TCAB1慢病毒表达载体的构建及其在人子宫内膜间质细胞中的表达(1)
[摘要] 目的 构建TCAB1基因的慢病毒过表达载体并验证在人子宫内膜间质细胞株(St-T1b)过表达效果。 方法 通过PCR获取TCAB1全长基因，进行TA克隆，进一步亚克隆构建TCAB1慢病毒表达载体，包被慢病毒后感染St-T1b细胞，通过观察荧光表达和实时定量PCR验证其表达。 结果 成功构建PLVX-TCAB1-IRES-ZsGreen1慢病毒表达载体并获得相应的慢病毒。荧光显微镜、实时定量PCR结果表明，TCAB1基因可在St-T1b细胞中实现过表达效果。 结论 成功构建人TCAB1基因特异性的重组慢病毒过表达载体，且证实包被的慢病毒可实现TCAB1基因在St-T1b细胞中的过表达，为后续人TCAB1在人子宫内膜间质细胞的功能研究奠定了基础。
[关键词] TCAB1；过表达；慢病毒；St-T1b细胞株
[中图分类号] R71 [文献标识码] A [文章编号] 1673-7210(2012)07(b)-0012-03
, 百拇医药
Construction of a lentiviral vector for TCAB1 and its stable expression in human endometrial stromal cell line
GU Lin1 SHI Yingli1* NI Jie1 CHEN Jie1 LI Ying2▲
1.The Nanjing MCH Hospital Affiliated to Nanjing Medical University, Jiangsu Province, Nanjing 210004, China; 2.The Family Planning Institute of Jiangsu Province, Jiangsu Province, Nanjing 210029, China
[Abstract] Objective To construct a lentiviral vector for TCAB1 and to establish a human endometrial stromal cell line (St-T1b) with TCAB1 over-expression. Methods Full length cDNA clone of TCAB1 gene was obtained by using methods of PCR, then TA clone was conducted to construct TCAB1 lentiviral vector. The recombinant lentiviral vectors were harvested and were used to infect St-T1b cells. Stable expression of TCAB1 in St-T1b cells was confirmed by immune-fluorescence staining and real-time PCR. Results The PLVX-TCAB1-IRES-ZsGreen1 lentiviral vector expressing TCAB1 gene was successfully established. The fluorescence and real-time PCR demonstrated that the expression level of TCAB1 in the lentivirus infected St-T1b cells was increased significantly. Conclusion A recombinant lentiviral vector expressing TCAB1 gene and St-T1b cells with TCAB1 stable over-expression were established successfully, laid an experimental basis on the studies of the functions and mechanisms of TCAB1.
, 百拇医药
[Key words] TCAB1; Over-expression; Lentivirus; St-T1b cells
TCAB1是人肿瘤细胞中近期发现的端粒酶蛋白组分，其表达缺失可使端粒酶复合物无法运送至染色体末端的端粒合成目的地，虽不影响端粒酶逆转录酶(hTERT)对自身携带RNA组分(hTR)的催化，但最终仍导致端粒形成受限。由于：①端粒机制与恶性肿瘤等细胞的增殖、迁移、侵袭属性密切相关[1-2]；②内异症虽为良性疾病，其远处种植和生长特性却与恶性肿瘤类似[3]。笔者前期研究中通过基因芯片实验发现，TCAB1异常表达于内异症异位内膜组织，提示TCAB1可能通过端粒机制参与了内异症异位内膜的生长、侵袭等病理机制，并介导了在内异症形成中的作用。为进一步研究TCAB1 功能及作用机制，笔者拟构建TCAB1的慢病毒表达载体。
1 材料与方法
1.1 实验材料
总RNA抽提试剂盒购自天根生化科技(北京)有限公司；PCR Taq酶购自TaKaRa公司；限制性内切酶(EcoRI和NotI)购自NEB公司；感受态细胞TOP10实验室自备；Agarose Gel DNA Purification Kit Ver2.0、TA克隆试剂盒购自TaKaRa；逆转录试剂盒(SuperScript ⅢFirst-Strand Synthesis System)购自invitrogen公司；测序委托上海英俊生物公司进行；慢病毒过表达载体(pLVX-IRES-ZsGreen)购自Clonetech公司；子宫内膜间质细胞株(St-T1b)购自ATCC。, 百拇医药(顾林 史颖莉 倪婕 陈捷 李瑛)
[关键词] TCAB1；过表达；慢病毒；St-T1b细胞株
[中图分类号] R71 [文献标识码] A [文章编号] 1673-7210(2012)07(b)-0012-03
, 百拇医药
Construction of a lentiviral vector for TCAB1 and its stable expression in human endometrial stromal cell line
GU Lin1 SHI Yingli1* NI Jie1 CHEN Jie1 LI Ying2▲
1.The Nanjing MCH Hospital Affiliated to Nanjing Medical University, Jiangsu Province, Nanjing 210004, China; 2.The Family Planning Institute of Jiangsu Province, Jiangsu Province, Nanjing 210029, China
[Abstract] Objective To construct a lentiviral vector for TCAB1 and to establish a human endometrial stromal cell line (St-T1b) with TCAB1 over-expression. Methods Full length cDNA clone of TCAB1 gene was obtained by using methods of PCR, then TA clone was conducted to construct TCAB1 lentiviral vector. The recombinant lentiviral vectors were harvested and were used to infect St-T1b cells. Stable expression of TCAB1 in St-T1b cells was confirmed by immune-fluorescence staining and real-time PCR. Results The PLVX-TCAB1-IRES-ZsGreen1 lentiviral vector expressing TCAB1 gene was successfully established. The fluorescence and real-time PCR demonstrated that the expression level of TCAB1 in the lentivirus infected St-T1b cells was increased significantly. Conclusion A recombinant lentiviral vector expressing TCAB1 gene and St-T1b cells with TCAB1 stable over-expression were established successfully, laid an experimental basis on the studies of the functions and mechanisms of TCAB1.
, 百拇医药
[Key words] TCAB1; Over-expression; Lentivirus; St-T1b cells
TCAB1是人肿瘤细胞中近期发现的端粒酶蛋白组分，其表达缺失可使端粒酶复合物无法运送至染色体末端的端粒合成目的地，虽不影响端粒酶逆转录酶(hTERT)对自身携带RNA组分(hTR)的催化，但最终仍导致端粒形成受限。由于：①端粒机制与恶性肿瘤等细胞的增殖、迁移、侵袭属性密切相关[1-2]；②内异症虽为良性疾病，其远处种植和生长特性却与恶性肿瘤类似[3]。笔者前期研究中通过基因芯片实验发现，TCAB1异常表达于内异症异位内膜组织，提示TCAB1可能通过端粒机制参与了内异症异位内膜的生长、侵袭等病理机制，并介导了在内异症形成中的作用。为进一步研究TCAB1 功能及作用机制，笔者拟构建TCAB1的慢病毒表达载体。
1 材料与方法
1.1 实验材料
总RNA抽提试剂盒购自天根生化科技(北京)有限公司；PCR Taq酶购自TaKaRa公司；限制性内切酶(EcoRI和NotI)购自NEB公司；感受态细胞TOP10实验室自备；Agarose Gel DNA Purification Kit Ver2.0、TA克隆试剂盒购自TaKaRa；逆转录试剂盒(SuperScript ⅢFirst-Strand Synthesis System)购自invitrogen公司；测序委托上海英俊生物公司进行；慢病毒过表达载体(pLVX-IRES-ZsGreen)购自Clonetech公司；子宫内膜间质细胞株(St-T1b)购自ATCC。, 百拇医药(顾林 史颖莉 倪婕 陈捷 李瑛)