野油菜黄单胞菌产胶基因GumD克隆与重组质粒的构建

目的:建立野油菜黄单胞菌产胶基因GumD的克隆与重组质粒构建的方法。方法:以优化PCR实验条件扩增得到野油菜黄单胞菌产胶基因GumD片断,与抗药性质粒pMD18-T连接,获得重组质粒,并用CaCl2法转化JM109大肠杆菌细胞。结果:DNA测序结果表明,成功克隆产胶基因和构建重组质粒。结论:此方法可用于克隆野油菜黄单胞菌产胶基因和构建产胶基因重组质粒。     

分子空物学与辐射研究

本文综述了近年来运用分子生物学的理论和技术研究放射生物学的主要进展:在DNA损伤研究方面用序列分析及限制性酶切技术对DNA链断裂及碱基损伤在分子内的定位获得了新的信息,基因探针有可能成为研究哺乳动物细胞DNA结构损伤的敏感的新方法;染色体畸变及细胞突变的分子机理已有所阐明;哺乳动物DNA修复基因的研究正在兴起阶段,已克隆出人的DNA修复基因;在哺乳动物细胞上进行外源基因转移的成就为辐射损伤的防治开辟了新的前景。     

Continuous release of interleukin-2 from liposomal IL-2 (mixture of interleukin-2 and liposomes) after subcutaneous administration to mice

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small and hydrophobic liposomes by simple mixing under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 displayed better distribution after intravenous administration in mice and improved therapeutic effect against experimental M5076 metastases, as reported previously.[1] In this study, the elimination of IL-2 from the dosing area was investigated when the liposomal IL-2 was administered to mice subcutaneously. The results suggest that the release of IL-2 from this liposome was continuous and almost complete. The mean residence time (MRT) of IL-2 in the dosing area was 11.0 ± 1.65 hr. This resulted in the 8-fold times enhancement of MRT in the systemic circulation by the presence of liposomes, and IL-2 was detected in the serum for 2 days. Using this liposomal IL-2 is expected to have the potential to decrease the number of injections and enhance the efficacy of IL-2 in immunotherapies and therapies against tumor.     

Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis

Two plasmids are described which can be used to remove the "loxP-markerMX-loxP" cassettes in strains lacking the ura3 mutation. Both contain the Cre-recombinase under control of the GAL1 promoter and the natMX cassette with the dominant marker nat, which gives yeasts resistance to the antibiotic ClonNat. pNatCre contains ARSH and CEN6 for maintenance in Saccharomyces cerevisiae. pKlNatCre has a Kluyveromyces lactis replication origin and centromere in addition.     

表达狂犬病毒糖蛋白的重组痘苗病毒对啮齿动物接种的反应

狂犬病毒糖蛋白重组痘菌病毒（V－RG）与非重组痘菌病毒比较，V－RG对小白鼠爆发性致死量下降10000LD50；对家兔眼粘膜及皮肤感染后，3～5天内呈现轻微的红、肿，3周内全部消退。所有局部损害仅限于表皮，未见其它不良反应。V－RG经口免疫的动物7天即产生抗体，于第14天能抵抗致死量狂犬病毒脑内或肌肉内攻击。     

^51Cr释放试验和细胞集落抑制试验测定LAK活性的比较研究

用于测定细胞毒研究的＾５１Ｃｒ释放试验与传统的细胞集落抑制试验相比呈高度相关性，相关系数ｒ＝０．９９１５（Ｐ〈０．００１）；在检测ＬＡＫ淋巴因子激活的杀伤细胞活性方面有简便和实用等优点。     

The maintenance of self-replicating plasmids in Saccharomyces cerevisiae: Mathematical modelling,computer simulations and experimental tests

A distributive model has been constructed to describe the maintenance of the native 2 μm and 2 μm-based plasmids in the yeast Saccharomyces cerevisiae. This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability. A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out. These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence. The form chosen involves an amplification system in which the RAF gene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of the FLP gene. At the same time an upper limit (or ‘ceiling’) was imposed on the number of plasmid molecules able to replicate. Maternal bias was accommodated by ‘tagging’ a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or ‘cleared’) by the Rep1/Rep2 dimers. This final form of the model makes specific predictions about the stability of 2 μm and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations. The predictions on stability have been subjected to experimental test and results provide good support for the model.     

KINETIC MODELING OF CELL GROWTH AND PRODUCT FORMATION IN SUBMERGED CULTURE OF RECOMBINANT ASPERGILLUS NIGER

The kinetics of cell growth and protein production for the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP]#11 were investigated. An unstructured kinetic model was developed to describe the cell growth and product formation mathematically. The dynamics of glucose consumption and biomass production can be well-described by the model. The assumption that the degradation of GFP by proteases is a first-order reaction can express the trend of the GFP profile reasonably well. The dynamics of protease production is also described reasonably well by considering the inhibition effect of glucose on protease production. Because of the high initial glucose concentration and keeping the pH value of the broth at 6, the protease activity could be kept low most of the time during the fermentation. The increase of protease activity near the end of the culture might be caused by cell lysis because the intracellular protease activity was much higher than the extracellular activity.     

大鼠活化STAT蛋白抑制剂1基因重组腺病毒质粒的构建及鉴定

目的构建大鼠活化STAT蛋白抑制剂1(Protein inhibitor of activated STAT1,PIAS1)基因重组腺病毒质粒,并进行鉴定。方法应用RT-PCR法从大鼠胰腺腺泡细胞AR42J细胞株中扩增全长PIAS1基因,经T-A克隆后,亚克隆至穿梭质粒pDC316中,利用同源重组将腺病毒骨架质粒Nad5/F35和穿梭质粒pDC316-PIAS1共转染293细胞,获得重组腺病毒质粒Ad5/F35-PIAS1,经包装和扩增后,获得重组腺病毒。RT-PCR检测PIAS1基因的表达;荧光显微镜观察病毒感染情况;Western blot检测PIAS1蛋白的表达;并计算重组腺病毒的滴度。结果从AR42J细胞中扩增出1 956 bp的PIAS1基因片段,重组腺病毒质粒Ad5/F35-PIAS1经双酶切鉴定证明构建正确。RT-PCR及Western blot分析显示,PIAS1基因和蛋白已在293细胞中表达;荧光显微镜观察显示,重组腺病毒的感染率达90%;病毒滴度为4.45×1010 PFU/ml。结论已成功构建了大鼠PIAS1基因重组腺病毒质粒,为进一步研究PIAS1基因在相关疾病中的作用及其临床应用奠定了基础。