本发明中低子量尿激酶突变体，具有序列表SEQ ID No．1所示的氨基酸序列。本发明的优点是：与天然HMW-uPA相比：（1）可提高产品的稳定性，使产品更均一，有利于产品的质量控制；（2）可避免体内凝血酶对pro-UK的灭活作用，提高血药浓...本发明中低子量尿激酶突变体，具有序列表SEQ ID No．1所示的氨基酸序列。本发明的优点是：与天然HMW-uPA相比：（1）可提高产品的稳定性，使产品更均一，有利于产品的质量控制；（2）可避免体内凝血酶对pro-UK的灭活作用，提高血药浓度以提高溶栓效率；可延长药物的体内半衰期；（3）可提高蛋白质的比活性，减少用药量。分子量较小的LMW-UK不仅容易表达，而且临床剂量还可减少，非常有利于哺乳动物细胞表达的生物技术药物的产业化。展开更多
AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and...AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.展开更多
Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light...Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.展开更多
Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell prot...Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.展开更多
Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gen...Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264.7 cell by using Lipofectamine TM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion:The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.展开更多
RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust an...RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.展开更多
As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expre...As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.展开更多
Cell communication affects all aspects of cell structure and behavior,such as cell proliferation,differentiation,division,and coordination of various physiological functions.The moving RNA in plants and mammalian cell...Cell communication affects all aspects of cell structure and behavior,such as cell proliferation,differentiation,division,and coordination of various physiological functions.The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication.The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids.When moving miRNA enters the target cell,it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells.Thus,moving RNA may act as a long-range modulator during development,organogenesis,and tumor metastasis.展开更多
文摘本发明中低子量尿激酶突变体，具有序列表SEQ ID No．1所示的氨基酸序列。本发明的优点是：与天然HMW-uPA相比：（1）可提高产品的稳定性，使产品更均一，有利于产品的质量控制；（2）可避免体内凝血酶对pro-UK的灭活作用，提高血药浓度以提高溶栓效率；可延长药物的体内半衰期；（3）可提高蛋白质的比活性，减少用药量。分子量较小的LMW-UK不仅容易表达，而且临床剂量还可减少，非常有利于哺乳动物细胞表达的生物技术药物的产业化。
文摘AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.
文摘Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.
基金The National Natural Science Foundation of China (30670094, 30700028)the Ph.D. Programs Foundation of Ministry of Education of China (2006-0023008)
文摘Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.
文摘Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264.7 cell by using Lipofectamine TM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion:The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.
文摘RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
基金The Knowledge Innovation Program of the Chinese Academy of Sciences,(No.KSCX2-EW-G-8)the National Basic Research Program of China program(No.2009CB118903)
文摘As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.
基金supported by the National Basic Research Program of China(2009CB825603,2012CB517605)National Natural Science Foundation of China(81272392)
文摘Cell communication affects all aspects of cell structure and behavior,such as cell proliferation,differentiation,division,and coordination of various physiological functions.The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication.The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids.When moving miRNA enters the target cell,it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells.Thus,moving RNA may act as a long-range modulator during development,organogenesis,and tumor metastasis.
