1．3倍乙型肝炎病毒全基因真核表达载体的构建及其在Bew0细胞中的表达

目的构建1．3倍乙型肝炎病毒（HBV）全基因的真核表达载体pcDNA3．1（＋）一HBV1．3。并观察其在Bewo细胞中的表达。方法以质粒pMD18T—HBV上的HBVDNA序列为模板,构建1-3倍HBV全基因序列,将其插入到真核表达载体pcDNA3．1（＋）,用酶切、PCR及测序鉴定,并把该载体转染Bewo细胞,以Western免疫印迹、微粒子酶免疫分析法（MEIA）和荧光定量PCR法分别检测胞内和上清中HBsAg、HBeAg蛋白的表达及HBVDNA水平。结果通过酶切、PCR及测序鉴定,成功构建1．3倍HBV全基因表达载体．该载体可以在Bewo细胞系中表达和分泌HBsAg与HBeAg。并可检测到高水平的HBVDNA。结论成功构建了1．3倍HBV全基因真核表达载体pcDNA3．1（＋）一HBV1．3,为研究胞内HBV宫内感染奠定了基础。     

荧光素酶标记的HBV细胞模型的建立

建立萤火虫荧光素酶(Fluc)为报告基因标记的乙型肝炎病毒(HBV)体外感染的细胞模型。利用分子亚克隆技术,以pAAV/1.2HBV质粒为模板,PCR扩增带有反向重复序列(ITR)的1.2倍HBV基因组片段,反向插入真核表达载体pGL3-CP-Fluc中,构建Fluc标记的HBV基因组表达质粒pGL3-CP-Fluc-HBV1.2,并将此质粒转染至Huh-7细胞中。全自动免疫分析仪进行HBsAg、HBeAg的定量检测,生物发光检测仪检测Fluc其在细胞的表达水平。成功构建了1.2倍HBV全基因真核表达载体,稳定转染Huh-7细胞后,质粒在细胞中高水平表达Fluc,并可以正常分泌HBsAg、HlBeAg。重组质粒pGL3-CP-Fluc-HBV1.2能在Huh-7细胞中表达,其稳定转染的细胞可作为一种新型的HBV体外感染模型,为抗病毒药物研究奠定基础。     

HBV基因组各基因真核表达载体的构建及转染

目的 构建HBV基因组各基因真核表达载体,转染HepG2细胞,建立稳定转染的HepG2细胞系.方法 采用PCR方法,以HBV全基因组质粒为模板扩增HBV基因的各基因片段,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1( ),经酶切和测序鉴定后,用脂质体转染法转染HepG2细胞,通过G418筛选,建立稳定转染的HepG2细胞系,用细胞流式技术及细胞免疫组化技术检测HBV各基因产物在细胞内的表达.结果 成功构建了pcDNA3.1( )/HBs、pcDNA3.1( )/HBc、pcDNA3.1( )/HBe、pcDNA3.1( )/HBp、pcDNA3.1( )/HB-preS1、pcDNA3.1( )/HB-preS2、pcDNA3.1( )/HBx真核表达载体,并建立了稳定转染的HepG2细胞系,成功地表达目的 基因.结论 真核表达载体成功构建和稳定转染HepG2细胞系的建立为进一步研究各基因的功能奠定良好的实验基础.     

TRAIL诱导HBV转染肝癌细胞BEL-7402凋亡的作用及机制研究

目的　研究肝癌细胞BEL- 74 0 2感染HBV前后,TRAIL诱导其凋亡的敏感度变化及作用机制。方法　构建含1.1倍HBV全基因组的真核表达载体pcDNA3 1.1HBV ,转染人肝癌细胞BEL -74 0 2 ,G4 18稳定筛选,建立HBVadr亚型体外转染的细胞模型。原位末端标记(TUNEL)检测TRAIL诱导的凋亡反应,并通过设立TRAIL联合其可溶性受体sDR5 (sDR5与TRAIL结合后,可特异性阻断TRAIL诱导凋亡) ,以证实该凋亡是由TRAIL特异性诱导的。琼脂糖凝胶电泳(DNAladder)检测染色体DNA的断裂情况。流式细胞术、半定量逆转录聚合酶链反应检测HBV感染前后细胞表面TRAIL各受体的表达。双荧光素酶报告基因系统检测抑制凋亡的核转录因子NF κB的活性。结果　成功构建了包含HBV全基因组的真核表达载体pcDNA3 1.1HBV ,转染BEL 74 0 2、经G4 18稳定筛选后得到HBVadr亚型转染的细胞模型BEL 74 0 2 1.1HBV。TRAIL诱导BEL 74 0 2、BEL 74 0 2 pcDNA3及BEL 74 0 2 1.1HBV的凋亡率分别为30 .5 %、2 9.8%和76 % (P <0 .0 1) ;经sDR5特异性阻断后,凋亡率分别为0 .9%、0 .8%和0 .9% ,证明凋亡是由TRAIL特异性诱导的。TRAIL作用2 4h后,琼脂糖凝胶电泳可见BEL- 74 0 2 1.1H-BV较BEL- 74 0 2、BEL 74 0 2 pcDNA3染色体DNA断裂的现象更为明显。无论RNA水平还     

应用RNA干扰抑制乙肝病毒X基因促HepG2细胞凋亡的作用

目的:构建靶向乙肝病毒X基因(HBX)的shRNA表达载体pSilencer3.1-shHBX,并应用其体外抑制HBX促HepG2细胞凋亡的作用.方法:设计并构建靶向HBX的shRNA表达载体pSilencer3.1-shHBX,脂质体转染法将HBX表达载体pcDNA3.1-HBX与pSilencer3.1-shHBX共转染人肝癌HepG2细胞,培养72小时后,分别以RT-PCR、Western blot和免疫荧光检测HBX的表达量.Annexin V-FITC/PI双染经流式细胞仪检测细胞的凋亡变化.结果:重组质粒pSilencer3.1-shHBX酶切鉴定和测序结果均与设计的一致.该质粒使HBX基因mRNA表达量降低了47.1%,进而使HBx蛋白表达量降低了58.9%,HBx蛋白荧光强度减弱,并使HBX 诱导的HepG2细胞凋亡率降低了52.11%.而阴性对照质粒无此作用.结论:成功构建靶向HBX shRNA表达载体pSilencer3.1-shHBX,并可应用其抑制HBX促HepG2细胞凋亡的作用.     

鸟苷结合蛋白1对柯萨奇病毒B3及乙型肝炎病毒体外介导的不同作用

目的构建人鸟苷结合蛋白1（hGBP-1）真核表达质粒，观察hGBP-1体外对柯萨奇病毒B3（CVB3）和乙型肝炎病毒（HBV）的抑制作用。方法长链RT-PCR扩增全长hGBP-1编码区基因，克隆到pCR2．1TA克隆载体，再亚克隆到pcDNA3．1（-）真核表达载体。体外转染HepG2细胞和Hela细胞，Western blot检测hGBP-1的表达。然后分别观察转染细胞中hGBP-1对HBV体外复制子pHBV1．3和CVB3的抑制作用。ELISA检测共转染HepG2细胞培养L清HBsAg、HBeAg水平；Southern blot检测细胞HBVDNA复制中间体。TCID50试验检测Hela细胞培养物中CVB3感染量。结果成功构建hGBP-1真核表达质粒，能在HepG2细胞和HeLa细胞进行高效表达。该质粒与pHBV1．3共转染HepG2细胞，不能抑制HBV复制，HBsAg、HBeAg及HBV DNA复制中间体水平与对照相比都无明显变化。转染质粒在HeLa细胞上对CVB3复制有明显的抑制作用，CVB3感染量显著降低，尤其在低剂量病毒攻击时能完全抑制CVB3复制。结论hGBP-1可能在IFN介导的抗CVB3中起重要作用，但不能抑制HBV的复制。     

MXA蛋白抑制HBV复制的体外研究

目的研究MXA蛋白抑制HBV复制的活性。方法将pcDNA3．1-MXA重组质粒和PU19-1．24HBV重组质粒分别按1：1、2：1共转染HepG2细胞（MXA组），对照组使用空pcDNA3．1、Salon DNA和PU19-1．24HBV重组质粒共转染，3d后Western blot检测MXA蛋白表达，Abbott法检测细胞上清HBeAg和HBsAg分泌量，定量PCR检测上清和胞内HBV DNA水平，统计学分析结果。pcDNA3．1-MXA与PU19-1．24HBV重组质粒共转染HepG2细胞，对照组为pcDNA3．1-MXA重组质粒、PU19空质粒和Salon DNA共转染．3d后裂解细胞Western blot检测MXA蛋白表达。结果Western blot显示MXA组有MXA蛋白表达：与对照组相比，pcDNA3．1-MXA和PU19—1．24HBV重组质粒按1：1转染时，MXA组HBeAg下降27％．上清HBV DNA和细胞内HBV DNA分别下降1个log值和0．6个log值；按2：1比例转染时MXA组HBeAg较对照组下降66％，上清HBV DNA和细胞内HBV DNA水平分别下降1．9个和1．7个log值，差异均具有统计学意义（P〈0．05）。Western blot检测显示MXA蛋白抑制HBV组与对照组MXA蛋白表达没有明显差别。结论MXA蛋白在HepG2细胞具有抑制HBV复制活性，抑制活性与蛋白的表达量相关；在抑制HBV复制过程中MXA蛋白自身可能不发生降解。     

40例乙型肝炎患者HBV逆转录酶基因耐药突变分析与表达载体构建

目的 分析40例慢性乙型肝炎患者血清中HBV逆转录酶区基因耐药相关突变,构建突变基因重组表达载体用于表型耐药分析.方法 从服用抗HBV核苷(酸)类似物的患者血清中提取病毒DNA,PCR扩增HBV RT全长基因,克隆到pGEM-Teasy载体中,随机挑选3～5个克隆进行DNA序列测定,以DNASTAR软件分析RT基因内与核苷(酸)类似物耐药相关的常见突变位点.用Xho Ⅰ和Nco Ⅰ双酶切pGEM-Teasy-RT及pTriEx-HBV(C)构建1.1表达载体,测序正确后转染Huh7细胞,检测HBsAg和HBeAg表达水平.结果 40例患者均分别检出拉米夫定、阿德福韦、恩替卡韦耐药相关的单一或联合突变;成功克隆了96条HBV RT基因,分别带有上述核苷(酸)类似物耐药相关变异的序列;挑选主要突变组合形式的40条RT基因构建pTriEx-HBV(C)1.1表达载体,转染Huh7细胞48 h后在培养上清中检测到HBsAg和HBeAg,表明表达载体构建成功.结论 本研究成功进行了临床患者HBV耐药相关突变分析与突变体重组表达载体构建,为进行表型耐药分析打下了基础.     

mTSARG3真核表达载体的构建及其稳定转染细胞系的建立

目的:构建小鼠mTSARG3基因真核表达载体,转染小鼠精原细胞系GC-1,建立稳定转染mTSARG3的GC-1细胞系,利用流式细胞技术(FCM)进行初步功能研究。方法:应用RT-PCR从小鼠睾丸cDNA文库中扩增mTSARG3的开放阅读框(ORF),并将PCR产物插入到pGEM-TEasy载体中测序验证。随后,经NotI和Hind III酶切后将目的片段进一步克隆到pcDNA3.1 Hygro(-)真核表达载体中。将经过测序验证的pcDNA3.1 Hygro(-)/mTSARG3表达质粒转染GC-1细胞,通过潮霉素筛选建立mTSARG3稳定转染的GC-1细胞系。RT-PCR和Western blot检测mTSARG3在稳定转染的GC-1细胞系中的表达;FCM观察转染pcDNA3.1 Hygro(-)/mTSARG3重组质粒)对GC-1细胞周期和凋亡的影响。结果:成功构建了pcDNA3.1 Hygro(-)/mTSARG3表达质粒,建立了稳定转染的GC-1细胞系。RT-PCR和Western blot检测结果发现,mT-SARG3在GC-1细胞系中成功表达。进一步通过FCM检测分析发现,转染mTSARG3可促进GC-1细胞增殖,抑制细胞凋亡。结论:mTSARG3真核表达载体的成功构建和稳定转染重组质粒GC-1细胞系的建立为进一步体外研究mTSARG3的功能奠定了基础。     

抗人B细胞淋巴瘤DNA疫苗的构建

目的 :探讨人B细胞淋巴瘤活检组织中肿瘤细胞膜表面免疫球蛋白VH(smIgVH)基因片段能否在动物体内激发特异性抗独特型抗体。方法 :以RT PCR法获得IgVH 基因片段 ,以小鼠单核细胞趋化因子 (MCP 3)基因作为佐剂分子 ,进行重组PCR获得MCP 3和VH 基因片段的融合基因 ,克隆在真核表达载体 pcDNA3.1中 ,构建DNA疫苗质粒 pcDNA/MCP BVH。通过脂质体转染验证该质粒在真核细胞COS 7中的表达。结果 :通过上述方法获得了以活检组织肿瘤细胞mIgVH区基因片段 ;成功地构建了DNA疫苗质粒 pcDNA/MCP BVH。体外瞬时转染实验证明 ,该质粒能够在真核细胞COS 7中正确表达。结论 :成功地构建重组表达质粒 pcDNA/MCPBVH,在体外能够正确表达 ,为进一步研制抗B细胞淋巴瘤基因疫苗奠定了基础     

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.