Objective To explore the differential expression and mechanisms of bone formation-related genes in osteoporosis(OP)leveraging bioinformatics and machine learning methodologies;and to predict the active ingredients of ...Objective To explore the differential expression and mechanisms of bone formation-related genes in osteoporosis(OP)leveraging bioinformatics and machine learning methodologies;and to predict the active ingredients of targeted traditional Chinese medicine(TCM)herbs.Methods The Gene Expression Omnibus(GEO)and GeneCards databases were employed to conduct a comprehensive screening of genes and disease-associated loci pertinent to the pathogenesis of OP.The R package was utilized as the analytical tool for the identification of differentially expressed genes.Least absolute shrinkage and selection operator(LASSO)logis-tic regression analysis and support vector machine-recursive feature elimination(SVM-RFE)algorithm were employed in defining the genetic signature specific to OP.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses for the selected pivotal genes were conducted.The cell-type identification by estimating rela-tive subsets of RNA transcripts(CIBERSORT)algorithm was leveraged to examine the infiltra-tion patterns of immune cells;with Spearman’s rank correlation analysis utilized to assess the relationship between the expression levels of the genes and the presence of immune cells.Coremine Medical Database was used to screen out potential TCM herbs for the treatment of OP.Comparative Toxicogenomics Database(CTD)was employed for forecasting the TCM ac-tive ingredients targeting the key genes.AutoDock Vina 1.2.2 and GROMACS 2020 softwares were employed to conclude analysis results;facilitating the exploration of binding affinity and conformational dynamics between the TCM active ingredients and their biological targets.Results Ten genes were identified by intersecting the results from the GEO and GeneCards databases.Through the application of LASSO regression and SVM-RFE algorithm;four piv-otal genes were selected:coat protein(CP);kallikrein 3(KLK3);polymeraseγ(POLG);and transient receptor potential vanilloid 4(TRPV4).GO and KEGG pathway enrichment analy-ses revealed that these trait genes were predominantly engaged in the regulation of defense response activation;maintenance of cellular metal ion balance;and the production of chemokine ligand 5.These genes were notably associated with signaling pathways such as ferroptosis;porphyrin metabolism;and base excision repair.Immune infiltration analysis showed that key genes were highly correlated with immune cells.Macrophage M0;M1;M2;and resting dendritic cell were significantly different between groups;and there were signifi-cant differences between different groups(P<0.05).The interaction counts of resveratrol;curcumin;and quercetin with KLK3 were 7;3;and 2;respectively.It shows that the interac-tions of resveratrol;curcumin;and quercetin with KLK3 were substantial.Molecular docking and molecular dynamics simulations further confirmed the robust binding affinity of these bioactive compounds to the target genes.Conclusion Pivotal genes including CP;KLK3;POLG;and TRPV4;exhibited commendable significant prognostic value;and played a crucial role in the diagnostic assessment of OP.Resveratrol;curcumin;and quercetin;natural compounds found in TCM;showed promise in their potential to effectively modulate the bone-forming gene KLK3.This study provides a sci-entific basis for the interpretation of the pathogenesis of OP and the development of clinical drugs.展开更多
AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T...AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.展开更多
We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltratio...We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.展开更多
Objective： To investigate the immunological function of a yeast expression system for thymosin α1 （Tα1）. Methods： A constructed Tetl yeast expression system was used to investigate the immunological function of ...Objective： To investigate the immunological function of a yeast expression system for thymosin α1 （Tα1）. Methods： A constructed Tetl yeast expression system was used to investigate the immunological function of orally administered Tα1. Dried yeast containing three different concentration of Tα1 was fed to normal Balb/c mice and other Balb/c mice whose immunities were inhibited in advance by cyclophosphamide. Synthesized Tα1 peptide was used as positive control and dried yeast with empty plasmid was used as negative control. CD4^＋ and CD8^＋ levels were detected by flow cytometry assay. TNF-α, IFN-γ, IL-2, IL-6 and IL-10 levels were detected by liquid chip. Results： In normal Balb/c mice or immune inhibition Balb/c mice, CD8^＋ levels were significantly increased. Especially in immune inhibition Balb/c mice, CD8^＋ levels in synthesized Tα1 group （18.77%±4.72%）, small dose group （13.48%±6.17%） and large dose group （22.74%±1.09%） were significantly higher than that in empty yeast control group （7.49%±2.14%）. Conclusion： Orally administered Tα1 has its certain immunomodulatory function.展开更多
Interaction between cytotoxic T lymphocyte-asso-ciated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1and B7-2) is of importance in the cellular events of lym-phocyte, including antigen-specific T-cell activation andi...Interaction between cytotoxic T lymphocyte-asso-ciated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1and B7-2) is of importance in the cellular events of lym-phocyte, including antigen-specific T-cell activation andinduction of autoreactive T-cell. We describe here the firstintroduction of a murine soluble CTLA4 gene, CTLA4Ig,to Mm1 cells, a macrophagic cell line. CTLA4Ig waJssuccessfully expressed on Mm1 cells and the expressedCTLA4Ig was found to be functionally active in their bind-ing to B7 molecules by flow cytometry and immunofluo-rescence studies- The biological activity of CTLA4Ig fromthe transfected Mm1 cells was studied and showed in-hibitory activity on mixed lymphocyte culture. A highCTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfec-tion and there had so far been no report on expression ofCTLA4Ig gene on Mm1 cells, these results suggested thatthe CTLA4Ig expressing Mm1 cells could be useful forExpression of CTLA4 on Mml and its biological activityanalysis of CTLA4 and B7 molecule interaction in bothmacrophage and T-cell.展开更多
The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor...The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.展开更多
基金National Natural Science Foundation of China(81960877).
文摘Objective To explore the differential expression and mechanisms of bone formation-related genes in osteoporosis(OP)leveraging bioinformatics and machine learning methodologies;and to predict the active ingredients of targeted traditional Chinese medicine(TCM)herbs.Methods The Gene Expression Omnibus(GEO)and GeneCards databases were employed to conduct a comprehensive screening of genes and disease-associated loci pertinent to the pathogenesis of OP.The R package was utilized as the analytical tool for the identification of differentially expressed genes.Least absolute shrinkage and selection operator(LASSO)logis-tic regression analysis and support vector machine-recursive feature elimination(SVM-RFE)algorithm were employed in defining the genetic signature specific to OP.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses for the selected pivotal genes were conducted.The cell-type identification by estimating rela-tive subsets of RNA transcripts(CIBERSORT)algorithm was leveraged to examine the infiltra-tion patterns of immune cells;with Spearman’s rank correlation analysis utilized to assess the relationship between the expression levels of the genes and the presence of immune cells.Coremine Medical Database was used to screen out potential TCM herbs for the treatment of OP.Comparative Toxicogenomics Database(CTD)was employed for forecasting the TCM ac-tive ingredients targeting the key genes.AutoDock Vina 1.2.2 and GROMACS 2020 softwares were employed to conclude analysis results;facilitating the exploration of binding affinity and conformational dynamics between the TCM active ingredients and their biological targets.Results Ten genes were identified by intersecting the results from the GEO and GeneCards databases.Through the application of LASSO regression and SVM-RFE algorithm;four piv-otal genes were selected:coat protein(CP);kallikrein 3(KLK3);polymeraseγ(POLG);and transient receptor potential vanilloid 4(TRPV4).GO and KEGG pathway enrichment analy-ses revealed that these trait genes were predominantly engaged in the regulation of defense response activation;maintenance of cellular metal ion balance;and the production of chemokine ligand 5.These genes were notably associated with signaling pathways such as ferroptosis;porphyrin metabolism;and base excision repair.Immune infiltration analysis showed that key genes were highly correlated with immune cells.Macrophage M0;M1;M2;and resting dendritic cell were significantly different between groups;and there were signifi-cant differences between different groups(P<0.05).The interaction counts of resveratrol;curcumin;and quercetin with KLK3 were 7;3;and 2;respectively.It shows that the interac-tions of resveratrol;curcumin;and quercetin with KLK3 were substantial.Molecular docking and molecular dynamics simulations further confirmed the robust binding affinity of these bioactive compounds to the target genes.Conclusion Pivotal genes including CP;KLK3;POLG;and TRPV4;exhibited commendable significant prognostic value;and played a crucial role in the diagnostic assessment of OP.Resveratrol;curcumin;and quercetin;natural compounds found in TCM;showed promise in their potential to effectively modulate the bone-forming gene KLK3.This study provides a sci-entific basis for the interpretation of the pathogenesis of OP and the development of clinical drugs.
基金Supported by National "863" Program of China , No. 2002AA214011
文摘AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.
基金Project supported by the National Key Technology Research and Development Program of China (No. 2007BAD59B06)the Interna-tional Science and Technology Cooperation Program of China (No. 2007DFA31260)the Science Foundation for the Excellent Youth Scholars of Guizhou Province, China (No. 20030312)
文摘We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.
基金Project (No. 2003C13015) supported by the Key Project from Sci-ence and Technology Department of Zhejiang Province, China
文摘Objective： To investigate the immunological function of a yeast expression system for thymosin α1 （Tα1）. Methods： A constructed Tetl yeast expression system was used to investigate the immunological function of orally administered Tα1. Dried yeast containing three different concentration of Tα1 was fed to normal Balb/c mice and other Balb/c mice whose immunities were inhibited in advance by cyclophosphamide. Synthesized Tα1 peptide was used as positive control and dried yeast with empty plasmid was used as negative control. CD4^＋ and CD8^＋ levels were detected by flow cytometry assay. TNF-α, IFN-γ, IL-2, IL-6 and IL-10 levels were detected by liquid chip. Results： In normal Balb/c mice or immune inhibition Balb/c mice, CD8^＋ levels were significantly increased. Especially in immune inhibition Balb/c mice, CD8^＋ levels in synthesized Tα1 group （18.77%±4.72%）, small dose group （13.48%±6.17%） and large dose group （22.74%±1.09%） were significantly higher than that in empty yeast control group （7.49%±2.14%）. Conclusion： Orally administered Tα1 has its certain immunomodulatory function.
文摘Interaction between cytotoxic T lymphocyte-asso-ciated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1and B7-2) is of importance in the cellular events of lym-phocyte, including antigen-specific T-cell activation andinduction of autoreactive T-cell. We describe here the firstintroduction of a murine soluble CTLA4 gene, CTLA4Ig,to Mm1 cells, a macrophagic cell line. CTLA4Ig waJssuccessfully expressed on Mm1 cells and the expressedCTLA4Ig was found to be functionally active in their bind-ing to B7 molecules by flow cytometry and immunofluo-rescence studies- The biological activity of CTLA4Ig fromthe transfected Mm1 cells was studied and showed in-hibitory activity on mixed lymphocyte culture. A highCTLA4Ig producing macrophagic cell line was obtained.As Mm1 cells were regarded as difficult for gene transfec-tion and there had so far been no report on expression ofCTLA4Ig gene on Mm1 cells, these results suggested thatthe CTLA4Ig expressing Mm1 cells could be useful forExpression of CTLA4 on Mml and its biological activityanalysis of CTLA4 and B7 molecule interaction in bothmacrophage and T-cell.
文摘The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.
