PTD-SARA融合蛋白的表达、纯化及鉴定

目的在大肠杆菌中表达PTD-SARA融合蛋白,并对其进行纯化和鉴定。方法使用大肠杆菌偏爱密码子合成PTD-SARA全长基因,将其克隆入载体pQE-30中,构建重组表达质粒pQE-30-PTD-SARA,转化感受态大肠杆菌M15,IPTG诱导表达。表达产物经Ni2+-NTA亲和层析纯化后,进行Western blot鉴定及N-端测序。结果重组表达质粒pQE-30-PTD-SARA经双酶切及测序鉴定证明构建正确。1.0mmol/LIPTG诱导4h,目的蛋白的表达量最高,约占菌体总蛋白的25%,主要以包涵体形式存在。纯化的融合蛋白纯度为94%,浓度为0.2mg/ml,且具有良好的反应原性,N-端有14个氨基酸。结论已成功表达并纯化了PTD-SARA融合蛋白,为其功能的研究奠定了基础,也为临床防治肾脏纤维化提供了一个新的策略。     

改性花生蛋白/PVA共混体系的流变性能

以花生粕为原料提取花生蛋白,对花生蛋白进行酰胺化改性,将改性花生蛋白与聚乙烯醇(PVA)共混制得改性花生蛋白/PVA共混原液。采用红外光谱仪对改性花生蛋白进行结构分析,并对改性花生蛋白/PVA共混体系的流变性能进行了研究。结果表明,马来酸酐对花生蛋白进行了酰胺化改性,改性花生蛋白/PVA共混体系为非牛顿流体,其表观粘度(ηa)随着温度的增加而下降,但随着PVA的含量增加而显著增加,改性花生蛋白的含量对ηa影响较小;当温度较低时,共混体系的剪切速率(γ.)对其ηa影响较为显著;共混体系的粘流活化能随γ.的增大而下降。     

Ursodeoxycholic Acid Suppresses Lipogenesis in Mouse Liver: Possible Role of the Decrease in β-Muricholic Acid,a Farnesoid X Receptor Antagonist

The farnesoid X receptor (FXR) is a major nuclear receptor of bile acids; its activation suppresses sterol regulatory element-binding protein 1c (SREBP1c)-mediated lipogenesis and decreases the lipid contents in the liver. There are many reports showing that the administration of ursodeoxycholic acid (UDCA) suppresses lipogenesis and reduces the lipid contents in the liver of experimental animals. Since UDCA is not recognized as an FXR agonist, these effects of UDCA cannot be readily explained by its direct activation of FXR. We observed that the dietary administration of UDCA in mice decreased the expression levels of SREBP1c and its target lipogenic genes. Alpha- and β-muricholic acids (MCA) and cholic acid (CA) were the major bile acids in the mouse liver but their contents decreased upon UDCA administration. The hepatic contents of chenodeoxycholic acid and deoxycholic acid (DCA) were relatively low but were not changed by UDCA. UDCA did not show FXR agonistic or antagonistic potency in in vitro FXR transactivation assay. Taking these together, we deduced that the above-mentioned change in hepatic bile acid composition induced upon UDCA administration might cause the relative increase in the FXR activity in the liver, mainly by the reduction in the content of β-MCA, a farnesoid X receptor antagonist, which suggests a mechanism by which UDCA suppresses lipogenesis and decreases the lipid contents in the mouse liver.     

Structure‐Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica

Hydratases provide access to secondary and tertiary alcohols by regio‐ and/or stereospecifically adding water to carbon‐carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure‐based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two‐electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio‐ and stereoselective hydration.     

Vif-ΔN28和Cullin5-N138蛋白的表达及纯化

目的获得高纯度的Vif-ΔN28和Cullin5-N138蛋白。方法以Vif/VR1012质粒为模板,PCR扩增出Vif-ΔN28基因并插入pRSETB质粒。将构建的原核表达载体Vif-ΔN28/pRSETB以及已有的Cullin5-N138/pRSETB质粒分别转化大肠杆菌BL21(DE3),经IPTG诱导表达,产物经Ni-NTA离子纯化柱纯化、复性。结果所表达的Vif-ΔN28和Cullin5-N138蛋白约占各自菌体总蛋白的20%,纯化后蛋白纯度均可达90%以上。结论已成功构建了Vif-ΔN28原核表达质粒,并在大肠杆菌中高效表达,获得了可溶性的纯化的Vif-ΔN28和Cullin5-N138蛋白。     

负载活性组分的核壳结构纤维膜的制备及性能

采用同轴静电纺丝技术将蛛丝蛋白（Ss）和美洲大蠊提取物（PAE）分别负载于纳米纤维的壳层与核层。随着Ss的增加,纤维直径从350 nm降至280 nm,核层直径由120nm升至140 nm,壳层厚度由115 nm降至70 nm。Ss的加入使纳米纤维膜具有良好的机械性能和亲水性,纳米纤维膜的拉伸强度可达到4.31 MPa,溶胀率可达到150%,水蒸气透过率可达到1834 g/(m2?24h),水接触角减小到 32.7 ?。纳米纤维膜核壳结构能够有效抑制药物突释,实现药物长效释放,7天内药物释放可达77%;纳米纤维膜能够有效抑制细菌生长,促进细胞增殖,相较于未负载Ss的纳米纤维膜,负载20%Ss的纤维膜的细胞增殖效果提高25%,说明Ss和PAE在伤口愈合过程中能够起到协同作用。     

聚合物接枝脂肪酶的合成及其对酶活的影响

利用原子转移自由基聚合方法将含C₂～C₄烷基链的单体化合物分别接枝到皱褶假丝酵母脂肪酶（CRL）表面合成了HEMA-g-CRL、GMA-g-CRL、nPMA-g-CRL和BMA-g-CRL四种聚合物接枝CRL。CD和荧光光谱学分析显示,聚合物接枝导致HEMA-g-CRL、nPMA-g-CRL和BMA-g-CRL分子的α-螺旋和β-折叠含量增加。与此同时,聚合物接枝CRL分子荧光发射光谱也发生了蓝移,意味着其具有更加紧密的分子构象。酶活测定结果进一步显示,聚合物接枝增强CRL酶活227%～278%。随着单体化合物烷基链长增加,聚合物接枝CRL的K _m值由0.17 mmol·L^-1降至0.09 mmol·L^-1。与此同时,聚合物接枝CRL的k _cat值则提高至106～182 s^-1。BMA-g-CRL的催化效率达到了野生型CRL的3.28倍。这反映出聚合物接枝助力了CRL“盖子”结构开启和CRL活性中心暴露,促进底物的转化。稳定性测试表明,聚合物接枝提升了CRL的热稳定性并拓宽了其pH操作范围。     

表面活性剂在生物工程中的应用之一—反胶团萃取

反胶团萃取分离蛋白质是一种新型的、有发展前途的生物产品的分离技术。它是利用表面活性剂在有机溶剂中形成反向胶团,从而实现了对蛋白质的萃取,是表面活性剂在生物工程中的一种成功应用。     

建筑类项目节能评估报告编制探讨

建筑类项目节能评估除体现全面性和深度外,还要针对建筑用能特点进行分析,作为评估结论的重要依据。在编制建筑类项目节能评估报告过程中,总结了该类型报告的主要内容,分析了一些容易出现的问题,并提出两点建议,以期对建筑类项目节能评估报告的编制提供一定的借鉴作用。