带Myc、His标签的SPAG4L真核表达载体的构建与表达

Construction of eukaryotic expression vector of SPAG4L tagged with Myc and His

摘要:

为了获得带有His、Myc 双标签的SPAG4L蛋白，建立能够稳定表达该蛋白的细胞系，本研究利用PCR技术从人睾丸cDNA中扩增SPAG4L开放阅读框，构建到pcDNA3.1(+)myc-his真核表达载体中进行测序和双酶切验证。将pcDNA3.1/myc-His(-)A/SPAG4L质粒和空白载体分别转染HeLa细胞，G418筛选后建立稳定转染细胞系。用Western blotting和免疫荧光技术对新建立的稳定转染细胞系进行检测。结果表明，成功扩增了SPAG4L基因，构建到pcDNA3.1/myc-His(-)A真核表达载体后，经酶切和测序验证所插入的SPAG4L序列完全正确；pcDNA3.1/myc-His(-)A/SPAG4L转染HeLa细胞后，经G418筛选后建立了稳定转染细胞系。Western blotting检测后发现，新建立的细胞系能够正确表达SPAG4L及其标签蛋白，进一步的免疫荧光实验发现，SPAG4L能够和内质网标签蛋白PDI共定位。研究结果所提供的稳定转染细胞系将为下一步进行免疫共沉淀和pull-down实验提供了有力的工具。

Abstract:

The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.