基于非靶向代谢组学的冠突散囊菌发酵代谢产物分析

冠突散囊菌在降糖降脂功能性食品和天然药物的开发中具有重要的应用价值。分析冠突散囊菌整体代谢产物信息和产生的降糖降脂活性成分,可以为菌株的开发和应用提供理论基础和数据支持。以冠突散囊菌菌株Ys-Ec-01为试验材料,采用基于超高效液相色谱-四级杆静电场轨道阱质谱(UHPLC-QE-MS)技术的非靶向代谢组学,结合主成分分析(PCA)和正交偏最小二乘法-判别分析(OPLS-DA)方法,分析Ys-Ec-01发酵代谢产物谱,寻找降糖降脂活性产物。结果表明:Ys-Ec-01发酵后代谢产物谱发生显著变化。从代谢产物谱中,鉴定出365种发酵产物,进而筛选出205种差异发酵产物和151种主要发酵产物,鉴定到13种降糖降脂活性成分,分别是金圣草黄素、山奈酚、异鼠李素、表儿茶素、辣椒素酯、烟酸、葫芦巴碱、1-脱氧野尻霉素、尿囊素、原儿茶酸、亚油酸、胆碱和L肉碱。这些活性成分为Ys-Ec-01的降糖降脂功能研究和开发应用提供更多靶点和基本信息。

Functional identification of C-type lectin in the diamondback moth,Plutella xylostella(L.)innate immunity

C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity in insects.In this study,we report a novel dual carbohydrate-recognition domain(CRD)CTL from Plutella xylostella which we designate PxIML.PxIML is a protein with a 969 bp open reading frame(ORF)encoding 322 amino acids,containing a signal peptide and a dual-CRD with EPN(Glu_(124)-Pro_(125)-Asn_(126))and QPD(Gln_(274)-Pro_(275)-Asp_(276))motifs.The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut.The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010(Bt8010)at 18 h,while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris.The recombinant PxIML(rPxIML)protein could bind to the tested pathogen-associated molecular patterns(PAMPs),and the bacteria of Enterobacter sp.IAE5,S.marcescens IAE6,Staphylococcus aureus,Escherichia coli BL21,and Bt8010 in a Ca^(2+)-dependent manner,however,it showed limited binding to the fungus,P.pastoris.The rPxIML exhibited strong activity in the presence of Ca^(2+) to agglutinate Bt8010,Enterobacter sp.IAE5 and S.aureus,but it only weakly agglutinated with E.coli BL21,and could not agglutinate with S.marcescens IAE6 or P.pastoris.Furthermore,the rPxIML could bind to hemocytes,promote the adsorption of hemocytes to beads,and enhance the phenoloxidase(PO)activity and melanization of P.xylostella.Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P.xylostella.

高职药学专业学生“1+X”考证意愿与评价性调查

目的对目前高职药学专业“1+X”考证存在的各种客观问题开展分析,并提出一些探索性的改革思路。方法利用问卷星软件对107名应届药学毕业生开展问卷调查。问卷项目内容包括参加“1+X”考证项目数量、项目名称、考证时间投入、证书类型与意愿期望、考证动机及原因、总体实用性评价等。结果通过调查发现,目前高职药学专业学生“1+X”考证项目中存在证书可选性过窄、适用性不强、技能性偏弱等问题。结论改革现有职业岗位证书的制度弊端、强化证书与岗位实践技能的有效融合、探索课证融合的职业教育新路径等措施将有助于实现高职药学专业“1+X”考证更高质量的发展。

东方肉座菌EU7-22纤维素内切酶Ⅰ的异源表达

[目的]实现东方肉座菌纤维素内切酶EGⅠ在毕赤酵母中的表达,获得重组EGⅠ。[方法]通过RT-PCR获得EGⅠ开放阅读框。将EGⅠ成熟肽和PHO1信号肽的DNA片段插入p PIC3. 5K后,重组表达载体电转化毕赤酵母。通过甲醇诱导表达和镍柱纯化获得EGⅠ。以羟甲基纤维素钠检测活性,以肽N-糖苷酶F分析N-糖基化,以SDSPAGE分析表达情况和糖基化修饰。[结果]获得EGⅠ分泌表达菌株,诱导96 h后上清液活性为0. 513±0. 002 U/m L,纯化后的EGⅠ活性为0. 558±0. 012 U/mg。SDS-PAGE表明EGⅠ分子量在100～180 k Da,远高于预测值47. 3k Da,经肽N-糖苷酶F处理后,降至63～75 k Da。[结论]实现了EGⅠ的分泌表达,获得活性为0. 558±0. 012 U/mg的糖基化重组EGⅠ。