H-2Kd基因siRNA表达质粒的构建及在小鼠LAK细胞的表达

山东大学学报(医学版)

H-2K^d基因siRNA表达质粒的构建及在小鼠LAK细胞的表达

庄学伟¹，夏西燕²，单宁宁¹，王洪春¹，张义¹，杨晓静¹，李晓丽¹，赵胜梅¹，邹雄¹

1. 山东大学齐鲁医院检验科, 济南 250012；2. 山东省济南市卫生学校免疫学教研室，济南 250022

收稿日期:2008-01-10 修回日期:1900-01-01 出版日期:2008-06-16 发布日期:2008-06-16
通讯作者: 邹雄

Construction of a plasmid expressing siRNA aimed at the H-2K^d gene and expression of H-2K^d in mouse LAK cells

ZHUANG Xue-wei¹, XIA xi-yan², SHAN Ning-ning¹, WANG Hong-chun¹, ZHANG Yi¹, YANG Xiao-jing¹, LI Xiao-li¹, ZHAO Sheng-mei¹，ZOU Xiong¹

1．Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan 250012, China；2. Department of Immunity, Jinan Health School, Jinan 250022, China

Received:2008-01-10 Revised:1900-01-01 Online:2008-06-16 Published:2008-06-16
Contact: ZOU Xiong

摘要/Abstract

摘要： 目的构建针对BALB/C小鼠H-2K^d基因siRNA表达质粒，观察其在小鼠LAK细胞的表达，为进一步研究H-2K^d基因功能奠定基础。方法设计siRNA干涉靶序列，体外合成两段互补的寡核苷酸，通过与线性化的pSilencer 3.0-H1连接,转化大肠杆菌DH5a扩增纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列。采用siRNA表达质粒封闭小鼠LAK细胞MHC-I（H-2K^d）的表达，同时设空转染组和无关序列组，流式细胞术检测不同组别靶蛋白的表达。结果纯化的质粒分子量为2.8Kb,插入的寡核苷酸序列与设计的序列完全相符, 流式细胞术检测证实siRNA能够抑制靶蛋白的表达。结论成功构建了针对H-2K^d基因的siRNA表达质粒并抑制了其表达。

关键词: H-2K^d基因, 小干扰RNA, 质粒

Abstract: To construct the siRNAs expressing plasmid aiming at the H-2K^d gene and to explore the H-2K^d expression in mouse LAK cells and function of H-2K^d with the RNA interference technology. MethodsFirst, a target sequence of the H-2K^d gene was selected, then according to the sequence, two complementary oligonucleotides were lisated into psilencer 3.0-H1, which was transformed into DH5a bacteria, and then it was amplified and purified to get the plasmid. The purified plasmid was identified by gel electrophoresis and sequencing. H-2K^d-targeting siRNA plasmid was used in spleen lymphocytes of BALB/C mice. Flow cytometry(FCM) was used to determine the expression of H-2K^d on the transfected and control cells. ResultsGel electrophoresis and sequencing results showed that the plasmid was about 2.8kb which was identical with the positive control, and the sequence was identical with what had been inserted. Also siRNA treatment significantly inhibited the expression of the targeted protein. Conclusion siRNAs expression plasmids aiming at the H-2K^d gene have been successfully constructed and could inhibit the H-2Kd expression.

Key words: Plasmid, H-2K^dgene, siRNA

中图分类号:

R730.4

庄学伟,夏西燕,单宁宁,王洪春,张义,杨晓静,李晓丽,赵胜梅,邹雄 . H-2K^d基因siRNA表达质粒的构建及在小鼠LAK细胞的表达[J]. 山东大学学报(医学版), 2008, 46(6): 590-593.

ZHUANG Xue-wei,XIA xi-yan,SHAN Ning-ning,WANG Hong-chun,ZHANG Yi,YANG Xiao-jing,LI Xiao-li,ZHAO Sheng-mei,ZOU Xiong. Construction of a plasmid expressing siRNA aimed at the H-2K^d gene and expression of H-2K^d in mouse LAK cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2008, 46(6): 590-593.

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