人DC-SIGN全长编码区基因的克隆及其胞外段的原核表达

目的:克隆人DC-SIGN全长编码区基因, 获得其胞外段的原核表达产物.方法:采用RT-PCR方法, 从健康产妇胎盘中克隆DC-SIGN全长cDNA, 扩增其胞外段基因并构建pET41a-sDC-SIGN重组表达质粒, 在大肠杆菌BL21(DE3)中表达, 以SDS-PAGE和Western blot鉴定表达产物.结果:从健康产妇胎盘总RNA中, 扩增获得约1 300 bp的DNA片段, 克隆至pGM-T载体获得重组质粒pGM-DC-SIGN.从pGM-DC-SIGN扩增DC-SIGN的胞外段基因, 构建重组表达质粒pET- 41a-sDC-SIGN;纯化表达产物sDC-SIGN-GST, 鉴定其相对分子质量( M r)为66 000, Western blot证明其可与抗DC-SIGN抗体特异性结合.结论:成功克隆DC-SIGN全长编码区基因, 并在大肠杆菌中成功表达其胞外段融合蛋白sDC-SIGN-GST, 为进一步研究DC-SIGN的功能奠定了基础.     

人源His-talin1原核表达载体的构建及其蛋白表达

目的克隆人源talin1 cDNA,构建其高效原核表达载体,并纯化得到高纯度His-talin1融合蛋白。方法 PCR法扩增talin1基因并连接到原核表达载体pET32a(+),筛选和测序鉴定阳性克隆。将重组质粒转化大肠杆菌BL21(DE3),经过IPTG诱导表达和亲和层析分离纯化表达产物,对纯化的蛋白进行SDS-PAGE和Western blot鉴定。结果成功扩增了2.4kb的talin1基因,构建了pET32a(+)-talin1重组质粒并在大肠杆菌中诱导表达出His-talin1融合蛋白。经SDS-PAGE和Western blot方法 ,验证了高纯度纯化的融合蛋白。结论建立了高效稳定的His-talin1表达体系,为进一步研究Talin1蛋白的结构及其与P-selectin之间的关系打下基础。     

结核分枝杆菌Zmp1基因原核表达载体的构建及表达鉴定

目的构建结核分枝杆菌锌离子依赖的金属蛋白酶1(Zmp1)基因的原核表达载体,并在大肠杆菌中进行表达。方法以卡介苗(BCG)基因组DNA为模板,采用PCR法扩增Zmp1基因;定向克隆到原核表达载体pET-32a(+)的多克隆位点中,构建重组原核表达质粒pET-32a(+)-Zmp1;转化入大肠杆菌BL21(DE3)中经IPTG诱导表达,表达产物经SDS-PAGE和Western blot法鉴定。结果PCR法扩增出Zmp1基因;重组表达质粒经双酶切及基因测序鉴定构建正确;表达的重组Zmp1融合蛋白相对分子质量(Mr)约为94 000,大小与预期融合蛋白一致;重组Zmp1融合蛋白可与His标签单克隆抗体特异性结合。结论成功构建了Zmp1基因原核表达载体,并在大肠杆菌BL21(DE3)中获得重组Zmp1融合蛋白表达。     

耐甲氧西林金黄色葡萄球菌细胞膜20肽重组蛋白SA0587的表达和鉴定

目的原核质粒表达耐甲氧西林金黄色葡萄球菌(MRSA)细胞膜20肽重组蛋白SA0587。方法构建p ET28aSA0587-GFP重组质粒,将其转化至大肠埃希菌BL21(DE3)中;挑取单菌落培养活化后诱导表达目的蛋白SA0587;SDS-PAGE及Western blot法鉴定SA0587蛋白。结果利用基因工程技术成功构建重组质粒p ET28a-SA0587-GFP,转染大肠杆菌后,成功表达出目的蛋白SA0587。结论成功在大肠杆菌表达MRSA细胞膜20肽重组蛋白SA0587。     

我国地方品种姜曲海猪TLR5基因的克隆、表达及鉴定

目的:克隆我国地方品种姜曲海猪TLR5基因,构建该基因的原核表达质粒,获得融合表达蛋白并鉴定其免疫特性,为进一步研制TLR5单克隆抗体(mAb)提供免疫原。方法:从全血中提取姜曲海猪的基因组,设计特异性引物,利用PCR方法扩增得到TLR5基因片段,PCR产物克隆到原核表达载体pET30a(+)中,构建重组表达质粒pET-TLR5,将重组表达质粒转化E.coli BL21,0.5 mmol/L IPTG诱导表达目的蛋白,应用SDS-PAGE和Western blot分析鉴定蛋白活性。结果:成功扩增出猪TLR5基因片段,大小为2 571 bp。酶切鉴定结果表明,猪的TLR5基因成功克隆入原核表达载体pET30a(+)中,重组质粒pET-TLR5在大肠杆菌中获得表达,SDS-PAGE结果显示出相对分子质量(Mr)大小约95 200;Western blot结果表明,表达产物与兔抗小鼠TLR5 mAb具有良好的反应性。结论:成功克隆并表达具有较好免疫原性的猪TLR5分子,为其mAb的制备提供生物材料。     

人精子相关抗原7的基因克隆及原核表达

目的为了阐明免疫性不孕不育的原因,研制人特异性精子抗原避孕疫苗,构建重组人精子相关抗原7(recombinant human sperm associated antigen 7,rhSPAG7)的原核表达质粒并在大肠杆菌中诱导表达。方法采用RT-PCR法,从人睾丸组织总RNA中扩增获得人SPAG7的c DNA,将其克隆入表达载体p BV220,构建人源性SPAG7的重组原核表达质粒p BV220/SPAG7。重组质粒经酶切和测序鉴定后,转化大肠杆菌JM109并在大肠杆菌中诱导表达目的蛋白。结果测序表明重组基因序列与人SPAG7基因完全一致。SDS-PAGE电泳显示,表达产物的相对分子量为25.8KDa与预期结果相符。结论获得了人SPAG7的编码基因,并在大肠杆菌中表达了人的SPAG7蛋白。     

欧蓍草花粉主要过敏原Parj1的重组表达及鉴定

目的:克隆、表达和纯化欧蓍草花粉主要过敏原Par j1。方法:根据Parj1.0102在GenBank中的序列号获得其核苷酸和氨基酸序列,确定开放阅读框,采用DNAstar软件优化密码子,合成全基因,并克隆到表达载体pET-44a中,转化表达宿主大肠杆菌Rosetta,优化蛋白表达条件并进行亲和层析纯化和Western blot鉴定。结果:PCR扩增及重组质粒测序结果表明成功地构建了pET-44a+/Par j1.0102原核表达质粒。对表达菌表达条件进行优化,最终确定在30℃,IPTG浓度为1.0 mmol/L,诱导4小时时蛋白表达量最高,重组蛋白经亲和层析纯化,SDS-PAGE分析纯化产物在23 kD处有明显的条带。Western blot表明重组蛋白具有与StrepII标签抗体结合活性。结论:国内首次获得融合StrepII标签的Par j1.0102重组蛋白,为欧蓍草花粉过敏诊断及特异性免疫治疗奠定基础。     

血管生成抑制因子arresten基因原核表达载体的构建及其在大肠杆菌中的表达

目的:构建血管生成抑制因子arresten基因的原核表达载体, 并在大肠杆菌中进行表达。方法:利用聚合酶链式反应(PCR), 由我们构建的重组质粒pGEM-Arr中扩增出arresten基因;采用基因重组技术, 将该基因定向克隆于原核表达载体pRSET中, 转化大肠杆菌BL21(DE3), 用IPTG诱导表达, 并对表达产物行SDS-PAGE分析。结果:酶切鉴定和DNA测序证实arresten基因正确地插入表达载体中。重组arresten在大肠杆菌中获得高效表达, 其分子量约为26kD, 表达量约占菌体总蛋白量的30%。结论:Arresten基因原核表达载体的成功构建和重组arresten蛋白在大肠杆菌中的高效表达, 为进一步研究其生物学功能奠定了基础。     

人CD154-GST融合蛋白基因在大肠杆菌中的表达

目的:为制备重组人CD154-谷胱甘肽巯基转移酶(GST)融合蛋白(hCD154-GST),用于人CD154单克隆抗体研制。方法:根据人CD154基因序列设计合成特异性引物,RT-PCR扩增人CD154基因,并插入融合蛋白原核表达载体pGEX-4T-1中,得到重组表达质粒pGEX-4T-1/CD154;用此重组质粒转化大肠杆菌BL21细胞,转化菌落经BamHⅠ、EcoRⅠ酶切鉴定。IPTG诱导大肠杆菌表达人CD154蛋白,SDS-PAGE电泳鉴定表达产物。结果:从人外周血淋巴细胞扩增出820bp的hCD154cDNA;将其克隆至pGEX-4T-1质粒中,经双酶切鉴定及DNA序列分析证实含有目的基因;IPTG诱导后的大肠杆菌经SDS-PAGE电泳鉴定出现明显的55kD蛋白带。结论:成功构建了人CD154-GST原核表达质粒,并在大肠杆菌中表达出人CD154-GST融合蛋白,为人CD154单克隆抗体的研制及进一步抗排斥反应的研究打下了基础。     

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.