重组铜绿假单胞菌外毒素PE38KDEL的克隆、表达与活性检测

目的对铜绿假单胞菌外毒素A的编码序列进行改造,以获得相对分子质量小、活性强的重组毒素PE38KDEL。方法基于铜绿假单胞菌外毒素A的编码序列的高GC含量,通过PCR反应和酶切技术,构建重组毒素PE38KDEL基因,并于大肠杆菌表达,用Westernblot分析表达产物。为检测其生物活性,将其与靶向分子IL4突变体cpIL4(13D)融合,构建融合蛋白表达载体,并于大肠杆菌表达,表达产物经亲和色谱和阴离子交换色谱纯化后,用MTT法检测其对表达IL4受体的黑色素瘤细胞LiBr的细胞毒性。结果成功构建了PE38KDEL基因,并在大肠杆菌BL21(DE3)中得到了高效表达,表达量占细胞全蛋白的21%左右;构建了融合蛋白cpIL4(13D)PE38KDEL,并于大肠杆菌AD494(DE3)中得到高效表达,纯化后融合蛋白的纯度达95%以上,细胞毒实验证明其对黑色素瘤细胞LiBr具有良好的杀伤作用,这说明改造后的PE38KDEL是具有细胞毒效应的。结论重组毒素PE38KDEL的构建,为各种导向药物的构建奠定了基础。     

一种新型融合毒素IL15-PEΔ293的构建与体外活性测定

目的构建IL15与铜绿假单胞菌外毒素突变体(PEΔ293)的融合蛋白IL15PEΔ293,并且对该融合毒素进行初步的体外活性测定。方法将人IL15基因片段与PEΔ293的基因按正确的阅读框架融合,定向克隆在pET16b表达载体T7启动子的下游,得到表达质粒pETIL15PEΔ293。从大肠杆菌相应重组菌株中通过Ni2 NTA亲和层析纯化出融合蛋白IL15PEΔ293。以IL15受体(IL15R)阳性红白血病细胞系K562、多药耐药细胞系K562AO2以及IL15R阴性细胞系Jurkat为靶细胞测定IL15PEΔ293的生物活性。结果限制性分析和测序鉴定证明融合蛋白IL15PEΔ293表达质粒pETIL15PEΔ293构建正确。该质粒在大肠杆菌BL21(DE3)pLysS中成功表达了融合蛋白IL15PEΔ293。利用Ni2 NTA亲和层析从大肠杆菌菌体总蛋白中纯化出重组蛋白。该融合蛋白对IL15R阳性细胞K562和K562AO2有明显的剂量依赖的细胞毒作用,而对IL15R阴性细胞Jurkat没有表现剂量依赖的细胞毒作用。结论融合蛋白IL15PEΔ293表达质粒构建成功,在大肠杆菌中表达纯化后的新型融合蛋白IL15PEΔ293对IL15R阳性细胞有良好的靶向杀伤活性。     

HSV-1 UL15 exon-Ⅱ的原核表达及抗体制备

目的 构建pQE-HSV-1 UL15 exon Ⅱ原核表达载体,在大肠埃希菌中高效表达并免疫家兔制备抗体.方法 以HSV-1感染后的Vero细胞总RNA为模板,通过RT-PCR扩增HSV-1 UL15 exon-Ⅱ基因,克隆入原核表达载体pQE-31;重组质粒经酶切、测序鉴定无误后转化大肠埃希菌JM109,IPTG诱导表达;SDS-PAGE检测蛋白表达情况;表达蛋白用Ni-NTA亲和层析纯化及透析复性处理后免疫家兔制备特异性抗体,抗体的免疫原性用Western blotting进行检测.结果 通过RT-PCR扩增获得了HSV-1 UL15 exon-Ⅱ全长基因;构建的pOE-HSV-1 UL15 exon Ⅱ重组质粒经酶切及测序鉴定无误;阳性转化菌株经WIG诱导后SDS-PAGE电泳显示表达蛋白的相对分子质量约为43 000,与理论值一致,蛋白表达率约为35%～40%;表达蛋白经纯化及透析复性后免疫家兔获得的抗血清经Western blotting鉴定具有较好的特异性和敏感性.结论 成功构建了pQE-HSV-1 UL15 exon Ⅱ原核表达载体,诱导蛋白表达并进一步用表达的蛋白制备了特异性抗体,为HSV-1末端酶全长基因表达情况的鉴定及最终构建抗病毒组装药物的分子筛选模型奠定了前期实验基础.     

H37Rv结核分枝杆菌蛋白脯氨酸-谷氨酸8(PE8)的表达及其兔多克隆抗体制备

目的克隆Rv1040c结核分枝杆菌脯氨酸-谷氨酸8(PE8)基因、构建pET28a-PE8重组载体和表达纯化PE8蛋白,制备抗PE8多克隆抗体。方法利用重组克隆技术,将PE8基因克隆至原核表达载体pET28a,测序鉴定后,转化至大肠杆菌BL21(DE3),用0.5 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达、稀释复性与纯化。用纯化PE8蛋白免疫新西兰大白兔,制备多克隆抗体,间接ELISA和Western blot法进行效价及特异性鉴定。结果成功构建pET28a-PE8原核表达载体, ITPG诱导后PE8蛋白在大肠杆菌主要以包涵体的形式表达,复性后纯化PE8蛋白纯度达90%,纯化多克隆抗体效价为1∶430 080以上,能与PE8蛋白发生特异性反应。结论大肠杆菌表达的PE8重组蛋白免疫新西兰大白兔获得高效价的多克隆抗体。     

重组人IL-6D24-PE40外毒素融合蛋白的构建及其生物学效应

目的 构建重组人白细胞介素6－绿脓杆菌外毒素融合蛋白IL－6D24－PE40,以选择性杀伤高表达IL－6受体（IL－6R）的肿瘤细胞和白血病细胞。方法 采用重叠延伸的基因融合技术将N－末端缺失24个氨基酸的重组人白细胞介素6（IL－6D24）cDNA与缺失细胞结合区的绿脓杆菌外毒素PE40基因进行融合,构建IL－6D24－PE40融合基因。利用HB101／pBV220表达系统,实现了IL－6D24－PE40融合蛋白在大肠杆菌中的高效表达,经MonoQ柱进行层析纯化,采用MTS法检测细胞毒活性。结果 IL－6D24－PE40融合蛋白在大肠杆菌中的表达水平达到40％－60％。包涵体蛋白经分离、复性及纯化得到纯度＞95％的融合蛋白。Western blot证明,纯化的融合蛋白与IL－6抗体及PEA抗体均发生特异性结合。细胞毒活性检测证实,IL－6D24－PE40融合蛋白能高度特异地选择性杀伤高表达IL－6R的U937细胞,ID50约为250ng/ml,而对不表达IL－6R的CEM细胞无杀伤作用。结论 IL－6D24－PE40融合蛋白能够选择性杀伤高表达IL－6R的靶细胞,有希望成为导向治疗高表达IL－6／IL－6R系统的某些白血病和其他肿瘤的新型导向药物。     

一种新型融合毒素IL15—PE△293的构建与体外活性测定

目的构建IL-15与铜绿假单胞菌外毒素突变体(PE△293)的融合蛋白IL15—PE△293，并且对该融合毒素进行初步的体外活性测定。方法将人IL-15基因片段与PE△293的基因按正确的阅读框架融合，定向克隆在pET16b表达载体17启动子的下游，得到表达质粒pET-IL15-PE△293。从大肠杆菌相应重组菌株中通过Ni^2 -NTA亲和层析纯化出融合蛋白IL15-PE△293。以IL-15受体(IL-15R)阳性红白血病细胞系K562、多药耐药细胞系K562／AO2以及IL-15R阴性细胞系Jurkat为靶细胞测定IL15-PE△293的生物活性。结果限制性分析和测序鉴定证明融合蛋白IL15-PF△293表达质粒pET-IL15-PE△293构建正确。该质粒在大肠杆菌BL21(DE3)pLysS中成功表达了融合蛋白IL15-PE△293。利用Ni^2 —NTA亲和层析从大肠杆菌菌体总蛋白中纯化出重组蛋白。该融合蛋白对IL-15R阳性细胞K562和K562／AO2有明显的剂量依赖的细胞毒作用，而对IL-15R阴性细胞Jurkat没有表现剂量依赖的细胞毒作用。结论融合蛋白IL15-PE△293表达质粒构建成功，在大肠杆菌中表达纯化后的新型融合蛋白IL15-PE△293对IL-15R阳性细胞有良好的靶向杀伤活性。     

重组汉滩病毒核蛋白的纯化及鉴定

目的：纯化重组表达的汉滩病毒核蛋白。方法：重组菌经IPTG诱导后，表达的目的蛋白为带有谷胱甘肽转硫酶（GST）标签的融合蛋白，并以包涵体形式存在。将包涵体变性、复性后，采用Glutathione Sepharose 4B亲和色谱对核蛋白进行纯化，并用夹心ELISA和Western blot检测纯化蛋白。结果：表达产物第一次过柱亲和层析后可去除杂蛋白，获得纯化的核蛋白与谷胱甘肽转硫酶的融合蛋白，再经凝血酶酶切，第二次过柱亲和层析后获得的穿过峰为核蛋白，洗脱峰为GST。纯化的融合蛋白和纯化的核蛋白均为SDS－PAGE单点纯，并具有良好的抗原活性。结论：Glutathione Sepharose 4B亲和色谱纯化重组汉滩病毒核蛋白是一种较为有效的方法。     

TAT-EGFP融合蛋白的表达纯化及穿膜活性的研究

目的在大肠埃希菌中高效表达TAT-EGFP融合蛋白并鉴定纯化,然后进行穿膜活性的研究。方法以本室前期构建的重组质粒pQE-EGFP为模板,采用PCR方法特异性扩增TAT-EGFP基因序列,随后克隆于原核表达载体pQE-31,所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109,IPTG诱导表达;SDS-PAGE和Western blotting鉴定表达蛋白质,经Ni2+-金属螯合亲和层析纯化,将纯化蛋白加入体外培养的小鼠黑色素瘤B16细胞中,荧光显微镜下观察TAT蛋白的穿膜活性。结果成功构建了pQE-TAT-EGFP重组质粒的,转化大肠埃希菌JM109,经IPTG诱导,目的蛋白表达率为25%,SDS-PAGE、Western blotting初步测定目的蛋白的相对分子质量(Mr)约为30160,纯化得到了目的融合蛋白TAT-EGFP。并在体外培养的B16细胞中证实TAT-EGFP融合蛋白穿透生物膜的能力。结论通过对TAT-EGFP融合蛋白穿膜活性的分析,证实了TAT具有穿膜活性,为TAT-PEⅢ融合蛋白抑瘤活性的研究提供了理论依据,也为生物大分子药物进入组织细胞内发挥治疗作用奠定了基础。     

大鼠IP-10-PE38 KDEL重组免疫毒素真核表达载体的构建及表达

目的 构建大鼠干扰素诱导蛋白10(rIP-10)基因和铜绿假单胞菌外毒素衍生物(PE38KDEL)基因的真核融合表达载体,并研究其在NIH 3T3细胞中的表达情况.方法通过PCR获得本实验需要的rIP-10基因片段,通过酶切原核表达载体PTKL459K-IL-4-PT38KDEL获得铜绿假单胞菌外毒素衍生物PE38KDEL基因,再通过适当的酶切及连接反应,构建rIP-10与PE38KDEL融合基因的真核表达载体pcDNA3.1( )-rIP-10-PE38KDEL.重组质粒经限制性性内切酶,PCR及DNA序列测定证实构建成功后,用PolyFect脂质体转染NIH 3T3细胞,然后用免疫荧光技术检测rIP-10-PE38KDEL融合蛋白在NIH 3T3细胞的表达.结果酶切分析、PCR及DNA序列测定证实,rIP-10-PE38KDEL融合基因被成功克隆人真核表达质粒载体pcDNA3.1( ),免疫荧光技术检测证实重组基因可在NIH 3T3细胞表达.结论 rIP-10-PE38KDEL融合基因可在NIH 3T3细胞中表达,为进一步研究其对自身免疫病的Th1细胞靶向毒性及临床应用奠定基础.     

抗铜绿假单胞菌 PcrV 蛋白单克隆抗体的筛选和功能验证

目的:获得能应用于治疗铜绿假单胞菌(Pseudomonas aeruginosa)感染的抗PcrV 蛋白全人源单克隆抗体。方法:以铜绿假单胞菌PAO1 基因组为模板,PCR 扩增PcrV 基因并构建重组表达载体pET-28a-PcrV,转化至大肠杆菌BL21(DE3),利用IPTG 诱导表达,并通过镍离子螯合树脂亲和纯化PcrV 蛋白。利用噬菌体抗体库筛选PcrV 结合的噬菌体克隆并进行序列测定,以测序正确的阳性克隆质粒为模板,PCR 扩增该抗体的重链可变区基因和轻链可变区基因并构建重组表达载体转染至293E 细胞,收取培养细胞上清,并利用Protein A 树脂亲和纯化抗体。利用ELISA 实验检测抗体亲和力,并通过小鼠铜绿假单胞菌感染肺炎模型验证抗体活性。结果:获得重组蛋白PcrV,通过噬菌体展示技术筛选并制备了一株全人源抗PcrV单克隆抗体YG5。ELISA 实验结果表明YG5 抗体具有较高的亲和力(EC50 =61 ng/ ml),进一步的实验结果表明,YG5 能有效抵抗铜绿假单胞菌感染,降低小鼠的死亡率。结论:靶向铜绿假单胞菌PcrV 蛋白的全人源单克隆抗体YG5 能够有效地抑制铜绿假单胞菌致病性,降低其对机体感染,有可能成为抗铜绿假单胞菌感染的治疗性抗体。     

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.     

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.     

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.     

'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 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.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.