独特型TCR Vβ2 DNA疫苗的构建和体外表达检测

目的:构建抗淋巴细胞白血病的独特型TCR Vβ2 DNA疫苗，并检测其转染K562细胞和体外表达情况。 方法:利用RT-PCR扩增表达TCR Vβ2的Molt-4细胞株的独特型TCR Vβ2基因片段，定向克隆于pIREs表达载体中，重组质粒转染至K562细胞中，利用间接免疫荧光法、SDS-PAGE和Western blotting等检测其表达情况。 结果:经PCR检测证实成功构建pIREs-TCR Vβ2重组子，转染至K562细胞后，利用Vβ2单抗可检测到K562细胞表面表达TCR Vβ2，SDS-PAGE分离检测为15 kD的蛋白质，并经Vβ2特异抗体Western blotting鉴定为TCR Vβ2蛋白。 结论:成功构建pIREs- TCR Vβ2 DNA疫苗，并可以在K562细胞株中表达特异蛋白。     

独特型TCR Vα1-pIRES-TCR Vβ8表达载体构建及体外表达

目的:构建Jurkat细胞株独特型TCR Vα1-pIRES-TCR Vβ8表达载体,转染后了解其体外表达情况.方法:根据聚合酶链反应-基因扫描(PCR-Genescan)检测Jurkat细胞株TCR Vα及Vβ亚家族表达情况,分别将其所表达的单克隆性的TCR Vα1及TCR Vβ8亚家族基因片段克隆至质粒pIRES的多克隆位点A(MCS A)和多克隆位点B(MCS B)中.通过限制性酶切分析、序列分析、RT-PCR、间接免疫荧光、流式细胞术(FCM)手段鉴定重组表达载体的正确性以及转染A549和Molt4细胞后基因及蛋白的表达情况.结果:构建了2组TCR Vα1-pIRES-TCR Vβ8表达载体,该表达载体转染A549和Molt4细胞后,在mRNA和蛋白水平检测到了TCR Vα1和TCR Vβ8的表达.结论:成功构建了2种独特型Vα1-pIRES-TCR Vβ8真核表达载体,为利用特异性TCR基因修饰T细胞的研究提供方法学依据.     

pTARGET-TCR Vβ5.2真核表达质粒的构建及表达

目的 构建含大鼠TCR Vβ5.2基因的重组真核表达质粒.方法 以RT-PCR法扩增Lewis大鼠脾脏淋巴细胞的TCR Vβ5.2基因片段,将目的基因直接克隆到真核表达载体pTARGET中,构建重组质粒pTARGET-TCR Vβ5.2.连接产物转化E.coli JM109细胞后进行蓝白斑筛选,并经菌落PCR和DNA测序进行鉴定.将重组质粒以肌注法免疫Balb/c小鼠,用RT-PCR和免疫组化方法分别检测重组质粒在注射部位的转录和表达.通过脂质体介导,将重组质粒转染COS-7细胞进行瞬时表达,用免疫细胞化学染色法检测目的基因在真核细胞内的表达.结果 测序鉴定证实,TCR Vβ5.2基因已被正确插入到pTARGET载体中,并检测到肌肉组织和COS-7细胞内目的蛋白的表达.结论 成功构建了重组真核表达质粒pTARGET-TCR Vβ5.2,为进行TCR VβDNA疫苗对胶原诱导性关节炎大鼠保护作用的研究提供了基础.     

PML-RARα/pIRES和PML-RARα/pSecTag重组载体的构建和鉴定

目的 构建PML-RARα基因重组表达质粒,为发展PML-RARα基因疫苗提供实验依据. 方法利用RT-PCR方法从急性早幼粒细胞白血病细胞株NB4细胞中扩增出465 bp 的位于PML-RARα融合基因的框内结构的片段,克隆至真核表达载体pIRES和分泌载体pSecTag后,经酶切和序列分析鉴定重组质粒的构建情况.结果 经酶切鉴定和序列测定表明,PML-RARα目的基因已正确插入真核表达载体中,构成了目的重组载体. 结论成功构建了PML-RARα/pIRES和PML-RARα/pSecTag重组真核表达质粒,可进一步用于PML-RARα基因疫苗的实验研究.     

PML-RARα/pIRES和PML-RARα/pSecTag重组载体的构建和鉴定

目的 构建PML-RARα基因重组表达质粒,为发展PML-RARα基因疫苗提供实验依据. 方法利用RT-PCR方法从急性早幼粒细胞白血病细胞株NB4细胞中扩增出465 bp 的位于PML-RARα融合基因的框内结构的片段,克隆至真核表达载体pIRES和分泌载体pSecTag后,经酶切和序列分析鉴定重组质粒的构建情况.结果 经酶切鉴定和序列测定表明,PML-RARα目的基因已正确插入真核表达载体中,构成了目的重组载体. 结论成功构建了PML-RARα/pIRES和PML-RARα/pSecTag重组真核表达质粒,可进一步用于PML-RARα基因疫苗的实验研究.     

大鼠TCR Vβ8.2基因真核表达质粒的构建及表达

目的：构建含大鼠TCR Vβ8．2基因的重组真核表达质粒，并检测其在体内外的表达。方法：以RT-PCR法分离Lewis大鼠脾淋巴细胞中的TCR Vβ8．2基因片段，插入到真核表达载体pTARGET中，构建重组表达载体pTARGET-TCR Vβ8．2，并转化E．coli JM109，经蓝白斑筛选阳性菌落，进行菌落PCR和测序鉴定。将重组质粒以肌注法免疫BALB／c小鼠，采集注射部位股四头肌处的肌肉，用RT-PCR及免疫组化染色法检测TCRVB8．2基因在注射部位的转录和表达：通过脂质体介导，将再组质粒导入COS-7细胞中进行瞬时表达，用免疫细胞化学染色法检测靶基因在真核细胞内的表达。结果：测序鉴定证实，TCR Vβ8．2基因已被正确插入到pTARGET载体中，并检测到在肌肉组织和COS-7细胞内重组蛋白的表达。结论：成功地构建重组真核表达质粒pTARGET-TCR Vβ8．2，并在体内体外均可检测到其转录和表达，为进一步进行TCR VβDNA疫苗的研究奠定了基础。     

EBV特异性CTL相关TCR Vα-pIRES-TCR Vβ重组质粒的构建及体外表达

目的 构建EBV特异性细胞毒T细胞(CIL)相关的TCR Vα-pIRFS-TCR Vβ真核双表达质粒.方法 通过RT-PCR和基因扫描技术分析EBV特异性CTL克隆的T细胞受体(TCR)Vα和Vβ谱系表达情况及T细胞克隆性,确定其优势表达的TCR Vα15和TCR Vβ1亚家族基因的核苷酸序列(包括CDR3互补决定区),PCR扩增出TCR Vα15和TCR Vβ1基因后,分别定向克隆入真核双表达载体pIRES,构建双顺反子重组质粒TCR Vα-pIRES-TCR、Vβ,转基因技术将其转染A549细胞和Molt4细胞,通过RT-PCR、实时定量PCR、流式细胞术和蛋白印迹分析检测TCR Vα15和TCR Vβ1基因的表达.结果 酶切分析和核酸序列测定证实重组质粒构建正确,转染重组质粒的细胞能够表达TCR Vα15和TCR Vβ1的mRNA及蛋白.结论 成功构建了EBV特异性的TCR Vα-pIRES-TCR Vβ双顺反子真核表达质粒,并在体外实现了共表达,为进一步的实验研究奠定了基础.     

人SYNOVIOLIN基因真核表达载体的构建及表达

目的：构建携EGFP的人SYNOVIOLIN基因真核表达载体。方法：应用基因重组技术,根据人SYNOVIOLIN基因序列和表达载体pIRES2-EGFP质粒上的多克隆位点设计引物,对含有SYNOVIOLIN基因的质粒pCDNA3-syno扩增,得到约1900 bp的目的片段,进行T-A克隆。SalⅠ/BamHⅠ双酶切测序正确的重组质粒,回收SYNOVIOLIN cDNA片段,将其亚克隆于pIRES2-EGFP载体的多克隆位点内得到质粒pIRES2-EGFP-syno。脂质体法转染HEK293细胞, 用激光共聚焦显微镜和Western blot检测EGFP和SYNOVIOLIN在HEK293细胞的表达。结果：PCR,酶切及测序结果表明pIRES2-EGFP-syno真核表达载体构建成功,激光共聚焦显微镜和Western blot显示EGFP和SYNOVIOLIN蛋白在HEK293细胞中成功表达。结论：成功构建pIRES2-EGFP-syno真核表达载体并在HEK293细胞表达,为抗肌腱粘连的　SYNOVIOLIN基因治疗研究奠定基础。     

抗原特异TCR Vα6-pIRES2-EGFP和TCRV β13-pIRES2-EGFP真核表达载体的构建及共转染研究

目的 构建携带弥漫大B型非霍奇金淋巴瘤(DLBL)相关抗原特异性的TCR Vα和TCR Vβ基因片段的真核表达载体TCR Vα-pIRES2.EGFP和TCR Vβ-plRES2-EGFP,将其共同转染Raji和Jurkat细胞.方法 前期研究从1例DLBL患者外周血T细胞中发现克隆性增殖T细胞受体(TCR)Vα6和Vβ13亚家族T细胞,在此基础上利用RT-PCR扩增TCR Va6和TCR Vβ13基因全长序列后,分别将其定向克隆入带有绿色荧光蛋白(EGFP)的真核表达载体pIRES2-EGFP,酶切和核酸序列测定分析方法鉴定重组质粒TCR Vα6-pIRKS2-EGFP和TCR Vβ13-pIRES2-EGFP的正确性;利用核转染技术(nueleofector)将其分别或共同转染Raji细胞和Jurkat细胞,24 h后利用激光共聚焦显微镜观察EGFP的瞬时表达情况,48h后利用实时定量PCR检测TCR Vα6和TCR Vβ13基因的表达情况,Western blot检测EGFP蛋白的表达情况.结果 获得来自DLBL患者的TCR Vα6和TCR Vβ13基因全长序列,酶切分析和核酸序列测定证实TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP重组质粒构建正确;转染24 h后,激光共聚焦显微镜下可观察到EGFP的表达,40%以上细胞发出绿色荧光,单独转染和共转染组荧光产生情况相似;实时定量PCR在单独转染和共转染组均町检测到TCR Vα6和TCR Vβ13基因的表达,共转染组2基因的表达水平稍低于单独转染组;Western blot检测在单独转染和共转染组均显示EGFP蛋白的表达,2组的蛋白杂交带强度相似.结论 成功构建了DLBL特异性的TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP真核表达质粒,两者可同时转染到细胞中,并实现了体外共表达.     

抗血管生成肽βpep-25重组原核表达质粒的构建及鉴定

目的： 合成抗血管生成肽βpep-25片断, 构建并鉴定含βpep-25肽的重组原核表达质粒.方法： 人工设计合成βpep-25肽序列片断, 经测序正确后, 利用基因克隆技术将其克隆到pGEM-T easy载体中, 构建重组质粒pGEM-T-βpep-25;用Nae I和BamH I双酶切pGEM-T-βpep-25后, 将T-βpep-25肽片断克隆到经Nae I和BamH I双酶切的重组载体pBV220-NT4中, 构建重组原核表达质粒pBV220-NT4-βpep-25, 并对其分别用BamH I酶切, EcoR I和BamH I双酶切分析.结果： 经酶切分析和DNA测序人工合成的肽βpep-25序列（113 bp）与文献报道结果一致;分别用BamH I酶切, EcoR I和BamH I双酶切重组原核表达质粒pBV220-NT4-βpep-25可以得到4 030 bp、 364 bp和3 666 bp的片段, 结果表明目的肽片段已经成功地克隆到了重组表达载体中, 说明重组原核表达质粒构建成功.结论： 抗血管生成肽βpep-25重组原核表达质粒的构建成功, 为进一步在细胞内外及动物模型研究其作用机制和抗肿瘤作用奠定了基础.     

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.     

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.     

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.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'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.