人源WNT16 HEK 293T稳转细胞株构建、蛋白纯化与活性检测方法研究  

Construction of Human WNT16 HEK 293T Stable Transforming Cell Line and Study on Protein Purification and Activity Assay

作　　者：王克芬 陈海迪 章嘉成 周鑫 邓成 武永康[1,2] Wang Kefen;Chen Haidi;Zhang Jiacheng(West China School of Medicine/Department of Laboratory Medicine,West China Hospital of Sichuan University,Chengdu,Sichuan 610044;Jintang First People's Hospital,Chengdu,Sichuan 610499,China)

机构地区：[1]四川大学华西医院实验医学科,前沿医学疾病分子网络前沿科学中心,四川成都610044 [2]四川大学华西金堂县第一人民医院,四川成都610499

出　　处：《四川医学》2023年第12期1233-1238,共6页Sichuan Medical Journal

基　　金：成都市科技局课题(编号:2019-GH02-00006-HZ)。

摘　　要：目的无翅型MMTV整合位点家族成员16(WNT16)是一种重要的信号调节蛋白,参与多种细胞生物学过程的调控。本研究旨在构建稳定转染WNT16基因的HEK 293T细胞株,并利用多聚赖氨酸标签(HIS-Tag)实现高效纯化WNT16蛋白。方法使用TK-PCDH-copGFP-T2A-Puro载体,将WNT16 CDS-HIS序列通过XbaI和BamHI位点连接载体,构建慢病毒载体质粒;将该质粒与pSPAX2和pMD2.G一起共转染HEK 293T细胞,制备携带WNT16基因的慢病毒;利用慢病毒感染HEK 293T细胞,并通过嘌呤霉素筛选和Western Blot鉴定稳定过表达WNT16的细胞株;利用镍螯合琼脂糖凝胶与HIS标签的高亲和性纯化WNT16蛋白,使用考马斯亮蓝染色和Western Blot法检测纯化效果;用纯化的WNT16蛋白刺激Jurkat细胞,使用Western Blot法检测Jurkat细胞中β-catenin信号通路蛋白的水平,以评估WNT16蛋白的活性。结果成功构建过表达WNT16基因的慢病毒表达载体,并用其感染HEK 293T细胞。经过嘌呤霉素筛选和Western Blot鉴定,我们获得了稳定表达WNT16的HEK 293T细胞株。使用HIS标签成功纯化了WNT16蛋白,并用考马斯亮蓝染色和Western Blot法检测了纯化效果。最后,我们用纯化的WNT16蛋白刺激Jurkat细胞,并用Western Blot法检测了Jurkat细胞中β-catenin信号通路蛋白的水平,WNT16蛋白能够显著增加Jurkat细胞中β-catenin蛋白的表达,表明纯化的WNT16蛋白具有激活WNT/β-catenin通路的生物学活性。结论本研究成功实现了WNT16蛋白的高效表达和纯化,并初步研究了其功能。这为进一步探究WNT16在细胞生物学中的作用提供了重要的实验基础。Objective Wnt family member 16(WNT16)is an important signaling regulator protein that participates in the regulation of various cellular biological processes.The aim of this study was to construct a stable transfected HEK 293T cell line with WNT16 gene and achieve efficient purification of WNT16 protein using HIS tag.Methods Using TK-PCDH-copGFPT2A-Puro vector,WNT16 CDS-HIS sequence was connected to the vector through XbaI and BamHI sites,and a lentiviral vector plasmid overexpressing WNT16 was constructed.The plasmid was co-transfected with pSPAX2 and pMD2.G into HEK 293T cells to prepare lentivirus carrying WNT16 gene.WNT16 gene was introduced into HEK 293T cells by lentivirus infection,and stable overexpression of WNT16 in HEK 293T cells was obtained by puromycin screening and Western Blot identification.WNT16 protein was purified by nickel chelating agarose gel and HIS tag with high affinity,and the purification results were detected by Coomassie brilliant blue staining and Western Blot method.Jurkat cells were stimulated with purified WNT16 protein,and the level ofβ-catenin signaling pathway protein in Jurkat cells was detected by Western Blot to evaluate the activity of WNT16 protein.Results We successfully constructed a lentiviral expression vector for WNT16 gene and infected HEK 293T cells with it.After puromycin screening and Western Blot identification,we obtained stable WNT16-expressing HEK 293T cell lines.We purified WNT16 protein using HIS tag and verified the purification by Coomassie brilliant blue staining and Western Blot.Finally,we stimulated Jurkat cells with the purified WNT16 protein and measured the level ofβ-catenin signaling pathway protein in Jurkat cells by Western Blot.WNT16 protein significantly increased the expression ofβ-catenin protein in Jurkat cells,indicating that the purified WNT16 protein had biological activity to activate WNT/β-catenin pathway.Conclusion This study successfully achieves efficient expression and purification of the WNT16 protein,and preliminary investigat

关 键 词：WNT16 HEK 293T细胞 过表达 稳转细胞株 纯化 HIS标签 

分 类 号：R789[医药卫生—口腔医学]