NT4-Apoptin-HA2-TAT融合基因重组腺相关病毒的构建及鉴定

目的:构建编码融合基因NT4-Apoptin-HA2-TAT的重组腺相关病毒表达载体.方法:利用限制性内切酶切相应载体后将Apoptin和HA2-TAT连入pUC19/NT4质粒, 再将融合基因NT4-Apoptin-HA2-TAT亚克隆至腺相关病毒的穿梭质粒内, 与辅助质粒pAAV/Ad、 腺病毒质粒pFG140共同转染HEK-293细胞, 通过同源重组获得NT4-Apoptin-HA2-TAT重组腺相关病毒载体, 收集病毒上清, Dot blot法测定其滴度.MTT比色法观察NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体, 对HepG2细胞存活率的影响.结果:经酶切及测序证实克隆出NT4-Apoptin-HA2-TAT融合基因; 得到高滴度的(3.14×1015 pfu/L)重组腺相关病毒表达载体.NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体, 对HepG2细胞有强烈的诱导凋亡作用, 与对照组比较, 处理组细胞的存活率明显降低.结论:通过分子克隆体外重组技术成功制备了NT4-Apoptin-HA2-TAT重组腺相关病毒载体, 为下一步的Apoptin应用于基因治疗奠定了基础.     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。 目的：构建可表达GLP-1的重组腺伴随病毒。 方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。 结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。     

低氧启动腺相关病毒载体的构建及高感染滴度质粒辅助重组腺相关病毒的获取

目的构建低氧启动重组腺相关病毒载体 ,优化重组AAV的条件 ,获取高感染滴度低氧启动重组腺相关病毒 ,为基因治疗缺血性脑血管病提供高效的特异性表达目的基因的载体。方法AAVHelperfree系统购自stratagene ,HEK293细胞购自中国协和医科大学细胞中心 ,XL -10 -GoldE.coli购自stratagene ,胎牛血清 ,细胞培养基DMEM、IMDM、MEM、Hepes均购自Gibco,PIRES -EGFP购自Clontech ,pGL3、荧光素酶检测系统、T4DNA连接酶购自Promega;BamHI等内切酶购自Takala。采用PCR和同尾酶的方法合成5拷贝HRE -CMV -mp,酶切连接构建荧光素酶低氧启动腺相关病毒载体 ;扩增提取病毒载体质粒、辅助质粒和Cap、Rep蛋白质粒 ;大量培养HEK293细胞 ;质粒磷酸钙共转染 ;转染48h收获病毒(氯彷 -PEG8000) ;测定生物滴度、物理滴度、电镜观察病毒和病毒蛋白电泳。测定低氧2 %和正常氧压下荧光素酶的效价。结果获取感染滴度2×1011的低氧启动腺相关病素 ,低氧6h荧光素酶表达量是正常氧压时的59倍。结论重组AAV病毒载体是通过对AAV病毒进行改造 ,将自身的编码基因去除 ,使其能装载治疗基因及表达元件而产生的。rAAV载体具有转染效率高、重复性强 ,不激活免疫反应 ,可重复应用 ,不诱导基因突变的优势。但腺相关病毒载体长期稳定表达目的基因     

融合基因CRT/vGPCR的构建及其在真核细胞膜上的表达

目的:将人钙网蛋白(CRT)基因与病毒G蛋白偶联受体(vGPCR)基因进行基因重组融合,由此将CRT携带到肿瘤细胞膜表面。方法:从pSG5-vGPCR质粒中获得全长vG-PCR基因片段并与CRT全长编码序列的3′端连接形成融合基因,然后将融合基因克隆到真核表达载体pcDNA3.1(+)中。用此质粒转染人A549肺癌细胞后,用RT-PCR检测重组融合基因的表达,用免疫荧光细胞化学和流式细胞术分析鉴定融合蛋白的表达及其在细胞膜上的定位。结果:成功获得重组融合质粒pcDNA3.1(+)-CRT/vGPCR,经DNA序列测定证实融合基因两阅读框(ORF)对接无误。将上述质粒转染A549细胞后,融合基因可在A549细胞中表达并定位到细胞膜上。结论:通过与CRT基因融合重组,具有膜定位能力的vGPCR能将CRT蛋白高效包被到肿瘤细胞膜上。     

pAAV-hSOX9-IRES-tdTomato重组质粒构建腺相关病毒包装

背景：实验为基因治疗退变椎间盘实验的前期部分,旨在构建含免疫荧光的pAAV-hSOX9-IRES-tdTomato重组质粒并应用腺相关病毒包装,为后期体内外实验打下基础。 目的：构建人SOX9基因过表达腺相关病毒pAAV- hSOX9-IRES- tdTomato的包装。 方法：用酶切法将质粒pAAV-IRES- tdTomato和质粒pUC57- hSOX9连接成pAAV-hSOX9-IRES- tdTomato,用质粒共转染方法包装腺相关病毒,感染293AAV细胞,腺相关病毒纯化及用生物学滴度测定法进行滴度测定。 结果与结论：经测序结果BLAST比对分析,pAAV-hSOX9-IRES-tdTomato完全与合成的基因序列hSOX9相符,滴度为1×107TU/mL。结果表明,人SOX9基因过表达腺相关病毒pAAV-hSOX9-IRES- tdTomato包装成功。      

人KLF4基因克隆及重组KLF4慢病毒表达载体的构建?

背景：KLF4基因在细胞分化过程中有重要作用。 目的：构建KLF4基因慢病毒过表达载体。 方法：聚合酶链反应扩增(PCR)目的基因KLF4后插入慢病毒表达载体pCDH中,构建重组载体pCDH-KLF4。通过PCR、双酶切和DNA测序方法对其鉴定,共同转染293NT细胞包装病毒,荧光显微镜观察报告基因的表达情况,收集病毒上清,测定病毒的滴度。将重组的慢病毒感染5637细胞,RT-PCR检测KLF4 mRNA的表达。 结果与结论：重组慢病毒表达载体质粒经限制性酶切和DNA测序分析证实重组慢病毒载体pCDH-KLF4的插入序列完全正确,重组慢病毒载体感染5637细胞后,细胞内KLF4 mRNA高表达。结果证实,实验成功构建KLF4基因慢病毒过表达载体。     

小鼠TFEB基因重组腺相关病毒的构建及鉴定

目的构建小鼠TFEB基因的重组8型腺相关病毒(AAV8),并通过感染293细胞观察TFEB表达情况。方法将pUC57-mTFEB及载体质粒分别进行酶切、连接、转化,得到重组质粒pAAV-CMV-mTFEB,并进行PCR鉴定及测序;将鉴定正确重组质粒,进行AAV8病毒载体包装;正确包装病毒载体后,感染293细胞,通过Western blot观察TFEB基因在293细胞表达情况。结果酶切鉴定和测序结果证明了pAAV-CMV-mTFEB病毒包装成功;病毒滴度4.65×10~(12)vg/m L;细胞实验提示相比对照组,实验组TFEB基因表达水平升高6倍余(P0.01)。结论 rAAV2/8-CMV-mTFEB构建及包装成功,并可以有效感染和表达。     

重组骨形成蛋白4/7融合基因腺相关病毒载体的构建与鉴定

背景：有研究表明骨形成蛋白(bone morphogenetic protein,BMP)异源二聚体比同源二聚体的活性高20倍,但相关BMP 4/7融合基因的相关病毒载体的构建至今少有报道。 目的：构建重组人BMP-4/7 融合基因腺相关病毒(adeno-associated virus,AAV)载体并检测其表达。 方法：扩增BMP-4、7成熟肽基因;分别构建质粒pGEM-BMP-4和pGEM-BMP-7;采用DNA连接法获取BMP-4/7融合基因,克隆获得pGEM-BMP-4/7质粒,进行基因测序。AAV颗粒转染HEK293细胞,收获病毒载体AAV-BMP-4/7。用SDS-PAGE电泳检测BMP-4/7融合基因蛋白在大肠杆菌的表达。用不同感染复数的AAV-BMP-4/7转染骨髓间充质干细胞3 d,提取细胞总RNA和总蛋白,进行RT-PCR和ELISA法检测融合基因BMP-4/7在骨髓间充质干细胞中的表达。 结果与结论：PCR 电泳,酶切鉴定及测序结果表明,装入pSNAV质粒中的BMP-4/7 基因正确,pSNAV-BMP-4/7重组成功。RT-PCR检测到骨髓间充质干细胞BMP-4/7融合基因的转录条带,ELISA检测显示经AAV转染的骨髓间充质干细胞中BMP-4/7蛋白随着感染复数的增加表达逐渐增高(P < 0.01)。结果证实,实验成功构建重组骨形成蛋白4/7融合基因腺相关病毒载体。     

1b型丙型肝炎病毒NS3-4b重组腺相关病毒载体的构建及表达

目的 构建1b型丙型肝炎病毒(HCV) NS34b基因重组腺相关病毒载体并了解其在HEK 293细胞中的表达,为进一步研究HCV重组腺相关病毒疫苗及其树突状细胞疫苗奠定前期基础.方法 收集基因1b型的丙型肝炎病人血清,用RT-PCR的方法扩增NS3-4b全长片段,与腺相关病毒的表达载体pAAV.CMV.eGFP重组,构建pAAV.CMV.HCV.NS3-4b重组表达载体,转染HEK293细胞,检测其蛋白表达情况.结果 PCR扩增获得的NS3-4b条带与预期大小(2838 bp)一致,重组质粒经双酶切和测序证实NS3-4b基因已重组成功,重组质粒转染HEK 293细胞后Western Blot图可见有目的蛋白表达.结论 成功构建腺相关病毒重组HCV NS3-4b载体并且其能在真核细胞中表达.     

mTOR shRNA慢病毒载体的构建及包装

目的:构建mTORshRNA慢病毒表达载体,为探讨RNA干扰技术抑制T细胞mTOR基因的相关研究奠定基础.方法:设计合成针对小鼠mTOR基因的shRNA片段,与酶切后的pLKD.UBC.GFP.U6载体片段进行连接并转化DH5α,提取阳性克隆质粒,测序,转染293T细胞.通过GFP表达水平测定病毒滴度.结果:DNA测序结果及PCR鉴定证实成功构建小鼠mTOR-shRNA重组慢病毒载体,对其进行包装后测定慢病毒滴度为1.38E+ 08 TU/ml.结论:成功构建并包装小鼠mTOR-shRNA重组慢病毒表达载体.     

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 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.     

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.