人睾丸特异性新基因hT279单克隆抗体制备及其应用

目的构建人睾丸特异性新基因hT279(GenBank登录号BC016750)的原核表达载体,纯化融合蛋白并以其为抗原免疫Balb/c小鼠制备抗人睾丸特异性hT279蛋白单克隆抗体(mAb)并进行特性鉴定。方法用PCR方法得到睾丸特异性新基因hT279并克隆至pET32a原核表达载体中,转化大肠杆菌BL21诱导融合蛋白表达;获得的可溶性蛋白经亲和层析纯化、SDS-PAGE鉴定后,免疫Balb/c小鼠,应用淋巴细胞杂交瘤技术制备抗hT279 mAb,并通过间接ELISA、Western blot和免疫组织化学法对mAb进行特性鉴定。结果实现了hT279的原核表达,获得了1株稳定分泌抗hT279 mAb的杂交瘤细胞株4B2,抗体亚型为IgG2b(κ),效价达到1×104。Western blot鉴定表明,该mAb在人正常睾丸蛋白中相对分子质量约为22 000处检测到特异条带。免疫组织化学显示hT279蛋白主要在正常人睾丸的精母细胞及圆形精子细胞中表达,而在男性不育患者睾丸组织中表达消失。结论成功制备出1株抗hT279的mAb 4B2,为进一步研究hT279在人类精子发生中的功能和男性不育症的诊断提供了特异的检测工具。     

抗SARS冠状病毒M蛋白片段单克隆抗体的制备与初步鉴定

目的:制备抗SARS冠状病毒(SARS-CoV)M蛋白N端1~43氨基酸(aa)单克隆抗体(mAb),并对其特性进行初步鉴定。方法:用纯化的SARS-M/GST融合蛋白抗原免疫BALB/c小鼠,采用杂交瘤技术制备抗M蛋白片段的mAb。用间接ELISA法筛选能分泌抗M蛋白片段mAb的杂交瘤细胞株。Westernblot和间接ELISA鉴定所获mAb的特异性,并用小鼠mAb亚类测定试剂盒检测所获的mAbIg亚类。为分析mAb识别位点,进一步将M蛋白片段截短为部分重叠的2段表达,以Westernblot初步定位mAb识别位点。结果:获得1株可分泌特异性抗SARS-CoVM蛋白片段的mAb杂交瘤细胞株(3H9),Ig亚类鉴定为IgG2a,轻链为κ型。Westernblot显示其mAb可特异识别SARS-CoVM蛋白N端1~43aa,间接ELISA证实mAb可与包被于聚苯乙烯微孔板上的SARS病毒全蛋白抗原发生特异性反应,其识别位点位于M蛋白N端16~28aa。结论:成功获得1株抗SARS-CoVM蛋白N端1~43aamAb杂交瘤细胞,并初步定位其识别位点。     

抗PSMA 膜外域单克隆抗体的制备及初步应用

目的:制备抗PSMA膜外域单克隆抗体。方法:应用生物信息技术预测PSMA膜外域多肽抗原片段,原核表达,免疫BALB/c小鼠,通过杂交瘤技术制备抗PSMA膜外域单克隆抗体,亲和层析纯化和Western blot及ELISA鉴定,并用于前列腺癌动物模型放射免疫显像。结果:经软件分析,获得含310aa序列特异性强的膜外域抗原片段,免疫、融合后,经过ELISA筛选获得到3株阳性杂交瘤细胞株(5E6、4A5和4D7),亚型鉴定结果为5E6、4A5为Ig G2a,4D7为Ig G1,腹水型抗体效价均在1∶256 000以上;Western blot结果表明,所制备的单克隆抗体均可与PSMA膜外域抗原特异性结合,竞争抑制ELISA试验显示制备的单抗与重组抗原及标准抗原PSMA能竞争结合。动物模型放射免疫显像结果进一步证实,制备的单克隆抗体在动物体内能与PSMA膜外域抗原特异性结合,并使肿瘤显像。结论:制备的抗PSMA膜外域单克隆抗体能特异性识别PSMA抗原,为前列腺癌的免疫诊断及免疫治疗奠定了基础。     

Cys C单克隆、多克隆抗体的制备及临床应用

目的：制备鼠抗人Cystatin C（Cys C）的单克隆抗体和兔抗人Cys C的多克隆抗体,并鉴定其特异性。方法：将PQE30-Cys C转化大肠杆菌BL-21,通过IPTG诱导蛋白表达,Ni-NTA agarose亲和层析纯化Cys C蛋白。通过免疫小鼠和大白兔,制备鼠抗人Cys C的单克隆抗体和兔抗人Cys C的多克隆抗体。结果：通过构建含Cys C的原核细胞表达质粒,表达及纯化了Cys C重组蛋白,建立了产生抗Cys C单克隆抗体的杂交瘤细胞株,及制备了兔抗人Cys C的多克隆抗体,末次抗血清效价为1：1000000,经过ELISA证实用CysC免疫的大白兔所制备的抗血清可与Cys C发生特异性的免疫反应。结论：本文成功制备鼠抗人Cys C的单克隆抗体和兔抗人Cys C的多克隆抗体,建立了ELISA检测Cys C,为用于临床奠定了基础。     

抗人心脏型脂肪酸结合蛋白单克隆抗体的制备、表位鉴定及其应用

目的 制备抗人心脏型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),鉴定其基本特性并通过配对检测临床样本血清,对已配对的mAb进行表位鉴定.方法 通过已制备的H-FABP抗原和合成肽免疫BALB/c小鼠,常规融合筛选后进行抗体亚型鉴定、效价和亲和力检测,纯化mAb后通过间接ELISA、Western blot法对mAb进行特异性检测;将获得的mAb分别作为捕获和检测抗体,经过配对建立检测重组H-FABP的ELISA体系,并初步用于临床血清检测;采用生物信息学分析设计2段HFABP表位,通过原核表达获得其短肽并纯化,通过Western blot法对配对的mAb进行表位鉴定.结果 成功获得4株抗HFABP特异性mAb,亚类分别为IgG2a和IgG2b,抗体效价为1:51 200～1:1024 000,亲和力最高可达到9.02×109 mol/L;经Western blot法和间接ELISA鉴定均能够特异检测重组H-FABP蛋白,经过配对发现mAb 3-H5和1-F10能够检测重组H-FABP,并在其临床血清样本检测中发现正常组和急性心梗组之间H-FABP水平具有显著性统计学差异;通过表位鉴定发现未知表位的mAb 1-F10能够特异识别位点为H-FABP的第86到第133位氨基酸.结论 制备了4株高特异性和高亲和力的抗H-FABPmAb,成功建立了检测临床样本血清中的ELISA体系,并对其表位进行了分析鉴定.     

FHL1抗体的制备与特异性鉴定

目的:制备FHL1及其剪接体FHL1B、FHL1C的多克隆抗体,并对其特异性进行鉴定。方法:融合表达GST-FHL1蛋白,常规免疫小鼠,制备多克隆抗体。分别构建带有FLAG标签的FHL1-5、FHL1B、FHL1C以及FHL1N端和C端缺失突变体的真核表达载体。Westernblot检测抗体的特异性。结果:获得了FHL1-5、FHL1B、FHL1C及FHL1(1-169aa)和FHL1(168-280aa)的真核表达载体;得到的多克隆抗体可特异识别FHL1、FHL1B和FHL1C,而不识别FHL2-5;抗体与FHL1N端反应,而不与其C端反应。结论:成功得到可特异识别FHL1、FHL1B和FHL1C的多克隆抗体,为进一步研究FHL1及其剪接突变体的功能奠定了坚实的基础。     

人血管内皮钙黏蛋白5单克隆抗体的制备及应用

目的制备并鉴定人血管内皮钙黏蛋白5(VECDH5)的单克隆抗体(m Ab)。方法用重组原核人VECDH5蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合;间接ELISA筛选阳性克隆,得到稳定分泌抗VECDH5抗体的杂交瘤细胞株,ELISA测定其效价;Western blot法、流式细胞术、免疫荧光细胞化学染色、免疫组织化学染色鉴定其特异性及抗原表位。结果制备并获得1株可分泌特异性抗VECDH5的杂交瘤细胞株(2C11)。ELISA测定腹水效价高达1∶10 000。多种方法均证实该抗体能特异性识别人VECDH5蛋白,并可在上述不同方法中应用。经抗原表位鉴定,研究制备的2C11识别的氨基酸序列为LDREVVPWYNLTVEA。结论成功制备了亲和力高、特异型好的抗人VECDH5的m Ab。     

戊型肝炎病毒新潜在中和性表位的发现

目的 探讨戊型肝炎病毒(HEV)衣壳蛋白是否存在除主要免疫优势中和表位aa459～606以外的其他中和表位.方法 通过对几株单克隆抗体及其表位的性质进行分析,对比位于主要免疫优势表位区aM59～606和位于该表位N端序列aa394～458区域的数个表位的中和活性.结果 发现位于aa423～437的表位对应的单抗具有潜在中和活性,不同于已知的HEV中和性表位(aa459～606),该表位是一个线性非免疫优势表位.结论 HEV ORF2 aa423～437为新的潜在的线性非免疫优势中和表位,该发现丰富了对HEV衣壳结构的认识,为HEV预防与治疗提供了新的针对靶点.     

Luffin P1-KDEL的构建、表达、纯化及鉴定

目的构建植物毒素Luffin P1的原核表达质粒PET32a(+)-Luffin P1,并对其进行诱导表达,以及表达蛋白的纯化及鉴定。方法采用基因工程技术将重组基因片段Luffin P1克隆到表达载体pET32a(+)中,经酶切及DNA序列分析正确后,转化至表达宿主菌BL21(DE3)中,经IPTG诱导表达Luffin P1融合蛋白,并用His标签抗体对该融合蛋白进行Western blot鉴定,对鉴定正确的融合蛋白进行纯化,肠激酶酶切及再纯化。结果成功的构建了原核表达载体pET32a(+)-Luffin P1,获得了纯度较高的Luffin P1蛋白。结论通过基因工程合成Luffin P1蛋白是成功的,并为进一步对Luffin P1功能研究及制备免疫毒素的弹头鉴定了实验基础。     

抗人木糖基转移酶蛋白多克隆抗体的制备及鉴定

目的：制备兔抗人木糖基转移酶（XT-I）蛋白的多克隆抗体，并对其特异性进行鉴定。方法：应用DNAssist软件分析XT-I蛋白的抗原表位，从中选择优势抗原表位，通过引物优化设计，PCR拼接并扩增相应区域DNA片段，将其插入原核表达载体pGEX-4T-2，转化大肠杆菌ER2566，获得融合表达产物。将纯化的可溶性蛋白GST-XT作为抗原，免疫新西兰大白兔，制备抗XT-I蛋白的多克隆抗体，ELISA间接法测定效价。GST柱亲和层析法纯化抗体，采用Westem blot法鉴定该抗体的特异性和生物学活性。结果：确定XT-I的N端氨基酸序列QKHQPELAKKPPSRQKELLKRKLEQQEKGKG（175-205）为抗原表位，成功构建了重组表达载体，表达融合蛋白GST-XT，并以其为抗原，免疫新西兰大白兔，制备了抗XT-I蛋白的多克隆抗体。ELISA证实其效价高达1：640000：Western blot结果显示：该抗体可与人涎腺腺样囊性癌肺转移细胞株ACC-M中表达的XT-I蛋白特异性结合。结论：获得具有高特异性的兔抗人XT-I蛋白多克隆抗体，为涎腺肿瘤性肌上皮细胞蛋白多糖生物合成的研究提供了特异性抗体。     

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.