摘要
目的建构对人类表皮生长因子样结构域7(EGFL7)基因真核表达载体以及稳定性转染EA.hy926细胞系的科学评价体系。方法采用实时荧光定量聚合酶链式反应(PCR)方法获得EGFL7的开放阅读框(ORF),借助双酶切方式,使得EGFL7能够被克隆至真核表达载体(pEGFP-N1)中并获取到已经重组好的质粒pEGFP-N1-EGFL7。在进一步接受测序和鉴定处理后,借助电转仪对EA.hy926细胞进行转染,然后通过G418筛查进而建构起稳定性转染EA.hy926细胞系。使用实时荧光定量PCR和免疫印迹法检测EGFL7基因及蛋白在EA.hy926细胞系中的表达。结果转染EGFL7的EA.hy926细胞的EGFL7基因和蛋白水平均明显高于对照质粒转染组和空白对照组(P<0.05)。结论成功建构人EGFL7真核表达载体并鉴定了EGFL7在EA.hy926细胞系中的过表达,为探讨EGFL7对早产儿支气管肺发育不良的影响奠定基础。
Objective To construct a scientific evaluation system for human epidermal growth factor-like domain 7(EGFL7)eukaryotic expression vector and stably transfected EA.hy926 cell line.Methods The open reading frame(ORF)of EGFL7 obtained by real-time quantitative PCR,and EGFL7 could be cloned into the eukaryotic expression vector peGFP-N1 by double enzyme digestion method,and then the recombinant plasmid PEGFP-N1-EGFL7 was obtained.After receiving sequencing and identification,EA.hy926 cells were transfected with the aid of electrotransduction apparatus,and stable transfection EA.hy926 cell lines were constructed after G418 screening.The expression of EGFL7 gene and protein in EA.hy926 cell line was detected by real-time quantitative PCR and western blotting.Results The level of EGFL7 MRNA and protein in EA.hy926 cells transfected with EGFL7 was significantly higher than that in control group and blank control group(P<0.05).Conclusion The successful construction of human EGFL7 eukaryotic expression vector will lay a foundation for the study of the role of EGFL7 in preterm infants of bronchopulmonary dysplasia.
作者
丁悦
黄为民
唐丽君
DING Yue;HUANG Weiming;TANG Lijun(Department of Neonatology,The South Hospital of Southern Medical University,Guangdong,Guangzhou 510515,China)
基金
广东省自然科学基金项目(编号:2018A0303130288)。
