摘要
目的构建含LRIG1胞外段+跨膜段(ET)及跨膜段+胞内段(TC)基因的真核表达载体,并研究它们的亚细胞定位。方法以人LRIG1全长的质粒p3XFLAG-CMV-9-LRIG1为模板,PCR扩增出3XFLAG-LRIG1-ET和LRIG1-TC,并将之分别亚克隆入真核表达载体pIRES2-EGFP-N1和p3XFLAG-CMV-9中。酶切和DNA测序鉴定,脂质体法将构建的质粒转染U251细胞,观察标记基因荧光蛋白EGFP的表达,间接免疫荧光检测标记基因3XFLAG的表达。结果构建的质粒序列正确,转染细胞后可检测到EGFP和3XFLAG基因的表达;LRIG1-ET和LRIG1-TC均主要表达在U251的胞膜上,少量表达在胞浆。结论包含LRIG1亚单位ET和TC的真核表达载体均构建成功,其表达产物均主要位于胞膜,少量位于胞浆。
Objective To construct and identify two chimeric eukaryotic expression vectors containing extracellular ＆- transmembrane region （ET） and transmembrane ＆ cytoplasmic region （TC） of a newly identified human LRIG1 gene. Methods The sequences of 3XFLAG-LRIG1-ET and LRIG1-TC were obtained by PCR from a plasmid containing full length of the LRIG1 gene, p3XFLACrCMV-9-LRIG1. The PCR products were cloned into pIRES2-EGFP-N1 and p3XFLAG-CMV-9 expression vector respectively. The accuracy of DNA sequence was confirmed by restriction endonuclease analysis and DNA sequencing. The constructs were transfected into U251 MG cell line and their subcellular localizations were determined by fluorescent microscopy and immunofluorescence assay. Results The sequences of the two constructed plasmids were correct. The expression of EGFP and FLAG could be detected in the U251 MG cell. Both LRIG1-ET and LRIG1-TC seemed to be located mainly on cells＇ surface and partly in cytoplasm. Conclusion The two eukaryotic expression vectors, pIRES2-EGFP-3XFLAG-LRIG1-ET and p3XFLAG-LRIG1-TC, were successfully constructed and could be used in latter researches.
出处
《华中医学杂志》
CAS
2006年第3期191-192,194,共3页
Central China Medical Journal
