珊瑚来源群体淬灭活性产生菌的筛选与抑菌活性研究

珊瑚病原菌的群体感应（Quorum Sensing，QS）是介导珊瑚疾病发生的重要因素，抑制病原菌的群体感应可以有效抵抗病原菌的侵染作用。为解决耐药性难题并维持珊瑚礁生态系统健康，本研究以紫色杆菌（Chromobacterium violaceum）为指示菌，从三亚市鹿回头半岛三亚湾（109°28''E，18°13''N）采集的珊瑚样品中，分离筛选具有较高群体淬灭酶活性的珊瑚共附生细菌，经16S rDNA测序初步鉴定其大部分为弧菌属。进一步研究群体淬灭活性菌株对两株珊瑚致病菌溶珊瑚弧菌V545（Vibrio coralliilyticus）和溶藻弧菌Z-14（V.alginolyticus）的生长、运动及生物被膜生成的影响。结果显示，5株群体淬灭活性菌株明显抑制溶珊瑚弧菌V545及溶藻弧菌Z-14的生长，8个菌株对溶藻弧菌Z-14的运动有明显抑制作用；9个菌株可显著抑制溶珊瑚弧菌V545生物被膜形成，13个菌株可显著抑制溶藻弧菌Z-14的生物被膜形成，其中11个菌株对溶藻弧菌Z-14生物被膜生长抑制率均超过85%；部分群体淬灭活性菌株对溶珊瑚弧菌V545和溶藻弧菌Z-14的生长、运动及生物被膜形成均有显著抑制作用。本研究筛选获得具有群体淬灭活性的细菌，并揭示其群体淬灭活性对珊瑚致病菌生长、运动、生物膜生成的影响，为解决珊瑚微生物抗病机制提供理论依据与原创材料。     

Bioactivity and constituents of several common seaweeds

Bioactivity and constituents of 8 common seaweeds from Dalian intertidal zone of northern Yellow Sea were investigated. In the anti-methicillin-resistant Staphylococcus aureus (MRSA) test, Symphyocladia latiuscula and Enteromorpha intestinalis showed obvious activities with MICs much lower than 1.0 mg·mL-1 . In the DNA damage repair test (DDRT), Chondrus ocellatu showed selective inhibitory activity against the DNA repair-defective E. coli strain vs. the wild-type E. coli strain; while Sym. latiuscula, Enteromorpha intestinalis and Sar. kjellmanianum showed significant anti-E. coli activity with MICs of 64-128 g·mL-1 . In the anti-Pyricularia oryzae test, Sym. latiuscula and Rh. confervoides strongly inhibited the germination of the spores of P. oryzae on agar plate. In the brine shrimp larvicidal test, Sym. latiuscula, Rh. confervoides and Sar. kjellmanianum exhibited potent toxicity against brine shrimp larvae, with LC50 much lower than 1 mg·mL-1 . The HPTLC analysis revealed their diversified secondary metabolites. The HPLC-DAD-MS analysis of the strongest species Sym. latiuscula and database searching showed that it can produce quite diversified metabolites, including halogenated ones, some of which may be new natural products. The results demonstrated the potentials of these seaweeds in the development of new antibiotics, antitumor drugs, agricultural fungicides and pesticides.     

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

This paper presents development of a quartz crystal microbalance （QCM） biosensor for real-time detection of E. coil O157：H7 DNA based on nanogold particles amplification. Many inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated sin- gle-stranded DNA （ssDNA） probes could be fixed through Au-SH bonding. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA of E. coli O157：H7 gene eaeA, then resulted in a mass change and corresponding frequency shifts （ △f ） of the QCM. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass. The electrochemical techniques, cyclic voltammetry （CV） and electrochemical impedance spectroscopy （EIS） were adopted to manifest and character each step. The target DNA corresponding to 2.0×10^3 colony forming unit （CFU）/mL E. coil O157：H7 cells can be detected by this biosensor, so it is practical to develop a sensitive and effective QCM biosensor for pathogenic bacteria detection based on specific DNA analysis. The piezoelectric biosensing system has potential for further applications, such as food safety and environment monitoring, and this approach lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.     

Expression of E1 gene of a hepatitis C virus inE. coli and protein purification

The cDNA containing full encoding region of E1 antigen of HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid pRSETE1 was introduced into the BL21 (DE3) strain ofE. coli. The engineering bacteria harbouring the pRSETE1 was cultivated in 2YT medium at 37°C. When the Expression of E1 protein was induced by 1 mmol IPTG, the bacteria was killed and the number of living cell was droped down from 10⁷ to 10³ cell/mL one hour post induction. Suggest that E1 protein is poisoned toE. coli. However, the 26kD polypeptide of E1 fussion protein still synthesized in appropriate condition. The expression level was about 10% of total protein 4 h after inducing. The E1 protin was purified by Ni²⁺-NTA-Agarose column chromatography to homogeneous. The purified E1 protein was sensitive and specific in reaction with anti-HCV antibody in sera. Supported by the Science Committec of Hubei Province Ye Linbai: born in Feb. 1948. Professor     

Time-resolved fluoroimmunoassay for quantitative determination of tylosin and tilmicosin in edible animal tissues

To quantitatively determine tylosin and tilmicosin in edible animal tissues,a time-resolved fluoroimmunoassay(TRFIA) has been developed and validated.For this purpose,desmycosin-O-carboxymethoxylamine-BSA was fixed onto microtiter plates,standards and samples were loaded and,finally,diluted europium-labeled anti-tylosin antibodies were added.Results show that the limit of detection for tylosin was 0.03 ng mL-1 and that for tilmicosin was 0.05 ng mL-1.The recoveries were 73.6% to 120.5%,with coefficients of variation below 15.6% in various biological matrices spiked with tylosin and tilmicosin at concentrations of 50-200 ngg-1.There was good correlation(R2>0.99) between the TRFIA,an enzyme-linked immunosorbent assay and high performance liquid chromatography data.In conclusion,the new TRFIA is applicable to the detection of tylosin and tilmicosin and is an effective and economical method that will enable high-throughput sample screening.The method is expected to be widely applicable.     

一种水产弧菌快速药敏检测方法的建立

【目的】实现弧菌药物敏感性的现场快速检测。【方法】以副溶血性弧菌、溶藻弧菌及灿烂弧菌3种弧菌为试验菌株,在Mueller-Hinton(MH)培养基基础上通过添加鱼肉蛋白胨及调节无机盐离子浓度,优选弧菌的增菌液。同时利用阿尔玛蓝作为颜色指示剂,选择11种水产常见药物并设置8个倍比稀释的浓度梯度,结合运用海藻糖作为包被保护剂,制备弧菌快速药敏检测微孔板,板内设有药敏检测区、生长对照区及质控区。【结果】利用药敏微孔板法及传统的试管稀释法分别对副溶血性弧菌、溶藻弧菌及灿烂弧菌进行药敏检测,结果显示微孔板法测定的所有药物对3种弧菌的MIC值与试管稀释法完全吻合。【结论】本研究建立的药敏微孔板法能够实现对3种弧菌药敏特性的快速、简便、准确检测,研究结果将为水产病原微生物的现场快速选药技术提供重要参考。     

广西北部湾大宗海水养殖鱼类卵形鲳鲹感染溶藻弧菌及其致病性研究

【目的】2016年广西钦州湾近海网箱养殖的卵形鲳鲹(Trachinotus ovatus)发生以体表皮肤溃烂、皮下出血、内脏器官病变为典型症状的细菌性疾病,通过对患病卵形鲳鲹中病原菌进行分离、鉴定与保存,为后续开展病原菌快速检测技术和防控技术等研究,以及控制相关病原菌在海水养殖中的暴发和流行奠定基础。【方法】利用LB平板和TCBS平板从患病卵形鲳鲹体表溃烂病灶、肾脏、肝脏组织中分离纯化疑似病原菌,并对其进行常规形态特征和生理生化反应的生物学检验,通过16SrDNA基因测序对疑似病原菌株进行分子生物学鉴定,在细胞和鱼体水平开展病原菌的致病性研究,同时进行人工感染的回接试验。【结果】分离得到4株疑似病原菌(TOQZ01,TOQZ02,TOQZ03,TOQZ04),基于其表型、分子生物学特征和进化关系,判定4株菌株均为弧菌属的溶藻弧菌(Vibrio alginolyticus),且细胞毒性试验和回归试验证实,分离得到的溶藻弧菌为此次卵形鲳鲹发病的病原菌。【结论】溶藻弧菌是引起广西沿海地区海水养殖鱼类发生细菌性鱼病的主要致病菌之一,未来将基于本研究中分离得到的溶藻弧菌开展病害快速检测技术和防控技术等现代海水生态健康养殖关键技术研究,以实现对广西海水养殖溶藻弧菌病的快速诊断、实时监控和有效预防。     

Characteristics of Baicalin Synergy with Penicillin or Notopterygium Ethanol Extracts Against Staphylococcus aureus

Introduction Staphylococcus aureus (S. aureus) resistance to most once powerful β-lactame antibiotics is now a severe problem world wide, so agents are urgently needed to reduce or moderate resistance to existing antibiotics. Traditional medicinal herbs …     

黄连水提物对卵形鲳鲹源溶藻弧菌的抑菌作用

卵形鲳鲹(Trachinotus ovatus)是一种生长快速且肉质鲜美的经济型海水养殖鱼类,但是近些年为满足人们的日常需求,在不断扩大养殖的同时病菌也逐渐泛滥。溶藻弧菌(Vibrio alginolyticus)是华南沿海地区海水养殖鱼类细菌性鱼病的主要致病菌之一,给水产养殖业带来巨大损失。本研究对黄连(Coptis chinensis Franch)的抗菌作用进行系统研究,目的是为由溶藻弧菌引起的爆发性细菌性鱼病的高效防治提供科学的用药依据。本研究采用二倍稀释法测定黄连水提物对卵形鲳鲹源溶藻弧菌的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和半数致死量(LD50),并使用BCA蛋白浓度测定试剂盒测定细菌超声破碎上清液中可溶性蛋白含量。结果表明,黄连水提物对溶藻弧菌具有明显的抑制作用,其对溶藻弧菌的MIC、MBC和LD50的值分别为7.800mg/mL、31.250mg/mL和15.625mg/mL。胞内可溶性蛋白含量变化的结果提示:黄连发挥抑菌作用的机制可能是通过损伤细菌的细胞壁导致细菌内容物的释放并引起菌体裂解死亡。黄连具有发展成为一种高效抗水产病害中草药制剂的潜力。     

Spectrofluorimetric determination of magnesium (II) with 7-(8-Hydroxy-3, 6-disulfonaphthylazo)-8-hydroxyquinoline-5-sulfonic acid

A new fluorescent reagent, 7-(8-hydroxy-3, 6-disulfonaphthylazo)-8-hydroxyquinoline-5-sulfonic acid (HDNHQ) for the determination of magnesium has been developed. It reacted with magnesium to form a 1∶1 fluorescent complex withλ _ex/λ _em immediately at room temperature in ammonia-ammonium chloride buffer (pH 10.7). A linear relationship was obtained in the magnesium concentration range of 0–160 ng·mL⁻¹ with the detection limit of 0.04 ng·mL⁻¹. The proposed method was simple, rapid and sensitive. It has been successfully applied to the determination of trace magnesium in blood serum with recoveries of 103.75% and 98.16%, respectively. Foundation item: Supported by the Zi-Qiang Foundation of Wuhan University Biography: Zhang Xian (1975-), female, Ph. D candidant, research direction: organic reagent synthesis and analysis.     

基于核酸适配体吸附检测技术的卵形鲳鲹源溶藻弧菌的快速检测

溶藻弧菌是引起广西等华南沿海地区海水养殖鱼类发生细菌性鱼病的主要致病菌之一,其引起的鱼病具有发病迅速、死亡率高、流行面广等特点,严重威胁着华南地区水产养殖业的健康可持续发展。着力发展操作便捷、成本低、耗时短、准确度高的水产致病菌快速检测技术,对于及早发现、确定病原,进而有的放矢地制定治疗方案来控制病原扩散、降低损失意义重大。在先前研究中,我们基于指数富集的配基系统进化技术(Systematic evolution of ligands by exponential enrichment technology,SELEX)筛选获得特异性识别卵形鲳鲹源溶藻弧菌的核酸适配体,本研究中我们基于核酸适配体VA8开展溶藻弧菌的快速检测诊断技术的研究,开发出一种新型的能够快速检测溶藻弧菌的核酸适配体吸附检测技术(Aptamer VA8-based enzyme-linked aptasorbent assay,VA8-ELASA),并对VA8-ELASA技术检测溶藻弧菌的特异性和灵敏性进行分析研究。VA8-ELASA技术可以用于溶藻弧菌的快速检测,具有特异性强、灵敏度高的特点。本研究基于核酸适配体VA8建立的新型核酸适配体吸附技术(VA8-ELASA),有望实现对广西卵形鲳鲹养殖中溶藻弧菌病的快速诊断、实时监控和有效预防。     

Thermoluminescence of Al2O3 crystals grown by temperature gradient techniques

Color center concentrations of the Al2O3 crystal grown by temperature gradient techniques were calculated from Gaussian fits to absorption spectra. The concentrations for F- and F + -centers at 204, 232, and 255 nm were determined to be 1.361×10 17 , 0.098×10 17 , and 0.325×10 17cm-3 , respectively. Studies have shown that the thermoluminescence (TL) glow curve exhibits a prominent 450 K peak that matches well the first-order fitting curve. The thermal activation energy E and frequency factor s of the trap were determined to be 0.94456±0.00545 eV and 5.8703×10 11 s-1 . With this theoretical analysis, a simple one-trap/one-center TL model is presented to provide a theoretical explanation of the TL process.     

Effective antibacterial and phase change PEG/Cu2O@A-HNTs composites for melt-spun difunctional PA6 fiber

Serving as the only defensive line between pathogens and human body, personal protective equipment (PPE) is increasingly attractive among researchers because of their strong antibacterial and temperature control abilities. However, efficient antibacterial properties and regenerative thermal control simultaneously remain unexplored in PPE. Here, one-dimensional halloysite nanotubes (HNTs) modified by acid etch method, are used to synthesis a multifunctional material of PEG/Cu₂O@A-HNTs via in-situ reduction and physical adsorption which serves the above two purposes. PEG/Cu₂O@A-HNTs showed rapid bacterial inactivation and achieved 96.3% bacteriostatic rate against E. coli in only 20 ?min. Meanwhile a broad and complete inactivation spectrum included both E. coli and S. aureus following a series of antibacterial mechanisms. Moreover, adsorption of 70 ?wt% PEG by acid etch HNTs is attributed to the nearly 3 times increased specific surface area compared with native HNTs. This enabled PEG/Cu₂O@A-HNTs to attain a phase change enthalpy of 108.4 ?J/g. In addition, using PEG/Cu₂O@A-HNTs as additives, antibacterial and phase change fiber (APCF) were melt-spun. Their efficiency factor against E. coli and S. aureus was above 99.99%, and retained a temperature control ability for 180s and 272s compared with PA6 fiber in hot and frigid environments respectively.     

Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

Highly stable N-containing polymer-based Fe/Nx/C electrocatalyst for alkaline anion exchange membrane fuel cell applications

A cost-effective electrocatalyst with high activity and stability was developed. The Fe-Nx and pyridinic-N active sites were embedded in nitrogen-doped mesoporous carbon nanomaterial by carbonization at high temperature. The electrocatalyst exhibited excellent electrochemical performance for the oxygen reduction reaction, with high onset potential and half-wave potential values (E_onset = 1.10 ?V and E_1/2 ?= ?0.944 ?V) than 20 ?wt % Pt/C catalyst (1.04 and 0.910 ?V). The mass activity of the Schiff base network (SNW) based SNW-Fe/N/C@800° electrocatalyst (0.64 ?mA ?mg^?1 @ 1 ?V) reached about half of the commercial Pt/C electrocatalyst (1.35 ?mA ?mg^?1 @ 1 ?V). The electrocatalyst followed the 4-electron transfer mechanism due to very low hydrogen peroxide yield (H₂O₂ ?< ?1.5%) was obtained. In addition, this electrocatalyst with dual active sites showed high stability during cyclic voltammetry and chronoamperometry measurements. More importantly, the electrocatalyst also demonstrated the power density of 266 ?mW ?cm^?2 in the alkaline anions exchange membrane fuel cell (AEMFC) test, indicating its prospective application for fuel cells.     

Spatial variability of cyanobacterial community composition in Sanya Bay as determined by DGGE fingerprinting and multivariate analysis

The cyanobacterial communities in the surface and bottom waters of Sanya Bay were investigated on April 24 and 25,2010.Flow cytometry showed that the total cyanobacterial abundance in the surface and bottom layers ranged from 0.7×10 4 to 2.38×10 4 cells mL-1 and from 1×10 4 to 1.8×10 4 cells mL-1,respectively.Cyanobacterial diversity was analyzed using a molecular fingerprinting technique called denaturing gradient gel electrophoresis(DGGE),followed by DNA sequencing.The results were then interpreted through multivariate statistical analysis.Differences in the compositions of cyanobacterial communities were observed in the surface and bottom waters at the same station,with some bands obtained from both the surface and bottom layers,whereas some bands were present only in one layer.The predominant cyanobacterial species of the excised DGGE bands were related to Synechococcus or Synechococcus-like species(56.2%).Other phylogenetic groups identified included Chroococcidiopsis(6.3%),Cyanobium(6.3%) and some unclassified cyanobacteria(31.2%).A redundancy analysis(RDA) was conducted to reveal the relationships between the cyanobacterial community composition and environmental factors.Analysis results showed that the spatial variations in the cyanobacterial community composition in surface waters was significantly related to chlorophyll a(Chla),the biochemical oxygen demand(BOD),nitrate and phosphate(P<0.05).Meanwhile,the spatial variations in the bottom waters was significantly affected by nitrate,nitrite,and phosphate(P<0.05).Environmental parameters could explain 99.3% and 58.3% of the variations in the surface and bottom layers,respectively.     

三丁基锡对热带爪蟾蝌蚪甲调基因mRNA表达的影响

用三丁基锡( TBT)对热带爪蟾(Xenopus tropicalis)NF51阶段蝌蚪进行48 h和96 h暴露.结果显示,50 ng·L-1和200 ng·L-1 TBTCl暴露对胚胎的存活率没有影响.50 ng·L-1TBTCl暴露组TRβ和DI 3 mRNA在蝌蚪尾巴中的表达比对照组增加了1～2.3倍.200 ...     

Synthesis and electrochemical properties of FeSbO4 nanorods

Well-crystallized FeSbO 4 nanorods with rutile-like structure are synthesized through a solid-state reaction and used as cathode material of Li-ion battery for the first time.The obtained nanorods can react with 11 Li-ions per FeSbO 4 unit with a specific discharge capacity of 1 100 1 mAh g between 0.1 and 2.0 V.Three discharge plateaus can be observed during the fully discharging process,but the reversible reaction with 1 Li occurs between 1.5 V and 4.5 V vs.Li + /Li,and the reversible capacity is only 50-80 1 mAh g.FeSbO 4 nanorods have a stable cyclic performance between 1.5 V and 4.5 V and it can be used as cathode material for rechargeable Li-ion battery.