筛选高表达CPEAN的H1299肺癌细胞株

筛选高表达CPEAN的H1299肺癌细胞株

背景与目的 N端截短的羧肽酶E（N-terminal truncated carboxypeptidase E, CPEΔN）是一个新的肿瘤转移相关蛋白。本研究旨在筛选高表达CPEΔN的H1299肺癌细胞株，为完成小鼠活体成像实验创造条件。方法构建CPEΔN的慢病毒表达载体。分别用CPEΔN慢病毒表达载体或对照慢病毒空载体转染H1299细胞，2μg/mL的嘌呤霉素加压筛选。Western blot分析CPEΔN蛋白的表达，荧光素酶报告基因实验分析荧光素酶对底物的分解作用。结果当感染倍数（multiple of infection, MOI）是20时，慢病毒对H1299细胞的转染效率可以达到80...

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Bibliographic Details
Published in:中国肺癌杂志 Vol. 18; no. 6; pp. 340 - 344
Main Author: 孙静 张桂荣 王虹月 申慧
Format: Journal Article
Language:Chinese
Published: 2015
Subjects:
N端截短体
慢病毒载体
羧肽酶E
肺肿瘤
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Summary:背景与目的 N端截短的羧肽酶E（N-terminal truncated carboxypeptidase E, CPEΔN）是一个新的肿瘤转移相关蛋白。本研究旨在筛选高表达CPEΔN的H1299肺癌细胞株，为完成小鼠活体成像实验创造条件。方法构建CPEΔN的慢病毒表达载体。分别用CPEΔN慢病毒表达载体或对照慢病毒空载体转染H1299细胞，2μg/mL的嘌呤霉素加压筛选。Western blot分析CPEΔN蛋白的表达，荧光素酶报告基因实验分析荧光素酶对底物的分解作用。结果当感染倍数（multiple of infection, MOI）是20时，慢病毒对H1299细胞的转染效率可以达到80%。CPEΔN高表达H1299细胞株（H1299-CPEΔN）和对照慢病毒载体表达H1299细胞株（H1299-control）中CPEΔN蛋白的表达量为4：1。H1299-CPEΔN和H1299-control均能够有效分解荧光素酶底物，可以满足活体成像实验的需求。结论筛选出高表达CPEΔN的H1299肺癌细胞株，为活体成像实验的开展创造了条件，也为进一步解释CPEΔN促进肿瘤转移的分子机制奠定了基础。
Bibliography:Jing SUN, Guirong ZHANG, Hongyue WANG, Hui SHEN （Biotherapy Research Center, Liaoning Cancer Hospital and Institute, Shenyang 110042, China）
Lung neoplsms; N-terminal truncated carboxypeptidase E; Lentiviral vector
Background and objective hTe N-terminal truncated carboxypeptidase E （CPEΔN） protein is a novel biomarker of tumor metastasis. hTis study screened the H1299 cell line with a highly expressed CPEΔN gene forin vivo imag-ing experiment.Methods Human CPEΔN gene was cloned into the luciferase lentiviral vector. H1299 cells transduced with CPEΔN or control lentiviral vectors were selected with 2 μg/mL puromycin. hTe expression of CPEΔN was identiifed through Western blot analysis, and luciferase activity was measured using luciferase reporters.Results hTe human CPEΔN lentiviral expression vector was successfully constructed. hTe transfection rate of H1299 cells by the lentivirus achieved 80%, with an infection multiplicity of 20. hTe H1299 cell line with high CPEΔN （H1299-CPEΔN） expression was established,
ISSN:1009-3419
1999-6187