鼠β.doc

1 鼠β作者:魏晓丽, 施桥发, 李虹, 李婉宜, 蒋忠华, 李明远【关键词】 mBD2;, 真核表达;, 免疫荧光;,RT Cloning and eukaryotic expression of murine betadefensin2(mBD2) [Abstract] AIM: To clone murine beta defensin2 gene (mBD2) and to express the mBD2 protEin eukaryotically. METHODS: Total RNA was isolated from the lungs of BALB/c mice which were injected with LPS in advance. The DNA fragment encoding mBD2 was amplified by RTPCR and inserted into the plasmid pcDNA31(+), which was then digested with EcoR Ⅰ and Xho I to construct the binant plasmid, pcDNA31(+)/mBD2. The pcDNA31(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA31(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBD2 was confirmed by immunofluorescent 2 staining and RTPCR. RESULTS: About 250 bp DNA fragment was amplified by RTPCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, (+)/mBD2, was essfully constructed after inserting the mBD2 fragment into pcDNA31(+). Most of SiHa cells transfected with pcDNA31(+)/mBD2 and screened by G418 could express the mBD2 protEIn, confirmed by immunofluorescent staining and RTPCR. CONCLUSION: The eukaryotic vector of pcDNA31(+)/mBD2 was essfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and antitumor mechanisms of the mBD2 protein. [Keywords]murine betadefensin2; eukaryotic expression; immunofluorescence; RTPCR [摘要] 目的: 对小鼠β防御素 2( mBD2 )基因进行克隆, 构建其真核表达载体, 筛选出稳定表达细胞株,并研究 mBD2 的生物学特性及其抗肿瘤机制。方法: 通过向 BALB/ c 小鼠腹腔注射内***(LPS), 建立小鼠急性时相反应, 取其肺组织提取总 RNA, 采用 RTPCR 方法扩增小鼠 mBD 2 基因,经 EcoR Ⅰ和 Xho Ⅰ双酶切后插入相同酶切的 3 (+ )真核表达载体, 对其进行酶切和测序鉴定。将构建好的真核表达质粒 (+)/mBD2 转染 SiHa 细胞, 采用 G418 进行稳定表达株的筛选, 用免疫荧光染色和 RTPCR 鉴定细胞内 mBD2 蛋白表达情况。结果: 提取小鼠肺组织总 RNA, 采用 RTPCR 方法扩增了 250 bp 左右的产物, 通过 EcoR Ⅰ和 Xho Ⅰ双酶切, 构建了真核表达质粒 (+)/mBD2 。 SiHa 被该质粒转染后,在 100 mg/L G41 8 浓度下筛选 20 d, 得到了稳定表达 mBD