蓄势待发的印度生物技术产业

目前正是印度生物技术产业发展的关键时期。虽然其产业规模不大,但它的国际地位正在提升。印度的自然资源和智力资源都很丰富,而且在生物技术应用领域已经取得了令人瞩目的成就。印度生物技术公司更加国际化,并且取得了显著的进步,公司将更加注重创新,以实现可持续发展。印度政府已经开始通过增加研发经费和开发人力资源来加速生物技术发展的步伐。     

质粒pBR322电注入E.coli HB101的研究

常用的转化方法是用CaCl_2或CaCl_2/RbCl_2处理受体菌使成感受态。转化率约为10~3—10~6个转化子/μg质粒DNA。近年发展起来的电转化技术可使转化率提高到lC~(?)—10~(10)个转化子/μg质粒DNA。我们以质粒pBR322和大肠杆菌HB101为实验材料,对常用的转化方法和电转化的结果进行了比较,对电转化中获得最佳转化效果和各种参数如电压、脉冲时间、脉冲次数、循环数等进行了探讨。     

人β干扰素重组质粒导入CHO—dhfr^—细胞

采用磷酸钙共沉淀技术,将人β干扰素编码区基因片段与pSV_2-dhfr载体质粒SV40早期启动子下游60碱基对(bp)处重组的pSVEHβ_1质粒导入中国仓鼠卵巢细胞二氢叶酸还原酶缺陷型株,简称CHO-dhfr~-细胞。共转化后,于选择培养基中长出的265个细胞克隆中随机挑出13株细胞克隆进行表达水平检测,其中7株细胞有表达,表达阳性细胞克隆株占54％,共转化率为1.33×10~(-4)。     

腾飞中的印度生物技术产业

Pharmaceutical Business News2003年442期14页报道：印度的生物技术产业已进人爆炸性发展阶段。统计表明，印度生物技术产业的年销售额，已从数百万美元猛增至数十亿美元。     

乳杆菌电转化条件的研究

用Pgk12、Pmg36c等质粒电转化不同的乳杆菌。研究了影响转化效率的多种因素。受体细胞经20μg/ml氨苄青霉素处理1h,可以提高转化效率200倍。同时,发现电击后的细胞必需在高渗培养基中才能存活,电击后2～3h的复苏表达期和用亚抑制抗生素浓度选择转化子,这些对电转化成功以及提高转化效率都是十分关键的。在改进电转化方法中,各种参数为电场强度8.75kV/cm,电阻100Ω或200Ω,电容25μF和2×磷酸缓冲液。     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3．1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1—egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45％。瞬时转染pcDNA3．1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

HIV-1gag/IFNα-2b的构建与表达鉴定

目的：构建艾滋病病毒核心蛋白(gag)与干扰素(IFNα-2b)融合基因表达质粒,观察其在痘苗病毒中共表达结果,研究其意义。方法：利用基因重组技术,将IFNα-2b基因片段插入到gag基因的nt531位点,经脂质体转染与血凝素阴性蚀斑筛选,挑出重组痘苗病毒。经免疫荧光、Western blot和Dot-ELISA鉴定表送产物。结果：间接免疫荧光实验结果显示,转染重组质粒的细胞表面有绿色荧光。免疫印迹实验与Dot-EILSA结果均显示重组质粒转染细胞的裂解物中存在表达的gag／IFNα-2b蛋白。结论：成功地构建了重组真核细胞表达质粒,表达的蛋白具有良好的免疫原性与免疫反应性。     

消除内生质粒对苏云金芽胞杆菌YBT-1463特性的影响

研究了经完全消除苏云金芽胞杆菌野生菌株YBT-1463的内生质粒对该菌部分形态、遗传及生理生化特性的影响。结果表明,消除内生质粒后的无质粒突变株不形成伴胞晶体,但电转化4种供体质粒,即pBMBl21、pBMB304-1Ab、pBMBLC和pBMB9748的效率显著提高,转化频率最高比出发菌株提高6.8×10 倍,而无质粒突变株对红霉素等10种抗生素的敏感性,对葡萄糖等19种碳源和谷氨酸等12种氮源的利用能力及生长性能与出发菌株无明显差异。     

Red体内同源重组介导的egfp、kan基因融合和重组质粒构建

重组工程是近年来建立的一种基于高效率体内同源重组的新型遗传工程技术,可应用于靶DNA序列的敲入、敲除和基因克隆等。在应用重组工程技术进行基因亚克隆时发现,体外重叠PCR法难以获得高质量的目的DNA打靶片段,严重影响重组效率。为了解决上述问题,根据Red重组酶介导的体内同源重组工作原理进行了技术改进。先用PCR方法合成egfp和kan两条末端互补的线性DNA片段,然后将其电击共转化进入携带Red重组酶和pcDNA3.1载体DNA的大肠杆菌DY331菌株内,经体内同源重组直接产生的pcDNA3.1-egfp-kan环状重组质粒DNA分子可通过抗生素标记筛选获得,阳性率可达到45%,瞬时转染pcDNA3.1-egfp-kan可获得绿色荧光蛋白在293细胞中的表达。     

生物信息学及其在环境微生物研究中的应用

生物信息学的快速发展,推动了微生物信息学的建立。模式微生物基因组学的研究,极大地丰富了生物信息学的内容。微生物结构基因组学和功能基因组学研究试图揭示基因结构与功能的内在联系,绘制出基因调控网络图。基因组功能注释是功能基因组学研究的主要目的。基因芯片技术的运用,成为环境微生物生态研究和功能酶基因定位的有力工具。生物信息学为环境微生物的研究和发展提供了一个崭新的信息平台和技术手段。介绍了一些相关数据库和专业网站。     

High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions

High efficiency transformation of Pseudomonas aeruginosa was achieved using frozen cell suspensions and high voltage electroporation. We have obtained frequencies as high as 5.8 x 10(8) transformants/micrograms of plasmid DNA using PA01 strain OT684 and a buffer of 15% glycerol-1 mM MOPS. The method allows for easy and reproducible production of frozen cell suspensions for rapid transformation of P. aeruginosa.     

融合标签技术及其应用

融合标签最初是作为一种有效的工具用于纯化重组蛋白质,近几年的研究表明,融合标签的作用并不局限于此。本文综述了融合标签技术的发展及在生命科学研究中的各种应用,包括重组蛋白质的纯化;目的蛋白质的检测、定向固定;体内生物事件的可视化;提高重组蛋白质的产量;增强重组蛋白质的可溶性及稳定性。     

Electroporation of Azospirillum brasilense with plasmid DNA

Abstract The feasibility of electric field mediated transformation of the nitrogen fixing bacterium Azospirillum was studied. The broad host range plasmid pRK290 was used throughout this study. Transformants were obtained with all A. brasilense strains tested, although with strain dependent efficiency. No transformants were obtained with an A. lipoferum strain. Transfer of the pRK290 plasmid DNA in the A. brasilense strains was confirmed by DNA extraction of the transformants and gel electrophoresis. The effects of the physiological status of the cells and the electric field strength during electroporation were studied in detail for one particular A. brasilense strain.     

重组胸腺素α1 pMAL-C2x-Tα1/TB1工程菌的构建与表达

目的构建表达重组胸腺素α1（Tα1）的pMAL-C2x-Tα1/TB1工程菌。方法将人工合成的Tα1序列进行PCR扩增,将扩增的片段和pMAL-C2x质粒载体分别经BamHI和EcoR I双酶切后,用T4 DNA快速连接酶连接构建pMAL-C2x-Tα1融合表达质粒,再经测序正确后,将重组体转化至大肠埃希菌TB1菌中,pMAL-C2x-Tα1/TB1菌在LB液体培养基中培养,经IPTG诱导表达麦芽糖结合蛋白与Tα1的融合蛋白（MBP-Tα1）,采用Westernblot对MBP-Tα1进行鉴定。结果 pMAL-C2x-Tα1/TB1工程菌能有效表达MBP-Tα1,融合蛋白占菌体蛋白的33.6%,分子量约为45×103。结论工程菌的成功构建和表达为重组Tα1的纯化、生物学活性等研究奠定了基础。     

Electroporation optimization to deliver plasmid DNA into dental follicle cells

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45–120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.     

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri

Abstract Two strains of Lactobacillus acidophilus Group A1, the neotype ATCC 4356 and a human isolate NCFM-N2, widely used as a dietary adjunct in milk and cultured dairy products, were transformed with plasmid DNA by electroporation. The transformation characteristics exhibited by the two L. acidophilus strains were found to differ markedly even though they appeared similar at the genomic level based on the DNA patterns of Sma I restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the A1 DNA homology group.     

基因芯片及其在环境微生物研究中的应用

基因芯片因其具有高密度、高灵敏度、快速 (实时 )检测、经济、自动化和低背景水平等特点 ,而广泛应用于不同的研究领域。目前 ,应用于环境微生物研究的基因芯片主要有功能基因芯片 (FGAs)、系统发育的寡核苷酸芯片 (POAs)和群落基因组芯片 (CGAs)。综述了基因芯片在环境微生物研究中的应用 ,包括自然环境中微生物的基因表达分析、比较基因组分析和混合微生物群落的分析等。讨论了基因芯片面临的挑战和前景展望     

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

Abstract The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.     

A cost-effective approach to microporate mammalian cells with the Neon Transfection System

Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold.