戊型肝炎病毒基因Ⅲ型病毒样颗粒的表达与抗原性分析

目的观察戊型肝炎病毒（HEV）基因Ⅲ型（GⅢ）ORF2全长片段在昆虫细胞中的表达和病毒样颗粒（VLP）的装配情况,对纯化的VLP进行抗原性分析。方法应用含Ⅲ型HEV全长ORF2的pVL 1393-GⅢ ORF2质粒,体外与杆状病毒同源重组后,转染昆虫细胞Sf9细胞与在rrn5细胞中表达,用聚丙烯酰胺凝胶电泳和western blot检测表达产物,超速离心法纯化,检测是否形成病毒样颗粒,用免疫电镜和ELISA分析病毒样颗粒的抗原性。结果Ⅲ型ORF2全长基因在Sf9和Tn5昆虫细胞中表达可获得72、64、58、56、53 kD的蛋白质,在Tn5细胞中有56、53kD的蛋白质分泌到培养液。纯化后可获得6．7mg／mL的蛋白质,分子量为53kD,电镜观察发现了直径约为25nm的VLP,浮密度为1．291g／cm^3。检测基因Ⅳ型戊肝感染病人血清,GⅢ VLP与GⅠVLP具有同样的灵敏度,均能检测到滴度分别为1：204800和1：102400的IgM和IgG抗体。同时GⅢ VLP在戊肝病人血清和IgM阳性猪血清孵育发生凝集反应。结论戊型肝炎病毒基因Ⅲ型ORF2全长基因片段可以在昆虫细胞表达系统高效表达。形成的VLP具有HEV病毒颗粒类似的抗原活性,可用于制备HEV抗体诊断试剂和开发预防用疫苗。     

3种戊型肝炎病毒抗原检测血清抗—HEV IgG的比例研究

目的：用3种抗原检测国家戊型肝炎参比血清和急性戊型肝炎病人血清抗－HEV IgG。方法：人工合成戊型肝炎病毒（HEV）ORF2和ORF3多肽抗原（BMU抗原）检测参比血清的灵敏度（90．0％），特异度（100．0％）和符合率（97．5％）均高于杆状病毒表达的HEV重组蛋白空粒子抗原（JP抗原）（分别为70．0％，93．3％和87．5％）及大肠杆菌表达的重组蛋白抗原（HKU抗原）（分别为70．0％，90．0％和85．0％），检测84例急性戊型肝炎病人血清抗－HEV IgG，用BMU抗在检测抗－HEV IgG的阳性率为1000％（84／84），高于JP抗原（83／84，98．8％）和HKU抗原（71．84，84．5％）。结果：不同HEV抗原检测国家参比血清和急性戊型肝炎病人血清抗－HEV IgG的灵敏度和特异性不同，其差异主要与编码该3种抗原的HEV基因片段不同有关，ORF3抗原在检测急性HE病人中具有重要意义。联合应用ORF2和ORF3抗原可提高抗－HEVIgG检出率，结论：抗－HEV IgG ELISA诊断试剂至少应包括ORF2和ORF3 2种抗原。     

卵巢癌相关基因HE4在杆状病毒表达系统的表达与鉴定

目的:使卵巢癌相关基因HE4在杆状病毒表达系统中获得表达。方法:将卵巢癌相关基因HE4克隆到pMagic-his-HE4中,通过转化E.coliDH10B筛选克隆,阳性克隆通过接合转移的方式与杆状病毒框架质粒体外重组,构建Bac-phes-HE4杆状病毒表达载体,后者经Lipofectamine2000介导转染Sf9细胞,获取重组病毒,扩增病毒并感染Sf9细胞进行表达,SDS-PAGE、Western blot分析鉴定表达蛋白。结果:经HE4抗体、6×His单抗鉴定,成功通过杆状病毒表达系统获得重组卵巢癌相关基因HE4。结论:卵巢癌相关基因HE4通过接合转移的方法在杆状病毒表达系统中成功表达。     

基于EV71交叉保护抗原P1的病毒样颗粒的构建

目的构建基于EV71交叉保护抗原P1(CP1)的病毒样颗粒。方法利用昆虫杆状病毒表达系统构建CP1和3CD的重组杆状病毒,两者共同感染Sf9细胞,组装形成CP1的EV71病毒样颗粒,并通过Western blot和电镜进行鉴定。结果 CP1、3CD重组杆状病毒共感染表达产物的Western blot结果显示,CP1蛋白经3CD蛋白酶酶切形成了大小约为39 k Da、34 k Da、26 k Da的VP1、VP0、VP3蛋白条带,电镜下可观察到形态规则的大小约为30 nm的CP1病毒样颗粒结构。结论 CP1病毒样颗粒构建成功,为下一步探讨CP1病毒样颗粒是否对多种亚型的EV71病毒具有交叉保护作用奠定基础。     

中国部分地区猪胆汁戊型肝炎病毒基因型和中和抗原位点变异研究

目的调查猪胆汁戊型肝炎病毒(HEV)携带率、基因型和中和抗原位点变异。方法在中国4个地区屠宰场采集成年商品猪的胆汁标本,采用real-time RT-PCR检测HEV RNA;采用巢式RT-PCR扩增HEV ORF2区部分基因片段,进行基因分型和中和抗原位点氨基酸序列分析。结果猪胆汁标本HEV RNA总阳性率为8.6%(46/532),4个地区阳性率在3.0%-12.5%之间。19株HEV基因分型成功,均为基因4型,其中11株HEV基因亚型分型成功,均为4a亚型。在该11株HEV的21个中和抗原位点中,2株HEV在ORF2区分别发生第487位氨基酸突变(Ser487Tyr)和第564位氨基酸突变(Thr564Pro)。结论中国部分地区猪胆汁HEV携带率较高,为中国人群中优势流行的基因4型,且部分中和抗原位点发生突变;应加强猪群HEV的持续监测。     

HPV16 L2肽段偶联AP205 VLP的原核表达及免疫原性分析

目的:将编码HPV16衣壳蛋白L2的65～71、112～120免疫优势表位连接到RNA噬菌体衣壳蛋白AP205氮端,组装形成病毒样颗粒,通过在大肠杆菌中实现表达及纯化,对其免疫原性进行研究.方法:合成编码AP205衣壳蛋白基因和HPV16 L2的65 ～71、112～120位氨基酸表位的基因序列,PCR连接并克隆至pET30a(+)原核表达载体,构建重组表达质粒pET30-AP205 -HPV16△L2,转化大肠杆菌BL21( DH3)感受态细胞,IPTG诱导表达.表达蛋白经凝胶层析纯化及SDS-PAGE、Western blot等理化性质检测,免疫接种ICR小鼠,通过间接ELISA法检测其免疫原性.结果:成功构建重组表达质粒,重组蛋白在大肠杆菌中以可溶性表达,透射电镜观察可见典型病毒样颗粒,该VLP在动物实验中表现出较好的免疫原性.结论:成功将HPV162表位偶联AP205以形成VLP,在大肠杆菌中实现可溶性表达.     

脊髓灰质炎病毒缺陷型重组载体系统

第一代脊髓灰质炎病毒重组载体系统由于受到构象的限制而不能有效地表达外源抗原，缺陷性重组载体系统则可利用外源基因置换脊髓灰质炎病毒结构蛋白Ｐ１编码区而插入较大片的外源基因，因此具有较广阔的前景，其基本原理是以我不基因置换脊髓灰质炎病毒的Ｐ１基因，同时以另一途径提供Ｐ１结构蛋白，形成缺陷性病毒颗粒，此颗粒可以感染新的细胞，并表达其重组的外源抗原，但不产生感染性病毒颗粒，此系统为利用脊髓灰质炎病毒表达大     

戊型肝炎病毒结构蛋白基因的真核表达

目的使用酵母表达系统表达戊型肝炎病毒结构蛋白基因。方法构建含戊型肝炎病毒(HEV)结构蛋白嵌合基因HEVORF23(由HEVORF2C端800bp片段与ORF3全基因组成)的重组表达质粒pPICZαA_HEVORF23,并通过电转移的方法将该基因整合入甲醇营养型酵母的基因组DNA中,利用甲醇进行诱导表达,表达产物经纯化后,进行Western印迹鉴定。结果pH6.0环境下诱导培养72h后,在细胞沉淀中获得一个分子量大约为69kDa的可溶性目的蛋白,纯化蛋白经Western印迹检测后发现能与戊型肝炎患者血清发生较强的特异性免疫反应。结论采用酵母表达系统诱导表达可以获得抗原性较强的HEV重组表达抗原,为进一步开发研制有效的诊断抗原和疫苗奠定了基础。     

GⅡ-4型诺如病毒ORF2基因克隆及序列分析

目的 获得中国流行株GⅡ-4型诺如病毒(NoV)ORF2基因序列,为进一步研究诊断、疫苗提供基础依据.方法 从GⅡ-4型NoV粪便中提取病毒RNA,经逆转录后PCR并克隆到simple PMD18-T载体获得完整开放读码柜架(ORF2)阅读框序列.结果 获得了正确序列的基因Ⅱ-4,亚型(genogroup Ⅱ-4,GⅡ-4)NOV ORF2基因序列,经软件分析,3株NOV ORF2序列编码的氨基酸大小约为54KD,与VA387(GⅡ-4)氨基酸同源性为92%～93%.ORF2编码的氨基酸序列存在3个相对保守序列.结论 获得GⅡ-4型NoV ORF2基因序列,为进一步表达病毒衣壳蛋白及Nov感染的诊断试剂及防治疫苗药物研制提供基础依据.     

对我国戊型肝炎防治对策的若干意见

戊型肝炎系由戊型肝炎病毒（HEV）所致，1983年首次用免疫电镜观察到HEV颗粒，1990年获得了HEVc DNA克隆。业已证明这种病毒属于嵌杯病毒属，颗粒直径为27-38nm的RNA病毒，由约7500个核苷酸组成，含有3个开放阅读框架（ORF），5‘端的ORF1为非结构基因，3‘端的ORF2为结构基因。     

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.     

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.