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Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

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Science China Chemistry 1995年第3期38卷 313-319页

作者：赛吉庆康良仪黄忠史春霖田波谢友菊 Beijing Agricultural University Beijing 100094 China Institute of Microbiology Chinese Academy of Sciences Beijing 100080 China

The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot.with an antiserum prepared against the native viral CP.

关键词： maize dwarf mosaic virus (MDMV) capsid protein (CP) sequence Western blot.

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

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Science China(Life Sciences) 2003年第1期46卷 1-9页

作者：刘惠卢芳陈建军任红玉陈常庆 Shanghai Research Center of Biotechnology Chinese Academy of Sciences

Two tumor necrosis factor-a mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser- 32Trp157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed in E. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot.indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-a. Compared to parent hTNF-a, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4—5 orders of magnitude but was equivalent to that of native hTNF-a on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-a.

关键词： human tumor necrosis factor, site-directed mutation, cytotoxicity, LD50, Western blot.

The screening and identification of endogenous retrovirus free CEMPs

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Science China(Life Sciences) 2004年第6期47卷 562-566页

作者： LU Quanzhi1,3, HAN Hongbing1,3, LIAN Zhengxing1,3, LI Ning2,3, ZHANG Qingcai1, ZHAO Zili4, PEI Dezhi1, ZHANG Xiaolan1 & WU Changxin1 1. College of Animal Science and Technology, China Agricultural University, Beijing 100094, China 2. State Key Laboratory of Agrobiotechnology, Beijing 100094, China 3. Laboratory of Animal Genetics and Breeding, Ministry of Agriculture, Beijing 100094, China 4. Sangzi Hu Pig Breeding Farm, Hubei 434010, China Correspondence should be addressed to Lian Zhengxing College of Animal Science and Technology China Agricultural University Beijing China Laboratory of Animal Genetics and Breeding Ministry of Agriculture Beijing China State Key Laboratory of Agrobiotechnology Beijing China Sangzi Hu Pig Breeding Farm Hubei China

The provirus DNA sequence of porcine endogenous retrovirus (PERV) distributed in the pig genome is the major obstacle that restricts the swine as the organ donors in xenotransplantation, and the copy number of PERV varies greatly among different breeds and individuals. In the experiment, 67 healthy, female Chinese Experimental Mini-Pigs (CEMPs) aged at 3—6 months were selected from the Animal Husbandry Station of China Agricultural University, the copy number of PERV and types of envelope protein gene (env) were then investigated by means of PCR analysis and Southern blot.ing. It is showed that the distribution of types of envelope protein gene in Landrace and CEMPs makes little difference, but the proportion of individuals carrying two types of envelope protein gene (env-A and env-B, which is de- noted as env-AB) is much larger than those which carry only one type of envelope protein gene (env-A or env-B). Meanwhile, two endogenous retrovirus free pigs were found for the first time during our research, and the copy number of others is relatively low, which is about 10 to 20. All the results illuminate the genetic diversity of indigenous pig breeds in China and the potential of CEMPs to serve as organ donors in xenotransplantation.

关键词： swine, CEMPs, porcine endogenous retrovirus (PERV), PCR, Southern blot.

Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis

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Science China(Life Sciences) 2005年第3期48卷 221-227页

作者： REN Yuanyuan 1,2 , DAI Xiuyu 1 , ZHOU Jian 1 , LIU Jingfang 1 , PEI Huadong 1,2 & XIANG Hua 1 1. State Key Laboratory of Microbial Resources and Center for Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China 2. Graduate School of Chinese Academy of Sciences, Beijing 100037, China State Key Laboratory of Microbial Resources and Center for Molecular Microbiology Institute of Microbiology Chinese Academy of Sciences Beijing China Graduate School of Chinese Academy of Sciences Beijing China

A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual ac- tivity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude en- zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L tre- halose is degraded within the same time period. Dot blot.ing revealed that the treP gene in T. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for indus- trial production of trehalose.

关键词： gene expression, Thermoanaerobacter tengcongensis, trehalose phosphorylase, dot blot.

Differential expression analysis of matrix metallo-proteinase 20(MMP20) in human esophageal cancer

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Chinese Science Bulletin 2001年第12期46卷 1009-1012,1057页

作者： YANG Cuihong LIU Zhihua WANG Qun LUO Aiping SUN Yuntian ZHANG Xun WANG Xiuqin ZHOU Chuannong WU Min National Key Laboratory of Molecular Oncology Cancer InstituteChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100021China Department of Pathology Cancer InstituteChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100021China

The aim of this study was to investigate the different expressions of MMP20 in the process of esophageal cancer. RT-PCR, immunohistochemistry and Western blot.were employed to detect the mRNA expression and protein expression of MMP20. Compared with the corresponding normal esophagus tissues, results showed the common trend of the dramatical overexpression of MMP20 in the esophageal cancer tissues. The analysis of MMP20 expression in different clinical early stages of esophageal cancer by RT-PCR indicated that MMP20 was much more overexpressed in the esophageal cancer with invasion than in the early stages. This suggested that MMP20 might play an important role in the progression of esophageal cancer. It probably plays a critical role in tumor invasion.

关键词： esophageal cancer MMP20 RT-PCR Western blot.

刚地弓形虫ROP2-P30复合基因在***21中的表达和鉴定(英文)

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中国人兽共患病学报 2006年第6期22卷 538-543页

作者：张佃波魏庆宽宰德富崔勇李瑾高红刚柏雪莲赵立江韩广东刘克义山东省寄生虫病防治研究所济宁272033

目的获得编码弓形虫RH株棒状体蛋白2和主要表面抗原1重组复合蛋白为弓形虫病快速诊断试剂盒及蛋白质疫苗的研制作准备。方法用PCR技术从弓形虫基因组DNA中扩增出ROP2和P30基因片段,分别克隆入pMD18T载体,并对重组入外源基因的质粒通过PC... 详细信息

目的获得编码弓形虫RH株棒状体蛋白2和主要表面抗原1重组复合蛋白为弓形虫病快速诊断试剂盒及蛋白质疫苗的研制作准备。方法用PCR技术从弓形虫基因组DNA中扩增出ROP2和P30基因片段,分别克隆入pMD18T载体,并对重组入外源基因的质粒通过PCR.,双酶切和测序鉴定,将pMD ROP2中的ROP2基因片段经EcoRⅠ和HindⅢ酶切,连接等反应,亚克隆入pET30a(+)原核表达载体构建pET ROP2载体,然后再将pMD P30中的P30基因片段与经BglⅡandEcoRⅠ酶切的pET ROP2载体连接,构建pET ROP2P30载体,经含卡那霉素的LB平板筛选,酶切和PCR鉴定。阳性重组质粒转化到大肠埃氏菌BL21(DE3)中,经IPTG诱导,表达产物用SDS PAGE进行鉴定。大量的表达融合蛋白经纯化和复性后,用Westernblot.析。结果从弓形虫RH株基因组DNA中扩增出特异的ROP2和P30基因片段,成功构建成pET ROP2和pET ROP2P30载体,成功表达了弓形虫棒状体蛋白2和弓形虫棒状体蛋白与主要表面抗原1的融合复合蛋白,表达出的蛋白经纯化复性后具有免疫反应性。结论ROP2和P30基因重组后,在原核表达载体中表达出的蛋白经纯化复性后具有活性,获得纯化和复性的弓形虫ROP2和ROP2P30的高效表达产物,为弓形虫病的诊断和疫苗研究奠定了基础。

关键词：刚地弓形虫 P30和ROP2基因融合蛋白 Western blot.