β-hCG蛋白在大肠杆菌系统的表达、纯化及活性鉴定

目的 获得β-人绒毛膜促性腺激素(β-hCG)融合蛋白β-hCG/GST并验证其抗原活性.方法 利用PCR法扩增β-hCG全长基因,将其克隆至原核表达载体pET-42a中,构建重组质粒pET-42a-β-hCG,将该重组质粒转化至大肠杆菌BL21(DE3)中IPTG诱导表达,经SDS-PAGE分析,亲和层析法纯化融合蛋白,目的蛋白用Western blot法鉴定,ELISA检测纯化得到的融合蛋白的抗原活性.结果 测序结果证实成功扩增出目的基因β-hCG;酶切和测序结果证实pET-42a-β-hCG原核表达载体构建成功;转化后可以成功诱导并纯化出大小与预期一致的蛋白;Western blot法及ELISA检测证实纯化后的β-hCG/GST融合蛋白具有良好的免疫原性.结论 成功获得β-hCG/GST融合蛋白,该蛋白具有良好的免疫原性,为以β-hCG为靶位的抗肿瘤主动免疫治疗研究奠定了基础.     

肾癌相关抗原G250的原核表达、纯化及抗原活性检测

目的 构建原核表达质粒pET-42a-hG250,表达并纯化肾癌相关抗原G250融合蛋白,并检测G250融合蛋白的抗原活性.方法 利用PCR从pGEM-T-G250质粒中扩增G250基因片段(112～1242 bp),测序正确后将其克隆至原核表达载体pET-42a中构建重组载体pET-42a-hG250.将其转化至大肠杆菌BL21(DE3)中,通过IPTG诱导G250蛋白的原核表达,随后将表达的融合蛋白进行纯化.经SDS-PAGE分析后,Western blot法检测纯化的蛋白,将纯化蛋白进一步包板后用ELISA对其抗原活性进行评价.结果 酶切和测序结果证实pET-42a-hG250原核表达载体构建成功;转化后可以成功诱导并纯化出大小与预期一致的G250融合蛋白;Western b1ot法和ELISA检测证实纯化的蛋白能与特异性的抗体发生反应,显示纯化后的G250融合蛋白具有良好的免疫原性.结论 成功构建了肾癌相关抗原G250基因的原核表达载体,纯化获得了G250融合蛋白,该蛋白具有良好的抗原活性.     

乳腺癌特异性转导小肽融合表达载体的构建以及目的蛋白的表达

目的构建乳腺癌特异性转导小肽PI-增强型绿色荧光蛋白融合蛋白的原核表达载体,并进行目的蛋白的表达、分离和纯化。方法合成PI的DNA序列,将其定向插入到p EGFPN2中,经双酶切获取PI-EGFP的核苷酸序列,再将其克隆入原核表达质粒pET-28a(+),构建出pET-28a(+)-PI-EGFP的原核表达载体;重组质粒转化BL21(DE3)pLysS感受态细胞,经IPTG诱导表达,利用His-tag对蛋白进行分离纯化,SDS-PAGE电泳和蛋白质印迹免疫分析(Western blot)鉴定纯化蛋白质。结果成功构建了pET-28a(+)-PI-EGFP的原核表达载体;其经诱导后,在大肠杆菌中高效表达,目的蛋白以可溶性形式存在;SDS-PAGE分析显示,在相对分子量约33kD的位置出现目的蛋白条带,与理论值相符;经His TALONTM Cartridge亲和层析纯化获得了高纯度的重组的PI-EGFP融合蛋白,Western blot鉴定结果显示获得了目的蛋白。结论成功构建出乳腺癌特异性转导小肽PI-增强型绿色荧光蛋白融合蛋白原核表达载体,并能够表达出PI-EGFP的融合蛋白,为进一步研究小肽的乳腺癌特异性转导功能奠定基础。     

人源N-末端脂多糖结合蛋白(NH-LBP)的原核表达及抗原性分析

目的 在大肠杆菌表达系统中大量表达、纯化N-末端脂多糖结合蛋白(NH-LBP)并检测其抗原性.方法 构建pET28a-NHLBP质粒,转入大肠杆菌BL21感受态细胞,IPTG诱导表达成功后扩大培养、提取包涵体、亲和层析分离纯化,并将纯化得到的NH-LBP融合蛋白经Western blot鉴定.结果 成功构建pET28a-NH-LBP表达质粒并转入BL21细菌进行诱导表达,目的 蛋白以包涵体形式表达为主;经扩大培养、分离纯化产物大小与NH-LBP融合蛋白理论计算值一致,经Western blot检测具有B细胞表位.结论原核表达人源N-末端脂多糖结合蛋白(NH-LBP)是制备抗原的一种可行方便的方法,为筛选针对NHLBP的人源性小分子抗体提供了材料.     

PBDC1蛋白的表达及其多克隆抗体的制备简

 目的 原核表达、纯化PBDC1蛋白,制备PBDC1多克隆抗体。方法 将PBDC1基因克隆到pET-28a(+)质粒中,构建成重组质粒pET-28a(+)-PBDC1。将此重组质粒转化大肠杆菌BL21(DE3)后,IPTG诱导其在宿主菌中大量表达,并进行SDS-PAGE检测。经镍离子亲和柱纯化得到PBDC1融合蛋白,并以此免疫新西兰大白兔制备多克隆抗体。结果 经质谱鉴定,获得了高纯度的PBDC1蛋白。经Western blot验证,获得了抗PBDC1蛋白的多克隆抗体。结论 成功获得抗PBDC1蛋白的多克隆抗体,为研究PBDC1在红系分化中的功能提供了有利工具。     

人磷脂转移蛋白的原核表达及其抗体的制备与鉴定

目的利用大肠杆菌表达人磷脂转移蛋白(PLTP),制备兔抗人PLTP的多克隆抗血清。方法采用RT-PCR技术,将人PLTP蛋白编码序列克隆入pET-30b(+)原核表达载体,转化大肠杆菌,IPTG诱导表达,切胶纯化蛋白后免疫家兔,制备PLTP抗血清。用间接ELISA法、Western blot、细胞免疫荧光检测对抗血清效价及特异性进行鉴定。结果SDS-PAGE分析表明,PLTP基因在大肠杆菌BL21(DE3)菌株的包涵体中高效表达,最佳诱导表达时间为3h;将纯化的蛋白免疫家兔,ELISA法测定的抗血清效价为1∶256000;Western blot及细胞免疫荧光检测显示,抗血清可以与PLTP原核及真核表达蛋白特异结合。结论成功地将PLTP进行了原核表达,制备了高效价、高特异性的兔抗人PLTP抗血清,为PLTP的临床检测及其结构与功能的进一步研究提供了有力工具。     

大肠杆菌丝状热敏蛋白Z相互作用蛋白A(Ec-ZipA)原核表达、纯化与大鼠多克隆抗体的制备

目的制备大肠杆菌丝状热敏蛋白Z相互作用蛋白A(Ec-ZipA)第185至328位氨基酸功能蛋白及其特异性大鼠多克隆抗体。方法以基因合成法合成Ec-ZipA第185至328位氨基酸功能蛋白的基因序列,利用DNA重组技术与p ET-30a(+)载体连接构建重组质粒。将重组质粒转化到E. coli BL21(DE3)中进行Ec-Zip A原核表达与表达优化。采用亲和层析方法分离纯化Ec-Zip A后,以荧光偏振(FP)实验进行生物学活性鉴定。以重组Ec-Zip A为抗原免疫大鼠制备多克隆抗体,采用ELISA和Western blot法检测抗体滴度和抗原特异性。结果 SDS-PAGE分析和Western blot实验证实大肠杆菌成功表达Ec-Zip A蛋白。FP实验表明纯化的Ec-Zip A具有良好的生物学活性。ELISA结果表明多克隆抗体具有较高的抗体滴度,效价可达1∶512 000。Western blot实验证实多克隆抗体具有良好的抗原特异性。结论成功进行了Ec-Zip A原核表达、分离纯化并制备了大鼠抗Ec-Zip A多克隆抗体。     

结核分枝杆菌rPstS1-HspX融合蛋白的表达、纯化及其免疫反应性分析

目的 构建结核分枝杆菌rPstS1-hspX (rph)融合基因及其原核表达载体pET-23b(+)-rPstS1-hspX[pET-23b(+)-rph],表达、纯化rPstS1-HspX (rPH)融合蛋白,并分析其免疫反应性.方法 采用基因拼接技术将PstS1和HspX编码基因通过多肽接头(GSGSG)的DNA序列进行连接,构建融合基因rph.将融合基因定向克隆入原核表达载体pET-23b(+),构建重组原核表达质粒pET-23b(+)-rph.将重组质粒转化大肠杆菌E.coli BL21 (DE3) pLysE感受态细胞,IPTG诱导融合蛋白表达.SDS-PAGE和Western印迹法鉴定其表达情况.用镍离子鳌合亲和层析柱纯化融合蛋白,Western印迹法初步评价融合蛋白的免疫反应性.结果 融合基因rph及其原核表达载体pET-23b (+)-rph构建成功.融合蛋白rPH主要以可溶性非包涵体形式表达,相对分子质量为51 000,表达量约占菌体总蛋白的23％.经亲和层析后得到了纯度达92％的融合蛋白.Western印迹证实融合蛋白能与结核病阳性血清发生特异性免疫反应.结论 成功构建了原核表达载体pET-23b(+)-rph,获得了rPH融合蛋白,为rPH融合蛋白在结核病诊断中的应用提供了依据.     

人核糖体蛋白S13的原核表达、纯化及多克隆抗体的制备

目的克隆人核糖体蛋白S13(ribosomal protein S13,RPS13)cDNA全长,构建原核表达质粒,诱导表达、纯化RPS13蛋白并制备其多克隆抗体。方法应用RT-PCR方法从胃癌多药耐药细胞SGC7901/VCR中扩增出RPS13的cDNA全长,利用DNA重组技术构建重组原核表达载体pET-28a( )-RPS13,双酶切及测序鉴定。将重组载体转化入大肠杆菌BL21中,筛选抗性克隆并经DNA测序证实。IPTG诱导融合蛋白的表达,利用镍离子亲和层析柱提纯融合蛋白,经SDS-PAGE和Western blot鉴定。用纯化的融合蛋白His-RPS13免疫BALB/c小鼠,ELISA法测定抗体效价,饱和硫酸铵沉淀法纯化抗体,Western blot检测抗体活性。结果RT-PCR扩增片段与预计目的基因片段大小一致;双酶切鉴定表明,重组表达载体pET-28a( )-RPS13构建成功,DNA测序结果显示所获基因序列与GenBank中收录完全相同。IPTG诱导3h后,经SDS-PAGE检测显示在Mr19000处出现一新生条带,与预计的融合白大小一致;利用抗His标签的抗体进行Western blot,结果证实融合蛋白表达成功。其免疫血清经Western blot证实可特异性的识别蛋白RPS13。结论成功地获得了RPS13的编码基因序列,并获得了纯化的RPS13蛋白质,为制备RPS13的单克隆抗体、进一步研究RPS13在肿瘤中的作用奠定了基础。     

人survivin蛋白的原核表达、纯化及抗原活性鉴定

目的 构建人肿瘤抗原survivin的原核表达载体,优化在大肠杆菌中的表达条件,并对survivin/His融合蛋白进行纯化和抗原活性鉴定.方法 设计针对survivin基因序列的特异引物,通过聚合酶链式反应(PCR)扩增人survivin全长基因序列(538 bp)克隆至原核表达载体pET28a(+),构建重组表达载体pET28 a-survivin,并将该载体转化大肠杆菌BL21(DE3),经IPTG诱导表达survivin/His融合蛋白,并采用Ni亲和层析凝胶纯化重组蛋白.纯化后的重组蛋白经Western blot法、ELISA鉴定其抗原活性.结果 重组表达载体经BamH Ⅰ和HindⅢ鉴定正确;IPTG诱导后经SDS-PAGE分析表明获得了相对分子质量(Mr)24000大小的重组蛋白;纯化后的蛋白纯度达到90％.Western blot法和ELISA检测证实纯化的survivin蛋白能够与特异性抗体发生反应,表明其具有良好的抗原活性.结论 成功构建了原核表达载体pET28 a-survivin,利用大肠杆菌表达系统实现了融合蛋白的可溶性表达并进行纯化,纯化后survivin蛋白经鉴定具备较高的抗原活性.     

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.