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The Construction of Schistosoma Japonicum Vaccine BCG-Sj26GST and Its Identification

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Journal of Huazhong University of Science and Technology(Medical Sciences) 1999年第3期19卷 161-165,169页

作者： HUANGFU Yongmu , ZHENG Bo , CHENG Jizhong , LIANG Juqing , FENG Zuohua department of Medical molecular biology, Research Center of Experimental Medicine, Tongji Medical University,Wuhan 430030 department of Medical molecular biology Research Center of Experimental Medicine Tongji Medical University Wuhan

Summary: The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette Guerin (BCG), Mycobacterium ( M. smegmatis ) and Escherichia coli ( E. coli ) were studied. The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E. coli as template. The Sj26GST cDNA was cloned into the downstream of human M. tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E. coli Mycobacteria shuttle plasmid pBCG 2000 to construct the expression shuttle plasmid pBCG Sj26. The recombinant BCG and M. smegmatis mc 2 155, which were electroplated with pBCG Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG Sj26GST was made. The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS PAGE at molecular weight of 26 ku. The content of rSj26GST accounted for 15 % and 10 % of total bacterial protein in BCG and M. smegmatis respectively. The results of Western blot showed the combination of rSj26GST with antibody of GST.

关键词： BCG, M. smegmatis shuttle plasmid Schistosoma Japonicum 26ku antigen gene vaccine gene expression

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

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Hepatobiliary & Pancreatic Diseases International 2006年第2期5卷 237-241页

作者： Jian-Lin Zhang, Jian-Jin Guo, Zi-Yan Zhang, Yi-Xin Jing, Lin Zhang, Rui Guo, Ping Yan, Niu-Liang Cheng, Bo Niu and Jun Xie department of Biochemistry and molecular biology, Shanxi Medical University ,Taiyuan 030001,China department of Biochemistry and molecular biology Shanxi Medical University Taiyuan 030001China.

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 stro

关键词： phage display technology phage antibody library hepatitis B virus surface antigen single-chain fragment variable

Expression of Foreign Gene in Mycobacterium Regulated by Human Mycobacterium Tuberculosis Heat Shock Protein 70 Promoter

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Journal of Huazhong University of Science and Technology(Medical Sciences) 1997年第4期17卷 193-199页

作者：程继忠皇甫永穆冯作化梁驹卿肖红 department of Medical molecular biology Research Center of Experimental Medicine Tongji Medical University Wuhan

The DNA fragments of 150bp length promoter 0f human Mycobacterium(M.) tuberculosis heat shock protein (hsp)7O and 650bp length foreign gene, the Schistosoma japonicum glutathione S-transferase(Sj26GST)gene,were obtained by amplification with polymerase chain reaction. And the 150p DNA sequence upstream initiation codon ATG of the human M. tuberculosis hsp7O promoter that contains the sequence TTGAG and ATCATA which consensus with E. coli promoter's -35 and-10 region respectively, as well as ribosome binding site GGAGG at position-12-8 upstream the ATG were determined by SangerDideoxyribonucleotide-mediated chain-termination method-Then, the human M. tuberculosis hsp70 promoter and Sj26GST cDNA were cloned into E. coli-mycobacteria shuttle plasmid pBCG-2000 to construct E. coli-Mycobacterium expression shuttle plasmid pBCG- Sj26 that can express Sj26GST gene.The M. smegmatis were electroporated and the positivecolonies were selected by kanamycin-The M.smegmatis containing the vector pBCG-Sj26 can be induced by heating and hydrogen peroxide (H2O2) to express GST. The molecular weight of the recombinant GST(rGST) was 26000. The rGST contents that were about 10 percent of the total bacterial protein were analyzed by density scanning after running SDS-PAGE. This study would provide scientific evidences for application of hsp70 promoter in expressing foreign gene in mycobacterium and development of mycobacterium as multiple-valent vectoral vaccine.

关键词： mycobacterium heat shock protein promoter shuttle plasmid gene expression

Effects of Shark Hepatic Stimulator Substance on the Function and Antioxidant Capacity of Liver Mitochondria in an Animal Model of Acute Liver Injury

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Acta Biochimica et Biophysica Sinica 2005年第8期37卷 507-514页

作者： Qiu-Ling FAN~(1,2) Cai-Guo HUANG~(2*) Yan JIN~2 Bo FENG~2 Hui-Nan MIAO~2 Wen-Jie LI~2 Bing-Hua JIAO~2 Qin-Sheng YUAN~(1*) ~1 State Key Laboratory of Bioreactor Engineering.East China University of Science and Techology,Shanghai 200237.China ~2 department of Biochemistry and molecular biology,Second Military Medical University.Shanghai 200433.China State Key Laboratory of Bioreactor Engineering East China University of Science and TechnologyShanghai 200237 China department of Biochemistry and molecular biology Second Military Medical UniversityShanghai 200433 China

This study was carried out to investigate whether shark hepatic stimulator substance(HSS)can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model ofthioacetamide(TAA)-induced liver injury.The acute liver injury was induced by two intraperitoneal injectionsof TAA(400 mg/kg)in a 24 h interval.In the TAA plus shark HSS group,rats were treated with shark HSS(80 mg/kg)I h prior to each TAA injection.In this group,serum liver enzyme activities were significantlylower than those in the TAA group.The mitochondrial respiratory control ratio was improved,and themitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group.The mitochondrialantioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in theTAA group.These results suggest that the protective effect of shark HSS against TAA-induced acute liverinjury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defensesand decreased oxygen stress.

关键词： shark hepatic stimulator substance acute liver injury mitochondrion respiratory function antioxidant capacity

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells

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Acta Pharmacologica Sinica 2005年第8期26卷 963-968页

作者： Min YANG~2, Qing-jun MA~(2,4), Geng-ting DANG~2, Kang-tao MA~3, Ping CHEN~3, Chun-yan ZHOU~(3,4) ~2department of Orthopedics, Peking University Third Hospital, Beijing 100083, China ~3department of Biochemistry and molecular biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China department of Orthopedics Peking University Third Hospital Beijing China department of Biochemistry and molecular biology School of Basic Medical Sciences Peking University Health Science Center Beijing China

Aim: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. Methods: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of lx 10～5 vector genomes per cell and subse-quent culture, C2C12 cells were assessed qualitatively for BMP7 production, alka-line phosphatase activity, osteocalcin production and Cbfal and MyoD expression. Results: C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. Conclusion: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

关键词： C2C12 adeno-associated virus bone morphogenetic protein-7 gene therapy

Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway

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Cell Research 2006年第5期16卷 489-498页

作者： Kaiming Cao State Key Laboratory of Genetic Engineering department of Biochemistry and molecular biology School of Life Sciences Fudan University Shanghai 200433 China

The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skpl-Cullin-F box protein)ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome.To investigate proteinsthat are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers,we compared the proteomes of theArabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE).Ten protein spots withhigher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to furthermass spectrometry (MS) analysis.The results showed that they were proteins involved in photomorphogenesis,circadianoscillation,post-translation process,stress-responses and cell expansion or elongation,suggesting that those processeswere affected in the ask1 mutant.The transcript levels of these genes were also compared based on the Affymetrix genechip microarray data.No significant difference was observed for most of the genes,suggesting that the proteins withelevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASKl-mediated proteolysispathway.These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes andalso demonstrate the importance and necessity of studying protein levels with respect to gene functions.

关键词： ASK1 two-dimensional polyacrylamide gel electrophoresis mass spectrometry proteolysis

BFP1:One of 700 Arabidopsis F-box proteins mediates degradation of JA oxidases to promote plant immunity

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molecular Plant 2024年第3期17卷 375-376页

作者： Claus Wasternack Bettina Hause department of molecular Signal Processing Leibniz Institute of Plant Biochemistry7 Weinberg 306120 Halle(Saale)Germany department of Cell and Metabolic biology Leibniz Institute of Plant Biochemistry06120 Halle(Saale)Germany

A switch off in jasmonate(JA)signaling by hydroxylation upon wounding was described in 2008(Miersch et al.,2008).This initiated the cloning of CYP450 enzymes involved in hydroxylation and carboxylation of JA and its amino acid conjugate JA-le,the most active form of JA compounds(reviewed by Wasternack and Feussner[2018]and Heitz et al.[2019]).Due to the central role of JA and JA-lle in plant stress responses and development(Wasternack and Hause,2013),catabolism of JA and its derivatives is an essential component in the sustainment of homeostasis of active JA compounds(Howe et al.,2018;Heitz et al.,2019).Catabolism of JA and JAIle takes place by JA-induced oxygenases(JOXs),also named JA-oxidases(JAOs),and by hydroxylation(CYP94B3/B1)followed by carboxylation(CYP94C1),respectively(Heitz et al.,2019).Another reaction performed by IAR3/ILL6 results in the deconjugation of JA-lle and 12-OH-JA-le to JA and 12-OH-JA,respectively(Heitz et al.,2019;Wasternack and Feussner,2018).

关键词： conjugate initiated media

A new oxidative pathway of nitric oxide production from oximes in plants

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molecular Plant 2024年第1期17卷 178-198页

作者： Pedro Lopez-Gomez Javier Buezo Marina Urra Alfonso Cornejo Raquel Esteban Jorge Fernandez de los Reyes Estibaliz Urarte Estefania Rodriguez-Dobreva Alejandro Chamizo-Ampudia Alejandro Eguaras Sebastian Wolf Daniel Marino Victor Martinez-Merino Jose F.Moran Institute for Multidisciplinary Research in Applied biology(IMAB) Department of SciencesPublic University of Navarre(UPNA)Avda.de Pamplona 12331192 MutilvaSpain Institute for Advanced Materials and Mathematics(INAMAT2) Department of SciencesPublic University of Navarre(UPNA)Campus de Arosadia31006 PamplonaSpain department of Plant biology and Ecology University of the Basque Country(UPV/EHU)Sarriena s/nApdo.64448080 BilbaoSpain Center for Plant molecular biology(ZMBP) University of TubingenGeschwister-Scholl-Platz72074 TubingenGermany Institute of Natural Resources and Agrobiology of Salamanca(IRNASA) Department of Plant-Microorganism InteractionsSpanish National Research Council(CSIC)Cordel de Merinas40-5237008SalamancaSpain department of Biochemistry Area of Molecular BiologyVeterinary FacultyUniversity of LeonCampus de Vegazanas/n.24071 LeonSpain

Nitric oxide(NO)is an essential reactive oxygen species and a signal molecule in plants.Although several studies have proposed the occurrence of oxidative NO production,only reductive routes for NO production,such as the nitrate(NO_(3)^(-))-upper-reductase pathway,have been evidenced to date in land plants.However,plants grown axenically with ammonium as the sole source of nitrogen exhibit contents of nitrite and NO3−,evidencing the existence of a metabolic pathway for oxidative production of NO.We hypothesized that oximes,such as indole-3-acetaldoxime(IAOx),a precursor to indole-3-acetic acid,are intermediate oxidation products in NO synthesis.We detected the production of NO from IAOx and other oximes catalyzed by peroxidase(POD)enzyme using both 4-amino-5-methylamino-2′,7′-difluorescein fluorescence and chemiluminescence.Flavins stimulated the reaction,while superoxide dismutase inhibited it.Interestingly,mouse NO synthase can also use IAOx to produce NO at a lower rate than POD.We provided a full mechanism for POD-dependent NO production from IAOx consistent with the experimental data and supported by density functional theory calculations.We showed that the addition of IAOx to extracts from Medicago truncatula increased the in vitro production of NO,while in vivo supplementation of IAOx and other oximes increased the number of lateral roots,as shown for NO donors,and a more than 10-fold increase in IAOx dehydratase expression.Furthermore,we found that in vivo supplementation of IAOx increased NO production in Arabidopsis thaliana wild-type plants,while prx33-34 mutant plants,defective in POD33-34,had reduced production.Our data show that the release of NO by IAOx,as well as its auxinic effect,explain the superroot phenotype.Collectively,our study reveals that plants produce NO utilizing diverse molecules such as oximes,POD,and flavins,which are widely distributed in the plant kingdom,thus introducing a long-awaited oxidative pathway to NO production in plants.This knowledge has essential implications for und

关键词： ammonium oxidation auxin nitric oxide oxime peroxidase root phenotype

Identifying genetic susceptibility to Aspergillus fumigatus infection using collaborative cross mice and RNA-Seq approach

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Animal Models and Experimental Medicine 2024年第1期7卷 36-47页

作者： Roa'a H.S.Yosief Iqbal M.Lone Aharon Nachshon Heinz Himmelbauer Irit Gat-Viks Fuad A.Iraqi department of Clinical Microbiology and Immunology Sackler Faculty of MedicineTel-Aviv UniversityTel-AvivIsrael School of molecular Cell biology and Biotechnology George S.Wise Faculty of Life SciencesTel Aviv UniversityTel AvivIsrael Institute of Computational biology Department of BiotechnologyUniversity of Natural Resources and Life SciencesMuthgasse 181190 ViennaAustria

Background:Aspergillus fumigatus(Af)is one of the most ubiquitous fungi and its infection potency is suggested to be strongly controlled by the host genetic back-ground.The aim of this study was to search for candidate genes associated with host susceptibility to Aspergillus fumigatus(Af)using an RNAseq approach in CC lines and hepatic gene expression.Methods:We studied 31 male mice from 25 CC lines at 8 weeks old;the mice were infected with Af.Liver tissues were extracted from these mice 5 days post-infection,and next-generation RNA-sequencing(RNAseq)was performed.The GENE-E analysis platform was used to generate a clustered heat map matrix.Results:Significant variation in body weight changes between CC lines was ob-served.Hepatic gene expression revealed 12 top prioritized candidate genes differ-entially expressed in resistant versus susceptible mice based on body weight changes.Interestingly,three candidate genes are located within genomic intervals of the previ-ously mapped quantitative trait loci(QTL),including Gm16270 and Stox1 on chromo-some 10 and Gm11033 on chromosome 8.Conclusions:Our findings emphasize the CC mouse model's power in fine mapping the genetic components underlying susceptibility towards Af.As a next step,eQTL analysis will be performed for our RNA-Seq data.Suggested candidate genes from our study will be further assessed with a human cohort with aspergillosis.

关键词： aspergillus fumigatus infection collaborative cross(CC)mice gene expression profile gene-network host susceptibility quantitative trait loci(QTL)mapping RNA-Seq