Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET w...Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.展开更多
Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in t...Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.展开更多
This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovir...This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.展开更多
目的:探讨重组腺病毒载体在真核细胞中稳定表达阳离子抗菌肽——β-防御素2的可行性。方法:采用RT-PCR方法扩增得到大鼠β-防御素2(rBD 2)基因片段,定向插入到腺病毒穿梭质粒pShuttle-CMV中,重组质粒pShuttle-CMV-rBD 2转化E.coli B J51...目的:探讨重组腺病毒载体在真核细胞中稳定表达阳离子抗菌肽——β-防御素2的可行性。方法:采用RT-PCR方法扩增得到大鼠β-防御素2(rBD 2)基因片段,定向插入到腺病毒穿梭质粒pShuttle-CMV中,重组质粒pShuttle-CMV-rBD 2转化E.coli B J5183-AD-1后,与腺病毒基因组质粒pA dE asy-1发生同源重组,重组质粒pA dE asy-rBD 2转染293细胞进行腺病毒表达载体的包装。将包装成功的腺病毒感染cos-7细胞,并建立SD大鼠腺病毒感染模型,进行β-防御素2多肽的体外和体内表达,采用W estern b lot与免疫组化方法检测其表达。结果:重组腺病毒表达载体构建成功,包含大鼠β-防御素2基因,滴度达到109PFU/m l,W estern b lot与免疫组化方法分别检测到β-防御素2在SD大鼠体外、体内的表达。结论:重组腺病毒载体能在真核细胞中稳定表达阳离子抗菌肽——β-防御素2。展开更多
Aim To construct an efficient recombinant viral vector for gene therapy.Methods(First-generation) adenovirus(Ad) vector was modified with the RGD peptide inserted into the fiber.Both in vitro and in vivo experiments o...Aim To construct an efficient recombinant viral vector for gene therapy.Methods(First-generation) adenovirus(Ad) vector was modified with the RGD peptide inserted into the fiber.Both in vitro and in vivo experiments of gene expression in different tumor cells with conventional and recombinant vectors were conducted.RT-PCR was used for detecting the expression of coxackievirus and adenovirus receptor and integrin at the surface of Meth-A cells.Results Fiber-mutant adenovirus vector showed a notably enhanced gene expression in A2058,B16BL6,OV-HM,and Meth-A tumor cells compared with that of conventional ones.In vivo study carried out using Meth-A tumor-bearing mice also demonstrated that the intra-tumoral injection of recombinant adenovirus induced strong gene expression in these CAR-deficient tumor cells. Conclusion The recombinant vector can be a promising one for effective cancer gene therapy.展开更多
基金Projects from China National Department of Science and Technology(81070465)a project from China Jiangsu Province (BM2008151)
文摘Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.
基金supported by projects from China National Nature Foundation (81170559)Jiangsu Health Foundation(XK02200901-NG09)
文摘Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.
文摘This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.
文摘目的:探讨重组腺病毒载体在真核细胞中稳定表达阳离子抗菌肽——β-防御素2的可行性。方法:采用RT-PCR方法扩增得到大鼠β-防御素2(rBD 2)基因片段,定向插入到腺病毒穿梭质粒pShuttle-CMV中,重组质粒pShuttle-CMV-rBD 2转化E.coli B J5183-AD-1后,与腺病毒基因组质粒pA dE asy-1发生同源重组,重组质粒pA dE asy-rBD 2转染293细胞进行腺病毒表达载体的包装。将包装成功的腺病毒感染cos-7细胞,并建立SD大鼠腺病毒感染模型,进行β-防御素2多肽的体外和体内表达,采用W estern b lot与免疫组化方法检测其表达。结果:重组腺病毒表达载体构建成功,包含大鼠β-防御素2基因,滴度达到109PFU/m l,W estern b lot与免疫组化方法分别检测到β-防御素2在SD大鼠体外、体内的表达。结论:重组腺病毒载体能在真核细胞中稳定表达阳离子抗菌肽——β-防御素2。
文摘Aim To construct an efficient recombinant viral vector for gene therapy.Methods(First-generation) adenovirus(Ad) vector was modified with the RGD peptide inserted into the fiber.Both in vitro and in vivo experiments of gene expression in different tumor cells with conventional and recombinant vectors were conducted.RT-PCR was used for detecting the expression of coxackievirus and adenovirus receptor and integrin at the surface of Meth-A cells.Results Fiber-mutant adenovirus vector showed a notably enhanced gene expression in A2058,B16BL6,OV-HM,and Meth-A tumor cells compared with that of conventional ones.In vivo study carried out using Meth-A tumor-bearing mice also demonstrated that the intra-tumoral injection of recombinant adenovirus induced strong gene expression in these CAR-deficient tumor cells. Conclusion The recombinant vector can be a promising one for effective cancer gene therapy.
