猪链球菌2型溶血素sly基因的表达及其产物的纯化和活性研究

目的 克隆并表达猪链球菌2型(SS2)溶血素基因(sly),为筛选SS2疫苗保护性抗原奠定基础.方法 用PCR方法从SS2临床分离株05ZYH33的基因组DNA中扩增出溶血素基因片段,将目的 基因插入表达载体pET-30b(+)中,构建重组表达载体pET30b-sly.重组载体经限制性内切酶酶切和DNA测序鉴定后,转化大肠埃希菌(E.coli Rosetta).IPTG诱导表达,镍离子亲和层析纯化重组蛋白,鉴定目的 蛋白的免疫学活性以及溶血活性.结果　PCR扩增的sly基因长度约为1500 bp,经测序分析,插入载体的sly基因序列准确并保持了正确的读框.经IPTG诱导的目的 蛋白表达量约占总蛋白的30%,亲和层析纯化后,蛋白纯度达80%以上.Western blot检测证实该蛋白能与感染SS2的人血清发生特异性结合.溶血试验表明重组蛋白溶血素能使猪红细胞发生溶解,溶血价为256.结论 成功构建了表达载体pET30b-sly,该载体可在大肠埃希菌中表达,表达蛋白具有免疫反应原性及溶血活性.     

梅毒螺旋体外膜蛋白Tp0136的制备及在血清学诊断中的应用

目的:为探索梅毒螺旋体(Treponema Pallidum,Tp)外膜蛋白Tp0136在早期梅毒诊断中的价值,利用基因重组技术制备Tp0136蛋白,用于血清抗体的检测。方法:采用PCR扩增Tp0136基因,构建不含信号肽序列的Tp0136基因原核表达载体,SDS-PAGE检测目的蛋白的表达情况,镍离子亲和凝胶分子筛二步法纯化目的蛋白,Western blot检测其免疫反应性,免疫日本大耳白兔评价其免疫原性,免疫双扩检测其效价,以rTp0136为包被抗原的间接ELISA检测早期梅毒血清抗体。结果:重组工程菌包涵体形式表达相对分子质量约为46 KDa的rTp0136,表达率为15%,制备得到纯度大于98%的rTp0136。纯化的rTp0136能诱导大耳白兔产生特异性免疫应答,免疫双扩测得其效价为1:32。Western blot检测重组蛋白能与兔抗Tp0136多克隆抗体发生特异性反应。间接ELISA检测正常人血清均为阴性,而早期梅毒血清抗体的阳性率为75.1%。结论:重组Tp0136具有良好的免疫活性,预示其在早期梅毒血清学诊断中具有良好的前景。     

人源性DNA聚合酶β原核蛋白的表达、蛋白纯化及其功能初探

[目的]利用人源性DNA聚合酶β原核表达载体,在大肠杆菌中表达并纯化DNA聚合酶β蛋白,并检测蛋白活性。[方法]将重组质粒pCR-BluntII-TOPO-DNApolβ转化入大肠杆菌E.coli中,IPTG诱导其表达并鉴定。利用金属螯合层析技术进行蛋白纯化,得到人源性DNApolβ融合蛋白,纯化的融合蛋白用EK酶酶切后,通过Ni2+柱纯化获得高纯度的人源性DNApolβ蛋白,电泳迁移率变动分析实验检测DNApolβ融合蛋白的AP修复活性。[结果]转化入大肠杆菌E.coliRosetta2的DNApolβ诱导表达最好,蛋白表达量高。金属螯合层析纯化后得到融合蛋白经EK酶切后得到高纯度的人源性DNApolβ蛋白,EMSA实验证明所纯化的DNApolβ融合蛋白不具有AP修复活性。[结论]利用原核表达载体pCR-BluntII-TOPO-DNApolβ在E.coliRosetta2中表达和纯化获得高纯度的DNApolβ融合蛋白,为下一步开展DNApolβ蛋白的生物学功能研究打下基础。     

结核分枝杆菌H37Rv株重组蛋白CFP-10的表达及纯化研究

目的表达、纯化结核分枝杆菌H37Rv株重组蛋白CFP-10(rCFP-10),为结核病血清学诊断价值的研究及亚单位疫苗的研制提供物质基础。方法重组质粒pET23b-CFP-10转化E.coli表达菌株BL21(DE3)pLysE,IPTG诱导rCFP-10表达。经SDS-PAGE电泳和Western印迹法鉴定后,优化表达条件用镍离子螯合亲和层析柱HisTrapTMHP纯化重组蛋白,最后用斑点金免疫渗滤法初步评价纯化蛋白的免疫反应性。结果成功构建了重组质粒pET23b-CFP-10,并且rCFP-10以可溶性形式高效表达,表达量约占菌体总蛋白的10%。经亲和层析后得到高纯度有免疫反应性的重组蛋白(纯度约为98.5%)。结论高纯度有免疫反应性的rCFP-10为新型亚单位疫苗的研发及其结核病血清学诊断价值的研究奠定了基础。     

幽门螺杆菌外膜36kDa蛋白重组表纯化及初步鉴定

[目的]构建幽门螺杆菌(H. pylori) NCTC11637菌株36kDa (OMP36)外膜蛋白基因原核表达系统,探索研制H. pylori疫苗的新途径.[方法]培养和收集H. pylori NCTC11637菌株,提取基因组DNA, PCR扩增目的基因片断.将其克隆到T载体并测序,再将目的基因克隆入PET32a( ),构建重组表达载体PET32a-OMP36.转化E.coliBL21后IPTG诱导表达,经SDS-PAGE鉴定、镍亲和层析纯化、Western blot检测表达蛋白.(结果)测序分析表明,插入的基因片断为987bp,表达的蛋白的相对分子质量(Mr)约为36kDa,并显示具有良好的抗原性.SDS-PAGE检测表明,表达量占细菌总蛋白的37%,镍亲和层析纯化率约92%.Western blot检测表达蛋白具有良好的抗原活性.[结论]成功克隆了外膜蛋白OMP 36kDa的编码基因,其表达产物OMP 36有望成为新的Hp疫苗候选分子,为H. pylori疫苗的研制和试剂盒的开发奠定了良好的基础.     

狂犬病毒糖蛋白基因在原核系统中的表达及纯化

[目的]选取狂犬病毒糖蛋白富集表位基因片段进行克隆、表达和纯化,以期获得能应用于血清学诊断的纯化重组蛋白。[方法]从狂犬病毒感染的Vero细胞上清液中提取病毒RNA,通过RT-PCR扩增CTN株糖蛋白表位富集区基因片段,与表达载体pPROEX HTb连接,构建重组表达载体,在BL21（DE3）中表达,重组蛋白经Western blot鉴定活性,最后用电洗脱法纯化重组蛋白。[结果]重组表达质粒在大肠杆菌中表达高产量的重组蛋白,Western blot结果显示,重组蛋白具有免疫活性,用电洗脱法纯化后的重组蛋白具有较高的纯度,测定蛋白含量为1.07 mg/ml。[结论]狂犬病毒糖蛋白富集表位基因片段能在大肠杆菌中成功表达,重组蛋白具有免疫活性,并可通过电洗脱法纯化,为狂犬病疫苗免疫血清抗体检测试剂的研制奠定了基础。     

幽门螺杆菌外膜36kDa蛋白重组表达、纯化及初步鉴定

[目的]构建幽门螺杆菌（H．pylori）NCTC11637菌株36kDa（OMP36）外膜蛋白基因原核表达系统，探索研制H．pylorl疫苗的新途径。[方法]培养和收集H．pylori NCTC11637菌株，提取基因组DNA，PCR扩增目的基因片断。将其克隆到T载体并测序，再将目的基因克隆入PET32a（＋），构建重组表达载体PET32a—OMP36。转化E．coli BL21后舢诱导表达，经SDS-PAGE鉴定、镍亲和层析纯化、Western blot检测表达蛋白。[结果]测序分析表明。插入的基因片断为987bp，表达的蛋白的相对分子质量（Mr）约为36kDa。并显示具有良好的抗原性。SDS—PAGE检测表明，表达量占细菌总蛋白的37％。镍亲和层析纯化率约92％。Western blot检测表达蛋白具有良好的抗原活性。[结论]成功克隆了外膜蛋白OMP36kDa的编码基因，其表达产物0MP36有望成为新的Hp疫苗候选分子，为H．pylori疫苗的研制和试剂盒的开发奠定了良好的基础。     

结核分枝杆菌分泌蛋白NPT64的克隆、表达、纯化和复性

目的 构建结核分枝杆菌分泌蛋白MPT 64的重组质粒,并在大肠杆菌中表达及纯化和复性重组蛋白.方法 用PCR方法从结核分枝杆菌H37Rv基因组中扩增出mpt64基因片段,插入PKRX-T载体中,再亚克降到表达载体PET30a中,在大肠杆菌中表达;应用组氨酸标签（His-Tag）纯化重组蛋白;在PBS中通过半透膜恢复重组蛋白的天然结构.结果 构建了含MPT64重组质粒的大肠杆菌工程菌,并以包涵体形式表达重组蛋白MPT 64,其占细胞总蛋白的20%～30%;重组蛋白变性后经镍柱纯化,其纯度达90%以上;通过半透膜完全恢复重组蛋白的天然结构.结论 具有天然结构和高纯度的重组融合蛋白有可能成为有效的结核病血清学诊断的候选抗原.     

结核分枝杆菌H37Rv株重组抗原ESAT-6的表达纯化研究

目的表达纯化结核分枝杆菌(简称:结核杆菌)H37Rv株重组抗原ESAT-6,为结核病血清学诊断价值研究及亚单位疫苗研制提供物质基础。方法重组质粒pET23b-ESAT-6转化E.coli表达菌株BL21(DE3)plysE,IPTG诱导重组抗原ESAT-6表达。经SDS-PAGE电泳和Western印迹鉴定后,优化表达条件用镍离子鳌合亲和层析柱纯化重组抗原。结果成功构建了重组质粒pET23b-ESAT-6并且重组抗原ESAT-6以可溶性形式高效表达,经亲和层析后得到高纯度有免疫原性的重组抗原(纯度>95%)。结论高纯度有免疫原性的重组抗原ESAT-6为新型亚单位疫苗的研发及其结核病血清学诊断价值研究奠定了基础。     

新型隐球菌Cap10蛋白的重组表达及多克隆抗体制备

目的:重组表达新型隐球菌Cap10蛋白并制备特异抗体,为新型隐球菌的检测和诊断提供特异抗原和抗体。方法:选择CAP10基因开放阅读框架(ORF)抗原表位集中、保守性高的片段,经过大肠杆菌偏嗜的密码子优化后进行基因合成,将目的片段克隆至原核表达载体pQE30中构建重组蛋白表达载体pQE30-CAP10并鉴定,IPTG诱导重组蛋白在M15中表达,并用SDS-PAGE和Western blot鉴定;镍柱纯化后,免疫家兔制备抗血清。结果:在大肠杆菌中成功表达Cap10,其纯度大于95%;二免后家兔血清抗体效价达到200万以上。结论:获得了高纯度的Cap10蛋白及高效价的多克隆抗体,为新型隐球菌的检测和致病机制研究奠定了基础。     

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.     

The human drama of genetics: 'hard' and 'soft' media representations of inherited breast cancer

This paper examines media coverage of 'breast cancer genetics', and explores its implications for public understanding. We present a content analysis of coverage in British newspapers and look at a variety of popular forms, including women's magazines, television soap opera and radio drama. Genetic/inherited risk receives a great deal of coverage across a wide range of media formats and outlets. Much of this attention has focused on individuals from 'high risk families' and dilemmas around prophylactic mastectomies. Through examining media coverage, combined with interviews with media personnel and their sources, we show why this story proved so attractive to the media and highlight the different production values which influence coverage. Finally, we introduce preliminary findings from focus group discussions to demonstrate how such 'human interest' framing has engaged audience attention and influenced public understandings. The paper concludes by highlighting the implications for analysing, predicting, and engaging with, media representations of science.     

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.     

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.     

Fatigue and health in a seafaring population

Background Occupational fatigue is relatively common withinthe general population and has been linked to reduced performance,injury and longer term ill-health. Despite growing acknowledgementof this problem in the maritime sector, little research hasbeen conducted into the risk factors, prevalence and consequencesof seafarers' fatigue. Aims To examine the prevalence of fatigue among seafarers, identifypotential risk factors and assess possible links with poor performanceand ill-health. Methods Cross-sectional questionnaire survey of seafarers workingin the offshore oil support, short-sea and deep-sea shippingindustries. A number of tools were used including the fatiguesubscale of the profile of fatigue-related symptoms, the CognitiveFailures Questionnaire, the General Health Questionnaire andthe SF36 General Health scale. Results In all, 1855 questionnaires were completed giving anoverall response rate of 20%. Fatigue symptoms were associatedwith a range of occupational and environmental factors, manyunique to seafaring. Reporting a greater number of risk factorswas associated with greater fatigue [e.g. OR = 2.53 (1.90–3.35)for those with three or four risk factors and OR = 9.54 (6.95–13.09)for those with five or more risk factors]. There was also astrong link between fatigue and poorer cognitive and healthoutcomes, with fatigue the most important of a number of riskfactors, accounting for 10–14% of the variance. Conclusions Seafarers' fatigue could impact on safety withinthe industry and may be linked to longer term individual ill-health.It can only be addressed by considering how multiple factorscombine to contribute to fatigue.     

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.