KCTD10基因慢病毒过表达载体的构建及病毒包装
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摘要
目的:构建KCTD10基因表达载体并包装成病毒颗粒。方法:合成KCTD10基因全长片段,构建p EZLv105-KCTD10载体,转化至大肠杆菌Stbl3中进行筛选。利用PCR及测序鉴定KCTD1-Lv105阳性克隆。将KCTD10-Lv105重组质粒和阳性对照e GFP-Lv105质粒同时与HIV包装质粒混合物共转染于293T细胞进行慢病毒包装,收集浓缩病毒颗粒。将所得病毒颗粒悬浮梯度稀释后感染293T细胞,利用Puromycin进行药筛滴度测定。结果:测序比对检测KCTD10-Lv105重组载体成功构建,荧光显微镜下观察e GFP-Lv105病毒包装成功,药筛检测KCTD10-Lv105浓缩病毒滴度为1.2×108TU/m L。结论:成功包装好过表达KCTD10的慢病毒颗粒,并为进一步研究KCTD10基因在胶质瘤的发生发展中的作用奠定了基础。
Objective To construct a lentiviral vector over-expressing KCTD10 and packaged it into viral particles. Methods Synthesized the full length of KCTD10 gene fragments, Constructed p EZ-Lv105-KCTD10 vector and conducted it into Stbl3 for screening. use PCR and DNA sequencing to identify the KCTD10-Lv105 positive clones. The recombinant plasmids of KCTD10-Lv105 and the positive control e GFP-Lv105 were co-transfected into 293 T cellswith the HIV packaging plasmid mixture, then viruses were collected, suspendedand dilutedto infect 293 T cells, Finally, viral titer was detected through Puromycin resistance screening. Results Lentiviral recombinant vectors KCTD10-Lv105 were successfully constructed, which was confirmed by sequencing analysis. e GFP-Lv105 virus was successfully packaged by fluorescenceobservation. The final titer detected by drug screening of concentrated viruses was 1.2×108 TU/m L Conclusion The KCTD10 overexpressed viruses were packaged successfully, which laid a foundation for further study of KCTD10 gene on the role of carcinogenesis and progress of gliomas.
Objective To construct a lentiviral vector over-expressing KCTD10 and packaged it into viral particles. Methods Synthesized the full length of KCTD10 gene fragments, Constructed p EZ-Lv105-KCTD10 vector and conducted it into Stbl3 for screening. use PCR and DNA sequencing to identify the KCTD10-Lv105 positive clones. The recombinant plasmids of KCTD10-Lv105 and the positive control e GFP-Lv105 were co-transfected into 293 T cellswith the HIV packaging plasmid mixture, then viruses were collected, suspendedand dilutedto infect 293 T cells, Finally, viral titer was detected through Puromycin resistance screening. Results Lentiviral recombinant vectors KCTD10-Lv105 were successfully constructed, which was confirmed by sequencing analysis. e GFP-Lv105 virus was successfully packaged by fluorescenceobservation. The final titer detected by drug screening of concentrated viruses was 1.2×108 TU/m L Conclusion The KCTD10 overexpressed viruses were packaged successfully, which laid a foundation for further study of KCTD10 gene on the role of carcinogenesis and progress of gliomas.
引文
[1]张敏,潘剑珍,刘彤,等.KCTD10在神经胶质瘤中表达的初步研究[J].湖南师范大学学报(医学版),2016,13(01):6-8.
[2]张敏.KCTD10对神经胶质瘤侵袭及迁移的影响[D].湖南师范大学,2016.
[3]Zhou J,Hu X,Xiong X,et al.Cloning of two rat PDIP1 related genes and their interactions with proliferating cell nuclear antigen[J].J Exp Zool A Comp Exp Biol,2005,303(3):227-240.
[4]Zhou J,Ren K,Liu X,et al.A novel PDIP1-related protein,KCTD10,that interacts with proliferating cell nuclear antigen and DNA polymerase delta[J].Biochim Biophys Acta,2005,1729(3):200-203.
[5]任凯群.KCTD10基因的功能研究[D].南京大学,2011.
[6]Hu X,Gan S,Xie G,et al.KCTD10 is critical for heart and blood vessel development of zebrafish[J].Acta Biochim Biophys Sin(Shanghai),2014,46(5):377-386.
[7]Ren K,Yuan J,Yang M,et al.KCTD10 is involved in the cardiovascular system and Notch signaling during early embryonic developmen[tJ].PLoS One,2014,9(11):e112275.
[8]Qu DW,Liu Y,Wang L,et al.Glial cell line-derived neurotrophic factor promotes proliferation of neuroglioma cells by up-regulation of cyclins PCNA and Ki-67[J].Eur Rev Med Pharmacol Sci,2015,19(11):2070-2075.
[9]Wang Y,Zheng Y,Luo F,et al.KCTD10 interacts with proliferating cell nuclear antigen and its down-regulation could inhibit cell proliferatio[nJ].J Cell Biochem,2009,106(3):409-413.
[10]Nabors LB,Suswam E,Huang Y,et al.Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells:a role for RNA stabilization and HuR[J].Cancer Res,2003,63(14):4181-4187.
[2]张敏.KCTD10对神经胶质瘤侵袭及迁移的影响[D].湖南师范大学,2016.
[3]Zhou J,Hu X,Xiong X,et al.Cloning of two rat PDIP1 related genes and their interactions with proliferating cell nuclear antigen[J].J Exp Zool A Comp Exp Biol,2005,303(3):227-240.
[4]Zhou J,Ren K,Liu X,et al.A novel PDIP1-related protein,KCTD10,that interacts with proliferating cell nuclear antigen and DNA polymerase delta[J].Biochim Biophys Acta,2005,1729(3):200-203.
[5]任凯群.KCTD10基因的功能研究[D].南京大学,2011.
[6]Hu X,Gan S,Xie G,et al.KCTD10 is critical for heart and blood vessel development of zebrafish[J].Acta Biochim Biophys Sin(Shanghai),2014,46(5):377-386.
[7]Ren K,Yuan J,Yang M,et al.KCTD10 is involved in the cardiovascular system and Notch signaling during early embryonic developmen[tJ].PLoS One,2014,9(11):e112275.
[8]Qu DW,Liu Y,Wang L,et al.Glial cell line-derived neurotrophic factor promotes proliferation of neuroglioma cells by up-regulation of cyclins PCNA and Ki-67[J].Eur Rev Med Pharmacol Sci,2015,19(11):2070-2075.
[9]Wang Y,Zheng Y,Luo F,et al.KCTD10 interacts with proliferating cell nuclear antigen and its down-regulation could inhibit cell proliferatio[nJ].J Cell Biochem,2009,106(3):409-413.
[10]Nabors LB,Suswam E,Huang Y,et al.Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells:a role for RNA stabilization and HuR[J].Cancer Res,2003,63(14):4181-4187.