Chen J F, Zhang Y F, Kang J M, Qin X Y, Wang M F, Wang G, Yang L H
Department of Hematology, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China.
Zhonghua Xue Ye Xue Za Zhi. 2016 Nov 14;37(11):971-975. doi: 10.3760/cma.j.issn.0253-2727.2016.11.009.
To construct pIRES2-ZsGreen1/F Ⅸ expression vector, using the pcDNA/FⅨ plasmid containing FⅨ cDNA as template, and express in HEK-293 cells. The total ORF of F Ⅸ gene was amplified from pcDNA/F Ⅸ plasmid, then the amplified fragment was clonded into the pIRES2-ZsGreen1 vector using the Infusion enzyme. The positive clones of eukaryotic expression vector of pIRES2-ZsGreen1/F Ⅸ were screened and expanded after transfection, then were constructed and confirmed by PCR and sequencing. Transient expression experiments were performed using HEK-293 cells transfected with the expression vectors and observed the expression of ZsGreen1 protein by confocal laser microscope. The relative expression levels of FⅨ mRNA, protein and FⅨ activity (FⅨ∶C) were detected by real time PCR (RT-PCR), immunofluorescence microscopy, One-Stage method, respectively. The expression vector, pIRES2-ZsGreen1/F Ⅸ, was successfully constructed and expressed in HEK-293 cells. RT-PCR detected the expression of F Ⅸ mRNA in HEK-293 cells and the immunofluorescence microscopy showed FⅨ protein distributed in the surrounding of nucleus. FⅨ∶C of cell lysates and cell culture fluid transfected with the expression vectors were (92.03 ± 0.29)% and (86.89 ± 8.78)%, respectively; while both F Ⅸ∶C of cell lysates and cell culture fluid transfected with or without the expression vectors were 0. The experimental results showed the expression vector, pIRES2-ZsGreen1/FⅨ, was successfully constructed , which provided experiment basement for the follow study on the location, function and molecular pathology of hemophilia B.
构建pIRES2-ZsGreen1/FⅨ表达载体，以含有FⅨ cDNA的pcDNA/FⅨ质粒为模板，在HEK-293细胞中进行表达。从pcDNA/FⅨ质粒中扩增FⅨ基因的全长开放阅读框（ORF），然后使用Infusion酶将扩增片段克隆到pIRES2-ZsGreen1载体中。转染后筛选并扩增pIRES2-ZsGreen1/FⅨ真核表达载体的阳性克隆，然后通过PCR和测序进行构建和鉴定。使用转染了表达载体的HEK-293细胞进行瞬时表达实验，并用共聚焦激光显微镜观察ZsGreen1蛋白的表达。分别通过实时荧光定量PCR（RT-PCR）、免疫荧光显微镜和一期法检测FⅨ mRNA、蛋白的相对表达水平以及FⅨ活性（FⅨ∶C）。成功构建了表达载体pIRES2-ZsGreen1/FⅨ并在HEK-293细胞中表达。RT-PCR检测到HEK-293细胞中FⅨ mRNA的表达，免疫荧光显微镜显示FⅨ蛋白分布在细胞核周围。转染表达载体的细胞裂解液和细胞培养液的FⅨ∶C分别为（92.03±0.29）%和（86.89±8.78）%；而未转染和转染空载体的细胞裂解液和细胞培养液的FⅨ∶C均为0。实验结果表明成功构建了表达载体pIRES2-ZsGreen1/FⅨ，为后续血友病B的定位、功能及分子病理学研究提供了实验基础。
