HIV-1 gp41 NHR结合性环肽的研究

目的 筛选可与HIV-1 gp41 NHR结合的环肽，为研制抗HIV-1早期感染的小分子药物奠定基础。方法 采用P＋LS方法，用源于gp41 NHR的合成肽N36肽筛选噬菌体环七肽库，ELISA鉴定噬菌体克隆，根据阳性克隆的DNA序列，合成环肽并鉴定其与N36肽结合。结果 经3轮筛选、鉴定，得到11个和N36肽结合的噬菌体克隆。DNA测序并推导氨基酸序列，表明这11个克隆展示同一序列CDRHQHKRC。根据此序列合成的环肽NA（Biotin—SACDRHQHKRCGG）经与载体蛋白BSA偶联后，EIJSA鉴定表明交联物可特异地与N36肽结合，这种结合可被游离N36肽、以及源于gp41 CHR的肽C34抑制。结论 sACDRHQHKRCGG环肽为HIV-1 gp41 NHR结合肽。     

HIV-1gp41核心结构模拟位的筛选与鉴定

目的：筛选并鉴定HIV-1 gp4l核心表位。方法：用识别HIV-1 gp41的构象特异性单克隆抗体NC-1筛选噬菌体12肽库，通过夹心ELISA、NC-1特异性阻断实验、竞争抑制实验鉴定阳性噬菌体克隆，DNA序列分析阳性克隆。结果：经3轮筛选，随机挑取24个噬菌体克隆，ELISA鉴定表明有lO个克隆可与NC-1结合，DNA序列分析并推导氨基酸序列，共5种序列：HDVHHRWVYLLS、ITVNEWLYTSEQ、HGRSHGMFKPKR、MGPIARPHWHLN、DMYRSPRPKPDT。其中gp41N肽和C肽所形成的复合物可特异性阻断表达HDVHHRWVYLLS，VNEWLYTSEQ和MGPIARPHWHLN的克隆与NC-1的结合。结论：所得序列HDVHHRWVYLLS，VNEWLYTSEQ及MGPIARPHWHLN模拟HIV-1 gp41六螺旋束核心表位。     

模拟HIV-1 gp41 CHR表位短肽的筛选及研究

目的 利用针对HIV-1跨膜蛋白gp41CHR序列合成肽C34的单克隆抗体1G1筛选噬菌体12肽库，旨在找寻模拟C34肽表位的序列，同时探索该短肽成为HIV-1gp41NHR与CHR结合抑制物的可能性。方法 以1G1为钓饵蛋白对噬菌体12肽库进行亲和筛选，以双夹心ELISA鉴定阳性克隆。结果 经3轮筛选后，随机挑选17个噬菌体克隆，其中6个克隆与1G1显示出较强的结合活性，上述6个阳性克隆经DNA测序，氨基酸序列相同；HYEFWAWNWEAN，其明显的疏水性质类似于G34N末端，特异性鉴定显示这些克隆均能够与HIV-1gp41N多肽结合，结论 该噬菌体克隆展示肽可模拟HIV-1gp41CHR多肽表位，并可与N多肽结合。     

我国HIV-1流行株gp41抗原表位筛选与串联表达

目的探讨HIV-1gp41抗原表位串联表达蛋白用于HIV抗体检测的可行性。方法用HIV-1gp41蛋白亲和层析柱制备HIV-1感染者血清中的多克隆抗体，用噬菌体展示随机十二肽库进行生物淘洗，反向吸附非特异性噬菌体。经ELISA鉴定阳性克隆，DNA测序，确定优势表位。将优势表位与文献报道的另一个优势表位串联，克隆人pQE30载体进行蛋白表达。结果成功筛选到位于HIV-1 gp41蛋白上的优势抗原表位(YGPKDAETTAIW)，串联表位(YGPKDAETTAIW-GGGS-SC-SAKFTCTTQI)在pQE30载体中实现可溶性表达。重组蛋白具有良好的抗原性，能与不同的HIV-1抗体阳性血清呈特异反应。结论HIV-1 gp41抗原表位串联表达设计足可行的，串联表位重组蛋白可用于HIV-1抗体检测，但检测灵敏性低于常规方法。     

模拟肽聚糖表位的序列分析及其抗原性鉴定

目的:在线预测肽聚糖(PGN)模拟抗表原位并鉴定其抗原性。方法:利用抗PGN单克隆抗体从12线性噬菌体随机展示肽库中筛选模拟PGN表位的阳性克隆,对阳性克隆进行DNA测序和推导氨基酸序列,结合生物信息学分析阳性序列GRWxHxVxWAGL的抗原性以及T细胞表位,根据分析对阳性序列进行修饰与合成,ELISA法鉴定阳性序列的抗原性。结果:经对噬菌体12线性肽库的3轮筛选,夹心ELISA鉴定得到16个与抗PGN单克隆抗体结合的克隆,DNA测序并推导氨基酸序列,该序列与前期具有抗金黄色葡萄球菌活性序列No.31(ATWxHxLxSAGL)含有保守序列WxHx…AGL。在线分析(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred)表明,含保守序列的阳性序列GRWxHxVxWAGL包含与人和小鼠MHC结合表位(-WxHxVxW-),C端加入AGGS后具有T细胞表位(DNAstar),据此合成线性肽Biotin-GRWxHxVxWAGLAGGS(命名为SP39)。ELISA结果显示,SP39能与抗PGN单抗及抗S.aureus全菌多抗结合,PGN能抑制SP39与抗PGN单克隆抗体结合。结论:经噬菌体肽库筛选获得阳性序列GRWxHxVxWAGLAGGS,该序列可能模拟PGN抗原表位。     

C1q结合十二肽的筛选与初步鉴定

目的　从噬菌体十二肽库中筛选结合C1q的短肽并进行初步鉴定。方法　以C1q为钓饵蛋白筛选噬菌体十二肽库 ,利用ELISA、U937细胞配体结合抑制试验、多聚IgG(AIgG)竞争抑制试验鉴定阳性克隆 ,再进行单链DNA测序和分析。结果　经 3轮筛选后 ,随机挑选 2 5个噬菌体克隆做ELISA鉴定 ,14个克隆显示与C1q有较强的结合。经过U937细胞配体结合抑制试验和AIgG竞争抑制试验鉴定后 ,将此 14个阳性克隆测序 ,从其展示肽DNA测序结果推导氨基酸序列 ,获得 9条氨基酸序列 :HWDPFSLSAYFP、WTPVRTNPFLLH、NGHLFSLSAYFP、RTQRNSPFFLCP、SPAFHPEHMGRG、SRAFHPFYRGRA、WYEGPFTLQTWP、LTQHNSPFFLLP和TSNPFFLWYPQP。结论　获得结合C1q的一些抑制性短肽 ,它们可能成为抑制补体经典途径激活的肽类先导化合物     

人泌尿系上皮肿瘤疫苗的初步研究

目的通过筛选噬菌体随机12肽库获得MUC1糖链抗原模拟表位。方法利用纯化获得的M a695抗体筛选噬菌体随机12肽库,通过夹心ELISA分析噬菌体克隆,测定阳性克隆DNA序列并进行同源性及氨基酸分析,竞争性抑制实验鉴定噬菌体克隆。结果经3轮筛选,获得了14个阳性克隆,DNA序列分析并推导出氨基酸序列:KHYDPFHHRMPQ,QADTARSVALAG,VPSKPDLHVRSI,MTPIHYWNHNRV。鉴定结果表明4个噬菌体展示肽克隆抑制率均在50%以上。结论所得序列KHYDPFH-HRMPQ,QADTARSVALAG,VPSKPDLHVRSI,及MTPIHYWNHNRV模拟了MUC1抗原表位。     

利用噬菌体肽库筛选IL-2Rα模拟表位肽的研究

目的 筛选IL-2Rα模拟表位肽,为研制高效、特异性强的小分子肽类免疫抑制剂奠定基础.方法 应用离子交换层析法从小鼠腹水中纯化抗人IL-2Rα单克隆抗体5G1,细胞ELISA方法检测其特异性.用5G1单抗筛选噬菌体环七肽库,并对噬菌体克隆进行抗原性鉴定.根据阳性克隆的DNA序列合成环肽并鉴定其生物活性.结果 经5轮筛选、鉴定,获得5个与5G1有较强结合特性的噬菌体阳性克隆.DNA测序并推导氨基酸序列,得到Lys-X-{X}-Lys-Gly保守序列.依此序列合成环七肽CP,小鼠淋巴细胞增殖试验证实其有免疫抑制活性.结论 环肽CP为IL-2Rα模拟表位肽,可作为IL-2Rα的拮抗剂发挥免疫抑制效应.     

从噬菌体随机环七肽库筛选可模拟C1qR的C1q结合肽

为获得能结合C1q并模拟C1q受体 (C1qR )配体结合位点的短肽 ,以C1q为钓饵蛋白筛选噬菌体环七肽库 ,采用C1q结合ELISA、U937细胞配体结合抑制试验、多聚IgG (AIgG )竞争抑制试验鉴定阳性克隆 ,再进行单链DNA测序和分析。结果经 3轮筛选后 ,随机挑选 2 3个噬菌体克隆进行ELISA鉴定 ,10个克隆与C1q有较强的结合 ;利用U937细胞配体结合抑制试验 ,得到了 7个阳性克隆 ;从其展示肽DNA测序结果推导氨基酸序列 ,获得 7个短肽序列 :QTPFQLW、NPFNWTS、SPFXLTS、FLTWLDP、FSTFLYP、GPMWWSY和NPFXLIL。     

广谱抗革兰氏阴性菌单克隆抗体3A8识别表位的初步研究

目的:利用噬菌体肽库技术筛选抗广谱革兰氏阴性菌与LPS单克隆抗体3A8的识别表位,以获得来源于不同LPS的保守表位.方法:用广谱抗G-菌、LPS的单克隆抗体3A8为靶,筛选噬菌体环七肽库,蚴鉴定阳性噬菌体克隆并测序.根据阳性保守序列合成短肽A2及A5,并与KLH载体交联,ELISA鉴定A2-KLH、A5-KLH与3A8单抗的结合.结果:随机挑选的33个噬菌体克隆中有15个可特异性与3A8结合,该结合可被LPS2630所抑制.根据阳性克隆DNA序列推导氨基酸序列,共有7种序列,均以疏水氨基酸为主,且包含保守序列Ser Pro Pro/Pro X Pro;选择所获阳性序列经设计与改造合成延长的两个环肽;经ELISA鉴定表明这些环肽可特异地与3A8结合.结论:得到可被3A8单抗特异性识别并含保守残基Ser Pro Pro/ProX Pro的阳性序列,据此合成的短肽可特异性结合3A8,提示该短肽可能模拟LPs共同表位的抗原性,有望成为疫苗候选表位.     

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.     

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 case for allele‐specific recognition by the TCR

There is a sharp difference in how one views TCR structure–function–behaviour dependent on whether its recognition of major histocompatibility complex‐encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele‐specific. The establishing of allele‐specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele‐specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.