幽门螺杆菌Mr 26 000外膜蛋白编码基因在DH5α中的表达

目的构建含幽门螺杆菌(H.pylori)Mr 26 000外膜蛋白编码基因的重组载体并在E. coli DH5α(PREP4)中表达.方法用PCR从H.pylori染色体中,扩增Mr 26 000外膜蛋白编码基因片段.将目的基因与pQE30同时经BamH Ⅰ、Hind Ⅲ双酶切、纯化、连接后,转化含有目的基因的重组载体.以含目的基因片段的重组载体转化大肠杆菌DH5α并表达.表达产物经纯化后,用ELISA法检测其抗原性.结果插入的基因片段为H.pylori Mr 26 000的外膜蛋白编码基因与Tomb等报道相比较,有1.1%的碱基发生变异, 1.5 1%的氨基酸残基改变;目的基因经表达其蛋白Mr为26×103 ,可溶性表达产物占全菌总蛋白的20%以上;目的蛋白经Ni2+-NTA琼脂糖树脂纯化后,其纯度达95%以上,且该目的蛋白可被H.pylori阳性患者的血清所识别,具有良好的抗原性.结论成功地克隆并表达了H.pylo ri Mr 26 000的外膜蛋白编码基因,为H.pylori蛋白质疫苗的研制和快速诊断试剂盒的研究打下了基础.     

人幽门螺杆菌18 000外膜蛋白与HspA双价疫苗的构建和表达

目的 :构建含人幽门螺杆菌 (Hp)热休克蛋白A(HspA)和Mr为 180 0 0的外膜蛋白编码基因的重组载体 ,进行核苷酸序列分析 ,并在E .coliBL2 1中表达。方法 :用PCR方法从HpDNA染色体中 ,扩增HspA编码基因片段。将目的基因HspA与载体 pET32a( )分别经kpnⅠ和BamHI双酶切后 ,进行连接、测序。同时将重组载体 pET32a( ) /HspA和pET32a( ) /Omp18,分别经HindIII和BamHI双酶切 ,通过凝胶电泳回收pET32a( ) /HspA和Mr180 0 0OMPDNA片段 ,经T4连接酶将HspA和Mr180 0 0OMP编码基因通过酶切粘端进行连接 ,而后转化并筛选含有两种目的基因的重组载体 ,并在大肠杆菌BL2 1(DE30 )中表达。表达产物经Ni NTA琼脂糖树脂纯化后 ,以Westernblot分析其抗原性。结果 :经酶切、测序表明 ,插入的基因片段为HpHspA和Mr 为 180 0 0OMP编码基因 ,由 891个碱基组成 ,与GenBank中登录的序列相比较 ,有 1.15 %的碱基发生变异 ,1.2 6 %的氨基酸残基改变。经SDS PAGE分析发现 ,融合基因表达的蛋白Mr 为 5 1× 10 3 ,其中 pET32a( )表达的蛋白Mr 约为 2 0×10 3 ,可溶性表达产物占菌体总蛋白的 18.96 %。重组蛋白经Ni NTA琼脂糖树脂纯化后 ,其纯度达 95 %以上。用Westernblot分析显示 ,该重组蛋白可被Hp阳性患者的血清及抗Mr为 180 0 0OMP单     

幽门螺杆菌oipA基因片段的克隆表达及抗原性分析

目的:分片段克隆表达幽门螺杆菌oipA基因,确定抗原性最强的重组蛋白肽段.方法:从Hp国际标准株NCTC11637中获取oipA全长基因,克隆至pGEM-T载体并鉴定正确,以重组pGEM-T/oipA为模板PCR扩增6个不同大小的oipA基因片段,与表达载体pET-42a连接,转化大肠杆菌BL-21,重组子经PCR、酶切、测序鉴定.IPTG诱导重组蛋白肽段的表达,经Western Breeze 化学发光法鉴定融合蛋白;优化诱导时间和IPTG浓度,诱导重组蛋白大量表达;Ni-NTA His*Bind树脂亲和纯化表达产物;羊抗Hp多克隆抗体,Western blot法检测纯化蛋白的抗原性,间接ELISA法筛选抗原性最强的重组肽段.结果:6条oipA基因片段在大肠杆菌中都得到了表达,表达的蛋白肽段均可被抗Hp多克隆抗体识别,具有抗原性,其中反应性最强的是最小的蛋白肽段.结论:oipA基因片段可在原核系统中表达,所获取的最小的肽段抗原性最强,可望制备检测血清相应抗体的试剂盒.     

人MBL糖识别域在大肠杆菌中的表达、纯化及鉴定

目的原核表达重组人甘露聚糖结合凝集素（MBL）糖识别域（CRD）。方法采用PCR技术,从含汉族人MBL全长编码区cDNA的重组质粒pGEM-mbl中扩增出CRD包括颈区的基因片段,将其插入原核表达载体pGEX-4T1中,经PCR、限制性酶切和测序确证后,以IPTG诱导其在大肠杆菌中表达。包涵体经变性、复性后以GSTtrap亲和层析柱纯化,融合蛋白经凝血酶切割后回收目的蛋白。分别以Western blot和ELISA分析表达产物的免疫学和糖结合活性。结果PCR扩增得到长约450bp的目的基因片段,插入pGEX-4T1载体所获重组表达载体pGEX4T1-CRD的酶切图谱和序列与预期的一致。重组子经诱导表达,表达产物主要以包涵体形式存在。以GSTtrap亲和层析柱纯化获得约Mr43000的GST-CRD融合蛋白,经凝血酶切割后得到约Mr17000的CRD蛋白。纯化的GST-CRD蛋白能与抗人CRD单克隆抗体特异性结合并具糖结合活性。结论获得了具生物学活性的人MBL CRD蛋白,为进一步探索MBL分子CRD的效应功能提供了实验材料。     

人幽门螺杆菌外膜蛋白18000 DNA疫苗的构建及表达

目的：构建含人幽门螺杆菌(Helicobacter pylori，H-pylori)18000外膜蛋白编码基因的真核重组载体，并在COS-7细胞中表达，为核酸疫苗的开发奠定基础。方法：从原核表达质粒pET32a( )／528中，酶切H-pylori 18000外膜蛋白编码基因片段，将目的基因与同样进行酶切、纯化的载体pcDNA3．1进行连接，而后转化并筛选含有目的基因的重组载体pcDNA3．1／528，并在COS-7细胞中表达，以RT-PCR，Western法检测其表达产物。结果：经单、双酶切证实插入的基因片段为H-pylori 18000外膜蛋白编码基因；采用RT-PCR方法，能够从转染的COS-7细胞中扩增出一条与目的基因大小一致的DNA片段，Western法等检测显示，该重组质粒能够在COS-7细胞中表达目的蛋白，而且具有生物活性。结论：成功地构建了核酸疫苗pcDNA3．1／528，并在COS-7细胞中表达，为H-pylori核酸疫苗的研制奠定了良好的基础。     

人HMGB1基因的原核表达、产物纯化及活性鉴定

目的　构建人高迁移率族蛋白 1(HMGB1)编码基因的表达载体 ,获得纯化的重组蛋白 ,研究其生物学功能。方法　通过RT PCR方法扩增出人HMGB1的编码基因 ,克隆于载体pGEM Teasy,再亚克隆于表达载体pGEX4T 2 ,经IPTG诱导可表达相对分子质量 (Mr)约 5 6× 10 3的融合蛋白GST HMGB1。经用GSTrapFF蛋白纯化柱和凝血酶行柱上酶切 ,得到纯化的重组蛋白HMGB1,用培养的人单核细胞系THP1检测蛋白活性。结果　构建了融合蛋白GST HMGB1的重组表达质粒 ,获得了Mr 约为 30× 10 3的纯化蛋白产物。该蛋白能刺激THP1细胞产生TNF α ,并能明显诱导THP1细胞的凋亡。结论　获得了纯化的人HMGB1原核表达产物 ,对研究其在脓毒症中的生物学功能具有重要的意义。     

幽门螺杆菌Catalase基因的克隆、表达及其抗原性鉴定

目的：构建含人幽门螺杆菌（Hpylori，Hp）过氧化氢酶（catalase，KatA）编码基因的重组质粒，测定、分析其核酸序列，并在E.coli中表达，研究其抗原性。方法：应用PCR技术从HpDNA染色体中扩增KatA编码基因片段，将其T-A克隆和测序，并与GenBank公布的其他Hp菌株的基因序列比较，再将目的基因插入至融合表达载体pGEX-4T-1中进行表达，用GST亲和层析对其进行纯化。纯化产物用于对29株小鼠抗Hp-全菌单克隆抗体（mAb）的鉴定及与Hp啊感染患者血清进行Western blot。结果：KatA基因全长为1 515 bp，并在GenBank上登录（No．DQ333889），与GenBank公布的其他Hp菌株的核酸的同源性为96％～97％，表达的KatA融合蛋白的相对分子质量（Mr）为85 000，29株小鼠抗Hp全菌mAb中有4株mAb是针对KatA的，表达产物可被Hp感染患者的血清特异性识别。结论：重组KatA具有较好的抗原性，为坳检测试剂和疫苗的研究奠定了基础。     

Balb/c小鼠MBL-C糖识别域的原核表达及其多克隆抗体的制备

目的 原核表达Balb／c小鼠肝型甘露聚糖结合凝集素（mMBL-C）糖识别域（CRD）并制备其多克隆抗体。方法应用PCR从含Baib／c小鼠MBL-C全长编码区基因的质粒pmMBL-C中扩增目的基因片段,构建重组表达载体,在大肠杆菌中表达。分离纯化表达产物并免疫动物,制备mMBL-C CRD的抗血清,采用ELISA和Western blot鉴定其效价和特异性。结果从pmMBL-C中扩增到约450bp的DNA片段,构建重组载体pET-CKS-mMBL-C-CRD和pET32a-mMBL-C-CRD,经酶切和测序鉴定,证实重组表达载体构建成功。将其导入大肠杆菌BL21（DE3）中表达,表达产物主要以包涵体的形式存在,相对分子质量（Mr）分别为44000和34000。利用pET-CKS载体表达产物免疫家兔,分离免疫血清后,使用pET32a（＋）载体表达产物进行ELISA及Westem blotting,显示多克隆抗体能够与mMBL-C CRD蛋白特异结合。结论获得了重组mMBL-CCRD蛋白及其多克隆抗体,为mMBL-C分子以及CRD的研究奠定了一定的基础。     

EB病毒gp85 N端片段的原核表达与初步鉴定

目的：构建EB病毒基因的原核表达载体，并在大肠杆菌中进行表达。分析非糖基化的EB病毒包膜糖蛋白gp85N端截短片段的抗原性。方法：采用基因工程技术，以B95—8细胞（美洲绒猴外周血B淋巴细胞经EBV转化后的细胞系）培养上清为模板，用PCR扩增EB病毒的BXLF2基因N端片段。PCR产物经Hind Ⅲ和XhoⅠ双酶切，插入原核表达载体pGEX-5T中，构建pGEX5T-85N重组表达质粒，并在大肠杆菌BL21中诱导表达gp85N蛋白。纯化表达的蛋白用Westernblot鉴定，并免疫BALB／c小鼠。结果：序列分析表明，插入片段的序列与GenBank登录的参考序列完全一致。重组表达的蛋白的相对分子质量（Mr）约为45000，同预期的大小相符。以纯化的可溶性重组蛋白免疫BALB／c小鼠，经ELISA检测获得了高效价的抗血清，且抗gp85单克隆抗体（mAb）可识别所表达的gp85N抗原。Westernblot显示，该抗原可与小鼠免疫血清起特异性反应。结论：表达并纯化的EB病毒截短的gp85N重组蛋白具有良好的抗原性，为下一步分析所产生抗体的生物学特性提供了条件。     

重组人N端缺失MBL的原核表达及其活性研究

目的 原核表达具生物学活性的重组人N端缺失甘露聚糖结合凝集素(rhMBLΔN)蛋白.方法 应用PCR技术,从含中国人野生型MBL cDNA的质粒pGEM-MBL中扩增rhMBLΔN基因片段,插入pET43.1a载体后,转化E.coli BL21(DE3)感受态菌诱导表达rhMBLΔN蛋白.应用Ni2 -NTA琼脂糖柱纯化目的 蛋白,以SDS-PAGE、Western blot和ELISA进行鉴定.结果 从pGEM-MBL中扩增到长约620bp的DNA片段,构建的重组表达载体经Bam HI和Eco RI酶切后出现约7270bp和620bp的片段,测序结果与预期的完全一致.表达的重组蛋白纯化后,经SDS-PAGE鉴定为Mr97000的蛋白.ELISA证实,纯化蛋白能与抗重组人MBL抗体结合,具备配体结合活性并能与人甘露聚糖结合凝集素相关丝氨酸蛋白酶1、2的N端片段结合.结论 获得了可表达rhMBLΔN的菌株和具活性的rhMBΔN蛋白,为进一步研究提供了条件.     

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.     

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.     

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.     

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.