抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达

目的 尝试将１株来源于人源性噬菌体抗体库的抗ＨＢｓＡｇ人Ｆａｂ,转换成完整的抗ＨＢｓＡｇ人ＩｇＧ。方法 采用重叠ＰＣＲ方法,在抗ＨＢｓＡｇ人Ｆａｂ和Ｖκ的ＶＨ基因的５’端接上人工合成的人κ链的前导序列,构建表达完整ＩｇＧ１的真核表达载体,转染ＧＨＯ（ＤＨＦＲ＾－）细胞,用ＥＬＩＳＡ、ＲＴ－ＰＣＲ和免疫印迹检测抗ＨＢｓＡｇ人ＩｇＧ的表达。通过ＤＨＦＲ／ＭＴＸ体系及金属离子诱导提高Ｉｇ表达。结果：获得     

随机化CDR3抗体库的构建及不经免疫制备抗体的初步探索

为探索利用噬菌体抗体库技术不经免疫制备抗体，在已有的抗乙肝病毒表面抗原（ＨＢｓ）人Ｆａｂ段基因的基础上，通过ＰＣＲ引入突变，对其ＶＨＣＤＲ３中１２个氨基酸残基（９５～１００Ｊ）进行了完全随机化，对９４和１００Ｋ位残基进行了有限随机化，构建了半合成噬菌体抗体库，库容为２×１０８，５８％含有随机化ＣＤＲ３序列，用无关抗原小鼠ＩｇＧ对该抗体库进行筛选，获得了三个可与小鼠ＩｇＧ结合而不与ＨＢｓ反应的克隆，经测定其ＣＤＲ３的ＤＮＡ序列，发现这三个克隆具有相同的ＣＤＲ３，证明单纯改变ＶＨＣＤＲ３可以得到新的特异性抗体，从而绕过免疫制备抗体。     

将抗体库所获抗-HBs Fab转换为全长人IgG

目的 将大肠杆菌表达的抗－乙肝表面抗原（ＨＢｓ）人Ｆａｂ转换为真核表达的全长人ＩｇＧ１。方法 用重叠ＰＣＲ法将人工合成的人Ｖｋ前导序列拼到抗－ＨＢｓ的ＶＨ和ＶＫ上,构建人ＩｇＧ１真核表达载体。转染真核细胞,通过ＥＬＩＳＡ、ＲＴ－ＰＣＲ、免疫印迹检测抗－ＨＢｓ人ＩｇＧ的表达。结果 用轻链和重链同时表达的单一载体在ＣＨＯ细胞中获得了抗－ＨＢｓ人ＩｇＧ的表达。结论 通过噬菌体抗体库所获Ｆａｂ段可经拼接人     

从人源性噬菌体抗体库分离出1株含有异常序列的抗HBsAg Fab克隆

曾从人源性噬菌体抗体库中筛选出１株抗人乙肝表面抗原的Ｆａｂ我隆，为了筛选出新的抗ＨＢｓＡｇＦａｂ段，采用抗原屏蔽法，用已得到的Ｆａｂ段封闭相应的抗原决定基，对该抗体库进行了再次筛选，得到了１株新的人抗ＨＢｓＡｇＦａｂ段克隆经序列分析发现其轻链可变区基因来源于Ｖｋ１亚群和Ｊｋ４基因，重链可变区基因来源于ＶＨ１亚群和ＪＨ４基因，但在ＶＨ第７７位和第７８位氨基酸基之间出现了７个多余的氨基酸残基，经对基因     

从噬菌体抗体库克隆抗人RBC单链抗体基因

目的用噬菌体抗体库技术克隆抗人红细胞（ＲＢＣ）的单链抗体（ＳｃＦｖ）基因。方法以人ＲＢＣ免疫小鼠，取脾细胞，ＲＴ－ＰＣＲ法扩增ＶＨ和Ｖκ基因，组装到ＳｃＦｖ－噬菌体抗体表达载体中，构建了噬菌体抗体库，以人ＲＢＣ为固相抗原，对该库进行了四轮“吸附－洗脱－扩增”筛选，获得了抗人ＲＢＣ的ＳｃＦｖ基因。结果ＤＮＡ序列分析表明其可变区基因分别属于小鼠ＶＨⅢ亚群和ＶκⅤ亚群。结论所表达的ＳｃＦｖ可与不同人ＲＢＣ结合，与ＡＢＯ血型抗原无关，可用于构建快速血凝诊断的工程抗体     

人源抗人免疫缺陷病毒1型噬菌体抗体库的构建及筛选

目的构建人免疫缺陷病毒１型（ＨＩＶ－１）特异性噬菌体抗体库，制备人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体。方法以半巢式聚合酶链反应（ＰＣＲ）从ＨＩＶ－１感染者外周血单个核淋巴细胞中扩增抗体重链Ｆｄ和轻链（ｋ）基因，与噬菌体载体ｐＣｏｍｂ３连接，构建噬菌体抗体Ｆａｂ组合文库。对抗体库进行３轮吸附－洗脱－扩增的亲和选择后，以ＥＬＩＳＡ法筛选抗ＨＩＶ－１ｇｐ１２０噬菌体抗体，并进行ＤＮＡ序列分析和Ｆａｂ的可溶性表达。结果半巢式ＰＣＲ有效地扩增出Ｆｄ和ｋ基因，以此构建成容量为１９５×１０７的噬菌体抗体库。３轮亲和选择使特异性抗体得到高度富集，抗ＨＩＶ－１ｇｐ１２０噬菌体抗体阳性克隆占３２％。对一阳性克隆抗体基因ＣＨ１和ＣＬ部分ＤＮＡ序列进行了测定，并在大肠杆菌表达出可溶性Ｆａｂ。结论抗ＨＩＶ－１特异性噬菌体抗体库的构建和人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体的制备为今后筛选抗ＨＩＶ中和抗体奠定了基础，具有重要的应用价值。     

嵌合抗CD20抗体Fab片段在大肠杆菌中的表达及活性鉴定

目的：构建抗ＣＤ２０嵌合抗体Ｆａｂ片段表达载体,并在大肠杆菌中进行高效可溶性分泌表达。结果：利用ＰＣＲ方法从抗ＣＤ２０单链抗体（ＳｃＦｖ）表达载体上扩增抗ＣＤ２０抗体轻链可变区基因（ＶＬ）、重链可变区基因（ＶＨ）,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ表达载体ｐＹＺＦ中,构建抗ＣＤ２０Ｆａｂ表达载体ｐＹＺＦ１ｃｄ２０,并在２７Ｃ７菌中高效表达。结果：经Ｆａｂ表达载体转化的２７Ｃ７菌株,进行表达培养,经分     

半合成抗体库的构建及鉴定

目的构建半合成抗体库,不经免疫制备抗体。方法通过ＰＣＲ法,从人胎肝ＤＮＡ扩增基因组ＶＨ段基因,从Ｆｄ基因制备含有不同长度随机化ＣＤＲ３的ＦＲ３－ＣＤＲ３－Ｊ－ＣＨ１片段,然后用重叠ＰＣＲ法构建Ｆｄ库,与人轻链基因库组合构建成半合成抗体库。结果通过各种组合多次建库,获得总容量为４×１０８的半合成抗体库,其Ｆｄ段和轻链基因的重组率均大于９０％,Ｆａｂ表达率为５０％。挑选２０个克隆测定ＣＤＲ３序列符合所设计的随机序列,用三种不同抗原进行筛选均得到了特异性噬菌体抗体。结论所构建的半合成噬菌体抗体库可以用于不经免疫制备抗体     

工程化人源性抗-HBs Fab抗体的制备、纯化和鉴定

用获得的表达人源性抗 ＨＢｓＦａｂ片段的大肠杆菌,发酵表达该抗体的可溶Ｆａｂ片段。以羊抗人Ｆａｂ抗体偶联ＨｉＴｒａｐ柱,用Ａｃｔｉｓｅｐｅｌｕｔｉｏｎｍｅｄｉｕｍ作为洗脱液,在ＦＰＬＣ上纯化。用ＳＤＳ ＰＡＧＥ及蛋白印迹法分析、鉴定,用固相放兔法检测其活性单位。蛋白印迹及ＥＬＩＳＡ显示转化菌株有抗 ＨＢｓＦａｂ片段的表达。经ＦＰＬＣ纯化的抗体Ｆａｂ片段呈单一峰,与抗 ＨＢｓ阳性血清相似。纯化后的抗体Ｆａｂ片段达到电泳纯,具有较高的活性。     

从人源单链抗体库中筛出具有抗原结合活性的重链可变区和更小抗体片段

目的：用噬菌体呈现技术结合体外致敏法构建了人源单链抗体库,并用大肠癌相关抗原ＣＡＨｂ３筛选出抗大肠癌噬菌体抗体克隆ＥＬ５Ｄ、ＥＬ６Ｄ和Ｃａ１２,测定３个克隆的核苷酸序列,并研究其免疫活性。方法：采用抗体克隆扩增、测序分析、免疫组化法测定活性。结果：抗体基因插入片段分别为２２０、５００、５００ｂｐ。测序结果表明,ＥＬ６Ｄ和Ｃａ１２属ＶＨ５ＤＪＨ４,仅为ＶＨ结构亚单位,而ＥＬ５Ｄ属Ｖκ的Ｊκ１,仅为ＦＲ３ＣＤＲ３ＦＲ４结构。三者均能与人结直肠癌细胞和组织反应,与其亲本鼠源单抗Ｈｂ３的结合特性一致。３个抗体克隆已列入国际基因库,序号为ＡＦ０５１１６５ＡＦ０５１１６７。结论：短的人源抗体片段具有潜在临床应用价值。     

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.