大肠杆菌热稳定肠毒素的攻毒最佳模型的确定

用致病性大肠杆菌C83916菌株,通过腹腔注射和经口灌胃两种途径感染小鼠,并且每一种途径分别按照体重给菌和按照分组给菌两种方式,最后确定最佳的给菌方式。结果:经腹腔注射并按照体重给菌可诱导小鼠腹泻,大便培养阳性,半数致死量为9.2×109个细菌/10g体重。最后确定经腹腔并按照体重注射致病性大肠杆菌C83916菌株是引起小鼠腹泻最佳模型。     

鸡沙门氏菌与大肠杆菌混合感染症的病原分离鉴定

对某鸡场送检的60日龄患呼吸道病的病鸡进行病理剖检及细菌分离鉴定。采用微生物学方法鉴定出沙门氏菌和大肠杆菌,确定该病是由两种细菌感染引起的。药敏试验指导用药,投以呋喃妥因、氧氟沙星治愈。药物治疗效果与药敏试验结果一致。     

肠道病毒71型外壳蛋白VP1在大肠杆菌中的表达

将扩增得到的肠道病毒71型外壳蛋白VP1基因克隆到测序载体pGEM-T,测序验证该序列为目的片段后,将目的基因克隆到原核表达载体pGEX-5x-1中,转化大肠杆菌BL21,IPTG诱导表达,产物经SDS-PAGE分析和Western blot验证。结果表明,在经IPTG诱导的BL21中检测到分子量与预期大小相符的大约60 kDa的融合蛋白。利用表达产物作为抗原,对EV71感染病人阳性血清的检测初步证实,重组蛋白VP1可以作为检测EV71感染的检测用抗原。     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coli

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metalloprotease as the mature protease was further confirmed by SDS-PAGE and immunoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

Statistical Optimization for Succinic Acid Production from E. Coli in a Cost-Effective Medium

Response surface methodology (RSM) was employed for optimization of medium components and cultural parameters in cost effective cane molasses based medium for attaining high yield of succinic acid. The important factors obtained by “one-variable-at-a-time-approach” (cane molasses, corn steep liquor, sodium carbonate, and inoculum density) were further optimized by RSM. The optimum values of the parameters obtained through RSM (cane molasses 12.5%, corn steep liquor 7.5%, and sodium carbonate 25 mM) led to almost double yield of succinic acid (15.2 g/l in 36 h) as against “one-variable-at-a-time-approach” (7.1 g/l in 36 h) in 500-ml anaerobic bottles containing 300-ml cane molasses based medium. Subsequently, in 10-l bioreactor succinic acid production from Escherichia coli was further improved to 26.2 g/l in 30 h under conditions optimized through RSM. This fermentation-derived succinic acid will definitely help in replacing existing environmentally hazardous and cost-intensive chemical methods for the production of succinic acid.     

利用微生物直接生产羟基丁酸单体

利用重组大肠杆菌E.coli HB101来进行直接生产羟基丁酸(HB)手性单体,研究了含pUCAB质粒的重组体E.coli HB101在各种条件下生长及积累HB单体的情况,研究了HB单体的积累随pH值变化的规律。结果表明,pH=6.8时,48 h内细菌可生产0.5 g/L以上的HB单体。     

Zwitterionic Cellulose Carbamate with Regioselective Substitution Pattern: A Coating Material Possessing Antimicrobial Activity

A polyzwitterion is synthesized by regioselective functionalization of cellulose possessing a uniform charge distribution. The positively charged ammonium group is present at position 6, while the negative charge of carboxylate is located at positions 2 and 3 of the repeating unit. The molecular structure of the biopolymer derivative is proved by NMR spectroscopy. This cellulose‐based zwitterion is applied to several support materials by spin‐coating and characterized by means of atomic force microscope, contact angle measurements, ellipsometry, and X‐ray photoelectron spectroscopy. The coatings possess antimicrobial activity depending on the support materials (glass, titanium, tissue culture poly(styrene)) as revealed by confocal laser scanning microscopy and live/dead staining.

     

基于场放大进样及Au纳米粒子双重富集用于大肠杆菌检测的毛细管电泳电化学免疫分析法

利用Au纳米粒子作为辣根过氧化物酶(HRP)标记抗体的载体,结合电堆积预富集技术,发展了一种基于场放大进样及Au纳米粒子双重富集的毛细管电泳电化学免疫分析技术用于大肠杆菌的检测.大肠杆菌与酶标抗体免疫反应后直接进行场放大进样预富集,免疫样品快速迁移并堆积在毛细管入口端,同时带负电荷的金纳米粒子向阳极端迁移,在样品与缓冲溶液的界面处吸附样品离子.金纳米粒子作为多酶载体使检测信号进一步放大.以标记在抗体上的HRP催化H2O2氧化邻苯二胺产生的电流信号来检测大肠杆菌.同常规电动进样毛细管电泳相比,该双重富集技术可使灵敏度提高1400倍.该方法对大肠杆菌检测的线性范围为2.0～2000.0 cfu mL-1,检出限为1.0 cfu mL-1,实现了对扇贝样品中大肠杆菌的快速、灵敏检测.     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.