人肿瘤坏死因子穿梭表达载体的构建

应用ＤＮＡ重组技术,将人肿瘤坏死因子（ｒｈＴＮＦ）ｃＤＮＡ插到质粒ＰＲＬ４３９的ＰｐｓｂＡ启动子下游,得到中间载体ＰＲＬ＿ＴＮＦ;再把ＰＲＬ＿ＴＮＦ上含ＰｐｓｂＡ和（ｒｈＴＮＦ）ｃＤＮＡ的片段连到穿梭载体ＰＤＣ＿８上,构建成穿梭表达载体ＰＤＣ＿ＴＮＦ．转化大肠杆菌ＴＧ１后,进行发酵培养．ＳＤＳ＿ＰＡＧＥ分析和免疫印迹检测结果显示ｒｈＴＮＦ获稳定表达,分子量为１７ｋｕ．本研究结果为ｒｈＴＮＦ在蓝藻中的表达提供了技术基础．     

大肠杆菌二硫键异构酶DsbA和脯氨酸异构酶PPIaseA双顺反子表达载体的构建及其在大肠杆菌中的表达

用ＰＣＲ方法获得大肠杆菌二硫键异构酶ＤｓｂＡ的编码基因ｄｓｂＡ和大肠杆菌脯氨酸异构酶ＰＰＩａｓｅＡ的编码基因ｒｏｔ,并将ＤｓｂＡ和ｒｏｔ以双顺反子形式克隆至含有Ｐｔａｃ启动子的表达载体ｐＫＫ２３３－２中。在ＩＰＴＧ的诱导下,ＤｓｂＡ和ＰＰＩａｓｅＡ获得了表达。ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：ＤｓｂＡ、ＰＰＩａｓｅＡ的表达水平分别为占菌体裂解上清液总蛋白质的４．３２％和４．０６％。     

小麦叶绿体psbA基因的克隆及表达

用ＰＣＲ直接从小麦叶绿体基因组扩增到完整的ｐｓｂＡ基因，并构建了ｐｓｂＡ基因的克隆ｐＴＤ１Ⅱ。为研究ｐｓｂＡ基因在Ｅ·ｃｏｌｉ中的表达，将完整的ｐｓｂＡ基因正向插入高表达载体ｐＫＫ２２３－３中构建表达克隆ｐＫＴＤ１。含有重组表达质粒的转化细胞经ＩＰＴＧ诱导后提取总蛋白，经ＳＤＳ－ＰＡＧＥ分析，小麦叶绿体ｐｓｂＡ基因能在Ｅ·ｃｏｌｉ中表达３２ｋＤ的Ｄ１蛋白，表达的Ｄ１蛋白占细菌总蛋白的３％以上。     

大肠杆菌二硫键异构酶DsbA和脯氨酸异构酶PPIaseA双顺反子表达载体的构建及其在大肠杆菌中的表达

用ＰＣＲ方法获得大肠杆菌二硫键异构酶ＤｓｂＡ的编码基因ｄｓｂＡ和大肠杆菌捕氨酸异构酶ＰＰＩａｓｅＡ的编码基因ｒｏｔ,并将ＤｓｂＡ和ｒｏｔ以顺反子形式克隆至含有Ｐｔａｃ启动子的表达载体ｐＫＫ２３３－２中。在ＩＰＴＧ的诱导下,ＤｓｂＡ和ＰＰＩａｓｅＡ获得了表达。     

EXPRESSION OF HUMAN TUMOR NECROSIS FACTOR α IN ANABAENA SP.PCC7120

通过三亲结合转移方式，将含人肿瘤坏死因子α（ＴＮＦ＿α）ｃＤＮＡ和ｐｓｂＡ启动子的鱼腥藻－大肠杆菌穿梭质粒ＰＤＣ＿ＴＮＦ导入单细胞丝状鱼腥藻７１２０中．Ｓｏｕｔｈｅｒｎ杂交结果表明，人肿瘤坏死因子αｃＤＮＡ在鱼腥藻７１２０细胞中能以自主复制形式存在于ＰＤＣ＿ＴＮＦ质粒上．ＳＤＳ＿ＰＡＧＥ和免疫印迹分析结果显示，转ＰＤＣ＿ＴＮＦ的鱼腥藻７１２０可表达分子量约为１７ｋｕ的人ＴＮＦ＿α，其表达量约占藻体蛋白的１６％左右．转ＰＤＣ＿ＴＮＦ的鱼腥藻７１２０粗提液的ＴＮＦ生物学比活性约为２５×１０４ｕ／ｍｇ．     

人胰岛素样生长因子—I的基因克隆融合表达及产物纯化

利用聚合酶链式反应（ＰＣＲ）方法,以ＩＧＦ－ＩｃＤＮＡ为模板,扩增ＩＧＦ－Ｉ的基因,在序更测定确证后,将其定向克隆到载体ｐＥｘＳｅｃｌ上,构建了ＩＧＦ－Ｉ融合表达载体ｐＥｘＩＧＦ,经ＩＰＴＧ诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出约３２．５８％的６ＫＤ融合蛋白,且主要以可溶性形式存在。经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ亲和层析一步纯化后,可获得纯度的９０％的融合蛋白,经Ｗｅｓ     

人粒细胞集落刺激因子在昆虫细胞中的表达

利用苜蓿尺核型多角 体病带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标志的非融合蛋白基因转移载体ｐＢＢ成功地构了重组杆状病毒ＡｃＮＰＶ－Ｇ－ＣＳＦ．在感染重组病毒的草地夜蛾细胞中ｈＧ－ＣＳＦ得到了高效表达。表达产物由ＷｅｓｔｅｒｎＢｌｏｔ检出，其分子量约为１９ＫＤａ。     

人胰岛素样生长因子—Ⅰ的基因克隆,融合表达及产物纯化

利用聚合酶链式反应（ＰＣＲ）方法,以ＩＧＦ－ＩｃＤＮＡ为模板,扩增ＩＧＦ－Ｉ的基因。在序列测定确证后,将其定向克隆到载体ｐＥｘＳｅｃＩ上,构建了ＩＧＦ－Ｉ融合表达载体ｐＥｘＩＧＦ,经ＩＰＴＧ诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出约３２．５８％的２６ＫＤ融合蛋白,且主要以可溶性形式存在。经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ亲和层析一步纯化后,可获得纯度约９０％的融合蛋白,经Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析,免疫学活性呈阳性反应。     

猫儿尿植物的化学成分及其药效学研究

首次从木通科猫儿尿（ＤｅｃａｉｓｕｅａｆａｒｇｅｓｉｉＦｒａｎｃｈ）中分离鉴定了１１种皂甙化合物，经光谱分析及化学方法鉴定，其中５种化合物为新化合物，分别命名为：ＤｅｃａｉｓｏｓｉｄｅＡ，Ｂ，Ｃ，Ｄ，Ｇ；另５种化合物为ＳａｐｏｎｉｎＰＧ，ＤｉｐｓａｃｏｓｉｄｅＢ，Ｋａｌｏｐａｎａｘ－ｓａｐｏｎｉｎＢ，Ｓａｐｏｎｉｎ１１，ＨｅｄｅｒａｓａｐｏｎｉｎＢ和ｓａｐｏｎｉｎＰＪ３，猫儿尿对小鼠移植性肿瘤Ｓ１     

A PROBE OF PROCESS WRITING IN THE TEFL FIELD

ＡＰＲＯＢＥＯＦＰＲＯＣＥＳＳＷＲＩＴＩＮＧＩＮＴＨＥＴＥＦＬＦＩＥＬＤ￥ＧｕｉＱｉｎｇｙａｎｇＡｂｓｔｒａｃｔ：Ｔｈｅｐｒｅｓｅｎｔｐａｐｅｒｉｓｉｎｔｅｎｄｅｄｔｏｍａｋｅａｓｔｕｄｙｏｆｔｈｅｎａｔｕｒｅｏｆｐｒｏｃｅｓｓｗｒｉｔｉｎｇａｎｄｔ...     

Heparin-Argarose亲和层析法分离人甲状腺肿瘤组织中的bFGFs

本文介绍了甲状腺肿瘤组织中碱性纤维母细胞生长因子(basis Fibroblast Growth Factors.bFGFs)的提取过程和分子量鉴定。研究表明:bFGFs对肝素具有强的亲和力,组织匀浆通过Heparin-Argarose亲和层析柱后,用2.0mol/L的NaCl即可洗脱下来,收集其亲和峰值的洗脱组分,经三氯醋酸沉淀后即可得到bFGFs,经Western-blot方法分析其分子量有三种,分别为14KD、17KD、18KD。     

人甲状腺组织中bFGFs的定位研究

目的：研究bFGFs在人甲状腺及其肿瘤组织中的组织学定位。方法：采用组织化学方法。结果：bFGFs存在于甲状腺滤泡上皮细胞中,同正常甲状腺相比较,甲状腺癌组织中存在大量bFGFs。结论：在甲状腺肿瘤（特别是恶性肿瘤）的发生、展过程中,bFGFs起重要作用。     

SDS-PAGE法检测人甲状腺及其肿瘤组织中bFGFs条件的选择

目的：探讨SDS－PAGE法检测人甲状腺及其肿瘤组织中bFGFs（basicFibroblastGrowthFactors）的条件。方法：采用SDS-PAGE电泳法检测人甲状腺及其肿瘤组织中的bFGFs．结果：分离胶浓度为12．5％,胶厚为0．5cm,电流强度为30mA,样品量为5－6ul,标准bFGFs上样量为20ul较适全人甲状腺及其肿瘤组织中各种分子量的bFGFs的分离及鉴定。结论：人甲状腺及其肿瘤组织中各种分子量的bFGFs的分离及鉴定与SDS－PAGE电泳时条件的选择密切相关。     

甲状腺癌组织端粒酶激活与p16基因失活的关系

目的探讨甲状腺癌细胞增殖、分化与端粒酶激活及抑癌基因p16失活（缺失突变）之间可能存在的关系。方法应用TRAP、多重PCR、免疫组化法检测42例甲状腺癌与16例癌旁组织端粒酶活性、P16基因外显子2缺失、P16蛋白表达。结果甲状腺癌组端粒酶活性90．48％,高于癌旁组织（P〈0．01）;甲状腺癌p16基因外显子2纯合缺失率28．57％,相应癌旁组未检出（P〈0．01）;甲状腺癌P16蛋白表达缺失率40．48％,高于癌旁组（P〈0．05）;甲状腺癌P16蛋白表达缺失率高于p16基因外显子2缺失率。结论端粒酶激活与p16基因失活以及P16蛋白表达下调可能是甲状腺癌变过程中的重要分子事件,甲状腺癌中p16基因失活可能是端粒酶激活的一种途径。     

Reduced Nm23/Awd protein in tumour metastasis and aberrant Drosophila development

Tumour metastasis is the principal cause of death for cancer patients. We have identified the nm23 gene, for which RNA levels are reduced in tumour cells of high metastatic potential. In this report we identify the cytoplasmic and nuclear Nm23 protein, and show that it also is differentially expressed in metastatic tumour cells. We also find that the human Nm23 protein has sequence homology over the entire translated region with a recently described developmentally regulated protein in Drosophila, encoded by the abnormal wing discs (awd) gene. Mutations in awd cause abnormal tissue morphology and necrosis and widespread aberrant differentiation in Drosophila, analogous to changes in malignant progression. The metastatic state may therefore be determined by the loss of genes such as nm23/awd which normally regulate development.     

体外培养人涎腺多形性腺瘤细胞及表面标记物的初步研究

体外培养15例人涎腺多形性腺瘤原发肿瘤组织,探讨体外培养的细胞与原发肿瘤组织的表面标志物表达情况。运用免疫组化法检测CK5/6、CK7、CK8/18、CK14、CK20,vimentin及肌上皮表面标记物α-SMA、calponin及p63在细胞和肿瘤组织中的表达。结果发现体外培养的多形性腺瘤细胞呈多样形,可同时表达角蛋白、肌上皮表面标记物及波形蛋白,与原发肿瘤表达基本一致。提示体外培养的多形性腺瘤细胞可能大部分来源于肌上皮细胞及导管上皮细胞等,这为研究涎腺多形性腺瘤肿瘤生物学多样性提供了实验依据。     

Insulin-like growth factor-II gene expression in Wilms' tumour and embryonic tissues

Wilms' tumour (nephroblastoma) is an embryonal neoplasm occurring in hereditary and spontaneous forms. Both types show rearrangements of the short arm of chromosome 11. The germ line of children with the rare inherited triad of aniridia, genito-urinary abnormality and mental retardation carry a chromosome 11 that has a deletion in its short arm (band 11p13) and these children are at increased risk of developing Wilms' tumour. Neonates with the Beckwith-Wiedemann syndrome, in which there may be duplication of the 11p13-11p15 region, are similarly predisposed. In the spontaneous form of the tumour a deletion of the 11p14 band in tumour cells, but not in normal cells, has been reported, and the development of homozygosity for recessive mutations in the 11p region is implicated in the aetiology of Wilms' tumour. In view of these chromosomal rearrangements and because Wilms' tumour is histologically indistinguishable from the early stages of kidney development, we have now examined the expression of genes localized to 11p in Wilms' tumour and human embryonic tissue. In 12 sporadic tumours examined, the expression of the gene coding for insulin-like growth factor-II (IGF-II), localized to the 11p15 region, was markedly increased relative to adult tissues, but was comparable to the level of expression in several fetal tissues including kidney, liver, adrenals and striated muscle. This may reflect the stage of tumour differentiation, but could also contribute to the malignant process, as IGF-II is an embryonal mitogen.     

中国人神经珠蛋白(NGB)基因克隆与序列分析

应用RT-PCR方法从中国人胎脑组织中扩增出神经珠蛋白(NGB)特异性编码基因片段,并将其克隆到pMD18-TVector中,构建了pMD18-T-hNGB克隆质粒,然后进行测序分析并与国外报道的NGB序列相比较.结果表明,本文获得的中国人NGB编码基因序列与国外序列的同源性为98%.     

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.     

突变型P53和肿瘤易感基因101在肺癌组织中的表达及意义

探讨了P53基因突变对肺癌组织中TSG101基因的影响,结果是:TSG101在正常组织中呈强阳性表达100%(30/30),在高分化肺癌(包括肺鳞癌和肺腺癌)组织、低分化肺癌组织、淋巴结转移组织的表达分别为77.97%(92/118)、25.37%(17/67)、18.95%(18/95);P53的表达则相反,在正常组织、高分化肺癌组织、低分化肺癌组织、淋巴结转移组织的表达分别为6.67%(2/30)、67.80%(80/118)、86.57%(58/67)、94.63%(90/95);P53基因PCR-SSCP分析谱检测P53第5~8外显子阴性56例占30.27%,阳性129例占69.73%,总突变率为69.73%(127/180);P53与TSG101呈现负相关.这些结果提示P53和TSG101可能共同参与了肺癌的发生、发展和转移.