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.     

蛭弧菌指示和净化海水的研究

厦门近岸海水海洋噬菌蛭弧菌的数量与海水中弧菌类的数量成正相关．灭菌海水中只加蛭弧菌，其数量出现负增长．若同时加入蛭弧菌和宿主菌鳗弧菌８９２７菌株，则蛭弧菌数增加、增加速率随宿主菌浓度增加而增加，并导致宿主菌数减少，此时宿主菌随培养时间延长而减少，但减少速度缓慢，１５ｄ仅减少２６％．     

新型显色基质法鲎试验对肺部革兰氏阴性菌感染患者的诊断意义

应用新型显色基质法鲎试验（ＮＣＬＡ）测定４１例肺部革兰阴性（Ｇ＾－）细菌感染患者）Ｇ组）；３２例肺部非革兰氏阴性细菌感染患者（非Ｇ组）；４２例无细菌感染的肺病患者（无感染组）的血浆内毒素浓度、并与１００例健康者（对照）比较，Ｇ组血浆内毒素水平明显高于对照组，非Ｇ组及无感染组（Ｐ均〈０．０１）。提示新型显色基质法鲎试验有助于肺部Ｇ细菌感染作出快速，灵敏的诊断。     

双抗原夹心ELISA检测抗鳗弧菌抗体效价的研究

建立检测抗鳗孤菌抗体效价的双抗原夹心ELISA法,评价其可行性。采用高碘酸盐法对鳗孤菌抗原进行辣根过氧化物酶标记,并通过血清抗体效价比较双抗原夹心ELISA与微量凝集法的优越性。结果显示：标记蛋白含量为2．5mg的抗原所需HRP的最适量为0．20rag。以超声破碎后鳗弧菌离心上清为最适标记抗原,其工作浓度为12．25mg／L．并且双抗原夹心ELISA操作简易具有较高的敏感性和特异性。     

仿刺参硫氧还蛋白基因在灿烂弧菌和鳗弧菌刺激下的应激表达特征

分别用灿烂弧菌(Vibrio splendidus)和鳗弧菌(V.anguillarum)对仿刺参(Apostichopus japonicus)进行注射感染试验,取注射后0、3、6、9、12、24 h的仿刺参体壁、肠道、呼吸树、触手和体腔细胞5种组织或细胞进行了实时荧光定量PCR检测,分析硫氧还蛋白(Thioredoxin,Trx)基因在这5种组织或细胞中的表达特征,探讨了硫氧还蛋白在仿刺参先天免疫中的作用。结果显示,Trx基因在仿刺参体壁、肠道、触手、呼吸树及体腔细胞中均表达;在灿烂弧菌刺激下各组织或细胞中Trx基因变化趋势相似,呈现一种"突增-下降-再突增"的双峰型模式;鳗弧菌刺激下Trx基因表达量变化同样呈现先升高后降低的趋势,但与灿烂弧菌组相比反应时间与上调幅度都不同。总而言之,Trx基因在仿刺参体内有组成型和诱导型两种表达模式,并存在表达的组织特异性和病原特异性;Trx基因参与了仿刺参对病原感染的免疫应答,使机体免受氧化胁迫,在抵御外界病原入侵、维持胞内氧化还原状态方面起到重要作用。     

羧基化多壁碳纳米管修饰电极测定依诺沙星

基于羧基化多壁碳纳米管修饰玻碳电极为工作电极,建立了无汞和高灵敏测定依诺沙星的电化学方法.在0.1 mol/L磷酸盐缓冲溶液(pH为5.91)中,在0.5 V富集5 min,然后以100 mV/s的扫描速度从0.5 V扫描至1.4 V,1次微分峰电流与依诺沙星在0.02～0.20 mg/L呈线性关系,检出限为0.01 mg/L.通过与药典方法比较,证实了方法的可靠性.该方法操作简单,灵敏度高,避免使用汞,可以用于依诺沙星胶囊含量的常规测定.     

杜仲提取物对鱼类病原菌作用的研究

采用二次水浸提法提取杜仲有效成分进行杯碟法抑菌试验.结果表明,杜仲提取物对嗜水气单胞菌、鳗弧菌、温和气单胞菌具有较好的抑菌效果,而对大肠杆菌的抑菌效果不明显.通过SDS-聚丙烯酰胺凝胶电泳分析病原菌在杜仲提取物作用下蛋白的差异表达,结果发现,药物抑菌效果越明显的菌种,其蛋白表达的差异性就越大,且受抑制的蛋白和诱导表达的特异蛋白的分布比较广;而抑菌效果较差的菌种,其蛋白表达差异性较小.     

Preparation and Identification of Monoclonal Antibodies Against Vibrio anguillarum

Monoclonal antibodies (Mabs) against V. anguillarum strain M3 are prepared, and their isotypes are also characterized. Among them, C1C5 is the only Mab which does not cross-react with other eleven non-V, anguillarum strains. The proteinase K digestion test shows that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contained protein. The periodate oxidation test showed that the epitopes recognized by Mabs except C1 C5 are glycosylated. In addition, results of additivity test indicate that the epitopes recognized by C6C3 and C6C32 Mabs are similar, and quite different from those recognized by MabC1C5.     

酸性铬兰K固体石蜡碳糊修饰电极溶出伏安法测定痕量钴

在pH为8.5,0．2mol／L,氨性缓冲底液中,以酸性铬兰K（ACBK）为修饰剂,固体石蜡为粘合剂,制备了ACBK碳糊修饰电极。在-1．0V电位下富集,将钴以Co－ACBK络合物的形式吸附在电极上,以阳极溶出伏安法制定Co2+,在十0．106V（VS．SCE,以下同）处有灵敏的氧化峰,其一次微分峰电流与Co2+浓度在1．0X10~-12～1．0X10~－5mol／L范围内的负对数呈良好的线性关系,此法的检测下限为5.0X10~13mol／L.     

双酚A在多壁碳纳米修饰电极上电化学性质及其测定研究

 制备出1种多壁碳纳米管化学修饰电极,详细研究了双酚A(SPA)在多壁碳纳米管化学修饰上的电化学行为,并提出了一种灵敏的、简便的直接检测双酚A的电化学分析方法.在0.30V富集后,双酚A在多壁碳纳米管修饰玻碳(MWNT-GCE)电极上出现一个灵敏度高、峰形好的氧化峰.在pH7.0的磷酸盐缓冲溶液中峰电位位于0.58V.多壁碳纳米管薄膜对双酚A的氧化表现出一定的催化作用,能显著提高双酚A的氧化峰电流.优化了测定参数如:底液的pH,修饰剂的用量、扫描速度,富集时间等.双酚A的氧化峰电流与其浓度在5.0×10^-8～2.0×10^-5mol·L^-1之间有很好的线性关系.富集3min后的检出限为2.0×10^-8mol·L^-1.用此方法测定了塑料中双酚A的含量,结果满意.     

南京市男男性行为人群基于抗体亲和力的HIV-1抗体阳转检测

评价了由美国疾病预防控制中心研制的基于抗体亲和力的用于HIV-1阳性血清阳转检测的方法——有限抗原亲和力酶联免疫吸附法(LAg-EIA)和亲和力指数酶联免疫吸附法(AI-EIA),并将其初步应用于南京市男男性行为(MSM)人群的阳转检测.方法:101份博卡HIV阳性血清和4份标准对照血清作为评价血清.检测一致性通过不同检测时间、板内、板间以及不同批次包被板之间的比较进行评价.用LAg-EIA法对2013年、 2015年及2016年男男性行为人群确证阳性血清阳转进行检测.结果:LAg-EIA法的总体一致性在97.22%以上(95%可信区间),检测时间一致性分别为97.22%, 98.94%和97.83%;板内一致性为99.78%;板间一致性为99.89%;不同批次一致性为99.99%. AI-EIA法的一致性在90.00%以上(95%可信区间),检测时间一致性分别为90.00%, 93.37%和93.56%;板内一致性为99.30%;板间一致性为97.06%;不同批次一致性为99.62%.南京市3年男男性行为人群HIV-1阳性中有42.8%, 65.7%和40.0%的血清为新近阳性,按照我国国家CDC平均阳转156 d来看,这些感染在156d以内;有57.1%, 34.2%和60.0%的血清为长期感染, HIV-1感染超过156d.每年平均阳转时间有波动,但确证感染检测总体较迟.结论:两种方法的一致性均高于90.00%,是适合于实际应用的好方法,其中, LAg-EIA法的一致性高于AI-EIA法.用LAg-EIA法检测南京市3年数据表明该人群确证感染检测总体较迟,不利于该病的预防和控制.     

方格星虫多糖抗菌和抗氧化活性研究

通过比较水提和碱提方格星虫粗多糖的抗菌和抗氧化活性,探讨这两种方法在方格星虫粗多糖提取中的优劣.结果,水提粗多糖对副溶血弧菌（Vib rio parahemol yticus）不敏感,对枯草芽孢杆菌（Bacillus subtilis）中度敏感,对其它细菌均为低敏感;碱提粗多糖对大肠杆菌（Escherichia coli）、金黄色葡萄球菌（Staphylococcus aures）和鳗弧菌（Vibrio an guillarum）低敏感,对白葡萄球菌（Staphylococcus cremoris）和副溶血弧菌中度敏感,对枯草芽孢杆菌和藤黄八叠球菌（Sarcina lutea）高度敏感.表明,碱提粗多糖的抗菌活性较水提粗多糖强.两种方法提取的方格星虫粗多糖均有一定的抗氧化性.水提和碱提多糖浓度均在800μg·mL-1时对羟自由基的清除能力最大,清除率分别为40.32％和39.86％;两种多糖浓度分别在800μg·mL-1和600μg· mL-1时对超氧自由基的抑制作用最大,抑制率分别为9.91％和5.86％.表明,水提方格星虫粗多糖的抗氧化性强于碱提粗多糖.     

Virus-specific effects of interferon in embryonal carcinoma cells

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).     

HRP-PAMAM-MCNT修饰电极的研制及其在油脂中过氧化物测定中的应用

以自组装方式,将多壁碳纳米管、聚酰胺-胺和辣根过氧化物酶修饰到玻碳电极表面,构建了H2O2生物传感器.研究了影响传感器工作性能的因素,确定了最佳分析条件,即pH值6.8的PBS缓冲溶液,工作电位为-0.15 V.在最佳工作条件下,电极对H2O2快速响应(响应时间<1 s),在9×10-7～1×10-4mol/L范围内,电极的电流响应值与H2O2浓度呈现良好的线性关系,线性回归方程为i(μA)=20.76 C(mmol/L)+0.223 0,R=0.999 0,检出限为5.0×10-7mol/L,电极的电流响应灵敏度为0.247 A mol-1.cm-2.电极应用于食用油脂中过氧化物测定,结果与碘量法一致.     

Preparation of a gene-engineering mutant of bacteriorhodopsin BR-D96V and corresponding poly(vinyl alcohol)-based functional composite films

Bacteriorhodopsin (BR) exhibits, as a membrane protein in Halobacterium salinarum, unique photoresponsive behaviors, and shows promise as a functional information material. A new mutant of BR with the 96th aspartic acid replaced by valine (BR-D96V) was obtained and then a composite film of BR-D96V in a synthetic polymer matrix was prepared in this research. The mutant BR-D96V was expressed in a bacterio-opsin deficient halobacterial strain (L33) by gene engineering. Although valine is very hydrophobic, this point mutant keeps the basic biological activities, namely, photoelectric and photochromic responses. Nevertheless, the lifetime of M intermediate in the BR mutant is nearly two orders of magnitude longer than that of wild-type BR in neutral aqueous solution, which benefits its potential application as an information material. The M lifetime is further significantly prolonged after embedding BR-D96V into poly(vinyl alcohol) (PVA). It was also found that BR-D96V is very sensitive to water content in comparison with wild-type BR and another BR mutant.     

The apoptosis and cell cycle changes of SUD4 and DOHH2 induced by topotecan

Topotecan (TPT), a semisynthetic analogue of the natural product camptothecin is a cell cycle-specific drug with antitumor activity. To clarify the effect of TPT on SUD4 and DOHH2 cell line in this study, we examined the apoptosis and cell cycle changes of the two human cancer cell lines by exposing to TPT for 18 hours at various concentrations. The linear relationship between apoptosis cell number and the concentration of TPT was observed by means of Flow Cytometry and Annexin V assay. Then, DOHH2 cell is much more sensitive to TPT than SUD4 cell. In addition, Cell Question Software Assay showed positive relationship between the frequency of cells accumulated in S-phase and the concentration of TPT. The least concentration of TPT to change cell cycle is 5 nmol·L⁻¹ in both cell lines. These results suggest that the inducing apoptosis of cancer cells is one of mechanism of TPT antitumor activity. Feng Xiaorong: born in 1965, Post-doctoral Research Fellow, Engaging in Cell and Molecular Biology.     

负载罗丹明B的活性炭吸附富集一分光光度法测定水中的痕量钒

本拟定了用负载罗丹明B的活性炭对水中的痕量钒（V）进行预算富集的方法,探讨了活性炭吸附钒（V）及有机试剂的作用机理。在试验条件下,100mg活性灰负载罗丹明B后可以大大提高对钒（V）的吸附效,同时除去干扰测定的金属离子,最后用分光光度法测定水中的痕量钒（V）。利用本方法对天然水样品中的痕量钒（V）进行分离富积测试,结果令人满意。     

四种海洋致病弧菌对抗生素敏感性的测定

测定了4种海洋致病弧菌对8种常用抗生素药物的敏感性．测试结果表明，鳗弧菌对氯霉素、庆大霉素具有敏感性，但对其他所测药物均表现出耐药性；副溶血弧菌对四环素、卡那霉素、青霉素表现出耐药性，而对氯霉素、头孢娄新、庆大霉素、红霉素和链霉素则表现出敏感性；河流弧菌对四环素、链霉素表现出耐药性，对氯霉素、卡那霉素、头孢娄新、庆大霉素、青霉素和红霉素则表现出敏感性；溶藻弧菌仅对四环素表现出耐药性，对其他7种抗生素均表现出敏感性．同时针对表现出抗药性的药物，相应地确定了它们的最小抑菌质量浓度值．     

常用抗菌渔药对致病性鳗弧菌作用效果比较

采用二倍稀释法测定7种常用抗菌渔药的最低抑菌浓度（MIC）,以5倍MIC下抑菌圈直径为参数,比较7种常用抗菌渔药单独用药与联合用药对鳗弧菌的抑杀菌效果．甲砜霉素的MIC最小为0．8μg／mL,庆大霉素最大为6．4μg／mL;在5倍MIC下,7种常用抗菌渔药抑菌圈分别为：庆大霉素1．90cm,氟哌酸1．50cm,链霉素1．43cm,土霉素1．43cm,烟酸诺氟沙星1．43cm,氟甲砜霉素1．40cm,甲砜霉素1．33cm．鳗弧菌对庆大霉素和氟哌酸高度敏感;对链霉素、土霉素、烟酸诺氟沙星、氟甲砜霉素和甲砜霉素中度敏感;烟酸诺氟沙星与甲砜霉素的联合效果最好。     

Highly sensitive detection of melamine based on reversed phase liquid chromatography mass spectrometry

In this work, we developed a highly sensitive method to detect melamine based on reversed phase liquid chromatography mass spectrometry. A mass spectrometry compatible ion pair, heptafluorobutyric acid(HFBA), was used to separate melamine by reversed phase liquid chromatography prior to electrospray mass spectrometry. The incorporation of isotope internal standard and multiple reaction monitoring improved the accuracy and linearity of quantification. Based on this strategy, the method limit of quantification was 0.1 ng/g. The limits of quantification were 8 ng/g for liquid milk and 15 ng/g for dry milk powder. This method provided a reproducible and stable approach to sensitive detection and quantification of melamine. Supported by the National Natural Science Foundation of China (Grant No. 30425021) and National High Technology Research and Development Program of China (Grant No. 2007AA02Z334)