人乳腺珠蛋白基因疫苗的构建及其免疫原性研究

目的:构建人乳腺珠蛋白(hMAM)基因重组质粒pcDNA3.1-hMAM,并观察重组质粒在COS-7细胞中的表达,为肿瘤DNA疫苗研究奠定基础。方法:利用PCR方法扩增hMAM基因,酶切测序后克隆入真核表达载体pcDNA3.1,构建真核表达载体pcDNA3.1-hMAM。将重组载体脂质体转染法转染入COS-7细胞,间接免疫荧光法和ELISA检测目的基因的表达。结果:成功扩增hMAM基因,酶切测序表明,重组质粒含有hMAM基因,在COS-7细胞中可检测到hMAM表达。结论:hMAM基因疫苗构建成功,为进一步进行DNA肿瘤疫苗研究提供了实验依据。     

白假丝酵母菌天冬氨酸蛋白酶真核表达质粒pcDNA3．1／SAP2的构建

目的构建白假丝酵母菌天冬氨酸蛋白酶真核表达质粒pcDNA3．1／SAP2．为以其作为基因疫苗进行免疫动物实验奠定物质基础。方法从白假丝酵母菌中提取基因组DNA，以其为模板采用聚合酶链反应（PCR）法获取SAP2基因，将真核表达质粒pcDNA3．1（＋）myc-HisC和SAP2基因行EcoRI和XhoI双酶切，琼脂糖凝胶纯化，连接酶切产物，转化TOP10感受态细菌，筛选菌落和测序鉴定。结果经PCR扩增获得的目的基因分子量与预计相同，并定向插入真核表达载体pcDNA3．1（＋）myc-HisC，电泳获得预期的SAP2条带,测序证实为正确的SAP2序列。结论利用真核表达质粒pcDNA3．1（＋）myc-HisC可以方便而高效地构建pcDNA3．1／SAP2重组质粒，该质粒能直接激活抗原提呈细胞，可以使重组质粒具有较强的免疫原性和免疫反应性。     

奈瑟氏淋球菌外膜蛋白PIA基因的克隆、真核表达与鉴定

目的克隆奈瑟氏淋球菌外膜蛋白PIA基因,构建稳定的真核重组表达载体,并在HELA细胞中表达。方法根据淋球菌PIA基因序列,利用Primer Premier5.0生物学软件设计一对特异性扩增引物,以淋球菌国际标准株基因组DNA为模板,PCR扩增除掉信号肽后PIA基因开放读码框（ORF）,将其插入真核表达载体pcDNA3.1（＋）,构建含目的基因的pcDNA3.1（＋）/PIA重组质粒,PCR及酶切鉴定重组载体,脂质体转染HELA细胞,以间接免疫荧光法检测PIA基因在HELA细胞中的表达。结果成功构建了pcDNA3.1（＋）/PIA重组质粒;pcDNA3.1（＋）/PIA能在HELA细胞中高效表达PIA蛋白。结论重组质粒pcDNA3.1（＋）/PIA构建成功并在HELA细胞中高效表达了PIA蛋白,为该蛋白的抗原性及功能学研究奠定了基础。     

汉坦病毒S基因真核表达载体的构建及表达

目的 将编码汉坦病毒76-118株核蛋白的S基因片段克隆到真核表达载体DTARGET TM，构建含汉坦病毒核蛋白S基因的重组真核表达质粒pTARGET-hans，并在体外进行瞬间表达。方法 采用PCR产物直接克隆方法，将核蛋白S基因插入到真核表达载体pTARGE TM中；酶切鉴定；用电穿孔法将重组质粒转染入Vero-E6细胞；用间接免疫荧光法检测其表达产物。结果 酶切鉴定证实S基因已被正确地插入pTARGET TM中；在部分转染重组质粒的Vem-E6细胞胞质内观察到中等强度的特异性荧光。结论 重组的真核表达载体pTARGET-hans已被成功地构建并可在体外表达．为下一步基因免疫打下基础。     

5种食源性致病微生物毒力基因重组质粒的构建、克隆表达与表达产物的研究

目的构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌5种主要食源性致病微生物毒力基因重组质粒载体并鉴定其表达产物,为探讨高通量磁性荧光纳米颗粒标记相应的抗体技术快速检测带毒力基因的致病微生物的可行性奠定基础。方法分别从5种食源性致病微生物毒力岛中选择特异性的毒力基因进行引物设计,分别提取5种目的细菌DNA片段,经PCR扩增、电泳回收目的基因片段,将目的基因片段与原核表达载体pET-23a、pET-28a、pET-32a构建各自的重组质粒,将重组质粒转化入大肠杆菌DH5α内并提取质粒载体,构建后的重组质粒进行酶切和测序鉴定,再转化入表达宿主E·coliBL21（DE3）。在0.1 mmol/L的IPTG诱导下,目的质粒载体在E·coliBL21（DE3）株中表达,SDS-PAGE电泳检测表达蛋白。结果实验成功构建大肠杆菌O157∶H7、副溶血性弧菌、金黄色葡萄球菌、沙门氏菌和志贺氏菌等细菌的毒力基因重组质粒载体pET-23a-eaeA、pET-28a-tdh、pET-28a-enterotoxin B、pET-32a-invA和pET-28a-ipaH,并克隆出969 bp的eaeA基因片段、567 bp的tdh基因片段、798 bp的enterotoxin B基因片段、1 041 bp的invA基因片段和771 bp的ipaH基因片段。重组质粒载体经酶切和测序与目标基因序列一致。在E·coliBL21（DE3）株中有质粒表达蛋白37.5 kDa（eaeA）、26 kDa（tdh）、34.5 kDa（enterotoxin B）、41 kDa（invA）、32 kDa（ipaH）。结论本研究成功构建了5种食源性致病微生物毒力基因重组质粒并在原核细胞中高效表达,为下一步获得相应的毒力基因抗体及快速诊断试剂的研制应用奠定了基础。     

人谷胱甘肽硫转移酶A1真核表达系统的构建及序列分析

目的 构建人谷肽甘肽硫转移酶A1真核表达系统，为毒理学、遗传药理学的研究等提供材料。方法 用RT—PCR技术从人肝脏组织总RNA中分离扩增人谷胸甘肽硫转移酶A1基因的cDNA序列，将其插入到真核表达载体pcDNA3多克隆位点中，构建重组表达质粒，并用PCR扩增、酶切分析及序列测定等方法对重组质粒进行鉴定。结果 人谷胱甘肽硫转移酶A1被正确地克隆到真核表达载体pcDNA3中，测序结果同Genbank序列比较，在152位点T→C，氨基酸由Met→Thr。结论 经酶切鉴定和PCR扩增，证实人谷胱甘肽硫转移酶A1真核表达系统构建成功。     

H5N1亚型禽流感病毒聚合酶酸性蛋白真核表达载体的构建与表达

目的构建甲型禽流感病毒H5N1亚型聚合酶酸性蛋白(PA)的真核表达载体,并表达其编码蛋白PA。方法采用RT-PCR法扩增PA基因,克隆至pMD18-T载体中构pMD18-T-PA质粒。双酶切pMD18-T-PA质粒与pXJ40-HA质粒后,胶回收并连接目的片段,构建真核表达载体pXJ40-HA-PA,鉴定后转染293T细胞。采用免疫印迹法(Western blot)鉴定重组PA蛋白的表达。结果成功构建了禽流感病毒H5N1亚型PA基因的真核表达载体,并在真核细胞中成功表达出分子量为75kD的重组蛋白。结论成功构建禽流感病毒H5N1亚型PA基因的真核表达载体,为后期进一步研究PA蛋白的功能奠定了基础。     

家兔pEGFP-N1/rCNP质粒的构建及其在人脐静脉内皮细胞中的表达

[目的]构建真核表达载体pEGFP-N1/rCNP并转染体外培养的人脐静脉内皮细胞,观察CNP基因在其中的表达。[方法]利用RT-PCR方法从家兔腹主动脉组织扩增获得CNP基因全场全长编码区,克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体pEGFP-N1上,构建重组载体pEGFP-N1/rCNP。利用LipofectaminTM 2000脂质体将重组质粒转染人脐静脉内皮细胞。[结果]重组质粒pEGFP-N1/rCNP构建成功;将其转染人脐静脉内皮细胞,观察到目的基因CNP表达。[结论]成功构建了家兔腹主动脉CNP基因的真核表达载体pEGFP-N1/rCNP,并可转染人脐静脉内皮细胞。     

潜伏活性的人可溶性TNFⅠ型受体融合蛋白真核表达载体的构建及表达

目的：采用基因工程技术，将转化生长因子B前体相关蛋白（LAP）通过基质金属蛋白酶（MMP）水解部位与人可溶性TNFI型受体（hsTNFRI）连接，构建pcDNA3，1／LAP-MMP-hs TNFRI融合蛋白真核表达载体，获得LAP-MMP-hsTNFRI融合蛋白。方法：将编码MMP水解部位氨基酸的正反义DNA序列退火形成互补双链后定向克隆插入真核表达载体pcDNA3．1（＋），得到pcDNA3．1／MMP重组体；将TGF-β1的LAP段和hsTNFRI与MMP水解部位连接，获得pcDNA3．1／LAP-MMP-hsTNFRI真核表达载体。测序鉴定后，脂质体介导重组质粒转染COS-7细胞，通过RT-PCR检测融合基因的表达。结果：酶切及测序结果证实pcDNA3．1／LAP-MMP-hsTNFRI重组载体构建成功，转染后RT-PCR结果表明重组质粒在COS-7细胞中得到有效表达。结论：成功构建了融合蛋白真核表达载体pcDNA3，1／LAP-MMP-hsTNFRI，并获得融合基因的瞬时表达，为进一步研究融合蛋白在子宫内膜异位症中的靶向治疗奠定了实验基础。     

miR-145真核表达载体的构建及表达

目的构建miR-145的真核表达载体,为研究miR-145在结肠癌中的生物学功能奠定基础。方法设计并应用PCR扩增miR-145基因片段,将其导入真核表达载体pCMV-myc中构建重组质粒pCMV-miR-145后,将重组质粒转染入结肠癌细胞系HCT116中,运用RT-PCR检测miR-145的表达情况。结果酶切及DNA测序证实miR-145被正确克隆入真核表达载体pCMV-myc中,该重组质粒能在HCT-116细胞中高效表达miR-145。结论成功构建了miR-145的真核表达载体pCMV-miR-145,该载体能有效高表达miR-145。     

Calcium intake in the elderly: validation of a dietary questionnaire

Epidemiological studies of calcium and osteoporosis have been hampered by the lack of a suitable tool for assessing calcium intake. This report describes a new frequency and amount questionnaire for measuring present and past calcium intake in the elderly. The validity of the questionnaire was tested against two commonly used standards of dietary assessment, five-day duplicate diets and seven-day weighed dietary inventories. The resulting correlation coefficients were, respectively, r = 0.76 and r = 0.69, while that for repeatability was r = 0.84. Furthermore, the questionnaire categorized subjects into thirds of the distribution of intake with almost no gross misclassification. It is suggested that the present findings may be extended to the majority of normal, healthy elderly subjects, implying wide application for the questionnaire in the assessment of calcium intake in the elderly.     

The occurrence of antimicrobial substances in toilet,sink and shower drainpipes of clinical units: A neglected source of antibiotic residues

Antibiotics represent one of the most important drug groups used in the management of bacterial infections in humans and animals. Due to the increasing problem of antibiotic resistance, assurance of the antibacterial effectiveness of these substances has moved into the focus of public health. The reduction in antibiotic residues in wastewater and the environment may play a decisive role in the development of increasing rates of antibiotic resistance. The present study examines the wastewater of 31 patient rooms of various German clinics for possible residues of antibiotics, as well as the wastewater of five private households as a reference.To the best of our knowledge, this study shows for the first time that in hospitals with high antibiotic consumption rates, residues of these drugs can be regularly detected in toilets, sink siphons and shower drains at concentrations ranging from 0.02?μg·L^?1 to a maximum of 79?mg·L^?1. After complete flushing of the wastewater siphons, antibiotics are no longer detectable, but after temporal stagnation, the concentration of the active substances in the water phases of respective siphons increases again, suggesting that antibiotics persist through the washing process in biofilms. This study demonstrates that clinical wastewater systems offer further possibilities for the optimization of antibiotic resistance surveillance.     

Self-reported health problems among Swedish miners one year after unemployment

Unemployment is considered to be a public health concern sincedeterioration in the health of the unemployed is often anticipated.However, for some groups, such as miners, unemployment mightimprove health due to a cessation of potentially harmful occupationalexposures. This study evaluates the health of 79 miners in oneSwedish iron-ore mine, and 226 age-matched controls from thegeneral population, during one year after the closure of themine. The participants received a questionnaire regarding medicalhistory and subjective symptoms at the beginning of the studyperiod, and after one year. Statistically significant negativeeffects on self-reported health attributable to unemploymentwere not found, although neuropsychiatric symptoms were morecommon among the unemployed miners. The miners reported a statisticallysignificant improvement in grip force (p=0.031). They had asignificantly higher prevalence of symptoms associated withmining related exposures when compared with the population controls;pain in the upper extremities [relative risk (RR)=2.27, 95%confidence interval (Cl)=1.44–3.59), back pain (RR=1.84;Cl=1.237–2.75), vasospastic disease of the fingers (RR=2.05;Cl=1.18–3.57) and obstructive respiratory symptoms (attacksof dyspnea and wheezing: RR=3.67; Cl=1.167–11.6).     

Excès de cas humains et animaux de tularémie en France au cours de l’hiver 2007–2008 : émergence ou phénomène isolé ?

Context

Tularemia is a zoonosis affecting humans and hares in France. We describe the results of surveillance in both species, in 2007 and 2008.

Methods

Human tularemia cases are mandatorily notifiable in France since 2003. In hares, surveillance relies on volunteer hunter associations in all districts of the country. Data from mandatory reports and volunteer surveillance in 2007/2008 were analyzed and compared with previous results.

Results

In 2007/2008, 144 cases were reported in humans and 117 cases in hares. This was a 100% increase compared to previous years. Human cases differed from those of previous years only by the frequency of contact with breeding animals. Human cases without any documented risk exposure were also more frequent.

Conclusion

An increase of tularemia cases occurred in 2007/2008 in both species. Complementary studies are needed to identify the species reservoir in France to understand the causes of this peak of cases.     

Occupational health hazards in mining: an overview

This review article outlines the physical, chemical, biological, ergonomic and psychosocial occupational health hazards of mining and associated metallurgical processes. Mining remains an important industrial sector in many parts of the world and although substantial progress has been made in the control of occupational health hazards, there remains room for further risk reduction. This applies particularly to traumatic injury hazards, ergonomic hazards and noise. Vigilance is also required to ensure exposures to coal dust and crystalline silica remain effectively controlled.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

深圳公立医院管理体制改革制度设计

深圳公立医院管理体制改革实行政事分开、管办分开,在理事会架构下按法定机构模式组建市医管中心,落实公立医院运营管理自主权.作者从当前公立医院管理体制的弊端入手,介绍了深圳市进行公立医院管理体制改革的基本思路及改革方案设计的主要举措,深入剖析了的改革方案的特点,并对改革效果进行了预测.     

Effects of nitrogen dioxide on red blood cells of rats: Changes in components of red cell membranes during in vivo exposure to NO2

Red cell membranes, prepared from red blood cells of rats exposed to 4, 10, or 20 ppm nitrogen dioxide (NO₂) for 1 to 10 days, were examined for evidence of changes in membrane components. Appreciable changes were not found in contents of phospholipid and cholesterol during exposure to 10 ppm NO₂. By contrast, protein content altered with the time of exposure. Moreover, changes in protein composition were observed by employing sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Twenty-four-hour exposure to NO₂ at the concentration above 10 ppm resulted in a marked increase in the percentage of lysophosphatidylethanolamine (LysoPE) to the total phospholipids. The prolonged exposure to 10 ppm NO₂ gave rise to a further increase in LysoPE, whereas the percentage of phosphatidylethanolamine (PE) showed a gradual decrease. A 1-day exposure to 4.0 ppm NO₂ also caused an increase in sialic acid content and decreases in those of PE and hexose. In addition to contents of these components the percentage of LysoPE increased 5 days after exposure and the elevated values were maintained up to the end of exposure period. These results demonstrate that red blood cells in circulation exhibit different membrane properties in terms of lipid and carbohydrate composition during 10 days of exposure to 4.0 ppm NO₂.     

Investigating clusters in the workplace and beyond

Clusters of disease are common and occur in the workplace and in the general community. They often arouse considerable concern among the population. Investigations have sometimes lead to exciting new knowledge, but in general the investigation of clusters is difficult and often unrewarding, especially for community clusters. In the workplace, investigations are more likely to find associations and even new causes, but still many clusters remain enigmatic. Despite this, there are many reasons for investigating clusters, including allaying community concern and identifying uncontrolled exposures. A structure for investigating clusters in the workplace is suggested.