用杆状病毒系统表达戊型肝炎病毒全长结构基因的研究

目的：获取含有戊型肝炎病毒（HEV）全长结构基因的重组杆状病毒，表达戊型肝炎病毒结构蛋白。方法：将HEV全长结构基因（5147－7126nt）插入杆状病毒表达载体pAcUW51，与线性化杆状病毒DNA（Baculo Gold DNA）共转染Sf9昆虫细胞，挑病毒斑进一步感染Sf9细胞，用SDS－PAGE，Western Blot及免疫荧光检测戊型肝炎病毒结构蛋白的表达及活性。结果：SDS－PAGE分析表明HEV结构基因在杆状病毒系统中高效表达，有相对分子质量约为73000和63000两种形式；Western Blot及免疫荧光检测证明表达产物可与HEV阳性血清特异反应，说明具有HEV特异抗原性。结论：应用杆状病毒表达系统成功表达了HEV结构蛋白。     

酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定

目的 对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定。方法 以原核细胞表达的重组HEV ORF2抗原作为对照，应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性；并测定不同保存时间对抗原稳定性的影响。同时，比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性。结果 应用酵母抗原可检测到HEV抗体的最低包被量为12．5ng／ml，可检测到HEV IgM、IgG抗体的血清最大稀释度皆为l：51200重组蛋白与其他类型肝炎患者血清无交叉反应，37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性。各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性，其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍。结论 应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位，具有良好抗原性、特异性和稳定性。提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原。     

重组杆状病毒表达HEV ORF2抗原片段

目的：用重组杆状病毒表达戊型肝炎病毒（hepatitis E virus,HEV)ORF2基因片段,并对其免疫学特性进行研究。方法：与中间转染载体pVL1393相连构建重组质粒,在Lipofectin介导下将其与杆状病毒线性DNA（BaculoGold^TM)共转染Sf9昆虫细胞得到重组病毒,用免疫荧光,Western blot等方法对表达产物进行免疫学特性初步研究,并进行动物免疫试验。结果：含ORF2结构基因片段的重组杆状病毒在Sf9昆虫细胞中获得了高效表达,经免疫荧光,Western blot,动物免疫试验证实该重组蛋白为HEV特异性蛋白,可刺激机体产生相应抗体。结论：重组杆状病毒能有效表达HEV抗原基因,所表达的ORF2重组蛋白具有HEV抗体识别的抗原表位,具有较好的抗原性和免疫原性。     

耐热可溶性戊型肝炎病毒基因工程抗原的表达

目的 利用硫氧还蛋白融合表达系统表达戊型肝炎病毒（Hepatitis E virus,HEV)结构基因片段，获得耐热，可溶、具有生物活性的HEV基因工程抗原。方法 将HEVORF26964-7126nt基因片段插入硫氧还蛋白融合表达载体pThioHisA，转化大肠埃希菌BL21，经IPTG诱导，表达融合蛋白。裂解细菌后，离心，取上清置于80℃处理10min，再次离心，取上清用ELISA方法测定表达产物的生物活性。结果 SDS-PAGE分析表明，HEV结构基因获得高效表达，相对分子质量约为20000，耐热，水溶性好，ELISA证实具有HEV特异抗原性。结论 应用硫氧还蛋白融合表达系统成功表达了耐热，可溶，具有生物活性的HEV基因工程抗原。     

戊型肝炎病毒Ⅳ型的ORF3及ORF2蛋白的分片段表达、纯化及抗原性分析

目的：对戊型肝炎病毒（HEV）Ⅳ型ORF3的全长基因片段和4个覆盖全长ORF2的互相重叠的基因片段进行了表达，对表达产物进行了纯化及抗原性分析。方法：将O3、FB5、E4、F2－2和E5基因片段分别连接到融合性表达质粒pThioHis，用IPTG进行诱导表达，并用反向、分子筛、离子交换和亲和层析方法进行纯化，用纯化的蛋白分别制备检测抗HEV IgG的诊断试剂，用该试剂检测急性散发性的戊肝病人和非戊肝病人血清。结果：位于ORF2－N端的FB5蛋白在急性期的戊肝病人血清中具有最强的反应性；而且仍有部分被HEV I型或／和Ⅱ型的诊断试剂排除的急性非戊肝病人血清对HEV Ⅳ型的多肽吴阳性反应。结论：为早期诊断戊肝病毒的感染，其诊断试剂应含有HEVⅣ型的ORF2N－端的蛋白片段。     

原核表达戊型肝炎病毒基因重组结构蛋白免疫保护恒河猴的初步实验研究

目的 观察基因重组戊型肝炎病毒（HEV）结构蛋白对恒河猴戊型肝炎野病毒攻击的保护作用。方法 用HEV重组结构蛋白免疫恒河猴，猴血清中抗-HEV升高后，与空白对照组一同用HEV野病毒攻击，采血观察野病毒攻击前后ALT和HEV抗体等的动态变化。结果 野病毒攻击后第3周，空白对照组5只猴ALT均出现明显异常，而免疫接种组5只猴ALT均正常，未观察到明显的肝脏炎症表现，结论 HEV重组结构蛋白免疫恒河猴之后，可以有效地保护HEV野病毒的攻击，该HEV重组结构蛋白可作为戊型肝炎基因工程疫苗的候选蛋白。     

一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性

目的以非包涵体形式表达含戊型肝炎病毒(HEV)中和抗原表位的新型HEV重组蛋白,并对其进行鉴定和分析。方法将HEV开放阅读框架2(ORF2)编码452~617位氨基酸的基因片段连接到载体pET28a( ),转化大肠杆菌,获取表达克隆。以Ni-NTA层析柱纯化表达的蛋白,并用SDS-PAGE、Westernblot和直接电镜负染等方法分析鉴定,最后免疫小鼠检测特异性抗体的产生水平。结果表达的重组蛋白天然可溶,相对分子质量(Mr)约为22000,并可形成直径为20nm左右的病毒样颗粒,能与戊型肝炎患者血清发生Westernblot阳性反应,免疫小鼠后可诱导产生高滴度的特异性抗体。体外中和试验显示产生的抗体具有中和HEV的活性。结论原核表达的、含HEV中和抗原表位的、长度仅166个氨基酸的HEVORF2近3′端编码蛋白能够形成病毒样颗粒,而且该新型病毒样颗粒具有良好的抗原性和免疫原性。     

4型戊型肝炎病毒重组衣壳研究及感染模型初探

目的 表达4型戊型肝炎病毒(HEV)开放式阅读框ORF2重组衣壳蛋白并初步建立用于4型HEV早期感染过程研究的细胞模型.方法 从猪胆汁中调取4型HEV ORF2的肽段(aa368-606)基因,并且利用大肠杆菌系统得到重组D66蛋白.结果 重组D66蛋白在水相中以二聚体为基本单位自组装成分子半径约为13 nm的颗粒.该颗粒与HEV感染的急性期、恢复期患者的血清均有较强的反应性.将D66颗粒与细胞共孵育,发现该颗粒可以与肝实质细胞系HepG2的细胞结合并进入细胞内,这种结合可被恢复期及急性期HEV患者血清所阻断.结论 这些结果表明重组D66蛋白颗粒,可以一定程度上模拟4型HEV的表面位点,并且可以特异性吸附并进入细胞,为进一步研究4型HEV感染的分子机制提供基础和平台.     

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

表位依赖型急性期戊型肝炎诊断抗原的制备与抗原性分析

目的 原核表达制备表位依赖型戊型肝炎诊断抗原,并对其抗原性及其在急性戊肝诊断的性能进行评价.方法 以包含密码子优化的GST、HEV优势表位的H15质粒为模板,PCR获取GST-HEV优势表位片段,将其插入表达质粒pET 43.1a中,在大肠杆菌中进行表达;利用亲和层析和阴离子交换层析纯化目的蛋白;利用Western Blotting技术对目的蛋白抗原性进行鉴定;建立HEV-IgM抗体检测ELISA方法,初步评价其对阴、阳血清样本的鉴别能力.结果 获取高度均质的表位依赖型急性戊肝诊断抗原;WB实验表明目的蛋白不但可以被anti-GST单克隆抗体识别,还可以被戊肝急性期血清相应抗体所识别;分别对60份戊肝急性期血清、阴性血清进行检测,发现其与临床和实验室诊断结果一致性较高.结论 原核表达与层析纯化可以获取抗原性优异的表位依赖型急性戊肝诊断抗原,偶联HRP后可应用于HEV-IgM ELISA血清学检测.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.