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目的：构建并包装针对HTRA1 基因以及其1091T>C 突变基因(HTRA1-Mut) 的过表达慢病毒载体，以及建立稳定表达 HTRA1 及HTRA1-Mut 基因的人脑血管平滑肌细胞(HBVSMC)株。方法：采用RT-PCR 方法扩增HTRA1 及HTRA1-Mut 基因片段并将其连接于GV287 载体质粒，采用慢病毒包装三质粒系统(GV287/pHelper 1.0/pHelper 2.0)转染293T细胞，收集富含慢病毒颗粒的细胞上清液并标定病毒滴度，慢病毒感染经培养和鉴定的HBVSMC 细胞株。结果：成功构建含HTRA1 及HTRA1-Mut基因的慢病毒重组载体，PCR鉴定阳性的克隆进行测序和BLAST比对分析显示与源基因序列一致，并能够有效的感染并在293T 细胞中表达。表达载体包装后测定病毒滴度为：2E+8 TU/mL。过表达慢病毒感染后HBVSMC 有荧光表达，并且荧光率达80%以上，细胞生长良好传后细胞几乎无死亡现象。结论：成功构建了过表达HTRA1 及HTRA1-Mut基因的慢病毒表达载体，得到了较高滴度的病毒悬液，建成了稳定表达HTRA1 及HTRA1-Mut 基因的HBVSMC细胞株，为进一步探讨HTRA1 基因及突变后细胞的功能变化提供了良好的研究工具。

英文摘要:

Objective:To construct the lentiviral vector containing HTRA1 as well as its mutant gene(HTRA1-Mut,1091T>C) and express themin HBVSMCs.Methods:HTRA1 and HTRA1-Mut gene regional fragment were amplified by RT-PCR and then cloned into the plasmid GV287.Lentiviral plasmid, pHelperl.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus. The lentiviral titer was detected. Collected lentivirus was applied to infect human vascular smooth muscle cells.Results:The recombinant lentiviral vectors were constructed and were confirmed by PCR and sequencing analysis. They could efficiently transfect 293T cells and express in the cells. The lentiviral titer was 2E+8 TU/ml. HBVSMCs produced fluorescent expression after lentivirus transfection, and HTRA1 gene expressed over 80%. Transfected HBVSMCs grew in order without inducing excessive death rate.Conclusion:HTRA1 and HTRA1-Mut recombined lentiviruses with high viral titer were successfully constructed and packaged, and the HTRA1 and HTRA1-Mut gene could be transfected into HBVSMCs with stable and high expression in infected cells, and these cells might be applied for mechanismresearches of HTRA1 gene.

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