CLONING AND EXPRESSION OF A GENE MEDIATING

［1］Nagata S. Apoptosis by death factor. Cell, 1997;88:355［2］Zou H, Henzel WJ, Liu X et al. Apaf-1, a human protein homologous to C. Elegans ced-4 participates in cytochrome c-dependent activation of caspase-3. Cell,1997;90(8) : 405［3］Nicholson DW, Thornberry NA. Caspases: killer proteases. Trands Biochem Sci, 1997;22(8) : 299［4］Hakem R, Hakem A, Duncan GS et al. Differential requirement for caspase 9 in apoptotic pathways in vivo.Cell, 1998;94(7) : 339［5］Kuide K, Lippke JA, Ku G et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta coverting enzyme. Science, 1995;267 : 2000［6］Kuida K, Zheng ZS, Na S et al. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature, 996;384 : 368［7］Li P, Allen H, Banerjee S. Mice deficient in IL-1 β-converting enzyme are defective in production of mature IL1 β and resistant to endotoxic shock. Cell, 1995;80:401［8］Bergeron L, Perez GI, Macdonald G et al. Defects in regulation of apoptosis in caspase-2-deficient mice.1998 ; (Genes Dev)12 : 1304［9］Wang S, Miura M, Jung YK et al. Murine caspase-11,an ICE-interacting protease, is essential for the activation of ICE. Cell, 1998;92(4) : 501［10］Woo M, Hakem R, Soengas MS et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev, 1998; 12 : 806［11］Kuida K, Haydar TF, Kuan CY et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell, 1998;94(7) : 325［12］Ibrado AM, Huang Y, Fang G et al. Overexpression of Bcl-2 or Bcl-xl inhibits Ara-C-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells. Cancer Reseach, 1996;56 : 4743［13］Hallan E, Blomhoff HK, Smeland EB et al. Involvement of ICE (caspase) family in γ-radiation-inducedapoptosis of normal B lymphocytes. Scand J Immunol,1997;46 : 601 (Continue on P14)［14］Roberts RA, Nebert DW, Hickman .JA et al. Perturbation of the mitosis/apoptosis balance: a fundamental mechanism in toxicology, Fundam Appl Toxicol,1997;38:107［15］Hendry JH, West CML. Apoptosis and mitotic cell death their relative contributions to normal tissue and radiation response. Int J Radiat Biol, 1997 ; 71(6) : 709     

HIGN LEVEL EXPRESSION OF HEPATITIS C VIRUS CORE GENE IN E.COLI

Hepatitis C virus (HCV) is the major agentfor the posttransfusinal and diffusive non- A,non-B hepatitis that could transform to chronic hepati-tis,and also has close relation to liver cancer andcirrhosis[1] .HCV is a single positive- stranded RNA viruswhose genome(about9.4kilobases) contains asingle uninterrupted open reading frame (ORF )which encodes a polyprotein precursor of 30 1 0～30 1 1 amino acid residues.The genome of HCV issimilar to flavivirus and pestivirus.In vitrostudieshav…     

bcl-2基因蛋白在外阴营养不良及外阴癌的表达

利用细胞凋亡抑制基因ｂｃｌ－２单克隆抗体及免疫组化ＬＳＡＢ法,对正常外阴皮肤、慢性外阴营养不良、不典型增生和外阴癌组织进行标记,发现ｂｃｌ－２蛋白在正常外阴皮肤基底层中有弱阳性表达;在增生型营养不良、不典型增生及外阴癌组织中表达率有不同程度增高,与外阴硬化苔癣型和混合型营养不良有显著差异（Ｐ＜０．０５,Ｐ＜０．０５,Ｐ＜０．０１）;而增生型与不典型增生ｂｃｌ－２基因蛋白表达无明显差异（Ｐ＞０．０５）;外阴癌的表达率最高;该基因蛋白表达与外阴癌细胞分化程度有关,与临床分期无关。     

p53蛋白在慢性宫颈炎及宫颈癌中的表达

采用免疫组织化学ＳＰ法分别对５７例宫颈癌、８例慢性宫颈炎ｐ５３蛋白表达情况进行了检测,并以１１例正常宫颈鳞状上皮组织作对照。结果∶ｐ５３蛋白在宫颈癌、慢性宫颈炎中的表达率分别为７８．９％、３７．５％。正常对照均为阴性。ｐ５３蛋白的表达率在宫颈癌病理Ⅲ级中明显高于病理Ⅰ、Ⅱ级（Ｐ＜０．０５）;在溃疡型中的表达率明显低于菜花型、结节型（Ｐ＜０．０５）。提示∶ｐ５３蛋白在慢性宫颈炎中的表达与细胞增殖率升高有关;ｐ５３蛋白的表达率不仅与癌细胞的分化程度有关,而且与宫颈癌的大体分型有关。     

人B7.2 cDNA的克隆及其真核表达

为克隆人 B7.2 c DNA,构建 B7.2真核表达质粒 ,并在哺乳动物细胞中表达。分离正常人淋巴结淋巴细胞 ,经 LPS诱导后 ,以 RT- PCR,克隆 B7.2 c DNA;构建真核表达质粒 p BCMGSNeo- B7.2 ,转染哺乳细胞 ,进行表达。结果成功克隆了 B7.2c DNA,并经测序证实 :所构建的 B7.2抗原真核表达质粒可在哺乳动物细胞中高效表达。表明经 LPS诱导的人淋巴细胞可表达 B7.2 m RNA,所建立的 p BCMGSNeo-B7.2哺乳动物真核表达质粒 ,可供进一步研究 B7.2结构和功能。     

THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E. COLI AGAINST HUMAN CERVICAL CANCER

Objective To obtain the gene of murine Single chain Fv fragment （ScFv） against haman cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridama cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDSPAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coll against haman cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.     

CLONING AND EXPRESSION OF A cDNA SEQUENCE FOR HUMAN THIOREDOXIN

Thioredoxin (TRX)isanoxidoreductaseen zyme,withamolecularweightof 1 2kD .Itwasi dentifiedoriginallyinE .coliasanhydrogendonorforribonucleotidereductaseanddeoxyribonucle otidesynthesis[1] .Thegeneofhumanthioredoxin(hTRX)islocatedonchromosome 3 p1 1 p1 2 ,withafulllengthof 1 3Kb .Theopenreadingframe ( 3 1 5nucleotideslong)codedforaproteinof 1 0 4aminoacids[2 ,3] .ManyfunctionsofTRXhavebeenre ported,whichincludethefollowing :TheTRXsystem (whichincludesHADPHasaprotondonor,TRXreductase ,…     

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Bone morphogenetic protein- 4(BMP- 4) is alow molecular weight glycoprotein ,classified as amorphogen.It is capable of inducing the formationof new cartilage and bone.This osteoinductiveability has led to the use of bone morphogeneticproteins as therapeutic agents for creation of newbone useful in treatment of skeletal injuries anddiseases,and in oral and maxillofacial applica-tions.Although many researchers have got the na-tive BMP from animal s demineralized bone by bio-chemical method,the…     

人肺癌p53及NDPK/nm23蛋白的研究

应用免疫组织化学法对56例肺癌组织进行p53蛋白及NDPK／nm23蛋白的研究。结果显示，p53蛋白总阳性率为55.35％，NDPK／nm23蛋白阳性率为78．57％，两者均与肺癌组织学类型及分化程度有关。并随着肿瘤恶性程度的增加，p53蛋白表达的阳性率增加，而NDPK／nm23蛋白的阳性率下降，在有转移的肿瘤中更为明显。p53与NDPK／nm23蛋白之间似乎存在着一定的负性相关关系。因此，我们认为同时检测p53及NDPK／nm23基因产物的表达情况，有助于对肺癌生物学行为的进一步了解及患者预后的判断。     

EXPRESSION CLONING OF A PROTECTIVE LEISHMANIA ANTIGEN

ＥＸＰＲＥＳＳＩＯＮＣＬＯＮＩＮＧＯＦＡＰＲＯＴＥＣＴＩＶＥＬＥＩＳＨＭＡＮＩＡＡＮＴＩＧＥＮＥ．Ｍｏｕｇｎｅａｕ，Ｆ．Ａｌｔａｒｓ，Ａ．Ｅ．Ｗａｋｉｌ，ＺｈｅｎｇＳｈｉｃｈｕｎ；Ｔ．Ｃｏｐｐｏｌａ，Ｚ．Ｗａｎｇ，Ｒ．Ｗａｌｄｍａｎｎ，Ｒ．Ｍ．Ｌｏｃ...     

P_(53)蛋白和增殖细胞核抗原在肾癌中表达的临床意义

采用免疫组织化学方法检测40例肾癌组织中P53蛋白和增殖细胞核抗原（PC-NA）的表达。发现P53蛋白阳性表达者共10例（25％）。P53蛋白表达与肿瘤病理分级、临床分期及预后无相关性。肾癌组织的PCNA增殖指数随肿瘤病理分级的上升而增加，PC－NA高表达组（增殖指数＞10％）预后不良，特别是临床早期但PCNA高表达者预后也很差。结果提示P53蛋白表达在肾癌生物学行为及预后评估中无重要意义，而PCNA可作为肾癌恶性度及预后评估指标。P53蛋白和PCNA表达无相关性。     

EXPRESSION OF NESTIN AND GLIAL FIBRILLARY ACIDIC PROTEIN IN DIFFERENT PERIOD AFTER SPINAL INJURY IN ADULT RATS

Objective To study the expression of Nestin and glial fibrillary acidic protein (GFAP) in different period after spinal injury in adult rats. Methods Animal moels were created by artery clamp. Expression of Nestin and GFAP in different period ( 1,3,5days; 1-8 weeks) and different area( injury locus and its surrounding tissue ) after spinal injury were observed pathologicaly using immunofluorescence and LeicaQ5001W imaging processing system.Results There was expression of Nestin and GFAP in injured area 1 day after injury. The expression increased not only in in injured area but its sourrounding 3--7 days later and gradually got to peak value. As the time went on, expression of Nestin and GFAP dereased. Conclusion Spinal injury can induce the expression of Nestin. Nerve stem cell has response to spinal injury. There is positive correlation between expression of Nestin and hyperplasia of reactivity astrocyte. Nerve stem cell maybe invovled in the repair of central nervous system (CNS).     

THE EXPERIMENTAL STUDY ON THE CELL APOPTOSIS AND EXPRESSION OF BCL-2 PROTEIN IN INTRACEREBRAL HEMORRHAGE IN MODEL OF RATS

Intra-cerebral hemorrhage is a common clinicaldisease,with a high mortality and morbidity.So itis one of the clinical hot topics.It has been foundinrecent years that there is a close relationship bet weenthe cell apoptosis and the course or prognosis of in-tra-cerebral hemorrhage.Bcl-2,as the apoptosis-adjusted gene,plays ani mportant role in the courseof cell apoptosis,but the mechanis min the cell ap-optosis in intra-cerebral hemorrhage remains un-clear.In this experi ment,with the model bui…     

局灶脑缺血再灌流后c-fos和bcl-2基因的表达

为探讨短暂局灶脑缺血后不同灌流期即刻早期基因（ＩＥＧｓ）与凋亡抑制基因在同一脑区的表达状况,采用不开颅血管腔内置线法制作鼠大脑中动脉阻塞（ＭＣＡ０）的局灶缺血再灌流模型,通过免疫组化法观察原癌基因ｃ－ｆｏｓ和ｂｃｌ－２蛋白在缺血３０ｍｉｎ再灌６ｈ和２４ｈ的表达。结果显示∶再灌６ｈ,二者在缺血同侧梨状皮层显著表达,而底节区表达很弱（Ｐ＜０．０１）;再灌２４ｈ,ｃ－ｆｏｓ蛋白在梨状皮层表达显著减弱（Ｐ＜０．０１）;ｂｃｌ－２蛋白表达仍显著（Ｐ＞０．０５）,且在底节区的表达有增强（Ｐ＞０．０５）。因而推论∶ｃ－ｆｏｓ与ｂｃｌ－２蛋白的表达可能与缺血损害的内源性保护机制有关。     

bcl-2蛋白和PCNA在乳癌中表达的定量测定及其预后

应用图像分析仪对６０例乳癌ｂｃｌ－２、ＰＣＮＡ和雌孕激素受体进行免疫组织化学及酶联亲和法定量研究。结果表明：ｂｃｌ－２蛋白定量高值组,乳癌组织学分级低,患者生存期长、生存率高（Ｐ＜０．０５）;ＰＣＮＡ表达定量高值组,组织学分级高,患者生存期短、生存率低（Ｐ＜０．０５）。显示了定量检测ｂｃｌ－２蛋白和ＰＣＮＡ对评估乳癌分化程度及判断预后具有一定价值。ｂｃｌ－２与雌孕激素受体间有显著的正相关性（Ｐ＜０．００１）,提示ｂｃｌ－２蛋白检测对乳癌患者内分泌治疗有重要参考价值。     

pSVL/GM-CSF真核表达质粒的构建及其表达活性观察

以真核瞬时表达质粒ｐＳＶＬ为载体构建ｐＳＶＬ／ＧＭ－ＣＳＦ真核表达质粒,用电穿孔法导入Ｃｏｓ－７细胞,以ＧＭ－ＣＳＦ依赖株ＴＦ１为靶细胞,检测其生物学活性;将不同剂量的ＰＳＶＬ／ＧＭ－ＣＳＦ质粒直接注射Ｂａ１ｂ／Ｃ小鼠股四头肌,动态观察小鼠血清中ＧＭ－ＣＳＦ的表达;将ｐＳＶＬ／ＧＭ－ＣＳＦ质粒导入Ｋ５６２细胞,观察ＧＭ－ＣＳＦ对瘤细胞生长状态的影响。结果表明：构建的ｐＳＶＬ／ＧＭ－ＣＳＦ真核表达质粒可在Ｃｏｓ－７细胞中有效表达ＧＭ－ＣＳＦ;小鼠骨骼肌直接注射ｐＳＶＬ／ＧＭ－ＣＳＦ（１００μｇ／及２００μｇ）,在血清中可检出ＧＭ－ＣＳＦ,在１０～１５ｄ达高峰（８Ｕ／ｍｌ）,为临床应用提供了实验基础;导入ＧＭ－ＣＳＦ基因的Ｋ５６２细胞可表达ＧＭ－ＣＳＦ,且经ＧＭ－ＣＳＦ基因修饰的Ｋ５６２细胞增殖状态明显优于对照组,提示对造血细胞肿瘤单用ＧＭ－ＣＳＦ应慎重,而与其它细胞因子联合应用可能是一个合理的方案。     

THE QUANTITATIVE MEASUREMENT OF BCL-2, P53 PROTEIN AND PCNA EXPRESSION IN BREAST CARCINOMA AND THEIR CORRELATION WITH PROGNOSIS

Recently,alargenumberofresearchesindicatedthatoccurenceanddevelopmentofbreastcarcinomaswerecorrelatedwithhighexpressionofblockingapoptosisoncogene,mutationofantioncogeneandexpressionofcontrollingcellproliferativeoncogene.Therewerefewreportsondocumentsofquantitativestudyofexpressionaboutblockingapoptosisoncogene--hcf--2(Bcellleukaemia/lymphomagene),productofP53proteinandproliferatingcellnuclearantigen(PCNA)inbreastcarcinoma.Ourpurposewastoinvestigatequantitativeindexinrelationtoclinicalpathol…     

THE CONSTRUCTION OF HPV16 L1 RECOMBINANT PLASMID AND ITS EXPRESSION IN MAMMALIAN CELLS

Ｐｒｅｖｉｏｕｓｓｔｕｄｉｅｓｈａｖｅｄｅｍｏｎｓｔｒａｔｅｄｔｈａｔｇｅｎｉｔａｌｉｎｆｅｃｔｉｏｎｗｉｔｈｈｉｇｈ－ｒｉｓｋｔｙｐｅｓｏｆｈｕｍａｎｐａｐｉｌｌｏｍａｖｉｒｕｓ（ＨＰＶ）,ｍｏｓｔｏｆｔｅｎＨＰＶ１６,ａｒｅｔｈｅｍｏｓｔｓｉｇｎｉｆｉｃａｎｔｒｉｓｋｆａｃｔｏｒｆｏｒｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆｃｅｒｖｉｃａｌｃａｎｃｅｒ．ＨＰＶ１６ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎｓａｎｄｎａｔｉｖｅｖｉｒｉｏｎｓｏｎｌｙｅｘｉｓｔｉｎｄｉｆｆｅｒｅｎｔｉａｔｅｄｓｕｐｅｒｆｉｃｉａ…