Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.     

基因芯片技术研究柽柳NaHCO3胁迫下基因的表达

运用基因芯片技术研究了NaHCO3胁迫下柽柳(Tamarix androssowii)基因的表达.将Cy5和Cy3两种荧光染料分别标记在NaHCO3处理和对照的柽柳cDNA上,将两种荧光探针混合,与载有柽柳基因的高密度芯片进行杂交并用芯片扫描系统进行扫描,通过Cy5与Cy3信号强度比值的计算研究基因的差异表达.共获得了89个差异表达的基因,其中,27个下调表达,62个上调表达.BlastX分析表明这些基因按功能可以分为光合作用、活性氧清除、渗透调节、信号传导与表达调控、代谢、发育相关、核糖体蛋白、蛋白质的分解与再生、转运类蛋白、水通道蛋白等几大类别.同时,发现了一些与盐胁迫相关的功能未知基因或未有任何功能信息的基因,这些基因可能在柽柳抗盐过程中具有重要作用.揭示了柽柳的抗盐胁迫涉及的几种重要途径,并获得了NaHCO3胁迫前后柽柳基因表达谱.     

基因芯片技术检测拉美夫定相关的HBV变异

In order to study the feasibility of gene chips technology in the detection of HBV mutation associated with lamivudine, we detected the mutation of HBV in peripheral blood of 30 patients treated with lamivudine for at least half a year by gene chips. The result was compared with that from direct sequencing. Both results are highly coincident. The rate reaches 100% while detecting single strain of virus infection, and 85% in multi-strains virus infection. Gene chip technology is quite valuable and practical in future clinic.     

Direct and nondestructive verification of PNA immobilization using click chemistry

The fluorogenic 1,3-Huisgen dipolar cycloaddition reaction was used as part of a novel immobilization strategy of PNA capture probes on a microarray. By using this click chemistry, azidocoumarin-anchored PNA probes were immobilized on phenyl acetylene-modified glass slides with the simultaneous generation of the fluorescent triazolylcoumarin moiety. Since the emitting moieties are generated in the immobilization reaction itself, fluorescent signals can be used to directly monitor the integrity of immobilization in a nondestructive manner. By using this strategy, PNA microarrays were prepared and successfully employed to perform microarray-based diagnosis of selected mutations in the breast cancer susceptibility gene BRCA1.     

基于遗传算法的基因芯片图像网格定位

对基因芯片荧光图像进行网格定位是进行芯片分析的前提与关键。利用为形模板匹配方法,基因芯片的自动网格定位问题转化为优化问题,从而用遗传算法求解。遗传算法是一种模拟自然界进化过程的启发式优化计算法,具高效及可收敛到全局最优的特点。实验证明该方法具有良好的准确性与可靠性。     

红花注射液对气虚血瘀证模型大鼠基因调控作用的研究

目的:采用基因芯片技术,分别构建气虚血瘀证大鼠和红花注射液给药处理后气虚血瘀证大鼠的差异基因表达谱,比较并分析,筛选出红花能够治疗气虚血瘀证的关键基因群,并推测其起治疗作用的基因组调控机制。方法:15只SD大鼠随机分为模型组、给药组、空白对照组。模型组和给药组采用疲劳游泳和饥饿饲养处理。造模一周后,给药组尾静脉注射红花注射液(100mg/kg/d),模型组给予相同体积生理盐水;对照组不做任何处理。造模进行两周后处死大鼠,取血检验血流变指标并评价造模情况;另抽取足够的血分离mRNA并逆转录杂交基因芯片;扫描信号分析确定受红花注射液调控的基因;并通过基因数据库查询相关基因功能,结合相关文献分析初步探讨红花作用的机制。结果:两周后经过检验和观察发现模型组大鼠在不同切率下的全血粘度增加,并且其体征表现出虚弱和瘀血的状态、体重下降,确定造模成功;给药组大鼠则相对于模型组的各项检测指标和状态有所改善,确认药物有疗效。在差异基因的比较中,空白组相对于给药组上调基因252条,下调基因54条;给药组相对于模型组上调基因196条,下调基因32条;两次差异表达基因中有16条相同基因,这些差异基因涉及到炎症损伤、免疫调节反应等方面。结论:红花注射液对于气虚血瘀证有治疗作用,在基因层次上是通过抗炎症损伤机制实现的。     

黑龙江地区400例乙型肝炎病毒基因分型与耐药突变研究

目的:运用基因芯片技术分析黑龙江地区乙型肝炎病毒(HBV)基因型分布特征及基因耐药变异情况。方法:随机选择2012年11月至2015年11月本医院乙型肝炎患者血清样本400例,应用PCR-反向点杂交的基因芯片技术对样本血清中HBV基因型及常见4类抗病毒药物耐药相关的多个位点进行检测,并进行数据分析。结果:400例样本中基因型分布以C型为主占83.25%(333例),B型7.25%(29例)、D型0.25%(1例)及混合基因型2.75%(11例);耐药突变位点检出188例,总耐药率为45.19%,其中突变位点236T(4.61%)提示阿德福韦酯单项耐药,耐药率为5.82%(10例),与拉米夫定耐药相关的为126例,突变位点以rt204I和(rt180M+rt240V)为主,显著高于其他抗病毒类药物,耐药风险较高。结论:黑龙江地区乙型肝炎基因分型以C为主,B型和其它混合型较少,且更容易对拉米夫定产生耐药。     

Determination of Biotinylated Proteins as an Index for Purification of Plasma Membrane using Surface Plasmon Resonance-based Optical Biosensor

Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.     

糖芯片制备技术研究进展

糖芯片是一种分析糖-蛋白质相互作用最直接有效的方法.对目前糖芯片的各种制备方法进行了综述,并分析了影响糖芯片质量的一些关键因素及其对底片材料和固定方法的整体要求,进而对该领域发展方向和广阔的应用前景进行了展望.