转基因红豆草植株的再生

红豆草子叶外植体能被含非致瘤性的Ti质粒的根癌农杆菌感染.该载体质粒含一个npt-Ⅱ基因和一个胭脂碱合成酶基因.对卡那霉素抗性植株的胭脂碱检测、NPT-Ⅱ酶活性测定和DNA分子杂交试验表明,外源的npt-Ⅱ基因和胭脂碱合成酶基因已导入了红豆草细胞并能在植株水平表达相应的性状.     

Construction,Purification, and Characterization of Anti-BAFF scFv–Fc Fusion Antibody Expressed in CHO/dhfr<Superscript>−</Superscript> Cells

Elevated levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, we have constructed a vector for the expression of a novel compact antibody composed of anti-BAFF single-chain antibody fragment (scFv) and the Fc region (the hinge region, CH2, and CH3 domains) of human IgG1 in Chinese hamster ovary cells. The scFv–Fc fusion protein, showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges, was affinity-purified on protein A Sepharose from culture supernatant. The scFv–Fc antibody was demonstrated to retain high binding affinity to antigen and prolonged clearance time in blood and to possess some human IgG crystallizable fragment effector functions such as protein A binding and antibody-dependent cellular cytotoxicity. These results suggest that this recombinant antibody may have therapeutic applications in the therapy of autoimmune disorders mediated by BAFF.     

限制性内切酶指纹-高效毛细管电泳激光诱导荧光法测定原核/真核质粒

采用线性聚丙烯酰胺修饰石英毛细管的高效毛细管电泳无胶筛分技术,对原核/真核质粒用不同限制性内切酶酶切,运用限制性内切酶指纹-高效毛细管电泳激光诱导荧光(REF-HPCE-LIF)检测法同时对内切酶酶切后多个和较长DNA片段进行了检测,电泳缓冲液为1×TBE(pH8.3),阴极电压进样(10kV,5s),分离电压13kV,25℃,激光诱导荧光检测器检测(λex=520nm)。结果表明,所建立的REF-HPCE-LIF方法可对原核/真核酶切后多个和较长DNA片段进行检测,获得了满意的限制性内切酶指纹图谱,能够检测片段大小相差不超过10bp。所建立的方法较琼脂糖电泳分辨率高,可应用在检测多个和较长DNA片段的突变,在诊断肿瘤方面有一定应用前景。     

新型抗HIV-1重组导向制剂SL41在毕赤酵母中的表达及活性实验

以酵母分泌型表达载体pPIC9k为基础, 通过一段柔性连接肽Linker构建含有人源化抗HIV-1 gp41单链抗体(ScFv41)和免疫诱导因子葡萄球菌肠毒素A(staphylococcal enterotoxin A, SEA)的融合表达质粒pPIC9k-SL41, 线性化后, 采用电转化法整合入巴斯德菌毕赤酵母GS115中, 经His+MutS表型鉴定、PCR分析以及G418筛选获得高拷贝重组转化子. 摇瓶培养、甲醇诱导表达、SDS-PAGE和Western Blot分析结果表明, 目的蛋白得到良好表达, 表达量最高可达到47.9 mg/L. 目的蛋白经初步纯化后, 用于制备的HIV-1感染靶细胞复制模型进行抗体亲和力测定、细胞结合活性测定和细胞杀伤活性研究, 结果显示, 目的蛋白能够很好地与靶细胞模型中的HIV-1外膜蛋白gp160发生结合反应, 并可介导特异的CTL反应, 对靶细胞具有明显的杀伤活性, 表明获得了具有生物活性的抗HIV-1重组导向制剂.     

大肠杆菌分泌型穿梭质粒P^#GTE5DNA的酶切图谱

本文采用碱性裂解法获得产率较高的分泌型穿梭质粒P~# GTE_5 DNADNA,并作限制性内切酶的单、双酶完全消化,通过对DNA电泳条带的分析,初步得出该质粒的限制性酶切图谱.     

在大肠杆菌中利用反义RNA抑制乙肝病毒（HBV）X基因的表达

将乙肝病毒（ＨＢＶ）ａｙｗ株完整的Ｘ基因正向重组到原核表达质粒ｐＢＶ－２２１的ＰＬ启动子下游，得到能表达Ｘ蛋白的重组质粒ｐＢＶ－ＨＢＶ（＋）；同时将Ｘ基因反向重组到原核表达质粒ｐＢＶ－２２０的ＰＬ启动子下游，得到能转录Ｘ基因反义ＲＮＡ的重组质粒ｐＢＶ－ＨＢＸ（－）．利用这两个质粒，构建出能同时转录Ｘ基因ｍＲＮＡ和反义ＲＮＡ的重组质粒ＰＥＸ．ＡＮ－ＨＢＸ，并在原核水平上，证实了反义ＲＮＡ对Ｘ基因的表达具有明显的抑制作用，从而为在真核水平上利用反义ＲＮＡ对Ｘ基因进行调控的研究提供了有利的基础．     

Studies on saccharomyces cerevisiae cinder carbon-limiting growth transformed with plasmid pcyg4 that carries the gene for NADP-GDH

The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned fromSaccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 μm bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon ratelimiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).     

Improved manufacture and application of an agarose magnetizable solid-phase support

A simple, semiautomated, nonhazardous procedure for the production of a magnetizable solid-phase support (MSPS) has been developed based on the extrusion of molten agarose-iron oxide mixtures, which enables manufacture of a range of differently sized spherical agarose-iron oxide beads. This system has enabled scale-up of an original manufacture procedure and reproducible preparation of kg quantities of MSPS suitable for biomolecular purifications. An improved protocol for the isolation of plasmid DNA directly from cell lysates using this MSPS, derivatized with diethylaminoethyl (DEAE) groups, is reported. This involves a modified alkaline lysis, followed by adsorption to and elution from the support, yielding plasmid DNA of a purity comparable with, or better than, other methods of plasmid isolation. Using the same procedure, plasmid DNA can be isolated from bacterial cell culture volumes of 1.5 mL and 100 mL with equal efficiency and purity.     

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K _m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K _m for βCD and its derivatives, with improved activity compared to wild type enzyme.