不含硫柳汞重组乙型肝炎疫苗(CHO细胞)的免疫原性及稳定性

目的观察不含硫柳汞重组乙型肝炎疫苗(CHO细胞)的免疫原性及其稳定性。方法配制12批不含硫柳汞的重组乙型肝炎疫苗(CHO细胞),观察其免疫原性及在4、22和37℃保存不同时间的稳定性。结果该疫苗各项检测指标均符合《中国药典》三部(2005版)要求,实验组与对照组的效价测定结果差异无显著意义。10μg/ml疫苗22℃放置6个月和4℃放置12个月效力均未下降;20μg/ml疫苗在37℃放置4周后,效力略有下降。结论为婴幼儿提供了一种更加安全有效的重组乙型肝炎疫苗。     

Bcl-2、C-Raf及双靶点RNAi质粒对HCT-8细胞株增殖的抑制作用

目的研究Bcl-2、C-Raf及双靶点的RNA干扰质粒对人结肠癌HCT-8细胞株增殖的抑制作用。方法利用Ambion公司网上设计软件,分别以Bcl-2和C-Raf为靶标,设计合成63个核苷酸的双链RNA,构建质粒pSilencer3·1-H1/Bcl-2、pSilencer3·1-H1/C-Raf及pSilencer3·1-H1/Bcl-2/C-Raf,分别转染结肠癌细胞株HCT-8,用MTT法检测其对细胞增殖的抑制作用,RT-PCR检测Bcl-2和C-Raf的mRNA转录。结果经MTT法检测,构建的Bcl-2和C-Raf基因及双靶点RNAi质粒3个转染组的细胞存活率均降低,双靶点质粒转染组较两个单靶点转染组细胞存活率明显降低,差异有显著意义。经RT-PCR检测,转染双靶点质粒后,HCT-8细胞中Bcl-2和C-Raf基因的表达均较对照组明显减弱。结论Bcl-2、C-Raf及双靶点RNAi质粒对HCT-8细胞株增殖有抑制作用。     

固定化基因工程菌的培养和hEGF表达

引　言人表皮生长因子 (humanepidermalgrowthfactor,hEGF)是由 5 3个氨基酸残基组成、相对分子质量为 6 0 4 5的单链多肽[1] .hEGF具有多种生物活性 ,并在医药、化妆品工业中具有广泛用途 .国内外已有很多重组表达hEGF的报道[2 ,3] .但是 ,各种hEGF表达系统的质粒稳定性随培养     

中国莱姆病螺旋体PD91重组外膜蛋白A的动物免疫效果分析

目的观察重组中国莱姆病螺旋体Borrelia garinii基因型外膜蛋白A(rOspA)的动物免疫效果。方法采用纯化的rOspA作抗原,以新西兰家兔、绵羊和犬为试验动物,分不同剂量组和对照组进行免疫,用间接免疫荧光法(IFA)检测其血清特异性抗体(IgG),并进行体外中和试验。结果用rOspA免疫新西兰家兔、绵羊和犬后,其血清抗体(IgG)效价较免疫前均显著升高,其几何平均滴度为1∶169,体外中和试验表明,每毫升兔抗rOspA血清可杀灭1.0×105个莱姆病螺旋体;绵羊和犬的抗rOspA血清杀菌能力因免疫剂量的不同而不同,每毫升绵羊和犬抗rOspA血清最高可杀灭1.0×106个莱姆病螺旋体。结论rOspA有较好的免疫原性,可作为中国莱姆病rOspA亚单位疫苗的一种有效成分。     

容迟网络中的节点转发策略

容迟网络结构为很多面临挑战的网络提供了互操作性,它没有稳定的端到端的传输路径,而且具有高延迟和频繁中断的特点,当前的Internet体系结构和网络协议无法适用于这样的网络。基因算法（GA）是启发式求解法,建立在生物器官的基因遗传过程的基础上。并行基因进化论（PGE）是一种新的有效探索某问题解决空间的方法。在并行基因进化论（PGE）中,群体被分为若干亚群体,基因算法在每个亚群体中独立运作。主要研究在容迟网络中利用分布式基因进化理论,研究基于并行基因进化论（PGE）的节点策略模型,构建以质粒迁徙为基础的区域性基因算法,研究高度分布式环境下的节点转发策略。     

表达MAGE-1/HSP70/MAGE-3工程菌的稳定性

目的检测重组工程菌pET28a-MAGE-1/HSP70/MAGE-3/BL21(DE3)生物学性状的稳定性。方法将工程菌菌株连续传50代,每隔10代挑取单克隆菌落进行诱导表达,并计算质粒丢失率。提取不同代次的质粒,扫描电镜观察,检测融合蛋白表达水平,并进行工程菌的染色镜检及生化鉴定。结果pET28a-MAGE-1/HSP70/MAGE-3/BL21(DE3)融合蛋白工程菌呈典型的大肠杆菌形态,各代次菌的各项检测结果与原始菌种差异无显著意义。结论重组工程菌pET28a-MAGE-1/HSP70/MAGE-3/BL21(DE3)生物学性状稳定,可作为生产用菌种。     

The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material

 The fate of ingested recombinant plant DNA in farm animals (cattle and chicken) being fed a diet containing conventional maize or recombinant Bacillus thuringiensis toxin-maize (Bt-maize) is described. The probability of the detection by polymerase chain reaction of chloroplast-specific gene fragments of different lengths (199 bp and 532 bp) and a Bt-maize-specific fragment [truncated version of CryIA(b)] is shown. First data indicated that only short DNA fragments (<200 bp) derived from plant chloroplasts could be detected in the blood lymphocytes of cows. In all other cattle organs investigated (muscle, liver, spleen, kidney) plant DNAs were not found, except for faint signals in milk. Furthermore, Bt-gene fragments possibly recording the uptake of recombinant maize, were not detected in any sample from cattle. However, in all chicken tissues (muscle, liver, spleen, kidney) the short maize chloroplast gene fragment was amplified. In contrast to this, no foreign plant DNA fragments were found in eggs. Bt-gene specific constructs originating from recombinant Bt-maize were not detectable in any of these poultry samples either. Received: 23 February 2000 / Revised version: 20 March 2000     

Development of a confirmatory method for detecting recombinant bovine somatotropin in plasma by immunomagnetic precipitation followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry

ABSTRACT

Recombinant bovine somatotropin (rbST), a synthetic growth hormone, is used to stimulate growth and enhance milk production in dairy cows. Both its use and the sale of dairy products from treated animals are prohibited in the European Union, as well as in Australia, Canada, Japan, and New Zealand, but authorised in several countries (e.g. Brazil, USA). Screening methods involve detecting anti-rbST antibodies (biomarkers) in treated cows. Confirmatory methods are required to prove rbST abuse. The major challenges in determining rbST are its potentially low levels, its high similarity to native bST, and matrix interferences. To overcome these obstacles, we have developed a method involving immunomagnetic precipitation followed by UHPLC-MS/MS for rbST detection. Briefly, protein G magnetic beads pre-coated with an in-house produced monoclonal antibody were added to plasma. Incubation at room temperature allowed rbST present in the sample to bind to the magnetic beads. After that, magnetic beads were isolated by centrifugation and thoroughly washed (PBS, PBS + 0.2% Tween 20). Finally, rbST was released by alkalinisation and the samples were trypsin digested prior to UHPLC-MS/MS analysis in the MRM mode. Validation was done in accordance with European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used. The decision limit (CCα) reached with this approach was 0.11 µg l^?1.     

Experimental determination of codon usage-dependent selective pressure on high copy-number genes in Saccharomyces cerevisiae

One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes, particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here, we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required, optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings deepen our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.     

“Recombinamers” as advanced materials for the post-oil age

Biotechnology offers powerful solutions to the challenges that arise during the design and development of new complex biomimetic materials to achieve specific biological responses. Recombinant DNA technologies, in particular, provide unique solutions in the biomaterials field, especially regarding the control of macromolecular architectures involving protein sequences with the aim of addressing the multiple functional requirements needed for biomaterials' applications. Here, elastin-like recombinamers are presented as an example of an extraordinary convergence of different properties that is not found in any other polymer system. These materials are highly biocompatible, stimuli-responsive, show unusual self-assembly properties and can include bioactive domains along the polypeptide chain. Applications of these engineered biomimetic polymers in nanotechnological systems, stimuli-responsive biosurfaces and tissue engineering will be discussed.