属螫合亲和层析技术(IMAC)获得较纯的目的蛋白，为进一步研究硫氧还蛋白的功能及其应用提供了条件．     

中国流行株HIV—1gp120及其与IFNα基因共表达核酸疫苗质粒的构建及表达

目的 研究pGP和pGPIFNα在真核细胞中的表达。方法 以表达中国流行株HIV－1gp120基因的核酸疫苗质粒pGP及其表达中国流行株HIV－1gp120基因与IFNα基因与IFNα基因的核酸疫苗质粒pGPIFNα。转染BHK－21细胞，以间接免疫荧光鉴定其表达产物。结果 荧光显微镜下，pGP和pGPIFNα转染细胞可见绿色荧光，而HK－21细胞和空质粒则不见绿色荧光。结论 pGP和pGPIFNα可在真核细胞中表达目的蛋白gp120。     

NF—IL6对iNOS基因表达的负调控

诱生型一氧化氮合酶基因的调控序列中含有ＮＦ－ＩＬ６的识别元件，但ＮＦ－ＩＬ６对ｉＮＯＳ基因的调控功能目前尚不清楚。研究发现ＰＳ２／０骨髓瘤细胞能低剂量持续表达ｉＮＯＳ，该利用该模型将含有ｉＮＯＳ调控序列的报告基因质粒与ＮＦ－ＩＬ６的表达质粒共转染至ＳＰ２／０细胞，观察到ＮＦ－ＩＬ６５的表达能抑制ｉＮＯＳ基因的表达，并呈剂量效应关系，初步认为ＮＦ－ＩＬ６是ｉＮＯＳ基因表达的负调控因子。     

大肠杆菌磷酸果糖激酶基因在野生型专性自养嗜酸性氧化硫硫杆菌Tt-Z2中的表达

利用PCR技术扩增E．coli K-12磷酸果糖激酶-1基因(pfkA)，将该基因与广泛寄主的IncQ质粒pJRD215连接构建重组质粒pSDK-1，该质粒可与pfk基因缺陷株E．coli DF1010互补．通过接合转移的方式将其导入专性自养极端嗜酸性氧化硫硫杆菌Tt-Z2中并得到表达．pSDK-1在宿主Tt-Z2中有较好的稳定性，在无选择压力条件下连续传50代仍可保留68％．酶活性测定表明，pSDK-1的pfkA基因在缺陷株E．coli DF1010中表达水平略高于出发菌株E．coli K-12；而在氧化硫硫杆菌中则以较低水平进行表达．葡萄糖可促进含pSDK-1的氧化硫硫杆菌Tt-Z2的生长，而对照菌株的生长则未受明显影响．说明重组菌可部分利用葡萄糖作为碳源而生长．     

在杂合启动子控制下乙肝病毒表面抗原SA-28融合基因在酵母中的表达及调控

采用酵母杂合启动子PADH2－CUP1或PADH2－GAPDH及终止子TADH1，构建了一系列酵母表达载体．在这些表达载体中插入乙肝病毒表面抗原S－preS1融合基因SA－28后，将合乙肝病毒表面抗原的表达单元克隆至高稳定质粒PHC11的BamHI位点．然后将表达质粒分别转化酿酒酵母Y16，Y19．对SA－28基因表达的研究表明，在酵母菌胞内实现了SA－28基因的高表达，且表达受葡萄糖浓度调控．     

Ri质粒与发状根的研究概况

本文从Ri质粒转化植物细胞的机理。发根农杆菌诱导能力的影响因素。Ri质粒的质应用，问题及展望四个方面对Ｒi质粒与发状根的研究进行了综述，详尽介绍了利用Ri质粒转移外源基因，接种。发状根除菌，发状根的增殖，发状根的选择与分化，再生植株的检测和利用发状根的培养技术生产天然有用物质及扩大植物基因资源和促进生根等方面的研究结果。     

限制性内切酶介导的整合技术

限制性内切酶介导的整合技术是近年发展起来的一种研究基因的新方法,与质粒拯救相结合,可快速分离质粒两侧的DNA序列,进而对该序列做进一步分析和研究．该技术已应用于基因突变、基因标记、寻找新基因等研究领域．本文重点介绍了限制性内切酶介导的整合技术的原理、影响因素及应用现状．     

IL-2与猪瘟病毒E_2基因真核双表达载体的构建及其免疫增强作用的研究

应用DNA重组技术构建了猪瘟病毒E2 基因与IL 2基因的双表达真核表达载体 ,观察其表达水平 ,并对其免疫增强效果进行了观察。结果是构建了猪瘟病毒E2 基因与白细胞介素 2的真核表达质粒pIRSTIL 2。将质粒转染BHK 2 1细胞 ,可在体外表达E2 和有生物活性IL 2。pIRSTIL 2质粒能诱导产生CSFV的特异性免疫反应。pIRSTIL 2所诱导的免疫应答反应比使用单表达质粒pIRST强。实验表明 :IL 2与猪瘟病毒E2 基因构建的双表达基因疫苗能有效提高基因疫苗的免疫效果     

PCR法快速鉴定阳性克隆实验的改进

分析了常规PCR扩增鉴定阳性克隆实验中存在的问题,进行了改进探索:反转录PCR(RT-PCR)获得目的基因片段,TA克隆连接质粒载体、转化大肠杆菌感受态细胞;利用目的基因的特异性引物,用菌液PCR法直接筛选重组克隆,在筛选的阳性菌液中扩增到与目的基因片段大小一致的阳性条带,与质粒PCR及酶切的阳性对照一致,验证了菌液PCR具有可靠性。实验证明,自身引物菌液PCR法用于定性筛选重组阳性克隆,是一种简便、快速、有效的方法。     

恶臭假单胞菌萘质粒ⅠNL基因的克隆及酶切图谱

恶臭假单胞菌（ＰｓｅｕｄｏｍｏｎａｓｐｕｔｉｄａＧ７）携带的质粒ＮＡＨ７，经限制性内切酶ＥｃｏＲＩ切割后，产生含有萘降解酶全部基因（２４，６ｋｂ）的片段，此片段插入到载体ｐＵＣ１１９的多克隆位点，构成了ｐＫＡＯ质粒，此质粒经ＨｐａＩ，ＥｃｏＲＩ酶切获得的５．１ｋｂ片段亚屯隆到ｐＵＣ１１９中，衍生出ｐＮＮ１质粒，绘制出内切酶图谱。从ｐＮＮＩ质粒上切下含目的基因ｎａｈＩ、ｎａｈＮ和ｎａｈＬ的ｋｐｎＩ－ｋｐｎＩ片段（２．３ｋｂ）．正向插入载体ＢｌｕｅｓｃｒｉｐｔⅡＳＫ＋中获得重组质粒ｐＮＮ２，反向插入为ｐＮＮ３，将其转化到大肠杆菌ＸＬ１－Ｂｌｕｅ中，目的基因得以大量增殖。     

The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.     

A transposon, Tn732, encoding gentamicin/tobramycin resistance.

Gentamicin and tobramycin are important antibiotics in the treatment of hospital infections because of their activity against a wide range of bacterial genera. With their increasing use, bacteria resistant to these drugs have appeared, the resistance being frequently plasmid determined. The resistance genes determine various enzymes that modify and inactivate the drugs and there is association between particular gentamicin/tobramycin resistance genes and plasmids of particular groups, implying that acquisition of such a gene by any plasmid is a rare event. We now report the identification of a transposon or 'jumping gene' encoding the gentamicin/tobramycin adenylylating enzyme, ANT(2"), on a plasmid of incompatiblity group FII (IncFII).     

通用型奶牛多位点基因打靶载体系统的构建

以奶牛为研究对象,以其重复的rRNA基因间的间隔序列为靶位点,基于BAC重组酶系统构建多位点基因打靶载体,为建立体内多位点基因打靶技术获得关键材料.首先构建BAC-TDN筛选载体,然后构建pYLVS-GD表达载体.将BAC-TDN筛选载体和pYLVS-GD表达载体共转化至大肠杆菌NS3529中,通过Cre重组酶的作用形成BAC-TDN-VS-GD质粒,采用归位内切酶I-SceI切除pYLVS载体骨架,构建奶牛多位点基因打靶载体BAC-TDN-GD,利用接头LS使之环化.BAC-TDN-GD打靶载体与pYLSV质粒组成了通用型奶牛多位点打靶载体系统.以重复序列为靶位点的多位点基因打靶技术,将部分解决目前存在的打靶效率低、安全性差等问题.     

Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.     

利用RIG质粒在大肠杆菌中有效表达转录因子类蛋白

 大肠杆菌是常用的基因工程蛋白表达系统宿主菌,而密码子偏倚性常常成为某些异种蛋白质在大肠杆菌中成功表达的障碍.利用含有编码多种稀有tRNA的重组质粒RIG,一个含有大量稀有密码子的人类线粒体转录终止因子样蛋白成功地在大肠杆菌中得到表达.证明使用RIG质粒是克服大肠杆菌密码子偏倚性的有效手段.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

海洋鳗弧菌铁载体合成的调控

以一株鳗弧菌pJM1质粒缺陷为研究对象，考察了儿茶酚类铁载体Anguibactin的合成代谢调控。研究发现：在由染色体和质粒共同编码介导的铁载体Anguibactin的合成途径中，其分界点位于2，3－二羟基苯甲酸合成，2，3－二羟基苯甲酸合成之前代谢途径受染色体编码调节,后由质粒编码介导；编码2，3－二羟基苯甲酸的染色体相关基因的表达受环境铁离子浓度的诱导调节。