A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonel...A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.展开更多
Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin...Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods： The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SI） cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results： The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29% ± 2.71% and 83.85% ±2.34%（P〉0.05）, which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion： Survivin shRNA successfully and got GBC-SD cells which are stable expression vector was constructed expression shRNA.展开更多
以潮霉素抗性和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为筛选标记,利用地衣型真菌Cladonia metacorallifera的菌丝,成功实现了根癌农杆菌介导的遗传转化,PCR检测证明转化子中存在潮霉素抗性基因,共聚焦显微镜...以潮霉素抗性和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为筛选标记,利用地衣型真菌Cladonia metacorallifera的菌丝,成功实现了根癌农杆菌介导的遗传转化,PCR检测证明转化子中存在潮霉素抗性基因,共聚焦显微镜检测到转化子菌丝能够产生绿色荧光,证明EGFP能够在trp C启动子控制下在地衣型真菌中表达。展开更多
AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T ...AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.展开更多
In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMN...In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.展开更多
MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the ...MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein （EGFP） gene was used as reporter gene. The Bornbyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot. Tests showed that the recom- binant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.展开更多
MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the ...MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction ofeukaryotic miRNA expression vector was described. The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein （EGFP） gene was used as reporter gene. The Bombyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression ofmiR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.展开更多
基金The Knowledge Innovation Program Key Project (KSCX1-YW-R-07)
文摘A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.
文摘Objective： To construct sarvivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods： The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SI） cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results： The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29% ± 2.71% and 83.85% ±2.34%（P〉0.05）, which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion： Survivin shRNA successfully and got GBC-SD cells which are stable expression vector was constructed expression shRNA.
基金the Scientific Research Foundation for Returned Overseas Chinese Scholars,Personnel Affairs Bureau of Zhejiang Province
文摘AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.
基金The knowledge innovation program of the Chinese Academy of Sciences (KSCX2-YW-Z-0938)
文摘In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.
文摘MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein （EGFP） gene was used as reporter gene. The Bornbyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot. Tests showed that the recom- binant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
文摘MicroRNAs （miRNAs） are small endogenous RNAs molecules, approximately 21-23 nucleotides in length, which regulate gene expression by base-pairing with 3＇ untranslated regions （UTRs） of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction ofeukaryotic miRNA expression vector was described. The cytoplasmic actin 3 （A3） promoter and flanked sequences of miRNA-9a （miR-9a） precursor were amplified from genomic DNA of the silkworm （Bombyx mori） and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein （EGFP） gene was used as reporter gene. The Bombyx mori N （BmN） cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression ofmiR-9a was evaluated by reverse transcriptase polymerase chain reaction （RT-PCR） and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
