人神经营养素-3成熟蛋白基因克隆及序列分析

应用 PCR技术 ,分别以 2个健康成人末梢血白细胞染色体 DNA为模板 ,扩增出人神经营养素 - 3( h NT- 3)成熟蛋白基因 ,并克隆至原核质粒 p UC1 9中进行基因序列测定。将所得序列与 Gen Bank提供的序列 ( M6 1 1 80 )进行对比 ,结果显示一序列与已知序列完全一致 ,另一序列中第 1 41、2 0 4和 2 1 9位碱基发生了改变 ,但改变的碱基不影响其编码的氨基酸。     

媒体与设计学院成立

9月 1 0日 ,上海交通大学隆重举行了第 1 9个学院——“媒体与设计学院”的成立揭牌仪式。媒体与设计学院下设新闻传播、影视艺术、工业设计、艺术设计等 4个系。媒体与设计学院现有本科生45 0人 ,硕士生 37人 ;现有设计艺术学和工业设计工程硕士两个硕士点。预计 2 0 0 3年 9月的招生规模为本科生 2 2 5人、硕士生 30人左右 ,至 2 0 0 4年学院将达到校本科生 1 0 0 0人 ,硕士生 1 0 0人 ,博士生 5～ 1 0人的办学规模。在学科建设方面 ,将设立设计艺术学博士点和设计艺术学、广播电视艺术学、电影学、美术学、传播学实验中心和上海交大数字…     

重组小鼠血管抑素基因在胆囊癌细胞中的表达及对血管内皮细胞增殖的抑制作用

目的　研究重组小鼠血管抑素基因真核表达质粒转染的胆囊癌细胞能否表达具有抑制血管内皮细胞生长活性的血管抑素蛋白。方法　应用脂质体LipofectAMINE 2 0 0 0将重组小鼠血管抑素基因的真核表达质粒 pcDNA3.1(+) angiostatin转染胆囊癌细胞株GBC SD ,G4 18抗性筛选 ;以Westernblot检测血管抑素的表达 ,以血管内皮细胞增殖分析检测其活性。结果　经Westernblot检测 ,得到高表达血管抑素的胆囊癌细胞克隆 ,其培养上清能有效抑制bFGF刺激的血管内皮细胞增殖 (P <0 .0 5 )。结论　小鼠血管抑素基因真核表达质粒 pcDNA3.1(+) angiostatin转导的胆囊癌细胞能够表达具有生物学活性的血管抑素蛋白 ,在胆囊癌的血管抑制基因治疗中有潜在临床应用价值     

人硫氧还蛋白cDNA的克隆及序列测定

目的　克隆人硫氧还蛋白 (HumanThioredoxin ,hTRX)cDNA序列并进行序列测定。方法　应用RT PCR技术 ,以 1 43(TK- )人骨肉瘤细胞RNA为模板 ,扩增出hTRX成熟蛋白的cDNA基因 ,并克隆至 pGEM TEasy载体中进行基因序列测定。结果　将所得序列与GenBanK(J0 4 0 2 6)提供的序列比较 ,其酶活性中心 (Trp Cys Gly Pro Cys)和基序与已知序列一致 ,第 1 80、2 84位碱基与已知序列不同 ,编码的氨基酸也发生了改变。结论　克隆得编码hTRX成熟蛋白的cDNA基因 ,为进一步探讨hTRX的生物活性和应用奠定了基础。     

甲状腺刺激性抗体对甲状腺细胞分泌功能的影响

目的　研究甲状腺刺激性抗体 (TSAb)对甲状腺细胞分泌T3 的作用及其机制 ,探讨TSAb致Graves病的分子机制。方法　以原代培养人甲状腺细胞为研究对象 ,应用放免法检测T3 ,信号传导抑制剂阻断各传导通道。结果　 2g·L-1TSAb作用 4 8h可使甲状腺细胞T3 的分泌增加约 4 10 % ;蛋白激酶 (PKA)抑制剂及胞内Ca2 +鳌合剂分别使TSAb作用的T3 分泌增加减少约 6 5 .3% ,32 .0 % (P <0 .0 5 ) ;而木黄酮对T3 分泌无明显影响 (P >0 .0 5 )。结论　TSAb通过激活cAMP/PKA及PIP2 (磷酸酰肌醇 ) /Ca2 +途径导致T3 分泌增加可能是TSAb致Graves病的主要机制之一     

协同因子β_2-GPI的分离、纯化和鉴定

目的　进一步研究β2 糖蛋白Ⅰ (β2 GPI)、心磷脂 (CL)和抗心磷脂抗体 (aCL)三者之间的关系 ,以及 β2 GPI在aCL致血栓形成中所起的作用 ,从人血清中提纯 β2 GPI。方法　采用高氯酸沉淀血清中的杂蛋白 ,过DEAE纤维素层析柱和DEAESephadexA 5 0层析柱。 结果　经SDS PAGE测定纯化样品的分子量为 5 0KD ;经皮免疫小鼠后 ,小鼠血清中可检测到高滴度的aCL。结论　上述组合方法方便可行 ,为β2 GPI的进一步研究奠定了良好的基础。     

NT4-ADNF-9融合基因原核表达载体的构建

目的　构建神经营养素 (NT4 )与活性依赖性神经营养因子 9(ADNF 9)融合基因的原核表达载体pBV2 2 0 /NT4 ADNF 9,为进一步对神经系统退行性疾病基因治疗的研究奠定基础。方法　采用非对称互补引物 /模板法 ,制备两端含有酶切位点的ADNF 9的cDNA ,将其连接到NT的信号肽和前导序列的 3′端 ,组成融合基因NT4 ADNF 9,再将该融合基因亚克隆于原核表达载体pBV2 2 0 ,构建为pBV2 2 0 /NT4 ADNF 9。结果　经DNA测序 ,限制性内切酶酶切等方法证实已成功地将ADNF 9重组到NT4信号肽和前导序列的 3′端 ,并将融合基因亚克隆于pBV2 2 0内。结论　成功构建了NT4与ADNF 9融合基因的原核表达载体pBV2 2 0 /NT4 ADNF 9。     

甘肃裕固族9个STR基因座遗传多态性研究

目的　研究中国甘肃裕固族STR遗传结构。方法　选择 9个STR基因座 (D3S135 8,VWA ,FGA ,TH0 1,TPOX ,CSF1PO ,D5S818,D13S317,D7S82 0 ) ,采用STR复合扩增及荧光标记STR基因扫描技术 ,同时检测 12 0个裕固族健康无关个体血液样本。结果　 9个STR基因位点共检出 6 5个等位基因 ,基因频率分布在 0 .0 0 5 7～0 .5 795 ;基因型共有 178种 ,频率分布在 0 .0 114～ 0 .30 6 8之间 ;9个STR位点基因型分布均符合Hardy Weinberg平衡定律 (P >0 .0 5 ) ;9个STR位点多态信息量 (PIC)均大于 0 .6 0 5 4 ,杂合度 (H)均大于 0 .6 15 8,个体识别力 (DP)均大于 0 .82 2 6 ,非父排除率 (EPP)均大于 0 .5 0 17。结论　获得了中国甘肃裕固族 9个STR基因座的遗传多态性数据 ,丰富了中华民族基因数据库 ,在人类群体遗传学及法医学研究领域有重要应用价值。     

UL2基因与HSV-1在三叉神经节中潜伏感染的相关性

目的　深入了解UL2基因与HSV 1在三叉神经节潜伏感染的相关性。方法　选择HSV 1国际参考标准株F株、HSV 1RE(株 ) (UL2基因插入突变株 )和其亲代HSV 1△ 30 5株 (TK- )三株病毒进行对比分析研究。对三株HSV 1病毒腹腔免疫接种小鼠双侧三叉神经节分别进行PCR扩增。结果　HSV 1 (RE)和HSV 1 (△ 30 5)组HSV 1潜伏感染率明显低于HSV 1 (F)组 (P <0 0 2 5) ,提示UL2基因可能在HSV 1形成潜伏感染过程中起一定作用。经HSV 1 (F)病毒攻击实验后三个实验组小鼠 ,其潜伏感染率无明显变化 ,而对照组小鼠潜伏感染率明显高于HSV 1 (RE)组和HSV 1 (△ 30 5)组 (P <0 0 1 )。结论　HSV 1 (RE)株的免疫接种可以预防HSV 1潜伏感染的建立。同时也为HSV 1RE株作为神经系统疾病基因治疗载体提供了理论依据     

Fine Mapping of a Deafness Mutation hml on Mouse Chromosome 10

Objective To map a mouse deafness gene.identify the underlying mutation and develop a mouse model for human deafncss.Methods Genetic lindage cross and genome scan wer used to map a novel mutation named hypoplasia of the membranous labyrinth (hml),which causes hearing loss in mutant mice.Results ① hml was mapped on mouse Chr 10 (～43 cM from the centromere) suggests that the homologous human gene is on 12q22--q24, which was defined on the basis of known mouse-human homologies (OMIM,2004). ② This study has generated 25 polymorphic microsateUite markers, placed 3 known human genes in the correct order in a high-resolution mouse map and narrowed the hml candidate gene region to a 500 kb area.     

A novel mutation-L539fs/47 of hERG in a Chinese long QT syndrome family

Objective To identify the mutation of human ether-a-go-go-related gene (hERG) and analyze the clinical characteristics of a Chinese family with long ST syndrome (LQTS). Methods The electrocardiogram and DNA samples were obtained from a Chinese LQTS family of 26 members. Genotype was performed with polymorphic short tandem repeat (STR) markers at the known LQT1, LQT2, and LQT3 loci. SSCP analysis was used to find aberrant conformers. hERG mutation was confirmed by cloning and sequencing. Results Three gene carriers were linked to chromosome 7q35-36, where the potassium channel gene hERG was encoded. A 19-base pair deletion was identified. The mutation was located at nucleotide position 1 619-1 637 between transmembrane domains S4 and S5. Furthermore, A1692G polymorphism was found both in the normal control and patients. Conclusion A novel 19 bp deletion mutation of hERG is identified in a Chinese family. All gene carriers are demonstrated to be typical LQT2 ECG phenotype.     

PCR-SSP检测人胰腺癌细胞株PC-2K-ras基因点突变及其方式

目的　检测人胰腺癌细胞株PC 2K ras基因点突变及其突变方式 ,明确基因治疗靶点的碱基序列。方法　针对K ras基因第 1 2位密码子点突变方式 (CGT、CAT、GTT)设计顺序特异性引物 (SSP) ,对人胰腺癌细胞株PC 2进行聚合酶链反应 (PCR) ,扩增产物借助聚丙烯酰胺凝胶电泳判定该细胞株有无K ras基因点突变及其突变方式。结果　人胰腺癌细胞株PC 2存在K ras基因点突变 ,突变方式为CGT。结论　PCR SSP法简便快速 ,特异性高 ,本研究结果为胰腺癌的下一步基因治疗奠定了基础     

K-ras基因点突变的反义寡脱氧核苷酸对人胰腺癌靶基因表达的抑制作用

目的　观察针对于胰腺癌K ras基因点突变的反义寡核苷酸对靶基因表达的抑制作用。方法　脂质体将人工合成的针对于K ras基因第 12位密码子点突变的反义寡核苷酸转染体外培养的人胰腺癌细胞株PC 2 ,用免疫组化和原位杂交技术检测靶基因的表达。结果　转染 4 8h后 ,反义寡核苷酸作用组胰腺癌细胞株PC 2的ras蛋白和K rasmRNA的表达程度较正义组和对照组均明显降低 (P <0 .0 5 )。结论　针对于K ras基因点突变的反义寡核苷酸作用于体外培养的胰腺癌细胞 ,对靶基因的表达有明显的抑制作用     

The cloning and expression of renalase and the preparation of its monoclonal antibody

To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase. Foundation item: the Scientific Research Project of Shanghai Municipal Health Bureau (2008Y034), and the Natural Scientific Research Project of Shanghai (05ZR14086)     

IDENTIFICATION OF HUMAN MURR1, THE HOMOLOGUE OF MOUSE IMPRINTED Murr1 GENE

Different fromthe biallelic expression mannerof most common autosomal genes,i mprinting is aspecial genomic expression phenomenon,of whichthe i mprinted genes show parental-specific expres-sion only according to their parental origin[1].Cor-rect i mprintingis i mportant inthe nor mal embryon-ic development,postnatal growth,as well as behav-ior of ani mals.Dysregulation of i mprinting hasbeen i mplicated in the pathogenesis of congenitaldiseases and cancer[2-3].To date,78kinds of mousei mprinte…     

Construction of expression vector for NT4-ADNF-9 fusion gene

Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neuraseusory deafness. Methods By means of asymmetrical prince/ template, double stranded eDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 eDNA was ligated to the signal and leader peptides of nenrotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was suheluned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restrtction enzymes digestion showed that we recombined ADNF-9 eDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcluned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 cbicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivtty is satisfactory.     

DETECTING LOW DENSITY LIPOPROTEIN RECEPTOR MUTANT GENE OF RABBIT BY PCR

Objective Watanabe Heritable Hyperlipidaemic （WHHL） rabbits with low density lipoprotein receptor （LDLr） gene mutation have provided unprecedented opportunities for the study of human atherusclerusis, in order to confirm LDL receptor gene status in rabbits, we developed a simple PCR technique to detect LDL mutations in rabbits. Methods Rabbits genomic DNA were extracted from ear biopsy, and amplified by PCR to detect 12 bp deletion mutation in WHHL rabbits. PCR products were directly digested with Bgl Ⅰ, and then applied to polyacrylamide gel electrophoresis. Results PCR products from homozygous LDLr ＋/＋ rabbits generated 2 bands of 212 and 94 bp after Bgl Ⅰ digestion, LDLr ＋/- rabbits generated 3 bands （294, 212, and 94 bp）, LDLr -/- animals, however, generated only 1 product （294 bp）. Conclusion This modified PCR method is simple and reliable.     

重组人单链白细胞介素12融合基因(rhscIL-12)的构建和真核表达

克隆人 IL - 1 2 ( h IL - 1 2 ) p40和 p35亚单位 c DNA,构建人单链 IL- 1 2 ( rhsc IL- 1 2 )融合基因 ,并在哺乳动物细胞中进行表达。方法　从经 PDBu刺激的 EBV转化的人 B淋巴母细胞株 NC37中提取 m RNA,经 RT- PCR分别获得 h IL- 1 2 p40和 p35亚单位编码序列的 c DNA,运用重组 PCR技术将两段基因通过一疏水性多肽接头 ( Gly4 Ser) 3 DNA序列进行体外基因重组 ,构建 rhsc IL- 1 2融合基因 ,将其插入 pc DNA3.1 ( + )真核表达载体 ,经脂质体转染 COS7细胞进行表达 ,Western blot进行分析。结果　所克隆的 h IL- 1 2 p40、p35c DNA序列和构建的 rhsc IL - 1 2融合基因 DNA序列均经测序得以证实 ,融合基因可在 COS7中表达其产物 rhsc IL- 1 2融合蛋白 ,其分子量为 70 KD,可与鼠抗人 IL - 1 2 p40 /p70单克隆抗体特异性结合。结论　本研究结果为进一步探讨 rhsc IL- 1 2融合蛋白的生物学活性和特性奠定了基础 ,也为 rhsc IL- 1 2融合基因在原核细胞中的表达提供了可能性