Pin1真核表达载体的构建及鼻咽癌稳定表达细胞的鉴定

Construction of Pin1 eukaryotic expression vector and identification of stable expression cells of nasopharyngeal carcinoma

目的构建Pin1真核表达载体并筛选鼻咽癌稳定表达细胞株。方法提取HEK293细胞RNA行逆转录,扩增Pin1基因编码区,将其克隆到真核表达载体pcDNA6B中,菌液PCR鉴定并测序;将成功克隆的质粒转染至CNE1细胞,杀稻瘟素筛选和Western Blot鉴定稳定表达Pin1细胞。结果成功扩增Pin1编码区,并克隆至真核表达载体pcDNA6B中;Pin1过表达载体转染至CNE1细胞并筛选稳定表达细胞后,Western Blotting检测结果显示Pin1表达水平明显增加。结论成功构建Pin1过表达载体并筛选出鼻咽癌稳定表达细胞,可用于后续研究。

Objective To construct the Pin1 eukaryotic expression vector and screen stable expression cells of nasophary-ngeal carcinoma.Methods The extracted HEK293 RNA was reversely transcripted.The coding region of Pin1 gene was amplified and then cloned into the eukaryotic expression plasmid pcDNA6B,followed by sequencing validation.The successfully constructed plasmid was transfected into CNE1 cells,and the stable Pin1 expression cells were selected with blasticidin and then determined by Western blot.Results The coding region of Pin1 gene was successfully amplified,and cloned into the expression vector pcDNA6B.After transfected with Pin1 overexpression vectors,stable Pin1 expression cells were identified.Expression of Pin1 was upregulated in CNE1 cells.Conclusion Pin1 overexpression vector is successfully constructed.Stable Pin1 overexpression cells of nasopharyngeal carcinoma is identified and could be used for the continued study.