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一个抗真菌蛋白在绿色木霉中的分泌表达

生物化学与生物物理学报 2003年第5期35卷 454-458页

作者：徐军龚蓁蓁中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室上海2.0031

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,... 详细信息

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 30bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3个氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。

关键词：绿色木霉巨大曲霉抗真菌蛋白分泌表达

Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

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Acta Pharmacologica Sinica 2004年第6期25卷 19-24页

作者： Lu-yang YU, Bo LIN, Zhen-lin ZHANG2, Li-he GUO3 instituteof biochemistryand cell biology, shanghai institutes for biological sciences, Chinese academy ofsciences, shanghai 2.0031 Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, 2 shanghai JiaoTong UniversityAffiliated Sixth People’s Hospital, shanghai 200233, china institute of biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 上海 200031 Center for Preventing and Treating Osteoporosis Osteoporosis Research Unit Shanghai JiaoTong University Affiliated Sixth People's Hospital 上海 200233

AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS: PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Western blots. Urine amylase, NO generation, and histological examination were examined. RESULTS: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen, duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

关键词： adenoviridae streptozocin insulin-dependent diabetes mellitus gene therapy nitric oxide glucose amylases reversed transcriptase polymerase chain reaction Western blotting

Identification and characterization of a new member of serpin family- Hon-grES1 in rat epididymis

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cell Research 2002年第Z2期 407-410页

作者： ZHI HONG HU, QIANG LIU, QUAN SHANG, MIN ZHENG, JUN YANG, YONG LIAN ZHANGstate key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, the Chinese academy of sciences, shanghai 2.0031, china

A full length cDNA named HongrESl was isolated and cloned by screening rat epididymis cDNA library using a mouse EST as a probe and 5’RACE followed. It contained 1590bp nucleotides and its predicted protein had 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain. Tissue distribution pattern showed it was specifically expressed in adult rat epididymis; moreover, in situ hybridyza-tion indicated this gene was expressed in a limited region of the cauda epididymis near vas deference. Such kind of expression pattern sugested that HongrESl had potential function in male reproduction.

关键词： HongrESl serpin epididymis α1-antitrypsin in situ hybridization.

Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

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cell Research 2003年第5期13卷 369-374页

作者： HENG CHUAN XIA, FENG LI, ZHEN LI, Zu CHUAN ZHANG~* key laboratory of Proteomics, institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese academy of sciences, shanghai 2.0031, china. state key laboratory of Reproductive biology Institute of Zoology Chinese Academy of SciencesBeijing 100080 China Department of Veterinary Pathobiology University of Missouri-Columbia MO 65211 USA

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin （Cucurbita moschata） has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of～29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

关键词： ribosome-inactivating protein (RIP) Moschatin immunotoxin.

Establishment of a Screening System for Selection of siRNA Target Sites Directed against Hepatitis B Virus Surface Gene

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Acta Biochimica et Biophysica Sinica 2005年第5期37卷 310-316页

作者： Xiu-Min ZHOU~(1.3) Ju-Sheng LIN~1 Yi SHI~3 De-An TIAN~2 Huan-Jun HUANG~2 He-Jun ZHOU~1 You-Xin JIN~3 1 institute of Liver Diseases. Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, china: 2 Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, china 3 state key laboratory of molecular biology, institute of biochemistry and cell biology,shanghai institutes for biological sciences,Chinese academy of sciences, {12., china institute of Liver Diseases Tongji Hospital Tongji Medical College Huazhong University of Science and TechnologyWuhan 430030 China state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Department of Gastroenterology Tongji Hospital Tongji Medical College Huazhong University of Science and TechnologyWuhan 430030 China

To study the inhibitory effects of plasmid-derived small interfering RNA(siRNA)andsynthetic siRNA on the expression of the hepatitis B virus surface(HBs)gene,three plasmid-derived siRNAsand one synthetic siRNA that complement the coding region of the HBs gene were *** HBsexpression plasmid pHBs-EGFP was also *** cells were co-transfected with pHBs-EGFPand the above *** HBs mRNA quantities were measured by reverse-transcription PCR,and thelevel of HBs-EGFP fusion protein was quantified by fluorescent *** concentrations of thehepatitis B virus surface antigen(HBsAg)derived from the culture supernatant of transfected HepG2.2.15cells were measured by an enzyme-linked immunosorbent assay(ELISA)*** results showed that thethree plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNAand *** plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of *** can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%.To furthersubstantiate the above observations,psiRNA1 was transfected into HepG2.2.15 cells(an HBV secreting cellline).The transfections resulted in almost complete blockage of HBsAg production,whereas control vector-transfected cells secreted high levels of HBsAg 7 days *** conclusion,our data suggeststhat RNA interference(RNAi)is an efficient approach for reducing the level of HBs transcripts and proteinsand for suppressing HBsAg production.

关键词： RNA interference(RNAi) small interference RNA(siRNA) hepatitis B virus surface gene(HBs gene)

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

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cell Research 2004年第4期14卷 315-323页

作者： Li YANG, Jin Bo YANG, Jia CHEN, Guang Yao YU, Pei ZHOU, Lei LEI, Zhen Zhen WANG, Catherine CY CHANG, XinYing YANG, Ta Yuan CHANG, Bo Liang LI state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese academy of sciences, Graduate School of the Chinese academy of sciences, 320 Yueyang Road shanghai 2.0031, {12.. Department of biochemistry, Dartmouth Medical School, Hanover, NH 03756 USA. state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai China Department of biochemistry Dartmouth Medical School Hanover USA

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-1-derived macrophages. The luciferase activity assay demonstrated that Dex coul

关键词： ACAT dexamethasone macrophage cholesteryl ester gene promoter.

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酵母蛋白质YJL084c的磷酸化分析

生物化学与生物物理学报 2002年第4期34卷 433-438页

作者：石晓钟敖世洲中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室上海2.0031

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190... 详细信息

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190w在一段区域内有一定的同源性 ,并且根据已公布公共数据库 ,YJL0 84c是PCL6的一个结合蛋白。利用双杂交系统分析了YJL0 84c与这三个细胞周期蛋白因子之间的相互作用 ,表明PCL7也可以结合YJL0 84c。利用免疫共沉淀证实它们在体内的相互作用 ;通过体外GST沉降分析 ,验证它们的体外相互作用。YJL0 84c的体外翻译产物可以被PCL7 PHO85复合物磷酸化 ,进一步分别表达纯化YJL0 84c的氨基末端、中间和羧端三部分的GST融合蛋白片段 ,PCL7 PHO85复合物可以磷酸化其中间部分 ,而对N末端和C末端则未表现出磷酸化。以该中间片段蛋白质为底物 ,进一步研究高低磷条件对其磷酸化的影响。PCL7 PHO85对YJL0 84c的磷酸化受到无机磷条件的明显影响 :高磷条件下 ,磷酸化程度高 ;而低磷条件下磷酸化程度低。有意思的是 ,PCL6 PHO85免疫沉淀复合物也表现出同样的磷酸激酶特征。于是分析PHO81与这三个细胞周期因子之间相互作用。此外 ,还构建了Yjl0 84c的缺失株 ,分析了缺失的可能影响。

关键词： PCL6 PCL7 YJL084c 磷酸化酵母双杂交系统

A mRNA molecule encoding truncated excitatory amino acid carrier l(EAAC1)protein(EAAC2)is transcribed from an independent promoter but not analternative splicing event

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cell Research 2002年第Z1期 257-262页

作者： XIAO PING JIN, JI BIN PENG, FANG HUANG, YA NI ZHU, JIAN FEI*, LI HE GUO*laboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese academy of sciences, 320 Yueyang Road, shanghai

Glutamate transporter EAACl removes excitatory neurotransmitter in central nervous system, and also absorbs glutamate in epithelia of intestine, kidney, liver and heart for normal cell growth. When a mouse cDNA was screened using EAACl cDNA fragment as probe in our lab, a transcript (GenBank U75214) encoding an EAACl protein with 148 residues truncated at N-terminal was cloned and named as EAAC2. Sequence analysis shows that EAAC2 has it’s own start code and unique 5’UTR that is different from that of EAACl. A mouse genomic library was screened and a positive clone including EAACl CDS was sequenced (GenBank AF 322393) and indicates that normal EAACl transcript (GenBank U73521) is transcribed from 10 exons in terms of exon Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, Ⅶ, Ⅷ,Ⅸ, Ⅹ, and EAAC2 transcript is consisted by exons from IV to IX as same as that of EAACl and with its unique exon β upstream to exon Ⅳ and exon δ downstream to IX. EAAC2 transcript has a cluster of transcriptional start sites not overlapping with the t

关键词： genomic organization glutamate transporter transcriptional start sites EAACl.

重组单链胰岛素在含有巯基试剂的变性剂中的解折叠

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生物化学与生物物理学报 2001年第4期33卷 431-436页

作者：郭占云乔志松冯佑民中国科学院上海生命科学研究院生物化学与细胞生物学研究所!分子生物学国家重点实验室上海2.0031

重组单链胰岛素 (PIP)含有 3对二硫键。在含有巯基试剂的变性剂中 ,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱 (HPLC)和圆二色性 (CD)光谱分析了PIP在含有 0 .2mmol/L 2 巯基乙醇的... 详细信息

重组单链胰岛素 (PIP)含有 3对二硫键。在含有巯基试剂的变性剂中 ,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱 (HPLC)和圆二色性 (CD)光谱分析了PIP在含有 0 .2mmol/L 2 巯基乙醇的尿素和盐酸胍中的解折叠程度。PIP二硫键异构体混合物通过胰蛋白酶酶解并用质谱测定酶解片段的分子量 ,证明PIP确实产生了二硫键交换。同时还分离纯化了PIP的一种主要非天然二硫键异构体并研究了它重新折叠成天然构象的情况。观察到PIP只有一种热力学稳定的二硫键配对方式 ,PIP的非天然二硫键异构体在巯基试剂存在的条件下可以高效转化为天然二硫键配对。还将PIP解折叠和再折叠的情况与胰岛素样生长因子 I(IGF I)及胰岛素做了比较 :胰岛素和PIP只折叠成一种热力学稳定的三级结构 ,IGF I却折叠成两种热力学稳定的二硫键异构体 ;胰岛素的双链重组需缓慢进行 ,而PIP却可以快速折叠

关键词：重组单链胰岛素变性解折叠二硫键再折叠