人HNA-3a基因的克隆及其在HEK-293细胞的表达

陈丽; 周浩; 何宏天; 管政

doi:10.13898/j.cnki.issn.1000-2200.2019.01.002

人HNA-3a基因的克隆及其在HEK-293细胞的表达

Cloning of human HNA-3a gene and its expression in HEK-293 cells

摘要

摘要:
目的构建人HNA-3a基因全长的真核表达载体，探索一种检测抗人HNA-3a抗体的实验方法，观察HNA-3a蛋白表达情况。
方法参照GeneBank中人HNA-3a基因序列，设计并合成引物，采用RT-PCR方法反转录HNA-3a基因外显子片段全长，定向克隆到pEGFP-N3载体中，然后采用Lipofectamine^TM 2000试剂将含目的基因的pEGFP-N3表达载体转染至HEK-293细胞中进行稳定表达，并以免疫荧光和蛋白质免疫印迹法（WB）鉴定目的基因的表达情况。
结果免疫荧光和免疫印迹结果显示，目的基因在HEK-293细胞中获高效表达。
结论成功构建了高效表达HNA-3a蛋白的真核表达载体，为检测人血浆中抗HNA-3a抗体奠定了基础。

Abstract:
ObjectiveTo construct an eukaryotic expression vector containing full-length sequences of human HNA-3a gene, explore an experimental method for detecting human anti-HNA-3a antibody, and observe the protein expression of HNA-3a.
Methods The primers were designed and synthesized according to human HNA-3a gene sequences in Genebank.The full-length of HNA-3a gene was reversely transcribed using RT-PCR, and cloned into pEGFP-N3 vector.The pEGFP-N3 expression vector containing human HNA-3a gene was transfected into the HEK-293 cells for transient expression using Lipofectamine^TM 2000 reagent, and the expression of the target gene was identified by Western blotting.
Results The eukaryotic expression vector containing the full-length sequences of human HNA-3a gene was successfully constructed, and the target gene was highly expressed in HEK-293 cells.
Conclusions The eukaryotic expression vector with high expression of HNA-3a protein is successfully constructed, which lays the foundation for the detection of the anti-HNA-3a antibody in human plasma.

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