Expression of CTB-human Insulin(BA) Fusion Protein in Gynostemma Pentapyhllum Makino Callus Cells and Its Hypoglycemic Effect in Mice

The plant expression vector of choleratoxin B subunit(CTB)-human insulin(BA) fusion protein pBI121/(CTB-BA) was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBI121/(CTB-BA) was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/(CTB-BA) via the freeze tha-wing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA se-quence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice.The sequences of synthetic CTB and human insulin genes(BA) were completely identical to those designed. Restriction map proved that the length of gene fragment in-serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The ex-pressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result.The animal test showed that only the G Penta-pyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher. The plant expression vector pBI121/(CTB-BA) was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans-formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.     

An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin

The aptameric enzyme subunit (AES) is an artificial enzyme subunit that can allosterically control partner enzyme activity. By means of an AES, target molecules can be detected by measurement of enzymatic activity in a homogeneous solution. We have developed a thrombin aptamer-based AES that can detect several targets by measuring clotting time in a fibrinogen solution. Measurement of the clotting activity in the fibrinogen solution is not suitable for a convenient biosensor. However, kinetics measurement of clotting activity is suitable for convenient detection of thrombin activity. The interference-enhanced reflection (IER) method is a simple, real-time technique to detect the thickness and/or refractive index of a thin film formed on a glass substrate surface. We demonstrated that clotting activity on a glass substrate with immobilized thrombin can be monitored in real time by IER. By means of the IER method, we were able to detect the target molecules of an AES. IER-based sensors have already been commercialized as portable sensors for the detection of organic compounds. Thus, portable biosensors could be developed by combination of the IER and AES technologies.     

Partial agonists and subunit selectivity at NMDA receptors

Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N-methyl-D-aspartic acid (NMDA) receptor class. Development of these ligands seems to be a difficult task because of the conserved region in the binding site of the NMDA receptor subunits. A few scaffolds have been developed showing potential to differentiate between the NMDA receptors.     

棕色固氮菌细菌铁蛋白捕获能力、稳定性和亚基相互作用的强度

选用柱层析、电泳和反相高效液相色谱(RP-HPLC)技术制备质谱纯棕色固氮菌细菌铁蛋白(Bacteri-al ferritin ofAzotobacter vinelandii,AVBF),并采用释放铁动力学和肽质量指纹图谱(Peptide mass fingerprint-ing,PMF)技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝(MB),其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度(40%~50%)的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白(Liver ferritin of shark,SLF)释放不稳定亚基,但在较低浓度(20%~30%)的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关。     

象山港黄墩支港菲律宾蛤仔种群COI和ITS1基因序列特征及遗传多样性分析

为了解象山港黄墩支港菲律宾蛤仔种质资源的遗传多样性现状, 采用COI和ITS1分子标记对该种群进行了基因序列特征和遗传多样性分析. 结果表明 在该种群30个个体中扩增得到的COI基因序列长度均为709bp, ITS1序列长度范围在693~729bp(共有746个位点). COI基因序列的位点中有670个保守位点(占94.50%)、39个变异位点(占5.50%); ITS1序列的位点中有保守位点671个(占89.95%)、变异位点62个(占8.31%)和缺失/插入位点13个(占1.74%). COI基因序列的保守性高于ITS1序列. 在COI基因序列中碱基(A+T)的占比(65.84%)高于(C+G), 而ITS1序列中则是碱基(A+T)占比(37.59%)低于(C+G). COI和ITS1序列的遗传距离分析均显示群体内遗传分化不明显. 基于COI基因序列和ITS1序列构建的遗传进化树显示 各科贝类分别相聚, 象山港菲律宾蛤仔位于帘蛤科的分支中, 其COI序列与杂色蛤进化关系最近. 遗传进化树中各种贝类的聚类关系与传统分类学结果相一致, 可作为分类的参考. COI和ITS1序列的平均核苷酸差异数分别为5.497和6.549, 核苷酸多样性指数分别为0.00775和0.00973, 单倍型数目分别为21和28, 单倍型多样性分别为0.963和0.993, 根据COI和ITS1两个序列计算得到的菲律宾蛤仔象山港群体单倍型多样性均大于0.5, 核苷酸多样性指数均大于0.005, 表明该种群遗传多样性丰富, 并处于稳定状态. 本研究结果补充了菲律宾蛤仔遗传多样性方面的研究资料, 并可为象山港菲律宾蛤仔种质资源保护和遗传育种提供理论基础.     

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.     

用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.     

Lateral gene transfer in phylogeny of azoreductase enzyme

This paper attempts to reconstruct the phylogeny of azoreductase enzyme from different organisms and compare it with the small subunit rRNA-based phylogeny of the organisms. The two phylogenies were found to be incongruent, indicating several events of lateral transfer of azoreductase gene between phylogenetically diverse organisms. However, the phylogenetic analysis methods have several limitations and a single method may not give the true pattern. Hence, it is necessary to corroborate the results with other complementary analysis tools. We used several tools to test our hypothesis of lateral transfer and found that it was supported not only by the analysis of the whole sequences, but also by the conserved motifs detected in these sequences. There were ample evidences for lateral transfer of azoreductase gene among enteric bacteria. There were also indications that azoreductase probably evolved in prokaryotes and then it was laterally transferred to eukaryotes in multiple events, resulting in some sequence variation among eukaryotic azoreductases. Finally, profile HMMs and conserved motifs extracted from these azoreductase sequences were found to provide sensitive tools for identifying potential azoreductases from the database. The analysis techniques used in this study can be extended to other gene trees to verify their evolutionary histories.