[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector fo...[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.展开更多
[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（...[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.展开更多
AIM:To investigate the function of the KISS-1 gene in gastric carcinoma cells and to explore its potential mechanism.METHODS:A KISS-1 eukaryotic expression vector was constructed and transfected into BGC-823 cells.Res...AIM:To investigate the function of the KISS-1 gene in gastric carcinoma cells and to explore its potential mechanism.METHODS:A KISS-1 eukaryotic expression vector was constructed and transfected into BGC-823 cells.Resistant clones were obtained through G418 selection.reverse transcription-polymerase chain reaction and western blotting were used to detect KISS-1 and matrix metalloproteinase-9(MMP-9)expression in transfected cells.The growth of transfected cells was investigated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)proliferation assays,and the cells'invasive potential was analyzed by basement membrane(Matrigel)invasion assays.The anti-tumor effects of KISS-1 were tested in vivo using allografts in nude mice.RESULTS:The expression level of KISS-1 mRNA andprotein in BGC-823/KISS-1 transfected cells were significantly higher than in BGC-823/pcDNA3.1 transfected cells(P<0.05)or the parental BGC-823 cell line(P< 0.05).The expression level of MMP-9 mRNA and protein in BGC-823/KISS-1 were significantly less than in BGC-823/pcDNA3.1(P<0.05)or BGC-823 cells(P< 0.05).MTT growth assays show the proliferation of BGC-823/KISS-1 cells at 48 h(0.642±0.130)and 72 h(0.530±0.164)were significantly reduced compared to BGC-823/pcDNA3.1(0.750±0.163,0.645±0.140)(P<0.05)and BGC-823 cells(0.782±0.137,0.685± 0.111)(P<0.05).Invasion assays indicate the invasive potential of BGC-823/KISS-1 cells(16.50±14.88)is significantly reduced compared to BGC-823/pcDNA3.1(20.22±14.87)(P<0.05)and BGC-823 cells after 24 h(22.12±16.12)(P<0.05).In vivo studies demonstrate the rate of pcDNA3.1-KISS-1 tumor growth is significantly slower than pcDNA3.1 and control cell tumor growth in nude mice.Furthermore,tumor volume of pcDNA3.1-KISS-1 tumors(939.38±82.08 mm3)was significantly less than pcDNA3.1(1250.46±44.36 mm3) and control tumors(1284.36±55.26 mm3)(P<0.05).Moreover,the tumor mass of pcDNA3.1-KISS-1 tumors(0.494±0.84 g)was significantly less than pcDNA3.1(0.668±0.55 g)and control tumors(0.682±0.38 g)(P <0.05).CONCLUSION:KISS-1 may inhibit the proliferation and invasion of gastric carcinoma cells in vitro and in vivo through the downregulation of MMP-9.展开更多
A stable transformation system for the expression of foreign genes in the unicellular green marine alga （Chlorella sp. MACC/C95）was established. Using electroporation, the alga was transformed with a plasmid contain...A stable transformation system for the expression of foreign genes in the unicellular green marine alga （Chlorella sp. MACC/C95）was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase （ npt ） as a selectable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytase gene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.展开更多
UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum int...UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum into PQE-30, the prokaryotic expression vector of PQE-UGP was successfully constructed. Then the vector plasmid of PQE-UGP was transformed into host bacteria M 15 and the expression of target gene was induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG）. The research laid foundation for study on the prokaryotic expression of UGPase.展开更多
Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea...Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea‘Sichuan Yellow Seed’by using the homologous cloning method.The expression vector of the GUS gene driven by the BjNAC102 promoter was constructed by seamless cloning technology.The results showed that the sequence of the promoter of the BjNAC102 gene contained many cis-acting elements involved in light responsiveness,gibberellinresponsive element,and auxin-responsive element.It was speculated that BjNAC102 played an important role in the abiotic stress response in Brassica juncea.The expression vector of the promoter of the BjNAC102 gene was constructed,which layed a foundation for further studies of the expression pattern of the BjNAC102 gene in Brassica juncea.展开更多
基金Supported by Natural Science Foundation of Liaoning Province(20072122)Projects Funding of Liaoning Provincial Education Office(05L339)~~
文摘[ Objective] The study was to report the construction of plant virus expression vector pCIYVV/CP/W and the expression of green fluorescent protein（GFP） with pCIYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein. [ Method] A section of multiple cloning sites among NIb/CP genes in pCIYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease Nla were cloned with infectivity full-length cDNA of clover yellow vein virus （CIYVV）, and pCIYVV/CP/W vector was constructed, GFP gene was inserted into pCIyVV/CP/W to construct the pCIYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot （WB）. [Result] The broad bean seedling inoculated with pCIYVV/CP/W/GFP expressed the same symptom as wild type CIYVV, morbidity was of 100%, the result showed that recombinant virus clone pCIYVV/CP/W/GFP didn＇t suppress, insertion of foreign gene didn＇t destroy the open reading frame of pCIYVV/CP/W. Foreign gene can keep living in F, progeny virus genorne steadily, recombinant virus clone pCIYVV/CP/W/GFP could steadily express GFP in progeny virus at least.[ Conclusion] The useful plant virus vector was provided for useful protein expressing.
基金Supported by National Natural Science Foundation of China(31070451)Qianjiang Talent Project of Zhejiang Province(2009R10016)Zhejiang Provincial Natural Science Foundation of China(Y5110067)~~
文摘[Objective] The aim was to construct the fusion expression vector of polyphosphate kinase（PPK） and green fluorescent protein（GFP） genes.[Method] In this study,the primers were designed based on PPK gene sequence（L03719） of E.coli DH5α in Genbank.Genomic DNA of E.coli DH 5α was extracted as template for the amplification of PPK gene by PCR method.By using In-Fusion@ HD Cloning Kit,the PPK gene was directionally cloned into NcoI site of the pCAMBIA1302 vector.[Result] Sequencing results showed that the 2.0 kb long fragment of PPK gene was inserted into the plant-based expression vector pCAMBIA1302 in front of GFP gene.[Conclusion] The fusion expression vector of PPK and GFP genes were successfully constructed.
基金Supported by The Natural Science Foundation,No.2011A310005the Science and Technique Foundation of Henan Province,No.112102310206
文摘AIM:To investigate the function of the KISS-1 gene in gastric carcinoma cells and to explore its potential mechanism.METHODS:A KISS-1 eukaryotic expression vector was constructed and transfected into BGC-823 cells.Resistant clones were obtained through G418 selection.reverse transcription-polymerase chain reaction and western blotting were used to detect KISS-1 and matrix metalloproteinase-9(MMP-9)expression in transfected cells.The growth of transfected cells was investigated by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)proliferation assays,and the cells'invasive potential was analyzed by basement membrane(Matrigel)invasion assays.The anti-tumor effects of KISS-1 were tested in vivo using allografts in nude mice.RESULTS:The expression level of KISS-1 mRNA andprotein in BGC-823/KISS-1 transfected cells were significantly higher than in BGC-823/pcDNA3.1 transfected cells(P<0.05)or the parental BGC-823 cell line(P< 0.05).The expression level of MMP-9 mRNA and protein in BGC-823/KISS-1 were significantly less than in BGC-823/pcDNA3.1(P<0.05)or BGC-823 cells(P< 0.05).MTT growth assays show the proliferation of BGC-823/KISS-1 cells at 48 h(0.642±0.130)and 72 h(0.530±0.164)were significantly reduced compared to BGC-823/pcDNA3.1(0.750±0.163,0.645±0.140)(P<0.05)and BGC-823 cells(0.782±0.137,0.685± 0.111)(P<0.05).Invasion assays indicate the invasive potential of BGC-823/KISS-1 cells(16.50±14.88)is significantly reduced compared to BGC-823/pcDNA3.1(20.22±14.87)(P<0.05)and BGC-823 cells after 24 h(22.12±16.12)(P<0.05).In vivo studies demonstrate the rate of pcDNA3.1-KISS-1 tumor growth is significantly slower than pcDNA3.1 and control cell tumor growth in nude mice.Furthermore,tumor volume of pcDNA3.1-KISS-1 tumors(939.38±82.08 mm3)was significantly less than pcDNA3.1(1250.46±44.36 mm3) and control tumors(1284.36±55.26 mm3)(P<0.05).Moreover,the tumor mass of pcDNA3.1-KISS-1 tumors(0.494±0.84 g)was significantly less than pcDNA3.1(0.668±0.55 g)and control tumors(0.682±0.38 g)(P <0.05).CONCLUSION:KISS-1 may inhibit the proliferation and invasion of gastric carcinoma cells in vitro and in vivo through the downregulation of MMP-9.
文摘A stable transformation system for the expression of foreign genes in the unicellular green marine alga （Chlorella sp. MACC/C95）was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase （ npt ） as a selectable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytase gene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.
文摘UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum into PQE-30, the prokaryotic expression vector of PQE-UGP was successfully constructed. Then the vector plasmid of PQE-UGP was transformed into host bacteria M 15 and the expression of target gene was induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG）. The research laid foundation for study on the prokaryotic expression of UGPase.
基金Supported by Natural Science Foundation of Hunan Province(2023JJ50083,2023JJ50084)Excellent Youth Project of Hunan Provincial Department of Education(22B0844)Hunan Provincial Graduate Research Innovation Project(CX20231274)。
文摘Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea‘Sichuan Yellow Seed’by using the homologous cloning method.The expression vector of the GUS gene driven by the BjNAC102 promoter was constructed by seamless cloning technology.The results showed that the sequence of the promoter of the BjNAC102 gene contained many cis-acting elements involved in light responsiveness,gibberellinresponsive element,and auxin-responsive element.It was speculated that BjNAC102 played an important role in the abiotic stress response in Brassica juncea.The expression vector of the promoter of the BjNAC102 gene was constructed,which layed a foundation for further studies of the expression pattern of the BjNAC102 gene in Brassica juncea.
