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Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant RNA Viruses and a Viroid

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Acta Biochimica et Biophysica Sinica 2007年第5期39卷 326-334页

作者： Zhiyou DU~(1,2), Bo JIN~3, Wenhong LIU~4, Liang CHEN~1, and Jishuang CHEN~(2,1*) 1 college of life sciences, zhejiang university, hangzhou 310029, china 2 Institute of Bioengineering, zhejiang sci-tech university, hangzhou 3.0018, china 3 college of life sciences, zhejiang Chinese Medical university, hangzhou 310053, china 4 college of Bioengineering, zhejiang Chinese Medical university, hangzhou 310053, china college of life sciences Zhejiang UniversityHangzhou 310029 China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China college of life sciences Zhejiang Chinese Medical UniversityHangzhou 310053 China college of Bioengineering Zhejiang Chinese Medical UniversityHangzhou 310053 China

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing32p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate （SSC） as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus （TMV） RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y （PVY） and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid （PSTVd） was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.

关键词： glass slide hybridization fluorescent labeling dot-blot hybridization virus viroid

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Study on Substrate Specificity at Subsites for Severe Acute Respiratory Syndrome Coronavirus 3CL Protease

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 807-813页

作者： Yu-Fei SHAN~1 Gen-Jun XU~(1,2)1 Institute of Bichemistry and Cell Biology.Shanghai Institutes for Biological sciences,Chinese Academy of sciences,Shanghai 200031,china 2 college of life science zhejiang sci-tech university,hangzhou 3.0018,china Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China.

Autocleavage assay and peptide-based cleavage assay were used to study the substratespecificity of 3CL protease from the severe acute respiratory syndrome *** was found that therecognition between the enzyme and its substrates involved many positions in the substrate,at least includingresidues from P4 to P2′.The deletion of either P4 or P2′residue in the substrate would decrease its cleavageefficiency *** contrast to the previous suggestion that only small residues in substrate could beaccommodated to the S1′subsite,we have found that bulky residues such as Tyr and Trp were also *** addition,based on both peptide-based assay and autocleavage assay,Ile at the P1′position could not behydrolyzed,but the mutant L27A could hydrolyze the Ile peptide *** suggested that there was astereo hindrance between the S1′subsite and the side chain of Ile in the *** 20 amino acids exceptPro could be the residue at the P2′position in the substrate,but the cleavage efficiencies were *** specificity information of the enzyme is helpful for potent anti-virus inhibitor design and usefulfor other coronavirus studies.

关键词： SARS coronavirus 3CL protease substrate specificity autocleavage binding site inhibitor design

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Quantitative Determination of Cucumber Mosaic Virus Genome RNAs in Virions by Real-Time Reverse Transcription-Polymerase Chain Reaction

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 669-676页

作者： Jun-Li FENG~1 Shao-Ning CHEN~1 Xiang-Shan TANG~1 Xian-Feng DING~1 Zhi-You DU~2 Ji-Shuang CHEN~(1,2*) 1 Institute of Bioengineering,zhejiang sci-tech university,hangzhou 3.0018,china 2 college of life sciences,zhejiang university,hangzhou 310029,china Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China college of life sciences Zhejiang UniversityHangzhou 310029 China

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green Ⅰ was developedfor determining the absolute and relative copies of cucumber mosaic virus （CMV） genomic RNAs containedin purified *** specific to each CMV ORF were designed and *** were thenamplified with length varying from 61 to 153 *** dilution series of CMV genome RNAs prepared by invitro transcription as the standard samples,a good linear correlation was observed between their thresholdcycle （C_t） values and the logarithms of the initial template *** copies of genomic RNA 1,RNA 2,RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method,and the ratios were about1.00:1.17:3.58:*** results were confirmed by Lab-on-a-Chip and Northern blot hybridization *** work is the first report concerning the relative amounts of different RNA fragments in CMV virions asa virus with tripartite genome.

关键词： real-time reverse transcription-polymerase chain reaction cucumber mosaic virus genomic RNA quantitative determination

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Expression and Identification of Mutated Osteoprotegerin in Culture Cells and Larvae of Silkworm

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Acta Biochimica et Biophysica Sinica 2004年第5期36卷 331-335页

作者： Hai-Long XIAO, Yong-Feng JIN, and Yao-Zhou ZHANG1* Institute of Biochemistry, college of life sciences, zhejiang university, hangzhou 310029, china college of life sciences, zhejiang university of science & technology, hangzhou 3.0018, china Institute of Biochemistry College of Life Sciences Zhejiang University Hangzhou 310029 China college of life sciences Zhejiang University of Science & Technology Hangzhou 310018 China

The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG- 372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivi- ties of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.

关键词： OPG-372 silkworm baculovirus expression system hypocalcemic effect

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In vitro insulin refolding:Characterization of the intermediates and the putative folding pathway

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science china(life sciences) 2007年第6期50卷 717-725页

作者： TANG YueHua1,2, WANG Shuai1, CHEN Yan1, XU GenJun1,2 & FENG YouMin1,2 1 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological sciences, Chinese Academy of sciences, Shanghai 200031, china 2 college of life science, zhejiang sci-tech university, hangzhou 3.0018, china Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China college of life science Zhejiang Sci-Tech University Hangzhou China

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B,; P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates; the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain; B chain individually formed the intermediates: two A chain intermediates (P1A; P3A),; four B chain intermediates (P2B, P4B, P5B,; P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II; III, each containing two native disulfides, were formed through the recognition; interaction of transient I with P4B or P6B; the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II; III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.

关键词： insulin, insulin refolding, folding intermediate, folding pathway, disulfide

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Satellite RNA-mediated Reduction of Cucumber Mosaic Virus Genomic RNAs Accumulation in Nicotiana tabacum

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Acta Biochimica et Biophysica Sinica 2007年第3期39卷 217-223页

作者： Qiansheng LIAO~(1,2) Liping ZHU~2 Zhiyou DU~2 Rong ZENG~(2,3) Junli FENG~2 Jishuang CHEN~(1,2*) 1 college of life sciences,zhejiang university,Hangzhon 310029,china 2 Institute of Bioengineering,zhejiang sci-tech university,hangzhou 3.0018,china 3 college of Biological Safety technology Hunan Agricultural university,Changsha 410128,china college of life sciences Zhejiang UniversityHangzhou 310029 China Institute of Bioengineering Zhejiang Sci-Tech UniversityHangzhou 310018 China college of Biological Safety technology Hunan Agricultural UniversityChangsha 410128 China

Satellite RNAs(satRNAs)are molecular parasites that interfere with the pathogenesis of thehelper *** this study,the relative accumulation of cucumber mosaic virus(CMV)-Fny genomicRNAs with or without satRNAs were quantitatively analyzed by real-time *** results showed thatsatRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumu-lation of CMV-Fny genomic RNAs,tested as open reading *** accumulation of CMV-Fny 1a,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at differentinoculation ***-FnyΔ2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the2a gene were deleted,caused only a slight mosaic effect on *** seedlings,similar to that of CMV-Fsat,but the addition of satRs to CMV-FnyΔ2b showed further decrease in the accumulation of CMV-FnyΔ2b genomic *** results indicated that the attenuation of CMV,by adding satRs or deleting the2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction ofCMV genomic RNAs accumulation in *** was possibly related to the 2b gene.

关键词： cucumber mosaic virus satellite RNA deletion of 2b gene relative accumulation

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Impact Factors of Energy Productivity in china: An Empirical Analysis

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Chinese Journal of Population,Resources and Environment 2007年第2期5卷 28-33页

作者： Wei Chu1,2, Shen Manhong2 1. college of Economics and Management, zhejiang sci-tech university, hangzhou zhejiang 3.0018, china 2. college of Economics, zhejiang university, hangzhou zhejiang 310017, china college of Economics and Management Zhejiang Sci-Tech University Zhejiang 310018 Hangzhou China college of Economics Zhejiang University Zhejiang 310017 Hangzhou China

This article developed a decomposition model of energy productivity on the basis of the economic growth model. Four factors were considered which may influence china’s energy productivity according to this model: technology improvement, resource allocation structure, industrial structure and institute arrangement. Then, an econometric model was employed to test the four factors empirically on the basis of china’s statistical data from 1978 to 2004. Results indicated that capital deepening con- tributes the most (207%) to energy efficiency improvement, and impact from labor forces (13%) is the weakest one in resource factor; industrial structure (7%) and institute innovation (9.5%) positively improve the energy productivity.

关键词： energy productivity impact factor economic growth china

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Recombinant Functional Human Lactoferrin Expressed in Baculovirus System

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Acta Biochimica et Biophysica Sinica 2006年第3期38卷 201-206页

作者： Tao LIU~(1,2) Yao-Zhou ZHANG~2* Xiang-Fu WU~31 Institute of Biochemistry,college of life sciences,zhejiang university,hangzhou 310029,china 2 Institute of Biochemistry,zhejiang sci-Tec university,hangzhou 3.0018,china 3 Institute of Biochemistry and Cell Biology,Chinese Academy of sciences Shanghai 200031,china Institute of Biochemistry College of Life Sciences Zhejiang UniversityHangzhou 310029 China Institute of Biochemistry Zhejiang Sci-Tec UniversityHangzhou 310018 China Institute of Biochemistry and Cell Biology Chinese Academy of SciencesShanghai 200031 China

Human lactoferrin (hLf) is a multifunctional iron-binding *** this study,weamplified hLfcDNA by reverse transcription-polymerase chain reaction from normal human mammary *** nucleotide sequence of the hLf was identical to the known *** constructed a recombinant virus,vBm-hLf,harboring the hLf gene and exploited the BmN cells as host to produce recombinant human lactoferrin(rhLf).It was found that a recombinant protein with a molecular mass of approximately 78 kDa was *** 13.5μg rhLf was purified from 1-2×10～5 BmN cells infected by vBm-hLf and the rhLfproved to be biologically *** method established in our study will pave the way for efficient productionof rhLf for further application of this protein in the future.

关键词： bacteriostatic activity baculovirus Bombyx mori human lactoferrin reverse transcriptionpolymerase chain reaction

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Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca,Ampullaria crossean

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 702-708页

作者： Yan-Hong LI~(1#) Rui GUO~(3#) Qiu-Yu YIN~1 Ming DING~1 Si-Liang ZHANG~3 Gen-Jun XU~(1,2) Fu-Kun ZHAO~(1,2*) 1 Key Laboratory of Proteomics,Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological sciences.Graduate School of the Chinese Academy of sciences,Chinese Academy of sciences,Shanghai 200031,china 2 college of life science,zhejiang sci-tech university,hangzhou 3.0018,china 3 Bioengineering Institute,East china university of science and technology,Shanghai 200233,china Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Graduate School of the Chinese Academy of Sciences Chinese Academy of SciencesShanghai 200031 China Bioengineering Institute East China University of Science and TechnologyShanghai 200233 China college of life science Zhejiang Sci-Tech UniversityHangzhou 310018 China

Two novel endo-β-1,4-glucanases,EG45 and EG27,were isolated from the gastric juice ofmollusca,Ampullaria crossean,by anion exchange,hydrophobic interaction,gel filtration and a secondround of anion exchange *** purified proteins EG45 and EG27 appeared as a single bandon sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa,*** optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for *** optimumtemperature range for EG27 was broad,between 50℃ and 60℃;for EG45 it was 50℃.The analysis on thestability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 evenwhen the incubation time was prolonged to 24 h at 30℃,whereas EG45 remained relatively stable at *** 85% of the activity of EG27 could be retained upon incubation at 60℃ for 24 ***,less than 10% residual activity of EG45 was detected at 50℃.Among different kinds of substrates,bothenzymes showed a high preference for carboxymethyl ***45,in particular,showed a carboxymethylcellulose hydrolytic activity of 146.5 IU/mg *** enzymes showed low activities to xylan (from oatspelt) and Sigmacell 101,and they were inactive to p-nitrophenyl-β-D-cellobioside,salicin and starch.

关键词： cellulase endo-β-1,4-glucanase Ampullaria crossean purification substrate specificity

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Noether-type theory for discrete mechanico-electrical dynamical systems with nonregular lattices

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science china(Physics,Mechanics & Astronomy) 2010年第9期53卷 1687-1698页

作者： FU JingLi1,CHEN LiQun2 & CHEN BenYong3 1 Institute of Mathematical Physics,zhejiang sci-tech university,hangzhou 3.0018,china 2 Department of Mechanics,Shanghai university,Shanghai 200072,china 3 Faculty of Mechanical-Engineering & Automation,zhejiang sci-tech university,hangzhou 3.0018,china Institute of Mathematical Physics Zhejiang Sci-Tech University Hangzhou China Department of Mechanics Shanghai University Shanghai China Faculty of Mechanical-Engineering & Automation Zhejiang Sci-Tech University Hangzhou China

We investigate Noether symmetries and conservation laws of the discrete mechanico-electrical systems with nonregular *** operators of discrete transformation and discrete differentiation to the right and left are introduced for the *** on the invariance of discrete Hamilton action on nonregular lattices of the systems with the dissipation forces under the infinitesimal transformations with respect to the time,generalized coordinates and generalized charge quantities,we work out the discrete analog of the generalized variational *** this formula we derive the discrete analog of generalized Noether-type identity,and then we present the generalized quasi-extremal equations and properties of these equations for the *** also obtain the discrete analog of Noether-type conserved laws and the discrete analog of generalized Noether theorems for the *** we use an example to illustrate these results.

关键词： Noether symmetry variational formula quasi-extremal equation conservation law discrete mechanico-electrical dynamical system

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