Construction and identification of eukaryotic expression plasmid of human estrogen receptor β1 and its variant

Abstract

Objective To construct eukaryotic expression plasmid of human estrogen receptor β1(ERβ1) and the 5th to 8th exon missing variant ERβ△5-8(in this paper referred to as ERβ△). Methods Genomic fragment of ERβ1 and ERβ△ was amplified by RT-PCR. The recombinant plasmid was cleared by restriction endonuclease and cloned into eukaryotic expression vector IRES2-EGFP, which carried EGFP gene and Myc report gene and then transformed into the E.coli DH5α. The recombinant plasmid was identified by agarose gel electrophoresis. The genomic fragment of ERβ1 and ERβ△ was determined and analyzed by DNA sequencing. Results Eukaryotic expression plasmid was identified by PCR and restriction endonuclease. Sequencing results show that the length of fragments is the same with the CSD of ERβ1 and its variant in the GeneBank. Conclusion This study successfully constructs the eukaryotic cell express cloning IRES2-EGFP-ERβ1 and IRES2-EGFP-ERβ△. Key words: Gene cloning; Plasmid; Estrogen receptor β(ERβ)