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家蚕核型多角体病毒遍在蛋白及结合肽的研究

生物化学与生物物理进展 2004年第11期31卷 1038-1044页

作者：张文波林蓉张耀洲吴祥甫浙江大学生命科学学院浙江理工大学生物化学研究所杭州310018 浙江大学动物科学学院浙江理工大学生物化学研究所中国科学院生物化学与细胞生物学研究所

从家蚕核型多角体病毒镇江株 (BombyxmorinuclearpolyhedrosisvirusZhenjiangstrain ,BmNPV ZJ)基因组DNA中克隆遍在蛋白 (BmVUB)基因 .序列分析结果显示 ,BmVUB基因长 2 34bp ,编码 77个氨基酸 .BmVUB氨基酸序列内有一致HTH序列 ,遍在... 详细信息

从家蚕核型多角体病毒镇江株 (BombyxmorinuclearpolyhedrosisvirusZhenjiangstrain ,BmNPV ZJ)基因组DNA中克隆遍在蛋白 (BmVUB)基因 .序列分析结果显示 ,BmVUB基因长 2 34bp ,编码 77个氨基酸 .BmVUB氨基酸序列内有一致HTH序列 ,遍在蛋白保守序列LRLRGG ,参与遍在蛋白蛋白酶复合体形成的 4个保守性功能位点 (Lys 2 9、Cys 4 8、Cys 6 3、Gly 76 )及保守的Gly Gly X (X是疏水氨基酸残基 )蛋白酶切信号序列 ,在遍在蛋白保守的Gly Gly后多出由一个氨基酸组成的延伸肽 .原核诱导表达表明BmVUB主要以可溶性形式存在 ,表达量占总菌蛋白的 5 0 %以上 .纯化的遍在蛋白浓度为 1 0 3～ 2 4 6 g/L ,制备抗体 ,效价在 3 2× 10 -5以上 .用噬菌体表面展示技术筛选遍在蛋白结合肽 ,所筛选的结合肽可与遍在蛋白特异性结合 ,其中结合肽VAPHHAYAPMRT对细胞增殖有明显的浓度调控作用 ,即低浓度促进细胞生长 ,高浓度强烈抑制细胞的生长 .

关键词：结合肽遍在蛋白白及性功能原核细胞增殖细胞生长诱导表达氨基酸序列特异性结合

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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 310018,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

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 310018, 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

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 310018, 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

Mutational Analysis of the Absolutely Conserved B8Gly: Consequence on Foldability and Activity of Insulin

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 673-679页

作者： Zhan-Yun GUO~1 Zhou ZHANG~1 Xiao-Yuan JIA~1 Yue-Hua TANG~(1,2) You-Min FENG~(1,2*) 1 Key Laboratory of Proteontics,institute of biochemistry,and Cell Biology,Shanghai institutes for Biological sciences.Chinese Academy of sciences,Shanghai 200031.china 2 Instutute of biochemistry,zhejiang Sci-Tech university,hangzhou 310018,china Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China institute of biochemistry Zhejiang Sci-Tech UniversityHangzhou 310018 China

B8Gly is absolutely conserved in insulins during ***,its correspondingposition is always occupied by a Gly residue in other members of insulin *** work showedthat Ala replacement of BSGly significantly decreased both the activity and the foldability of *** effects of substitution are complicated,and different replacements sometimes cause significantly *** analyze the effects of B8 replacement by different amino acids,three new insulin/single-chaininsulin mutants with BSGly replaced by Ser,Thr or Leu were prepared by protein engineering,and both theirfoldability and activity were *** general,replacement of BSGly by other amino acids causes significantdetriment to the foldability of single-chain insulin:the conformations of the three B8 mutants are essentiallydifferent from that of wild-type molecules as revealed by circular dichroism;their disulfide stabilities inredox buffer are significantly decreased:their in vitro refolding efficiencies are decreased approximatelytwo folds;the structural stabilities of the mutants with Ser or Thr substitution are decreased significantly,while Leu substitution has little effect as measured by equilibrium guanidine *** far as biologicalactivity is *** replacement of B8Gly has only a moderate effect:its insulin receptor-bindingactivity is 23% of native *** Thr or Leu replacement produces significant detriment:the receptor-binding potencies of the two mutants are less than 0.2% of native *** present results suggest thatGly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability andactivity are concerned.

关键词： insulin foldability activity disulfide stability

Asymptotics of discounted aggregate claims for renewal risk model with risky investment

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Applied Mathematics(A Journal of Chinese Universities) 2010年第2期25卷 209-216页

作者： JIANG Tao School of Finance, zhejiang Gongshang university, hangzhou 310018, china School of Finance Zhejiang Gongshang University Hangzhou China

Under the assumption that the claim size is subexponentially distributed and the insurance surplus is totally invested in risky asset, a simple asymptotic relation of tail probability of discounted aggregate claims for renewal risk model within finite horizon is obtained. The result extends the corresponding conclusions of related references.

关键词： Discounted aggregate claims ruin probability within finite horizon renewal risk model risky investment subexponential class.

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Isolation and Partial Characterization of a Novel Pollen-specific cDNA with Multiple Polyadenylation Sites from Wheat

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 467-476页

作者： Yong-Feng JIN* and Teng-Fei BIANinstitute of biochemistry, zhejiang university, hangzhou 310029, china institute of biochemistry Zhejiang UniversityHangzhou 310029 China

A novel pollen-specific full-length cDNA clone PSG076 was isolated using suppression subtractive hybridization and 5’/3’ RACE techniques. PSG076 was shown to exhibit multi-site polyadenylation by sequencing the 3’ ends of the cDNAs. At least six transcripts with different length were produced from the single gene based on different poly(A) tail attachment sites. However, polyadenylation consensus sequence AAUAAA was not seen at the 3’-untranslated sequence. PSG076 contained a 299 bp 5’ untranslated region and an open reading frame of 663 bp encoding a 221 amino acid peptide with pI of 4.31. A blast search revealed that this sequence did not show a significant similarity to any genes deposited in the public database. Southern blot indicated that PSG076 was a single copy gene. Northern blot and RT-PCR analysis indicated that PSG076 transcripts showed specific expression in mature pollen, and weak or undetectable signals in uninucleate microspore, immature seed, stem, young leave, root and ovary. Further analysis of the expression pattern in gametophyte showed that PSG076 transcripts were undetectable in uninucleate, binucleate microspore and pollen at early stage, and were first detectable and increased rapidly at middle and late stages of pollen development with the maximum level in mature pollen and also expressed in germinating pollen in vivo, suggesting that PSG076might play a role in pollen germination and pollen tube growthin addition to its function in maturation. The evidences gathered in this work indicated that the six different transcripts from the single gene were differentially expressed during pollen development.

关键词： wheat (Triticum aestivum L.) pollen-specific cDNA suppression subtractive hybridization multiple-site polyadenylation differential expression

Targeting Gene-Virotherapy for Cancer

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Acta Biochimica et Biophysica Sinica 2005年第9期37卷 581-587页

作者： Xin-Yuan LIU~(1,2*) Jing-Fa GU~1 Wen-Fang SHI~1 1 institute of biochemistry and Cell Biology,Shanghai institutes for Biological sciences,Chinese Academy of sciences,Shanghai 200031,china 2 Xinyuan institute of Medicine and Biotechnology,School of Life sciences,zhejiang Sci-Tech university,hangzhou 310018,china institute of biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Xinyuan institute of Medicine and Biotechnology School of Life Sciences Zhejiang Sci-Tech UniversityHangzhou 310018 China

Gene therapy and viral therapy for cancer have therapeutic effects,but there has been nosignificant breakthrough in these two forms of ***,a new strategy called “targeting gene-virotherapy”,which combines the advantages of gene therapy and viral therapy,has been *** therapy has stronger antitumor effects than either gene therapy or viral therapy.A tumor-specific repli-cative adenovirus vector ZD55 (E1B55KD deleted Adv.)was constructed and various single therapeuticgenes were inserted into ZD55 to form *** are the targeting gene-virotherapy *** showed that a single gene was not effective in eliminating the tumor mass,and therefore twogenes were separately inserted into *** strategy is called “targeting dual gene-virotherapy”(withPCT patent).Better results were obtained with this strategy,and all the xenograft tumor masses were com-pletely eliminated in all mice when two suitable genes producing a synergetic or compensative effect ***-six papers on these strategies have been published by researchers in our ***,an adenoviral vector with two targeting promoters harboring two antitumor genes has beenconstructed for cancer *** results have been obtained with this adenoviral vector and anotherpatent has been applied *** antitumor strategy can be used to kill tumor cells completely with minimumdamage to normal cells.

关键词： cancer therapy gene targeting viral vector targeting gene-virus

Irrigation Water Use Efficiency of Farmers and Its Determinants: Evidence from a Survey in Northwestern china

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Agricultural sciences in china 2010年第9期9卷 1326-1337页

作者： WANG Xue-yuan College of Economics, zhejiang Gongshang university, hangzhou 310018, P.R.china College of Economics Zhejiang Gongshang University Hangzhou 310018 P.R. China

Irrigation water shortage is becoming an increasingly serious problem in agricultural production. In this case, it is very important for policy makers to take measures to improve irrigation water use efficiency, especially in the water-scarce areas. In this paper, the data envelopment analysis （DEA） techniques, based on the concept of input-specific technical efficiency were used to develop farm-level technical efficiency measures and sub-vector efficiencies for irrigation water use. The Tobit regression technique was then adopted to identify the factors that influence irrigation water efficiency differentials under the shortage of water resources. Based on a sample data of 432 wheat farmers in northwestern china, our experimental results of the DEA analysis showed the average technical efficiency of 0.6151. It suggested that wheat farmers could increase their production by as much as 38.49% by using inputs more efficiently. Further, the mean irrigation water efficiency of 0.3065, suggested that wheat farmers could produce the same quantity of wheat using the same quantity of inputs but with 69.35% less water. The results of the Tobit regression analysis showed that the farmer＇s age, income, education level, and the farm size tended to affect the degree of irrigation water efficiency positively, and the channel conditions and different irrigation methods made a significant impact on irrigation water use efficiency. Furthermore, the arrangements of exclusive water property rights and competitive water price mechanism have effectively encouraged the water saving behavior of farmers. These results are valuable for policy makers since it could help to guide policies towards high irrigation water use efficiency.

关键词： irrigation water efficiency determinants data envelopment analysis Tobit regression

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