Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of...Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.展开更多
Early detection of pregnancy is advantageous to breeders. However, tools that were used in the early pregnancy detection are expensive, laboratory-based and not efficient and applicable enough in the field to farmers....Early detection of pregnancy is advantageous to breeders. However, tools that were used in the early pregnancy detection are expensive, laboratory-based and not efficient and applicable enough in the field to farmers. Thus, there is a need to find a biomarker which not only can detect pregnancy but also could be applied to the livestock on the field. Proteomic approach was used in this study as to search for early pregnancy biomarker. When goat sera at day 14 of gestation were resolved by using two dimensional gel electrophoresis, five differentially expressed proteins were detected. Four of the proteins were identified as albumin precursors, immunoglobulin lambda light chain and zinc-alpha-2-glycoprotein. Pregnancy associated glycoprotein-1 was detected by using liquid chromatography mass spectrometry and the protein was validated by immunoblotting. These proteins have potential to be used as a biomarker for early pregnancy detection, however, more extensive analysis are needed to validate its possibility.展开更多
Objective： To investigate the proteornic differences between the high-sensitivity （HS） group and low-sensitivity （LS） group of cervical cancer treated by radiotherapy and confirm the radiotherapy sensitivity asso...Objective： To investigate the proteornic differences between the high-sensitivity （HS） group and low-sensitivity （LS） group of cervical cancer treated by radiotherapy and confirm the radiotherapy sensitivity associated proteins in early cervical cancer. Methods： The fresh carcinoma tissues were collected from 10 untreated cervical cancer patients and preserved in the -80 ℃ refrigeratory. The tissues were classified into two groups： high sensitivity group （HS） and low sensitivity group （LS）, according to their response to radiotherapy. In the first part of our experiment, protein separating was performed by using two-dimensional gel electrophoresis （2-DE） with Amersham 18 cm linear pH 3-10 immobilized pH gradient （IPG） strips. The images of the gels were acquired by the scanner and then analyzed by using PD-quest7.3 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots was incised from the gels and digested by trypsin. The peptide mass fingerprintings （PMF） was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and the proteins were identified by data searching in the Mascot-database. Part of differentially expression proteins were assayed by Western Blot. Results： Most of the gels were clear and successfully analyzed by PD-quest7.3 software. Most of the protein-spots concentrated on the area of 20-100Kda （Mw） and pH4-8. The average number of the protein-spots was 754 ± 64 in HS group and 777 ±48个 in LS group. The match rate was 87.6% between two groups. Five high expression proteins were found in HS group which were low expression in LS group, 3 high expression protein were found in LS group which were low expression in HS group. Reselts of Western Blot were in coincidence to proteomic result. Conclusion： The 2-DE gels image of HS group and LS group with early cervical cancer tissues treated by radiotherapy are successfully acquired. Some differentially expression proteins between the two groups are further confirmed by immunohistochemical assay.展开更多
基金Supported by the National Natural Science Foundation of China (No. 30800473)
文摘Objective:To reconstruct pEGFP-C2-L539fs/47,a HERG nonsense mutant in eukaryotic expression plasmid,and observe the fusion protein expressed in HEK293 cells(human embryo kidney cells).Methods:After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I,the small product fragment,from pcDNA3-L539fs/47,was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase.pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing.pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect,respectively.The expression of fusion protein in HEK293 cells was detected through immunofluorescence,laser confocal imaging scanning in vivo,Western blot and PCR.Results:Mutation region cDNA fragment(about 1 kb) and target vector fragment(about 7.2 kb) were ligated after purification and gel recovery.Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47,constructed approximately 8.2 kb,sequencing consistent with template gene.The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than60%.Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD,the expression of pEGFP-C2 fusion protein size of approximately 90 KD.The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis.Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells.Conclusion:pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells,which laid a foundation for the further study on L539fs/47.
文摘Early detection of pregnancy is advantageous to breeders. However, tools that were used in the early pregnancy detection are expensive, laboratory-based and not efficient and applicable enough in the field to farmers. Thus, there is a need to find a biomarker which not only can detect pregnancy but also could be applied to the livestock on the field. Proteomic approach was used in this study as to search for early pregnancy biomarker. When goat sera at day 14 of gestation were resolved by using two dimensional gel electrophoresis, five differentially expressed proteins were detected. Four of the proteins were identified as albumin precursors, immunoglobulin lambda light chain and zinc-alpha-2-glycoprotein. Pregnancy associated glycoprotein-1 was detected by using liquid chromatography mass spectrometry and the protein was validated by immunoblotting. These proteins have potential to be used as a biomarker for early pregnancy detection, however, more extensive analysis are needed to validate its possibility.
基金Supported by grants from the Hunan Natural Science foundation (No.06JJ4199)the Hunan Science Technology Foundation (No.2007SK3010)
文摘Objective： To investigate the proteornic differences between the high-sensitivity （HS） group and low-sensitivity （LS） group of cervical cancer treated by radiotherapy and confirm the radiotherapy sensitivity associated proteins in early cervical cancer. Methods： The fresh carcinoma tissues were collected from 10 untreated cervical cancer patients and preserved in the -80 ℃ refrigeratory. The tissues were classified into two groups： high sensitivity group （HS） and low sensitivity group （LS）, according to their response to radiotherapy. In the first part of our experiment, protein separating was performed by using two-dimensional gel electrophoresis （2-DE） with Amersham 18 cm linear pH 3-10 immobilized pH gradient （IPG） strips. The images of the gels were acquired by the scanner and then analyzed by using PD-quest7.3 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots was incised from the gels and digested by trypsin. The peptide mass fingerprintings （PMF） was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and the proteins were identified by data searching in the Mascot-database. Part of differentially expression proteins were assayed by Western Blot. Results： Most of the gels were clear and successfully analyzed by PD-quest7.3 software. Most of the protein-spots concentrated on the area of 20-100Kda （Mw） and pH4-8. The average number of the protein-spots was 754 ± 64 in HS group and 777 ±48个 in LS group. The match rate was 87.6% between two groups. Five high expression proteins were found in HS group which were low expression in LS group, 3 high expression protein were found in LS group which were low expression in HS group. Reselts of Western Blot were in coincidence to proteomic result. Conclusion： The 2-DE gels image of HS group and LS group with early cervical cancer tissues treated by radiotherapy are successfully acquired. Some differentially expression proteins between the two groups are further confirmed by immunohistochemical assay.
