赤红球菌组氨酸激酶的融合表达和功能分析

对赤红球菌的组氨酸激酶基因进行密码子优化，将优化后的组氨酸激酶基因（rhks）构建重组表达质粒pGEX-4T-2-rhks。将此质粒导入到大肠杆菌BL21（DE3）中进行异源表达。在25 ℃和1 mmol/L异丙基-β-D-硫代吡喃半乳糖苷诱导条件下，组氨酸激酶融合蛋白（GST-RHK）获得成功表达，并具有催化活性。经谷胱甘肽琼脂糖亲和层析纯化，获得电泳纯的GST-RHK，其中纯化倍数为3.1，得率为19.5%。该蛋白大小约为72.75 kDa，Km、Vmax和Kcat值分别为20.92 μmol/L、0.17 μmol/（L·min）和1.4 min－1。野生型赤红球菌、组氨酸激酶基因增强株sdrhkE和组氨酸激酶基因敲减株sdrhkD在分别含有苯酚、甲苯、氯苯、异辛烷4 种有机溶剂的培养基中培养，菌株sdrhkD的生长情况都优于野生型赤红球菌，菌株sdrhkE的生长情况都低于野生型赤红球菌。本研究为进一步揭示赤红球菌SD3中组氨酸激酶涉及的信号转导途径与赤红球菌有机溶剂耐受性的关联机制提供一定参考依据。     

雪菊精油对希氏肠球菌产酪胺及相关基因表达的影响

探讨不同质量浓度雪菊精油对希氏肠球菌（Enterococcus hirae）N47产酪胺的影响机制。利用反转录实时定量聚合酶链式反应分析E. hirae在雪菊精油作用下酪氨酸脱羧途径相关基因的表达情况；利用高效液相色谱法检测不同质量浓度雪菊精油对E. hirae产酪胺的影响。并将E. hirae接入到含不同质量浓度雪菊精油的熏马肠中发酵，评估香肠pH值、菌落总数和酪胺积累量。结果表明：在E. hirae纯培养体系和熏马肠体系中，雪菊精油通过抑制微生物的生长和酪氨酸脱羧途径中tyr DC、tyr P基因的表达，降低酪胺的积累量（P＜0.05）。当雪菊精油添加量为1/2最小抑菌浓度（minimal inhibitory concentration，MIC）和MIC时，熏马肠中酪胺的含量分别为78.52 mg/kg和45.83 mg/kg，较对照组分别减少了64.72%和79.41%。     

Comparative evaluation of different statistical tools for the prediction of uniaxial compressive strength of rocks

In this study, uniaxial compressive strength (UCS), unit weight (UW), Brazilian tensile strength (BTS), Schmidt hardness (SHH), Shore hardness (SSH), point load index (Is₅₀) and P-wave velocity (V_p) properties were determined. To predict the UCS, simple regression (SRA), multiple regression (MRA), artificial neural network (ANN), adaptive neuro-fuzzy inference system (ANFIS) and genetic expression programming (GEP) have been utilized. The obtained UCS values were compared with the actual UCS values with the help of various graphs. Datasets were modeled using different methods and compared with each other. In the study where the performance indice PI_at was used to determine the best performing method, MRA method is the most successful method with a small difference. It is concluded that the mean PI_at equal to 2.46 for testing dataset suggests the superiority of the MRA, while these values are 2.44, 2.33, and 2.22 for GEP, ANFIS, and ANN techniques, respectively. The results pointed out that the MRA can be used for predicting UCS of rocks with higher capacity in comparison with others. According to the performance index assessment, the weakest model among the nine model is P7, while the most successful models are P2, P9, and P8, respectively.     

重组人淋巴细胞活化基因-3蛋白胞外段的真核表达及鉴定

目的真核表达重组人淋巴细胞活化基因-3(lymphocyte activation gene-3,LAG-3)蛋白胞外段,并进行鉴定。方法用植物血球凝集素(phytohaemagg lutinin,PHA)刺激Jurkat细胞,流式细胞术检测Jurkat细胞中LAG-3蛋白的表达;提取Jurkat细胞总mRNA,RT-PCR法扩增人LAG-3蛋白胞外段基因片段,同时在蛋白C-末端引入His标签,将其克隆入载体pcDNA3. 1+,构建重组质粒,转染Expi293F真核细胞,当细胞活率低于50%时收获细胞上清,经镍柱亲合层析纯化。纯化产物进行4%~20%SDS-PAGE、HPLC及Western blot分析,BCA法测定浓度。结果经菌液PCR、双酶切及测序鉴定,表明质粒构建正确。重组表达蛋白的相对分子质量约60 000,纯化后纯度达95%以上,与鼠抗LAG-3单克隆抗体可发生特异性结合,浓度为2. 4 mg/mL。结论成功构建了重组真核表达质粒LAG-3/pcDNA3. 1+,并于Expi293F细胞中表达,纯化获得了纯度较高的LAG-3蛋白,为后期LAG-3蛋白的相关研究及其单抗的制备奠定了基础。     

Discovery and characterization of tyrosinases from sea anemone pedal disc

Abstract

The formation of underwater adhesion is a complicated physiological process and many different types of enzymes are found to be essential apart from structural proteins. Previous studies have shown that various tyrosinases were present in marine adhesives, but little information is available about the over-expression and enzymatic characterization of these enzymes. Specifically, this study first identified four significantly up-regulated tyrosinases in the pedal disc of Haliplanella luciae by means of multi-omics technology, and made preliminary bioinformatics predictions. Sequence alignment showed that the Tyr1_Hl contained six conserved His residues that bind to copper ions, of which a tyrosinase with diphenolase activity named as Tyr1_Hl^Δ, was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Enzymatic characterization showed that the activity of Tyr1_Hl^Δ was Cu²⁺ dependent and maximum catalytic activities were in 20?mM Tris–HCl (pH 8.0) at 37?°C. In summary, we identified novel tyrosinases in the pedal disks of sea anemone for the first time and the Tyr1_Hl^Δ was successfully recombinant expressed. Our study will provide basis for future exploration of bio-adhesion mechanism and design of bio-adhesives derived from sea anemones.     

基于深度语义分割的多源遥感图像海面溢油监测

针对遥感图像海面溢油区域通常受到斑噪声以及强度不均等因素的影响,从而导致溢油区域监测效果较差的问题,本文引入了深度语义分割的方法,将深度卷积神经网络与全连接条件随机场相结合,形成端对端连接。以Resnet结构为基础,首先通过深度卷积神经网络对多源遥感图像粗分割并作为输入,然后经过改进的全连接条件随机场,利用高斯成对势和平均场近似定理,建立条件随机场形成递归神经网络作为输出。通过多源遥感图像对海面溢油区域进行监测,并利用可见光图像估计溢油区域面积。实验在所建立的多源遥感图像数据集上与其它先进模型进行对比,结果表明本文方法提高了溢油区域的分割精度以及精细细节程度,平均交并比为82.1%,监测效果具有明显地改善。     

静不定结构的热应力分析

分析了受约束机构内材料热应力存在的危害,给出静不定结构内热应力的定量描述方式:弹性变形状态时,热应力仅与线膨胀系数、弹性模量及温度变量成正比关系;当热应力达到材料在某温度下的屈服强度时,其热膨胀发生弹塑性转变,热应力为当时温度下的屈服强度。采用热模拟试验机测试了材料的温度与热应力关系,分析结果与数值模型基本吻合,测出刚性约束下45钢在升温至620℃时热应力等于其屈服强度约200 MPa。     

一类两性分枝过程条件均值增长率的极限性质

本文主要研究随机环境中配对依赖人口数两性 Galton-Watson 分枝过程的条件均值增长率的极限性质．利用上可加函数的性质，得到配对单元平均增长率的极限性质和该过程条件均值的上界和下界．文中给出了关于过程条件均值增长率的两个序列，利用配对单元平均增长率的性质，获得了这两个序列的极限性质．随机环境中配对依赖人口数两性分枝过程比较复杂，本文的结论推广了现有的研究成果．