禽巴氏杆菌C48-3株成熟黏附蛋白基因cpm39的克隆及序列分析

目的：对禽巴氏杆菌C48-3躺株编码成熟黏附蛋白的基因cpm39进行克隆和序列分析。方法：通过PCR从禽巴氏杆菌C448-3。基因组DNA中扩增出cpm39基因,克隆到pMD18-T载体中,转化大肠杆菌DH5d,并对目的基因进行核苷酸序列测定;用Clustal X和Mega 2．1软件将测定的序列与GenBank中已登录的16种血清型巴氏杆菌株核苷酸序列进行同源性分析。结果：测序结果表明cpm39基因大小为1002bp,与已知的16个血清型巴氏杆菌cpm39基因核苷酸序列的同源性为81．5％～100％。结论：克隆得到禽巴氏杆菌C。躺株编码成熟黏附蛋白的cpm39基因,该基因在不同血清型巴氏杆菌中具有很高的同源性,该蛋白可以作为研制预防巴氏杆菌病亚单位疫苗的候选抗原。     

温度、盐度对强壮箭虫耗氧率和窒息点的影响

研究了不同温度、盐度下强壮箭虫的耗氧率和窒息点.结果表明:温度和盐度均对强壮箭虫的耗氧率和比耗氧率有显著影响.试验温度在5℃-25℃,强壮箭虫个体耗氧率(IO)和比耗氧率(SO)开始随温度的升高而升高,之后呈明显下降趋势.其回归方程分别为y=0.0058x3 -0.2956x2+4.415x-8.7816(R2=0.99,P＜0.05)和y=0.0011x3 -0.0546x2+0.8161x-1.6232(R2=0.99,P＜0.05),数值分别为6.30～11.71μg·ind-1·h-1和1.22 ～2.16μg· mg-1·h-1,窒息点为4.18～6.87 mg·L-1.盐度10 ～40条件下,IO和SO随盐度的升高逐渐下降,回归方程分别为y=-0.0068x2 -0.1412x+21.702(R2=0.89,P＜0.05)和y=-0.0013x2-0.0261x+4.0114( R2 =0.89,P＜0.05),数值分别为4.98～17.73μg ·ind-1·h-1和0.92～3.56 μg·mg-1 ·h-1,窒息点为4.02～6.24 mg·L-1.对强壮箭虫与其他水生动物的耗氧率和窒息点进行比较得出,强壮箭虫是一种狭氧性浮游动物.     

银杏叶提取物对糖尿病大鼠心肌损伤的防护作用

目的:研究银杏叶提取物(EGb)对糖尿病大鼠心肌的防护作用.方法:用光镜和透射电镜观察EGb对糖尿病大鼠心肌的形态学改变,并测定心肌组织内超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)、结构型一氧化氮合酶(cNOS)、诱导型一氧化氮合酶(iNOS)的活性及一氧化氮(NO)、丙二醛(MDA)的含量.结果:糖尿病大鼠心肌光镜下主要表现为心肌细胞空泡变性及心肌纤维局灶性溶解;电镜下主要表现为心肌线粒体肿胀,嵴变短,肌原纤维溶解;SOD活性下降,NOS、iNOS活性及MDA、NO含量增高.EGb治疗组病变明显减轻,EGb治疗组心肌组织内SOD活性明显高于糖尿病组,NOS、iNOS活性及MDA、NO含量低于糖尿病组.结论:EGb可能通过抗脂质过氧化作用和降低NO水平而对糖尿病心肌产生保护作用.     

流行性乙型脑炎病毒E基因的克隆、表达及初步应用

参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a( )中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。     

土壤中脂肪酶产生菌的筛选、鉴定及脂肪酶基因的克隆

以橄榄油为惟一碳源进行富集培养、以罗丹明B为指示剂的平板进行初筛,摇瓶复筛得到产脂肪酶菌株;利用滴定法、平板扩散法以及转酯反应试验,最终筛选出具有较高转酯活性脂肪酶的菌株B2.通过对B2进行形态学观察,生理生化测定以及16S rDNA特征片段比较分析,初步确定B2为克雷伯氏菌属(Klebsiella sp.).通过特定引物PCR扩增得到B2菌株两个脂肪酶基因K1和K2.利用丙酮法和(NH)2SO4沉降法得到B2菌株体内的胞内酶,以叔丁醇为溶剂,催化棉籽油与甲醇反应,经过薄层层析和高效液相检测产物为生物柴油.     

基于蛋白互作网络识别非小细胞肺癌相关基因功能模块

本研究对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)基因表达数据进行差异表达分析,并与蛋白质相互作用网络(PPIN)数据进行整合,进一步利用Heinz搜索算法识别NSCLC相关的基因功能模块,并对模块中的基因进行功能(GO term)和通路(KEGG)富集分析,旨在探究肺癌发病分子机制。蛋白互作网络分析得到一个包含96个基因和117个相互作用的功能模块,以及8个对NSCLC的发生和发展起到关键作用候选基因标志物。富集分析结果表明,这些基因主要富集于基因转录催化及染色质调控等生物学过程,并在基础转录因子、黏着连接、细胞周期、Wnt信号通路及HTLV-Ⅰ感染等生物学通路中发挥重要作用。本研究对非小细胞肺癌相关的基因和生物学通路进行预测,可用于肺癌的早期诊断和早期治疗,以降低肺癌死亡率。     

Insulation and wiring specificity of BceR‐like response regulators and their target promoters in Bacillus subtilis

BceRS and PsdRS are paralogous two‐component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P_bceA or P_psdA, resulting in a strong up‐regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross‐regulation has been observed between them. We therefore investigated the specificity determinants of P_bceA and P_psdA that ensure the insulation of these two paralogous pathways at the RR–promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high‐affinity, low‐specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low‐affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities.     

控释肥料对稻田氧化亚氮排放的影响

采用静态箱法研究了控释肥料和常规肥料处理对华南赤红壤发育的稻田N2O排放的影响．结果表明,施用控释肥处理与非包膜复合肥处理,在水稻移栽后10d内水层中NH4^+-N和NO3^--N浓度问差异达极显著水平,各处理水层中NO3^--N浓度与2d后或当天N2O排放量间的偏相关系数达极显著水平．包膜型控释肥比未包膜复合肥能极显著地降低稻田N2O的排放量．在施肥后100d内,控释肥的N2O累积排放量仅为未包膜型复合肥料的13．45％～21．26％,是尿素处理的71．17％～112．47％．复合肥处理的N2O排放主要集中在施肥后1～25d和水稻晒田期间,控释肥在此时期的排放量显著降低,尿素处理则延缓并减小了N2O排放峰．控释肥一次施用和尿素分次施用都能减少N2O排放．     

双向凝胶电泳-飞行时间质谱技术鉴定小鼠胚泡黏附时子宫内膜nm23-M2／NDPK B的表达

运用双向聚丙烯酰胺凝胶电泳(2DPAGE)分析未交配小鼠子宫内膜和妊娠第五天(D5)小鼠子宫内膜胚泡黏附时植入位点及其旁组织蛋白质组。差异蛋白质组学显示,等电点(isoelectric point,pI)约7．1、分子量(molecular weight,Mw)约18kDa的蛋白质点在D5小鼠子宫内膜特别是植入位点表达上调。对此蛋白质点用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption／ionization time of flying mass spectrometry,MALDI—TOF—MS)测定其胶内酶解后的肽质量指纹谱(Peptide Mass Fingerprint,PMF),经Mascot：Peptide Mass Fingerprint中SWISS-PROT数据库查询后,鉴定该蛋白质为鼠源性nm23-M2／NDPKB。RT—PCR和免疫组织化学结果也显示D5小鼠子宫内膜nm23-M2／NDPK B mRNA和蛋白表达明显增加。提示nm23-M2／NDPKB参与胚泡着床这一重要生命活动过程。     

High-level expression of human TFF3 in Escherichia coli

A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14-15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.